山东近海主要养殖动物的弧菌检测与现害防治的研究

本文从１９９９年秋季采集的山东近海鲈鱼、牙鲆、黑鲷、梭子蟹、牡蛎等５种主要养殖动物中分离出１１０个菌株,经形态、生理、生化测定,由计算机数值分类和《伯杰氏细菌鉴定手册》相结合,鉴定出溶藻胶弧菌、副溶血弧菌等９种菌属弧菌１种嗜水气单胞菌。文中尚对养殖生物弧菌病害的防治作了初步探讨。     

海水网箱区沉积物中硫酸盐还原菌（SRB）的分离纯化和鉴定

目前网箱养殖水域的硫酸盐还原菌（SRB）的危害表现日益严重,海水网箱沉积物中SRB分离鉴定尚属空白,针对不同属种的SRB开展生物修复具有一定的意义。在大亚湾大鹏澳网箱养殖沉积物中分离出25株硫酸盐还原菌纯菌株,通过对菌株的形态观察,生理生化特征和遗传特征的一系列研究,结果表明所有菌株均含有脱硫弧菌素,被鉴定为脱硫弧菌属（Desulfovibrio）其中CSRB2、CSRB4、ESRB2鉴定为脱硫脱硫弧菌（Desulfovibrio Desulfovibricans）,ASRB4、BSRB1菌株鉴定为脱硫脱硫弧菌河口亚种（Desulfovibrio Desulfovibricans subsp.aestuarii）,其余菌株鉴定为需盐脱硫弧菌种（Desulfovibrio salexigens）。此海区SRB种类数量特征是需盐脱硫弧菌〉脱硫脱硫弧菌〉脱硫脱硫弧菌河口亚种。     

养殖大黄鱼病原弧菌多重PCR检测技术的建立和应用

溶藻弧菌(Vibrio alginolyticus)、副溶血弧菌(vibrio parahaemolyticus)和哈维氏弧菌(Vibrio harveyi)是浙江省养殖大黄鱼(Pseudnosciaena crocea)弧菌病的主要致病菌.本研究选择针对溶藻弧菌和副溶血弧菌的胶原酶基因,哈维氏弧菌的部分ToxR基因的特异性,优化设计了3对特异性引物,通过进行多重PCR反应体系优化,多重PCR产物的测序鉴定与特异性和敏感性实验,建立了一种检测致病性弧菌的多重PCR检测方法.经过琼脂糖凝胶电泳后的条带分析判断,可以在一个PCR管中同时成功地检测这3种病原细菌,含溶藻弧菌、哈维氏弧菌和副溶血弧菌3种致病弧菌核酸的阳性对照样品分别扩增出大小为737 hp、382 bp和271 bp的预期产物,其灵敏度是102～102 CFU/mL.将该方法应用于检测人工感染后的养殖大黄鱼病鱼肝脏和肾脏,结果在6份组织样本中,5份检出原始感染菌株,与API 20E鉴定结果相符;对弧菌病流行季节采集的未发病的16份养殖大黄鱼组织样本和16份水体样本进行抽检,结果在1份大黄鱼组织样本中检出哈维氏弧菌,7份水体样本中检出这3种弧菌中的1种或2种,鉴定结果与API 20E鉴定结果符合率为93.75%.说明该方法不仅可以检测发病鱼,还可以检测无病症带菌大黄鱼以及带菌水样,且说明海洋水体中存在着大黄鱼弧菌病的致病菌.结果说明,多重PCR检测方法具有较高的敏感性与特异性,可以缩短检测时间,降低检测成本,该方法的建立对养殖大黄鱼弧菌病的快速诊断和分子流行病学的调查具有重要意义.     

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。     

九孔鲍消化道及养殖水体中弧菌胞外毒力因子的分析

从汕尾健生鲍鱼养殖场成鲍养殖水体和消化道中分离筛选到22株弧菌,其中14株来自成鲍消化道,8株来自养殖水体。本文对这两种不同来源的菌株进行了致病因子(胞外酶及溶血毒素)的分析比较,同时采用API条带法对其进行了种类鉴定。结果发现,消化道中除1株溶藻弧菌和3株最小弧菌外,其余均为河流弧菌;而水体中除副溶血弧菌和创伤弧菌各1株外,其他也均为河流弧菌。实验还发现,在5株胞外酶分泌能力最强的弧菌中,只有Sh031株是副溶血弧菌,另外4株(Bh14、Sh02、Sh08、Sh05)均为的河流弧菌。结果显示,无论是养殖水体还是消化道,河流弧菌都应视为一种海水养殖贝类(鲍)的主要条件致病菌,是能力较强的胞外致病因子生产者。     

长毛对虾“红腿病”的防治研究

随着对虾养殖业的迅速发展,对虾常见的病害的防治已成为急待解决的课题。从1987年以来,我们调查了福建沿海养殖场的对虾病害,发现由弧菌引起的“红腿病”是一种常见的、严重的流行病,死亡率高、造成经济损失大。有关养殖对虾弧菌病的研究,国外虽有过一些报道,但主要集中于病原细菌的分离、鉴定的研究上。近年来,我国养殖对虾弧菌病的研究也取得了进展,证实了溶藻性弧菌和副溶血性弧菌等能引起对虾的流行性弧菌病。最近郑国兴等报告鳗弧菌是中国对虾“红腿病”的主要病原菌。     

Biolog GN法对不同地区养殖对虾弧菌区系的比较研究

采用Biolog细菌鉴定技术,分析来自5个国家的4个对虾养殖品种苗期及部分养成期虾体上的185株弧菌(其中24株来自成虾).结果表明:来源、种类不同的养殖对虾苗期的主要弧菌的区系组成相似,溶藻弧菌(Vibrio alginolyticus)和鲨鱼弧菌(V. carchariae)(即哈维氏弧菌V. harveyi)是普遍存在的种类,同一种对虾在不同地区养殖,其区系组成略有差异;哈维氏弧菌多为对虾苗期致病菌,副溶血弧菌(V.parahaemolyticus)主要为成虾致病菌;在健康虾苗和发病虾苗体内都可分离到溶藻弧菌.     

养殖大黄鱼溶藻酸弧菌病病原菌传染途径的研究

从养殖大黄鱼病鱼的肝脏和脾脏内分离、纯化到溶藻酸弧菌，经回归感染试验证实它是该病的致病菌。使用法国生物一梅里埃公司生产的自动细菌鉴定仪(ATB)进行细菌鉴定，该菌株的生理生化特性符合溶藻酸弧菌的特性。研究结果表明，养殖大黄鱼溶藻酸弧菌病病原菌传染途径，主要是从水经伤口传染到体内，在试验条件下传染率为60％。病原菌在30℃时生长很快，20℃时生长慢，10℃时基本不生长，对氟苯尼考、四环素和乙酰螺旋霉素等7种药物敏感。     

养殖牙鲆致病性弧菌的鉴定及药敏分析

2020年丹东东港市某养殖厂养殖1龄牙鲆(Paralichthys olivaceus)突发病害,表现为腹水病症状。取患病的5尾养殖牙鲆的鳃、肝、脾、肠道、腹水为材料进行细菌分离及纯培养,选择优势菌株通过全自动细菌分析仪及16S rDNA序列分析对该菌株进行鉴定,结果表明分离纯培养的14株菌(DS200401-DS200414)共鉴定出2种,其中1种为该病的致病菌:弧菌属灿烂弧菌(Vibrio splendidus)。随后进行人工感染试验和药敏试验。用12种抗菌药物对3种菌进行药敏试验,结果显示在不同菌株间无明显的敏感与耐药性差异。     

50%过硫酸氢钾对溶藻弧菌抑菌效果的研究

正弧菌病是海水鱼、虾、蟹、贝类等品种养殖过程中普遍流行的一种细菌性疾病,对水产养殖对象的危害较大,给水产养殖业造成极大的经济损失。大部分弧菌对养殖动物具有较强的侵袭能力,同时可以产生多种有害蛋白质酶和外毒素等,影响养殖对象健康。溶藻弧菌是其中一种较为常见的弧菌,在TCBS(硫代硫酸盐柠檬酸盐胆盐     

Growth inhibition of the salmon pathogen Vibrio ordalii by a siderophore produced by Vibrio anguillarum strain VL4355

Abstract. Thirty strains of V. anguillarum were tested for the production of inhibitory substances against closely-related bacteria using the deferred antagonism test. Only one strain, Vibrio anguillarum VL4355, inhibited strains of V. ordalii and this effect was blocked by the addition of iron salts to the culture medium. Siderophore production was investigated for this strain. Results from bioassays suggested that strain VL4355 produced a siderophore related to anguibactin, the plasmid-encoded phenolate siderophore produced by V. anguillarum strain 775. However, when plasmid DNA was compared for strains 775 and VL4355 the Bam HI-generated restriction profiles were different, although hybridization experiments indicated some homology. Using the chrome-azurol sulphate assay to measure siderophore production, strain VL4355 yielded significantly higher values than other V. anguillarum strains. Amberlite XAD-2 was used to produce concentrated siderophore preparations from strains VL4355 and 775. Both preparations were inhibitory to the growth of strains of V. ordalii , but not V. anguillarum , as were solutions of the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid). The difference in sensitivity to iron-limiting conditions for V. ordalii and V. anguillarum , coupled with the inability of V. ordalii to utilize ferric-anguibactin, could reflect different mechanisms of iron uptake for these two organisms.     

Vibrio harveyi as a causative agent of mass mortalities of megalopa in the seed production of swimming crab Portunus trituberculatus

Outbreaks of mass mortalities among cultured megalopa of swimming crab (Portunus trituberculatus) occurred in a commercial hatchery during the spring of 2012 in Jiangsu province, P. R. China. Dominant bacteria were isolated and characterized by biochemical reactions, enzyme production, and sequencing analysis of the 16S rRNA, gyrB (DNA gyrase B subunit) genes, and two strains (DY1 and DY2) were selected as representative strains for virulence tests by immersion. The results showed that the characteristics of identified strains consist with Vibrio harveyi; the 16S rRNA and gyrB genes of the tested strains exhibited high similarity with V. harveyi; and the phylogenetic trees constructed using the neighbor-joining method based on 16S rRNA and gyrB genes. Two strains (DY1 and DY2) clustered with the V. harveyi and were supported by a higher bootstrap value, and two strains (DY1 and DY2) were found lethal to the healthy megalopa and juvenile crab. LD ₅₀ of DY1 and DY2 to megalopa and juvenile crab were 2.4 × 10⁶, 3.0 × 10⁶, 1.9 × 10⁶ and 2.5 × 10⁶ CFU/ml, respectively. The results confirmed that the diseased megalopa were infected by V. harveyi. In addition, we analyzed the tested strains (DY1 and DY2) by PCR for the presence of virulence genes that were known in V. harveyi, and the results showed that five virulence genes (luxR, toxR, vhhA, vhhB and pap6) were detected by a specific PCR assay, further supporting the assignment of isolates to V. harveyi and its pathogenicity. To our knowledge, this is the first report of V. harveyi as pathogenic bacteria of megalopa of swimming crab.     

Human Vibrio vulnificus infections and environmental isolates in the Netherlands

Abstract A 63-year-old man was admitted to hospital for septicaemia and severe metastatic skin infection, 24h after he had eviscerated fresh eels. V. vulnificus was isolated from his blood and wounds. The strain was indole negative, was ornithine decarboxylase positive and grew at 42°C. A strain of V. vulnificus with these characteristics was isolated in 1987 from diseased eels. The characteristics differed from those of V. vulnificus strains (biogroup 1) that have been reported from patients world-wide. V. vulnificus biogroup 1 was isolated from 3 of 11 seawater samples collected along the coast of the Netherlands, but indole negative strains of V. vulnificus were not isolated. We conclude that an indole-negative variant of V. vulnificus is pathogenic for eels and for human beings and that eels may transmit V. vulnificus to humans.     

中国对虾糠虾幼体病原哈氏弧菌的鉴定及毒力基因检测

2011年春季对江苏连云港某对虾育苗场中国对虾Fenneropenaeus chinensis病死糠虾幼体分离到优势生长菌,对分离菌进行致病性、形态与生理生化特征及16S rRNA和gyrB基因同源性与系统发育分析。结果显示,引起糠虾幼体大量死亡的病原为哈氏弧菌Vibrio harveyi,菌株kx1对中国对虾仔虾和日本对虾仔虾的半数致死量LD50分别为2.0×106CFU/ml和7.0×105CFU/ml。为进一步明确分离菌株毒力基因的携带情况,进行了分离鉴定的4株病原菌对群体效应调节基因(luxR)、毒力调控基因(toxR)、溶血素基因(vhhA和vhhB)、金属蛋白酶基因(vhpA和vhpB)、毒力相关基因(toxS)、鞭毛结构基因(flaA)及锌金属蛋白酶基因(pap6)共9种毒力基因的检测,结果表明,4株病原菌均可检测到luxR、toxR、vhhA、vhhB和pap6毒力基因,扩增片段大小分别为679、390、1324、216和355 bp,其他4种毒力基因未检测到。分离鉴定的4株病原哈氏弧菌携带相同的毒力基因,这些毒力基因可作为检测致病性哈氏弧菌的生物学标记。     

创伤弧菌、溶藻弧菌外膜蛋白特性的比较研究

对用Sarkosyl法分离的创伤孤菌、溶藻弧菌、副溶血弧菌、鳗弧菌的外膜蛋白进行了初步的比较分析.这4种弧菌外膜蛋白的SDS-PAGE和Western blotting的图谱有相似性亦有差异.SDS-PAGE显示,4种弧菌中除副溶血弧菌外均能分离到47、38 ku的外膜蛋白;创伤弧菌和溶藻弧菌存在4种共同的外膜蛋白,大...     

Recent update on the prevalence of Vibrio species among cultured grouper in Peninsular Malaysia

Vibrio infections are common among marine fish and lead to serious problems in the aquaculture sector. This study reports a recent occurrence of Vibrio species (spp.) isolated from cultured groupers in Peninsular Malaysia using the gyrB and pyrH genes. A total of 147 Vibrio strains were successfully isolated from 77 (64%) groupers using culture method and subjected to gyrB and pyrH sequencing for species identification and confirmation. Results showed that 89% of Vibrio strains were identified and clustered to six groups of Vibrio spp., while 11% were not clustered to any Vibrio spp. using the gyrB sequences. Meanwhile, by analysis of the pyrH sequences all the 147 Vibrio strains (100%) were successfully identified and clustered into 11 groups of Vibrio spp., including the gyrB non‐identified strains. The pyrH gene provides a better resolution for identification of Vibrio spp. compared with the gyrB gene. Thus, the pyrH gene was more suitable for a rapid determination of Vibrio spp. distribution in Peninsular Malaysia. Using the pyrH gene, our study found higher prevalence of Vibrio vulnificus (33%), V. alginolyticus (24%) and V. parahaemolyticus (22%), followed by V. rotiferianus (5%), V. harveyi (3%), V. tubiashii (2%), V. campbellii (2%), V. ponticus (1%), V. diabolicus (1%), V. owensii (1%) and others Vibrio sp. (7%). Thus, the results of this study revealed that the occurrence of pathogenic vibrios among grouper fish is still high in Malaysian aquaculture. In addition, the pyrH gene was proved as a suitable marker for rapid identification of Vibrio species compared with the gyrB gene.     

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.     

Serotyping of Vibrio anguillarum isolated from diseased freshwater fish in Japan

Abstract. During the period from 1965 to 1980, 263 Vibrio anguillarum strains from ayu, Plecoglossiis altivelis (Temminck & Schlegel), two from rainbow trout. Salmo gairdneri Richardson, and two from eel, Anguilla japonica (Temminck & Schlegel), were collected from fish suffering from vibriosis in various parts of Japan. On the basis of cross-agglutination and cross-absorption tests with thermostable (O) antigens, six distinct serotypes (A, B, C, D, E and F) were established among 12 selected strains of V. anguillarum . 241 strains isolated from ayu and two strains from rainbow trout belonged to serotype A, six strains from ayu and one strain from eel to serotype B, 12 strains from ayu to serotype C, three strains from ayu to serotype D, one strain from ayu to serotype E, and one strain from eel to serotype F. V. anguillarum strains belonging to serotypes D, E and F have not been detected from ayu, rainbow trout and eel since 1973; these serotypes appear to be minor types. V. anguillarum strain NCMB 6 and 1669 belong to our serotype A and V. anguillarum 813 to our serotype C.     

Vibrio vulnificus serovar A: an emerging pathogen in European anguilliculture

The spread of the emerging pathogen Vibrio vulnificus biotype 2 serovar A in Danish anguilliculture is reported. Serovar A was originally isolated in a Spanish eel farm in 2000 and occurred in Denmark in the summer of 2004, affecting eels of 5-10 g body weight cultured in fresh water. The Danish eels showed clinical signs different from those reported for Spanish eels, such as severe haemorrhages in the head and gill region with necrosis of the soft tissues. Danish isolates were biochemically and serologically identical to Spanish serovar A strains and also highly virulent for eels by both intraperitoneal injection and immersion challenges. Vaccination with Vulnivaccine, a vaccine against V. vulnificus serovar E, cross-protected eels against serovar A. The LD(50) for experimentally infected vaccinated animals was significantly higher than for non-vaccinated animals.     

Siderophores produced by Vibrio anguillarum in vitro and in infected rainbow trout, Oncorhynchus mykiss (Walbaum)

Abstract. Ten strains of Vibrio anguillarum produced three different types of iron-binding compounds when cultured under different conditions. These were (1) a common phenolate siderophore produced by all strains. (2) a hydroxamate siderophore produced by three strains and (3) a second phenolate siderophore, tentatively identified as anguibactin, produced by V. anguillarum strain 775 and two other strains, all of which contained a plasmid of 45–50 Md. The relative affinities of these siderophores, determined by competition for ⁵⁵Fe was: anguibactin < hydroxamate siderophore < common phenolate siderophore. However, under these conditions, none removed iron from purified aerobactin. Experimental infection of rainbow trout. Oncorhynchus mykiss (Walbaum), showed that only the common phenolate siderophore was detected in the kidney and spleen of fish infected with strains 91079 and NCIMB6. The hydroxamate siderophore produced in vitro by strain NCIMB6 was not detected in vivo. However, in the kidney of fish infected with strain 775, both the common phenolate siderophore and anguibactin were detected, showing that a second uptake system is required by strain 775 in vivo and that the iron-uptake system based on the common phenolate siderophore is defective.