罗非鱼链球菌病研究进展

链球菌（Streptococcus spp．）是一种广泛分布于自然界的革兰氏阳性菌,是人类的重要病原之一,也是其他多种脊椎动物包括猪、牛、鱼等的重要病原菌。目前已有多个国家报道了鱼类链球菌病的暴发与流行,受感染的鱼类包括多种海水和淡水鱼类,以温水性鱼类最为严重。近年中国罗非鱼链球菌感染的报道也呈现增加趋势,尤其在2009年,广东、海南、福建和广西地区养殖罗非鱼的链球病发病率为20％～50％,死亡率达50％～70％,甚至更高。链球菌病已严重危害着中国罗非鱼养殖业的健康发展。文章对罗非鱼链球菌病的病原、流行及防治方法作一综述,以期为今后开展罗非鱼链球菌病防控研究提供参考。     

罗非鱼链球菌病的诊治

根据目前资料报道,引起罗非鱼链球菌病的病原菌有β-溶血性链球菌和非溶血性链球菌。近几年池塘养殖罗非鱼,网箱养殖罗非鱼不时发生此病,此病多流行于夏秋高温季节,也危害虹鳟、斑点叉尾□、香鱼等鱼类。笔者在2004年8月下旬诊治过罗非鱼链菌球病。现将诊治的相关情况报告如下:     

12种药物对罗非鱼无乳链球菌的抑菌试验

无乳链球菌(Streptococcus agalactiae)普遍存在于养殖水体中,可产生外毒素,对动物具有致死性,由无乳链球菌和海豚链球菌(Streptococcus inia)引起的罗非鱼链球菌病被认为是对罗非鱼危害最严重的细菌性疾病,而且链球菌还可随伤口感染人类.近几年来,因罗非鱼链球菌病的暴发流行,给罗非鱼养殖业造成极大的危害,甚至威胁到罗非鱼养殖产业的稳定发展.2011年5月～10月,广西多地暴发罗非鱼链球菌病,危害各种规格的养殖罗非鱼,共造成7127吨鱼死亡,经济损失8610.8万元,已严重制约了广西罗非鱼养殖业的健康发展.研究结果表明,近两年广西养殖罗非鱼链球菌病的病原菌为无乳链球菌,且大多表现为多重耐药菌株.而生产实际中,药物防治仍是目前养殖户针对罗非鱼链球菌病普遍采用的主要控制措施.本试验选取12种常用国标渔药,研究其对无乳链球菌的抑菌作用,旨在为罗非鱼无乳链球菌病的药物防治提供参考.     

罗非鱼链球菌病的防治技术

<正>鱼类的链球菌病已然成为养殖鱼类重要细菌性疾病之一,在不少的国家和地区的鱼类养殖业都有链球菌病害爆发的案例。一、鱼类链球菌病受到链球菌感染的鱼类,出现明显症状的部位首先是头部,致病菌能破坏鱼体的脑神经,继而通过血液循环破坏肝、肾、脾等器官引发全身性出血病,是一种传染性极强的细菌性疾病,而且疾病一旦发生,患病鱼体的死亡率比较高,而且水温越高病情就越重。由海豚链球菌引起敏感鱼类感染死亡率可以达到20%~50%以上。     

罗非鱼链球菌病防治策略新思考

罗非鱼链球菌病有2种病原,即海豚链球菌和无乳链球菌。在5年前,罗非鱼链球菌病的主要病原是海豚链球菌,且疾病的发生多呈局部性的零星暴发流行。但随着罗非鱼养殖密度增大、养殖环境恶化、抗病性能退化,罗非鱼链球菌病呈现大面积、高死亡的急性流行暴发,且病原主要是无乳链球菌。近3年来,罗非鱼链球菌病的广泛暴发,给罗非鱼养殖业造成极...     

罗非鱼链球菌病病原鉴定及中草药药敏试验

从广东省肇庆、广州、番禺三个地区发病罗非鱼肝、肾、脑中分离出3株海豚链球菌（streptococcus iniae）。中草药药敏试验显示,茵陈蒿散、黄连解毒散、黄连解毒散改方、三黄散、黄连有良好的抑菌效果。随着罗非鱼养殖业的迅猛发展,近年在广东、海南、福建、广西等省的养殖罗非鱼中暴发一种流行病—链球菌病,无论是网箱,还是池塘养殖的罗非鱼,都有此病的发生,主要危害亲鱼及100克以上的幼鱼和成鱼。罗非鱼链球菌病是一种暴发性流行病,主要病理特征为眼球突出或混浊发白,眼眶充血,腹部膨大,肛门红肿,采食减少或绝食,游姿平衡失调,翻滚,转圈,解剖病鱼可见胆囊肿大,胆汁稀薄,色浅,肠腔充满淡黄色液体,肝脏增大。近期,华南地区养殖罗非鱼出现链球菌病,其发病面积之广,危害程度之重很快成为业内关注的焦点,无论是罗非鱼饲料经销商、罗非鱼出口企业还是政府相关部门都表示了高度的重视。本文对广东省部分地区发病鱼体中进行罗非鱼链球菌病原进行分离鉴定及中药药敏试验,为该病的防治提供试验依据。     

热带观赏鱼海豚链球菌病的防治技术研究

海豚链球菌,属于链球菌科的链球菌属,呈圆形或卵圆形,成对或呈不同长度的链状,无鞭毛,不运动,有荚膜,不形成芽孢,革兰氏染色阳性,是一种条件性人畜共患病病原菌,它不仅能感染几乎所有的海、淡水鱼类,还可感染人类,引起局部蜂窝组织炎,心内膜炎。海豚链球菌病是感染性极强的细菌性疾病,目前已成为热带鱼类主要致病菌之一。     

牛蛙链球菌病的诊断和防治

链球菌病近年来逐渐成为危害水产养殖行业的主要疾病之一,已报道的有海豚链球菌、无乳链球菌、停乳链球菌,常引起鲑鳟鱼类、罗非鱼、梭鱼、叉尾鮰等多种海、淡水鱼类发病。2009、2010年浙江湖州、嘉兴等地养殖牛蛙暴发链球菌病,给养殖户造成了巨大的经济损失。     

罗非鱼无乳链球菌与嗜水性单胞菌病的防治初探

罗非鱼是华南地区鱼类养殖中数量最多的品种之一,因其抗病力强、生长快、繁殖力强、适应性广、营养价值高、适合内销与外销等优点而受广大养殖户青睐,使我国成为世界最大的罗非鱼生产国,罗非鱼也成为我国对外出口的主要养殖品种.然而近年来由于养殖水体环境恶化、气候变化异常及养殖密度大、饲料品质参差不齐等诸多因素,使其病害发生率明显增加,其中细菌病的爆发是造成罗非鱼经济损失的重要原因之一.自2009年7月,广东、广西、海南等华南地区爆发链球菌病（海豚链球菌、无乳链球菌）以来,近几年一直呈现高发态势;与此同时,嗜水气单胞菌作为主要的病原菌之一,其发病率及耐药性也逐年增加,给广大养殖企业和养殖户造成重大损失,也给罗非鱼产业带来严重危害.     

观赏鱼海豚链球菌病的防治技术

正海豚链球菌,属于链球菌科的链球菌属,呈圆形或卵圆形,成对或呈不同长度的链状,无鞭毛,不运动,有荚膜,不形成芽孢,革兰氏染色阳性,是一种条件性人畜共患病病原菌,它能感染几乎所有的海、淡水鱼类,还可感染人类,引起局部蜂窝组织炎,心内膜炎。海豚链球菌病是感染性极强的细菌性疾病,目前已成为热带鱼类主要致病菌之一。此病也可以导致鱼慢性持续性死亡,最终造成重大的经济损失。该     

Evaluation of the link between gyrodactylosis and streptococcosis of Nile tilapia, Oreochromis niloticus (L.)

Streptococcus iniae and Gyrodactylus niloticus are two common pathogens of cultured Nile tilapia, Oreochromis niloticus. We studied concurrent infection of tilapia by G. niloticus and S. iniae and evaluated whether parasitism in tilapia with Gyrodactylus increased susceptibility and mortality following immersion infection with S. iniae. Results showed that death mainly occurred in fish with G. niloticus and challenged with S. iniae (G-S group). The accumulative mortality (42.2%) was significantly higher in the G-S group than in fish not infected by the parasite (6.7%), but exposed to S. iniae. Bacteriological examination revealed S. iniae from > or =92% of dead or moribund fish challenged with S. iniae. Gyrodactylus not only damaged fish epithelium and provided entry for invasive bacteria but also was found to harbour viable cells of S. iniae for 24 and 72 h. Streptococcus iniae was isolated from 60% and 40% of G. niloticus collected from fish infected by intraperitoneal injection or immersion, respectively, at 24 h post-challenge. The present study confirms that parasitism of tilapia by G. niloticus increased host mortality following exposure to the bacterial pathogen S. iniae.     

红尾皇冠鱼(Aequidens rivulatus)病原无乳链球菌的分离、鉴定与特性分析

为查明天津地区养殖红尾皇冠鱼(Aequidens rivulatus)大量死亡的原因,取患病红尾皇冠鱼进行细菌分离,从病鱼肾脏中分离到优势菌株071901,人工感染试验证实其对红尾皇冠鱼有较强的致病性;采用形态学观察、生理生化特性、16S r RNA基因序列系统发育树分析及cfb(GBS-specific gene cfb,CAMP factor)基因检测对菌株071901进行鉴定。结果显示,菌株071901为革兰氏阳性球菌,生化特性与链球菌属的无乳链球菌(Streptococcus agalactiae)相近,基于16S r RNA基因序列的系统发育分析将菌株071901与无乳链球菌聚为一支,以071901为模板特异性扩增无乳链球菌的cfb基因,也得到了预期的900 bp的核酸片段,进一步证实菌株071901为无乳链球菌。对30种抗生素的药敏结果显示,菌株071901对红霉素、阿奇霉素等19种药物敏感,对多粘霉素、罗红霉素、呋喃唑酮等11种药物耐药。     

Antigenicity of Streptococcus agalactiae extracellular products and vaccine efficacy

Streptococcus agalactiae is a major bacterial pathogen that is the cause of serious economic losses in many species of freshwater, marine and estuarine fish worldwide. A highly efficacious S. agalactiae vaccine was developed using extracellular products (ECP) and formalin-killed whole cells of S. agalactiae. The vaccine efficacy following storage of S. agalactiae ECP and formalin-killed S. agalactiae cells at 4 degrees C for 1 year was determined. The stored ECP containing S. agalactiae formalin-killed cells failed to prevent morbidity and mortality among the vaccinated fish, and the relative percentage survival was 29. Serum antibody responses of the stored ECP and freshly prepared ECP against soluble whole cell extract of S. agalactiae indicated that significantly less antibody was produced in fish immunized with stored ECP and S. agalactiae cells than in those fish immunized with freshly prepared ECP and S. agalactiae cells at day 31 post-vaccination. Silver staining of sodium dodecyl sulphate-polyacrylamide gels and immunostaining of Western blots with tilapia antiserum to S. agalactiae revealed that predominant 54 and 55 kDa bands were present in the freshly prepared ECP fraction. The 55 kDa band was absent from the stored ECP and new bands below 54 kDa appeared on the Western blot. The results of this study on S. agalactiae ECP provide evidence for a correlation between protection and antibody production to ECP and for the importance of the 55 kDa ECP antigen for vaccine efficacy.     

豚链球菌和停乳链球菌类M蛋白及其抗原性分析

分别用热酸抽提法、碱抽提法和胃蛋白酶消化法分离豚链球菌NUF849的类M蛋白，通过SDS-PAGE和Western-blotting印迹法分析比较三种方法所提类M蛋白的组成和抗原性差异。结果表明，热酸抽提法分离制备的类M蛋白得率和纯度都较其它两种方法高，蛋白的抗原性保持较好。采用热酸提取法提取5株豚链球菌NUF633、NUF693、 NUF701、 NUF812、 NUF849和2株停乳链球菌SD、L2的类M蛋白，其SDS-PAGE图谱明显不同，种间差异明显, 豚链球菌主要类M蛋白的分子量分别为60、48、37、30、27、24和15kDa，停乳链球菌主要类M蛋白的分子量分别为68、48、42、37、29、26、23、21、19、17、15、14和11kDa。Western-blotting结果显示，兔抗豚链球菌血清可与豚链球菌的两条类M蛋白带结合，分子量分别为60和37kDa；与停乳链球菌的两条类M蛋白带结合，分子量为68和37 kDa。两种链球菌主要类M蛋白组成及其抗原性差异明显。     

罗非鱼海豚链球菌16S rRNA基因的序列测定和系统进化分析

为了从分子水平上对1株致病性罗非鱼链球菌进行分类学鉴定,利用原核生物16SrRNA基因通用引物对分离纯化的罗非鱼致病性链球菌进行16S rRNA基因的克隆及序列分析。结果扩增出长约1.5 kp目的片段,测序得到1条长度为1 447 bp核苷酸序列。核苷酸相似性分析表明,序列与NCB I公布的海豚链球菌(Streptococcus iniae,S.iniae)SCCF5L菌株16SrRNA基因核苷酸序列相似性最高(99.4%),暂称为中国广西株(S.iniae-CGX)。同时,亲源关系较近的S.iniae、S.difficilis和S.agalactiae代表菌株构建的系统发育进化树显示,所得菌株与S.iniae代表菌株组成同一进化分支,与S.agalactiae代表菌株组成的另一进化分支距离较近(95.5%),而与S.difficilis代表菌株组成的进化分支距离较远(92.3%)。上述研究证实,本试验从发病罗非鱼脑组织分离到的致病性链球菌为海豚链球菌。     

Passive immunization of tilapia, Oreochromis niloticus (L.), with anti-Streptococcus iniae whole sera

Passive immunization of tilapia, Oreochromis niloticus, was conducted to determine whether anti- Streptococcus iniae whole sera (ASI), heat inactivated anti- S . iniae whole sera (HIASI) and normal whole sera (NWS) were protective when intraperitoneally (i.p.) injected into tilapia. The ASI was produced in tilapia actively immunized (challenged) with virulent S. iniae by i.p. injection. An antibody response against S. iniae was demonstrated by enzyme linked immunosorbent assay (ELISA) and 18% of the immunized fish died because of the S. iniae infection. The actively immunized tilapia demonstrated a secondary antibody response and immunity to S. iniae after challenge with S. iniae by i.p. injection. Survival was 100% in the actively immunized fish. The NWS was obtained from tilapia free of ASI antibody and susceptible to S. iniae infection (40% mortality). In two separate experiments, significantly higher mortality was noted in tilapia passively immunized with NWS (33 and 53%) and phosphate buffered saline (PBS) (30 and 60%), in comparison with mortalities of 0 and 10% or 3.3 and 6.7% in the fish passively immunized with ASI or HIASI 14 days after S . iniae infection by i.p. injection ( P  = 0.0003 and 0.0023). Results suggest that immunity provided by ASI and HIASI was because of antibody against S. iniae . Inactivation of complement in the HIASI treatment further suggests that ASI antibody plays a primary role in immunity against S. iniae infection.     

中国南方地区罗非鱼无乳链球菌的分子流行病学研究

从广东省以及海南省等地区养殖的患病罗非鱼体内分离、收集到多株致病菌,经生化分析和分子生物学鉴定,均为无乳链球菌。对这些菌株分别进行了耐药谱测定、分子分型试验以及分子血清型分析。药敏试验结果表明,2007—2010年分离到的无乳链球菌耐药谱基本相似;多位点可变数目串联重复序列分析(MLVA)试验中,选择5个高变异指数的可变数目重复位点(VNTR)进行分子分型,结果表明,所有鱼源无乳链球菌菌株为同一MLVA型,而作为对照的牛源无乳链球菌则明显不同;为了对这些菌株进一步分型,分别进行了分子血清型和表面蛋白抗原基因的检测,结果表明,鱼源无乳链球菌的分子血清型均为Ⅰa型,表面蛋白抗原均为alpha-C蛋白。这进一步说明了不同年份和不同地区的鱼源无乳链球菌在基因水平上为同一分子类型,具有相同的起源或传染源。同时也说明,我国南方地区罗非鱼无乳链球菌在这几年中未发生明显的遗传变异。这些结果为罗非鱼无乳链球菌病疫苗研制,疫病监测及药物防治的研究提供理论依据。     

罗非鱼源无乳链球菌溶血素基因体外表达及其免疫原性

为研究罗非鱼源无乳链球菌溶血素(Hemolysin,Hly)对鱼体的免疫保护作用,根据已获得的无乳链球菌ZQ0910全基因组序列设计引物扩增hly基因,定向克隆于原核表达载体p ET-28a中,构建原核重组质粒p ET-28a-hly,经IPTG诱导表达后,制成亚单位疫苗免疫吉富罗非鱼,并分析疫苗的免疫保护力。结果显示,hly基因产物大小1335 bp,编码444个氨基酸,经测序与Gen Bank报道的链球菌属Hly氨基酸序列同源性可达99%。经IPTG诱导表达后,SDS-PAGE分析可见一条51.7 k D的特异条带;Western blotting分析结果说明表达的Hly蛋白能与His-Tag单抗特异性结合;制备的亚单位疫苗免疫鱼体后第14天即可检测到抗体产生,并在第28天达到峰值,抗体效价为1∶4096,免疫保护率为70%。由此证实,该亚单位疫苗有望成为预防由无乳链球菌引起的罗非鱼链球菌病的基因工程类疫苗。     

广东省养殖罗非鱼、海鲈、尖吻鲈海豚链球菌感染调查

利用细菌分离培养方法结合特异PCR技术,对广东省珠三角地区养殖罗非鱼(Oreochromis spp.)、海鲈(Lateolabrax japonicus)及尖吻鲈(Lates calcarifer)的海豚链球菌(Streptococcus iniae)感染情况进行了周年调查。每月固定时间在特定养殖区域采集目标鱼的脑、肝、脾、肾和肌肉等组织,并对其进行海豚链球菌的细菌分离培养鉴定。仅从已经患病的尖吻鲈中分离到3株链球菌,经生理生化鉴定和16S rDNA测序确定为海豚链球菌。利用海豚链球菌特异PCR技术对上述养殖鱼类不同组织进行检测,发现罗非鱼、海鲈、尖吻鲈的海豚链球菌感染率分别为30.21%、23.53%、14.55%,其中罗非鱼脑和肌肉的感染率明显较其他组织高(P<0.05),分别为20.65%和23.75%;海鲈的脑部和肌肉感染率也较其他组织高(P<0.05),分别为12.1%和10%;而尖吻鲈各组织感染率没有较大差异(P>0.05)。另外,研究结果还表明采集样本的海豚链球菌感染率随着其体长的增加而呈现下降趋势。     

First report of Streptococcus iniae in red porgy (Pagrus pagrus, L.)

This work describes the first isolation of Streptococcus iniae in red porgy, Pagrus pagrus (L.), and the first European isolation of this pathogen in gilthead seabream, Sparus aurata (L.). In both farmed fish species, infection resulted in lethargy, anorexia, abnormal swimming, exophthalmia and sudden death, with mortality rates of over 25% in red porgy and 10% in gilthead seabream. Beta-haemolytic Gram-positive cocci, catalase negative and oxidase negative, were isolated in pure culture from internal organs. Conventional and rapid identification systems, and 16S rRNA gene partial sequencing were used to identify the causative agent of the natural disease. LD50 trials were carried out to show the virulence of this isolated strain in these species, with values of 1.7 × 104 CFU per fish in red porgy and 1.32 × 105 CFU per fish in gilthead seabream. The most prominent lesions were meningoencephalitis and multifocal infiltration of macrophage cells in the kidney and spleen.