养殖刺参溃疡病病原菌RH2的鉴定及其生物学特性分析

从患溃疡病养殖刺参(Apostichopus japonicus)病灶处分离出3株优势菌(RH1、RH2、RH3),以浸浴、体腔注射、体壁肌肉注射等方式进行感染实验,证实菌株RH2为养殖刺参溃疡病的病原菌,其对刺参的LD50(半数致死量)每尾为5.68×106CFU,并证实浸浴、体腔注射的方式无法感染刺参,该菌可能通过体表创伤侵入的方式感染刺参。经形态学观察、生理生化特性分析、16SrRNA和HSP60基因测序确定菌株RH2为溶藻弧菌(Vibrio alginolyticus)。基于16S rRNA基因构建的系统发育树显示,菌株RH2与溶藻弧菌(AF513447)亲缘关系最近,置信度为50%;HSP60基因序列分析表明RH2与溶藻弧菌(AY332570、AF230931)聚为一个分支,且置信度高达99%。菌株RH2的最适生长盐度为25~35,最适生长pH为7.2~8.0,最适生长温度为14~22℃。对18种药物的敏感实验证实菌株RH2对诺氟沙星、复方新诺明、壮观霉素等较为敏感。     

养殖大菱鲆出血病病原菌的分子生物学鉴定

对青岛某养殖场发病大菱鲆分离的5株病原菌进行鉴定。使用法国生物梅里埃公司的全自动微生物鉴定仪VITEK2Compact的革兰氏阴性菌鉴定卡(GNtest kit),结合生物梅里埃公司的API细菌鉴定系统API 20E进行生理生化反应测试。为了进一步确定5株菌的分类学地位,测定了16SrDNA基因序列,与相关细菌序列进行比对,构建了系统进化树。生化反应和16S rDNA基因序列表明其中4株菌与溶藻弧菌亲缘关系最近,人工感染试验表明溶藻弧菌对养殖大菱鲆的致病力是相当强的。     

养殖大黄鱼低温弧菌病病原的研究

浙江沿海深水网箱养殖大黄鱼冬天发生大量死亡，从病鱼体内分离出致病力较强的细菌GYC0227。人工感染试验证实这株菌为大黄鱼的病原菌。对细菌进行了形态、生理生化特性的测定，并进行了16S rRNA分子鉴定。PCR扩增获得了GYC0227菌株大小约1．5kb的16S rDNA部分基因片段，产物经回收纯化，克隆到pMD18-T载体，转化大肠杆菌TG1，对阳性克隆子进行酶切及PCR鉴定，并测序，将测定的序列递交NCBI进行BLAET同源序列比对，与溶藻弧菌的标准菌株的16S rDNA具有96％的同源性。结合菌株的生理生化特性，GYCD227菌株属于溶藻弧菌。该菌适应于低温生长，生长温度范围为7—25℃，并测定了其对14种药物的敏感性。     

养殖大菱鲆肠道中大菱鲆弧菌的分离鉴定及药敏试验

2014年11月,大连地区某大菱鲆养殖场发生病害并伴有死亡,主要症状为吻部下颌明显出血,腹腔积水,肝脏充血,肠道出血有白便。从患病大菱鲆肠道中分离出1株可在硫代硫酸盐—柠檬酸盐—胆盐—蔗糖琼脂培养基上生长的菌株HZ-C1,人工回接感染试验证明其对健康大菱鲆具有较强致病性。通过形态学观察、16SrDNA序列同源性分析及生理生化试验确定菌株HZ-C1为大菱鲆弧菌。药敏检测结果显示,大菱鲆弧菌HZ-C1对氯霉素、丁胺卡那及头孢曲松等11种抗生素敏感,但对青霉素、四环素及诺氟沙星等15种抗生素耐药。本研究为大连地区养殖大菱鲆细菌性病害防治提供了一定病原学依据。     

斜带石斑鱼3种致病性弧菌的分子生物学鉴定

从患病斜带石斑鱼分离到3株病原菌EcGS020802、EcGS021001、EcGS020801,经常规生理生化鉴定均属于弧菌属的种类。为了进一步确定其分类地位,测定了3株病原菌的16SrRNA和HSP60(heat shock protein,HsP60)基因部分序列。16SrRNA基因系统进化分析表明,3株病原菌与哈维氏弧菌、溶藻弧菌、副溶血弧菌亲缘关系较近,相互之间同源性均大于98．6％,差异不明显。HSP60基因序列分析表明,菌株EcGS020802、EcGS021001、EcGS020801 HSP60基因序列分别与哈维氏弧菌(AF230934)、溶藻弧菌(Ab230931)、副溶血弧菌(AF230951)HSP60基因的同源性最高,依次为95．7％、99．8％和99．8％,而与其它弧菌HSP60基因的同源性均低于90．6％,3株病原菌相互之间的同源性低于91．0％。HSP60基因构建的系统进化树表明,EcGS020802、EcGS021001、EcGS020801分别与Vibrio harveyi、Vibrio alginolyticus、Vibrio parahaemolyticus聚类。综合上述结果,菌株EcGS020802、EcGS021001、EcGS020801可分别鉴定为哈维氏弧菌、溶藻弧菌、副溶血弧菌。结果表明,HSP60基因比16SrRNA基因更适合用于海水鱼致病性弧菌种问的分类研究。     

养殖刺参“化板症”病原菌的分离与鉴定

本研究对辽宁4家刺参育苗场患“化板症”的稚参进行了病原学分析,从患有“化板症”的稚参体表病灶处分离得到一株优势菌Aj2010072802A90.人工回接感染试验证实,该细菌能使稚参发生厌食、萎缩、降低附着力、溃烂、死亡等现象,具有较强的致病性,为此次“化板病”的病原菌.在此基础上,利用细菌形态观察、生理生化及16S rDNA分子生物学方法对该菌株进行了鉴定,结果显示,该菌为副溶血弧菌Vibrio parahaemol yticus.这是副溶血孤菌导致海参感染的首次报道.此外,本研究针对该病原菌进行了药敏学测试,证实所分离的副溶血弧菌菌株对氟苯尼考等药物高度敏感,相关研究结果将为刺参疾病防控和健康养殖提供了理论依据和技术支撑.     

梭子蟹牙膏病病原菌——溶藻弧菌的鉴定及其系统发育分析

从山东潍坊池塘养殖的患有牙膏病的三疣梭子蟹(Portunus trituberculatus)中分离到1株病原菌(PX25),经回接感染证实为该病的病原菌。经生理生化试验及形态结构观察显示,该菌株革兰氏阴性,短杆状,直或稍弯曲,两端钝圆;固体培养基上培养后进行染色具有周生侧毛,液体培养基中培养后进行鞭毛染色具一极生单鞭毛;悬滴法观察PX25菌株具有很强的运动性。对其16SrRNA基因进行PCR扩增、测序和系统发育分析。结果表明,该菌株与溶藻弧菌亲缘关系最近,相似性达99.35%。综合菌体在形态、生理生化、API20NE与API20E自动鉴定结果、16S rRNA同源性等方面的特性,确认PX25为溶藻弧菌(Vibrio alginolyticus)。     

养殖大菱鲆(Scophthalmus maximus)腹水病的病原多样性及其耐药性分析

为了解引起养殖大菱鲆(Scophthalmus maximus)腹水病的病原多样性及其耐药性情况,针对2002-2010年由不同地区病样分离的27株细菌性病原进行了16S rDNA鉴定,并采用K-B法测定了27株细菌对22种抗生素的耐药性,分析了病原菌的耐药谱及耐药率变化.结果显示,大菱鲆腹水病病原菌主要有大菱鲆弧菌(Vibrio scophthalmi)、迟钝爱德华氏菌(Edwardsiella tarda)、鳗弧菌(Vibrio anguillarum)、哈维氏弧菌(Vibrio harveyi)、假交替单胞菌(Pseudoalteromonas espejiana).山东青岛地区以大菱鲆弧菌为主,威海地区以迟钝爱德华氏菌为主,烟台地区菌株种类分布平均.5类细菌对青霉素类、头孢菌素类、大环内酯类、复方新诺明耐药率高于50％.只有1株迟钝爱德华氏菌对氟苯尼考产生了耐药,其余菌株对其均没有耐药性,且在长期使用中不易产生耐药性,证实氟苯尼考为当前防治腹水病的一种良好抗菌药物.27株病原菌的耐药谱数量为27个,每个菌株具备自己独特的耐药谱,74.1％的菌株对10种以上的抗菌药物产生了耐药性,均有多重耐药性.     

养殖大菱鲆细菌性腹水病及其防治方法研究进展

大菱鲆(Scophthalmus maximus)是我国北方沿海地区重要的养殖经济鱼类,在北方沿海区域经济的快速发展中发挥了重要作用.但随着养殖的规模化和集约化发展,大菱鲆病害问题已成为产业可持续发展的主要瓶颈之一.文章综述了大菱鲆腹水病的临床症状、病原菌和致病条件,并结合养殖现状,提出在实际生产中采取免疫接种等综合预...     

养殖大菱鲆黑瘦症的病原菌鉴定及杀菌中草药筛选

从患黑痩症的大菱鲆幼体内分离得到一株优势菌株,人工感染实验证实其为大菱鲆致病菌,并经过生理生化及16SrDNA序列分析鉴定为灿烂弧菌Vibrio splendidus。组织病理学显示,病鱼的肝脏、肾脏、脾脏、肠道及脑均呈现不同程度的组织破坏。用煎煮法提取11种常用中草药成分,测定该致病菌对中草药的杀菌效果。实验表明,五倍子和白头翁的杀菌效果最好,黄柏、穿心莲次之,当归、党参、杜仲、秦皮、金银花、乳香和没药的杀菌效果最差。以五倍子、白头翁为主要成分的初步治疗试验证明,药浴和口服给药可有效治愈大菱鲆苗期的黑瘦症。     

大菱鲆源杀鱼爱德华氏菌(Edwardsiella piscicida)的分离鉴定

从患腹水病大菱鲆Scophthalmus maximus的肝脏中分离到一株优势细菌G1,经人工感染实验表明G1为引发大菱鲆腹水病的致病菌,且半致死浓度为LD50=1.21×105 CFU·g-1。采用常规的生理生化鉴定方法及分子生物学方法对G1进行分析,结果表明,G1的16SrRNA基因序列与杀鱼爱德华氏菌Edwardsiella piscicida的同源性达100%,系统发育分析表明菌株G1与杀鱼爱德华氏菌分支聚为一支,结合生理生化鉴定结果确定G1为杀鱼爱德华氏菌。药敏试验表明菌株G1对头孢曲松、环丙氟哌酸、左氧氟沙星等8种抗菌药物高度敏感。     

Isolation of a highly pathogenic Vibrio pelagius strain associated with mass mortalities of turbot,Scophthalmus maximus (L.), larvae

A bacterial strain, characterized as Vibrio pelagius (Hq 222), was isolated from a turbot, Scophthalmus maximus (L.), larvae mass mortality in a commercial fish farm in Spain. Turbot larvae, post-larvae (0.2 g) and juveniles (5 and 15 g) were experimentally infected. The bacterium appeared to be very virulent for larvae and post-larvae, LD50 being < 5 bacteria mL(-1) for larvae 1 week post-infection and 3.9 x 10(5) bacteria mL(-1) in post-larvae at day 12 post-infection. The bacterial strain was recovered in pure culture from the internal organs of infected fish. Histological lesions in post-larvae exhibited swelling and necrosis of gill secondary lamellae, sloughing of intestinal mucosa and necrosis of haematopoietic tissue in the kidney. Vibrio pelagius (Hq 222) was able to grow in sterile sea water when incubated at room temperature or at 15 degrees C. Vibrio pelagius (Hq 222) was more adherent to the turbot cell lines TV-1 and TF than Escherichia coli. In both cell lines, the number of adhered bacteria increased with incubation time.     

养殖大菱鲆主要疾病及防治技术

在流行病学调查的基础上,对目前我国大菱鲆养殖中常见疾病进行了较全面的介绍,包括病毒性、细菌性和寄生虫性疾病。系统地描述了这些疾病的流行特征、发病症状、危害程度、感染率及致病原等。同时为各种疾病提供了具体的防治技术和措施,对大菱鲆养殖生产与疾病防治具有理论指导意义和重要的参考价值。本文系国内首次对养殖大菱鲆疾病进行的全面性报道。     

Non-specific immune response of turbot,Scophthalmus maximus (L.), experimentally infected with a pathogenic Vibrio pelagius

The effect of a pathogenic Vibrio pelagius, isolated during a mass mortality of turbot larvae, on the non-specific immune response of turbot, Scophthalmus maximus (L.), macrophages was studied both in vitro and in vivo. The in vitro treatment of head kidney (HK) macrophages with viable V. pelagius caused a significant inhibition of the chemiluminescence (CL) response in comparison with untreated macrophages, while incubation with heat-killed bacteria did not affect this response. In vivo, the intraperitoneal injection of V. pelagius resulted in a significant inhibition of the CL response in infected fish at days 1 and 4 post-infection compared with the control fish response. The HK macrophage nitric oxide (NO) production was enhanced by in vitro incubation with intermediate doses of viable V. pelagius (5 x 10(3) and 5 x 10(4) bacteria mL(-1)) and higher doses of the heat-killed bacteria (5 x 10(4)-5 x 10(6) bacteria mL(-1)). In both cases, the NO inhibitor N-omega -nitro-L-arginine was capable of down-regulating the specific NO induction caused by incubation with the bacterial treatments. In contrast, incubation with ECPs at higher doses caused a reduction in NO production. In vivo, a significant enhancement in NO production was also observed in macrophage supernatants at day 10 post-infection. Lysozyme concentration in the serum was also significantly increased in the experimentally infected fish at days 4 and 10 post-injection. In addition, viable V. pelagius and its ECPs significantly reduced HK macrophage viability in vitro, whereas no significant differences in viability were observed during the incubation with heat-killed bacteria. As NO production was enhanced in the experimentally infected fish, the inhibitory effect of the NO donor, S-nitroso-acetyl-penicillamine (SNAP), was tested in vitro in a cell-free assay. The results showed that growth of V. pelagius was significantly inhibited using SNAP at a high concentration (1 mM).     

Identification and Detection of the Pathogenic Bacteria Responsible for Swollen Abdomen Disease in Cultured Turbot,Scophthalmus maximus,and Flounder,Paralichthys olivaceus

Swollen abdomen disease (SAD) seriously threatens the aquaculture of turbots and flounders. Two dominant bacterial strains, FS1 and FS2, were isolated from the livers and kidneys of fish with diagnosed SAD. Applications of biochemical analyses, sequence analyses of 16S ribosomal RNA gene and heat shock protein 60 gene revealed two distinct pathogenic bacterial species, Edwardsiella tarda and Vibrio alginolyticus. These two hypothesized SAD‐causing pathogens were validated by challenge trials on flounder, Paralichthys olivaceus. Challenges with E. tarda and V. alginolyticus demonstrated lethal dose 50 (LD₅₀) values at 1.51 × 10⁵ colony‐forming units (CFU) and 1.05 × 10⁵ CFU, respectively. To develop a rapid SAD diagnosis method in flounders and turbots, a multiplex polymerase chain reaction (PCR) assay method was developed to simultaneously detect E. tarda and V. alginolyticus. Our multiplex PCR assay successfully detected as low as 10⁵ CFU/mL E. tarda and V. alginolyticus in flounders and turbots. No other common fish pathogens were detected with the multiplex PCR, suggesting a high specificity of this assay. The multiplex PCR assay developed in this study showed a great reliability in detecting SAD‐causing bacterial pathogens. Further optimization of this assay may contribute to the development of a novel SAD diagnosis tool in aquaculture.     

大菱鲆内脏结节病病原的分离与鉴定

2017年8月,天津市滨海新区某养殖场大菱鲆(Scophthalmus maximus)发生病害,累计死亡率约为25%,患病鱼体表无明显症状。通过肉眼和病理组织切片观察发现,患病鱼肾脏、脾脏、肝脏和肠道存在大量圆形结节,肾脏、肝脏和脾脏组织病变明显,肾小管坏死,肝细胞脂肪变性,脾脏中存在大量的坏死细胞。此外,在脾脏和肾脏组织中发现大量的抗酸杆菌。利用传统病原菌分离方法,从具有典型症状的濒死大菱鲆肾脏组织分离到优势菌株myco-10,利用该菌株注射攻毒能导致健康大菱鲆66.7%的死亡率,且表现出与自然发病鱼相同的症状。采用16S rDNA、Hsp65、ropB基因序列分析,构建系统发育树并结合细菌形态特征、生理生化测试对菌株myco-10进行鉴定,结果显示,菌株myco-10的16SrDNA、Hsp65、ropB基因序列与分枝杆菌属细菌(Mycobacteriumspp.)相似度最高,且在系统发育树中与海分枝杆菌(Mycobacteriummarinum)和溃疡分枝杆菌(Mycobacteriumulcerans)聚为一簇,生理生化反应与海分枝杆菌一致。综合生理生化特征和基因序列分析结果,将菌株myco-10鉴定为海分枝杆菌。这是中国首例分枝杆菌引起大菱鲆病害的报道,可为大菱鲆内脏结节病的防治提供参考资料。     

弧菌对养殖大菱鲆的危害及其防治方法研究新进展

弧菌是细菌性疾病的常见病原,不但会感染大菱鲆(Scophthalmus maximus),还会对人类健康造成重大危害.大菱鲆是我国重要的海水养殖鱼类,具有较高的经济价值,然而疾病问题严重阻碍了其养殖业的发展.近年来,有关大菱鲆弧菌病检测及防治方面的研究有了新的突破.文章介绍了几种常见弧菌病原对养殖大菱鲆的危害,对新的检...