红鳍东方皮肤溃烂病病原菌的分离与鉴定

从体表溃烂的养殖红鳍东方鲀(Fugu obscurus)病灶处分离到1株优势生长菌,编号为H-06091.分别通过创伤浸泡和注射方式感染健康红鳍东方鲀,发现这两种途径均可引发皮肤溃烂,两种途径的感染率均为100%,2×108 cell/mL.菌浓度注射组死亡率为100%,4×107 cell/mL菌浓度注射组死亡率为50%,创伤浸泡感染未见死亡.药敏实验表明,复方新诺明、呋喃妥因、链霉素、环丙沙星、利福平、红霉素、氟哌酸、氟嗪酸、庆大霉素、阿米卡星、四环素、青霉素G等抗菌素对H-06091有较强的抑制作用.按照<常见细菌系统鉴定手册>进行菌种鉴定并测定其16S rRNA基因序列,结果表明,该菌呈革兰氏染色阴性,氧化酶阳性,接触酶阳性,V-P试验阴性,硝酸盐还原反应阳性等特征,符合哈氏弧菌(Vibrio harveyi)特征,其16s rRNA基因序列与GenBank中哈氏弧菌同源性为99%,因此将H-06091鉴定为哈氏弧菌.     

红鳍东方鲀3个白介素基因的序列特征及表达分析

本试验分析体质量(190.34±2.13)g的红鳍东方鲀白介素基因IL-1b、IL-8和IL-10的序列特征,检测其在红鳍东方鲀脑、鳃、心脏、肌肉、肝脏和脾脏中的表达,以及每尾红鳍东方鲀注射密度1×107 cfu/mL的哈维氏弧菌菌液0.1 mL后0、12、24、48 h在肝脏和脾脏中的表达.试验结果表明,IL-1b、...     

水产动物6种主要病原菌与抗血清的免疫交叉反应

采用ELISA、试管凝集、Western-blot等方法,分析了鳗弧菌(Vibrio anguillarum)、哈维氏弧菌(V. harveyi)、溶藻胶弧菌(V. alginolyticus)、副溶血弧菌(V. paraheamolyticus)、迟钝爱德华氏菌(Edwardsiella tarda)和荧光假单胞菌(Psedomonas fluorescens)等水产养殖中主要病原细菌与抗血清之间的免疫交叉反应.结果表明,弧菌属细菌之间的交叉反应程度比较大,而与其他两属的细菌之间存在的交叉反应程度小,或不存在交叉反应;Western-blot分析结果显示,哈维氏弧菌、溶藻胶弧菌和副溶血弧菌抗血清分别与其他3种弧菌在分子量为135.6 kD和121.5 kD;95.6 kD,48.4 kD,39.2 kD和34.9 kD;55.1 kD的蛋白带处存在交叉反应,而这些分子量的蛋白带与其他两属的抗血清均不发生反应.     

红鳍东方鲀、菊黄东方鲀及其杂交F1幼鱼耗氧率与窒息点研究

利用封闭呼吸室对同一日龄、不同规格的红鳍东方鲀、菊黄东方鲀和菊黄东方鲀(♀)×红鳍东方鲀(♂)杂交F1代幼鱼进行耗氧率和临界窒息点的研究。试验结果表明,水温14.8～15.6℃时,体质量(37.24±3.64)g的红鳍东方鲀幼鱼耗氧率为(0.3385±0.0161)mg/(g.h),体质量(14.45±1.08)g的菊黄东方鲀幼鱼耗氧率为(0.2327±0.0241)mg/(g.h),体质量(27.96±1.38)g的杂交F1代东方鲀幼鱼耗氧率为(0.2282±0.0219)mg/(g.h);同一日龄不同规格红鳍东方鲀、菊黄东方鲀和杂交东方鲀幼鱼的耗氧量分别为(12.5243±0.6720)、(3.3544±0.2975)、(5.8469±0.9537)mg/(h.尾);3种东方鲀的耗氧率呈明显的昼夜节律,白天平均耗氧率显著高于夜晚。水温为14.8～15.6℃时,红鳍东方鲀、菊黄东方鲀及杂交东方鲀幼鱼的临界窒息点分别为0.665、0.882mg/L和0.774mg/L。     

红鳍东方鲀体表溃烂病优势菌的分离、鉴定及治疗

2021年11月，辽宁大连某养殖场红鳍东方鲀(Takifugu rubripes)出现死亡。通过对患病红鳍东方鲀病灶部位进行细菌分离培养、细菌革兰氏染色、以及细菌16S rDNA序列分析，从患病红鳍东方鲀中共分离出3株优势菌且均为弧菌，分别为哈维弧菌(Vibrio harveyi)、大菱鲆源弧菌(V.scophthalmi)、费氏弧菌(Aliivibrio fischeri)。药敏检测结果表明，费氏弧菌对复方新诺明、氟苯尼考、多西环素3种抗生素敏感；大菱鲆源弧菌仅对恩诺沙星敏感；哈维弧菌对环丙沙星、复方新诺明、氟苯尼考、多西环素、恩诺沙星5种抗生素敏感。3种优势菌对16种抗生素的药物敏感实验结果表明3种优势菌均为多重耐药菌。结合药敏实验结果与相关研究，推荐联合多西环素与山苍子植物精油对红鳍东方鲀体表溃烂病进行防治。     

MS-222对红鳍东方鲀幼鱼血液生化指标的影响

通过研究MS-222对红鳍东方鲀(Takifugu rubripes)幼鱼血液生化指标的影响,以对其使用安全性及麻醉机理的分析提供依据。分别使用20、40、60mg/L等不同浓度的MS-222麻醉红鳍东方鲀幼鱼,测定麻醉后1/4、4、8、24h和复苏后24h鱼体血糖(GLU),血清中总蛋白(TP)、谷丙转氨酶(ALT)、谷草转氨酶(AST)、总胆红素(T-BIL)、尿素氮(BUN)等血液生化指标和血清溶菌酶(LZM)的活性。结果表明,使用MS-222麻醉对红鳍东方鲀幼鱼的大部分血液生化指标和血清溶菌酶活性均有显著影响,且在高麻醉浓度下,这种影响更加显著。使用MS-222麻醉复苏24h后,红鳍东方鲀幼鱼的大多数血液生化指标和血清溶菌酶活性尚未恢复至麻醉前水平。     

菊黄东方鲀♀×红鳍东方鲀♂杂交后代早期形态特征及生长速度的比较

对菊黄东方鲀、红鳍东方鲀及其杂交后代的早期形态特征和生长速度进行了比较。在全长5 cm和10 cm时,形态上的主要差异是:(1) 菊黄东方鲀的尾鳍为浅黄色,无黑杂色;红鳍东方鲀的尾鳍为黑色,其杂交后代与菊黄东方鲀相似,尾鳍浅黄色,略带黑色;(2) 菊黄东方鲀背部皮刺无或极少超出侧线,超出部分与体背黑斑不相交;红鳍东方鲀背部皮刺超出侧线,超出部分与黑斑相交,向下延伸至腹部,其杂交后代背部皮刺超出侧线,与体背黑斑部分相交,向下未能延伸至腹部;(3) 菊黄东方鲀背部皮刺向前延伸至两眼连接线,红鳍东方鲀向前延伸至两鼻孔连接线,其杂交后代超过两眼连接线而未达鼻孔连接线。以尾鳍颜色和皮刺的分布特征可以区分幼鱼阶段的菊黄东方鲀、红鳍东方鲀及其杂交后代。在体长为5 cm及10 cm时分别选取了2个和4个框架参数建立判别函数,判别菊黄东方鲀、红鳍东方鲀及其杂交种的准确率达到96.8%和100%。经过110 d的饲养,红鳍东方鲀、杂交东方鲀以及菊黄东方鲀的平均体长分别达到(110.24±3.78)、(101.16±6.56)和(82.92±4.29) mm,体质量分别为(35.68±5.04)、(33.00±6.24)和(20.99±3.00) g,无论体长还是体质量,都是红鳍东方鲀>杂交东方鲀>菊黄东方鲀,差异极显著。研究表明,菊黄东方鲀♀与红鳍东方鲀♂杂交,其后代早期形态与母本菊黄东方鲀相似,生长比红鳍东方鲀慢,而比菊黄东方鲀快,具有显著的经济杂交价值。     

日本的红鳍东方鲀人工养殖技术

<正> 红鳍东方鲀是日本海水养殖的主要鱼类之一,在日本市场上成为颇受欢迎的水产品。一、生态习性红鳍东方鲀是一种温水性海洋鱼类,分布在日本本州中部以南沿海和我国黄渤海,栖息于沿岸砂质海域。日本的红鳍东方鲀生存水温范围是4～29℃,最适水温范围16～25℃,在水温14℃摄食活动开始降低,10℃停止摄食;当处于28℃以上高水温和10℃以下低水温不适环境时,潜入沙中(可能由于种群的差别,产于我国黄渤海的鲀鱼其适温范     

一例红鳍东方鲀盾纤毛虫病的诊治

<正>红鳍东方鲀(Takifugu rubripes),为鲀形目、鲀科、东方鲀属的暖水性鱼类。近年,我国红鳍东方鲀养殖业蓬勃发展,在辽宁、河北、山东、天津等地均有养殖,其产品主要出口日本、韩国。随着养殖规模的扩大,各种病害问题频发。在2016年10月,本实验室接诊了一个以体表各部位尤其头部出现不规则隆起为主要病征的红鳍东方鲀     

福尔马林药浴防治红鳍东方鲀异沟虫病的研究

异沟虫是红鳍东方鲀常见寄生虫之一,对红鳍东方鲀养殖业危害极大.为有效防治异沟虫病,在观察异沟虫虫卵孵化规律的基础上,研究评价福尔马林药浴对异沟虫的杀灭效果及其对红鳍东方鲀的安全性,形成红鳍东方鲀异沟虫病的防治方案.试验结果发现,异沟虫虫卵的孵化过程大致可分为孵化初期、孵化中期、孵化后期和破卵4个阶段,在17～19℃条件...     

Pathogenicity of Vibrio harveyi to salmonids

Out of 19 Vibrio harveyi isolates obtained from a diversity of hosts and geographical locations, 14 were pathogenic to rainbow trout, Oncorhynchus mykiss (Walbaum), and Atlantic salmon, Salmo salar L., with mortalities of up to 100% following intraperitoneal injections of 106 cells fish?1. The extracellular products (ECPs) of only five pathogenic isolates were harmful to fish. Both pathogenic and non‐pathogenic cultures produced ECPs containing caseinase, gelatinase, phospholipase, lipase and haemolysins. Vibrio harveyi VIB 645, which was the most pathogenic isolate, produced ECPs with a maximal effect on salmonids from preparations obtained by using cellophane overlays on tryptone soya agar supplemented with 1% (w/v) sodium chloride with incubation at 28 °C for 24 h. This preparation contained the highest titre of haemolytic activity to Atlantic salmon (1:256) and rainbow trout (1:32) erythrocytes.     

A comparison of the antigenicity of the extracellular products and whole-cell sonicates from Mycobacterium spp. in rabbits, mice and fish by immunoblotting and enzyme-linked immunosorbent assay

The antigenicity of extracellular products (ECPs) derived from Mycobacterium spp. isolated from snakehead, Channa striata (Bloch), and Siamese fighting fish, Betta splendens (Regan), were examined by immunoblotting and enzyme-linked immunosorbent assay (ELISA) using sera collected from immunized rabbits, mice and fish (rainbow trout). All three species responded to a 65-kDa protein present in both the ECPs and whole cell sonicates (WCSs) from a variety of Mycobacterium spp. Cross-reactivity of anti- M. tuberculosis and anti-human heat-shock protein monoclonal antibodies (MAbs), and the presence of fibronectin binding proteins secreted into ECPs of mycobacteria were also examined. The MAbs against human 60-kDa heat-shock protein cross- reacted with the band at 65 kDa in the ECPs of TB1 (isolated from snakehead fish) and the type strain M. marinum, while the anti- M. tuberculosis MAb F29–47 elicited a strong reaction with a band at 21 kDa with most of the ECPs from mycobacterial strains examined. The major fibronectin-binding proteins were located between 21 and 25 kDa. The 65-kDa protein from ECPs of Mycobacterium spp. proved strongly immunogenic to rabbits, mice and fish. Rabbit antiserum against the 65-kDa protein from strain TB267 reacted with many non- Mycobacterium WCSs, and therefore, the 65-kDa protein from Mycobacterium spp. is believed to be a common protein found in many fish bacterial pathogens.     

Characterization of extracellular products from an isolate of Vibrio harveyi recovered from diseased post-larval Penaeus vannamei (Bonne)

Vibrio harveyi recovered from diseased post-larval Penaeus vannamei produced a thermostable exotoxin, which was lethal to Dublin Bay prawns, Nephrops norvegicus L., when injected intramuscularly. The extracellular products (ECPs) concentrated from tryptone soya broth supplemented with 1% (w/v) sodium chloride or from cellophane overlays on marine 2216E agar with incubation at 15, 22 and 27 °C were toxic, with the lethal dose 50% of the crude ECPs estimated to be 4.4 μgprotein prawn^−1. Proteolytic, haemolytic and cytotoxic activities were detected, although the occurrence and quantity of these activities were influenced by cultural conditions. The ECPs which had been heated (100 °C for 10 min) or digested with protease K produced the same pathology as crude, untreated ECPs. Western blotting demonstrated that all the ECP preparations contained low molecular weight lipopolysaccharides, which may constitute the lethal toxin of V. harveyi.     

哈维氏弧菌(Vibrio harveyi)ML01株胞外产物及分泌性蛋白的分离与特性分析

本研究以引起珍珠龙胆石斑鱼[Epinephelus fuscoguttatus(♀)×Epinephelus lanceolatus(♂)]幼鱼“皮肤溃疡病”的哈维氏弧菌(Vibrio harveyi) ML01株为研究对象,采用平板膜覆盖技术和柱层析技术,分离、纯化了ML01株的胞外产物及分泌性蛋白.应用毒性试验、质谱分析与分子克隆技术,对纯化的胞外产物和3种主要的分泌性蛋白进行了特性分析与鉴定.结果显示,哈维氏弧菌ML01株的胞外产物(Extracellular products,ECPs)具有酯酶、明胶酶、淀粉酶、酪蛋白酶活性,无脲酶活性.ECPs对羊红细胞无溶血性,对斑马鱼(Danio rerio)的半数致死剂量(LD50)为19.55μg/g鱼体重.从ML01株中分离到3种主要的分泌蛋白P42、P36、P31,其分子量分别为42、36、31 kDa.经质谱鉴定和分析,这3种蛋白分别为哈维氏弧菌的外膜蛋白OmpU和OmpN,以及一种功能未知的蛋白.利用同源克隆,成功地从ML01株基因组中扩增到了P42、P36、P31的基因.序列测定和比对结果显示,ML01株的这3个基因与哈维氏弧菌ATCC 33843(GenBank CP009467)的相应基因相比,其开放阅读框(Open reading frame,ORF)序列的相似性分别为97.08％、100％、99.67％,其编码的多肽序列的相似性分别为99.71％、100％和99.93％.本研究对进一步分析哈维氏弧菌ML01株的致病机理、研发该菌的亚单位疫苗具有重要的参考价值.     

The effect of in vivo growth on the cellular and extracellular components of the marine bacterial pathogen Photobacterium damsela subsp. piscicida

Photobacterium damsela subsp. piscicida, the causative agent of fish pasteurellosis, was grown in vivo. Bacterial cells and extracellular products (ECPs) were analysed via electrophoresis and immunoblot analysis, using specific sea bass antisera. Growth in vivo induced the synthesis of unique bacterial cell proteins at > 206, 206, 21.3, 18, 7.6 and < 7.6 kDa. Sea bass serum raised against live bacterial cells of the pathogen and especially a sea bass serum raised against formalin-inactivated bacterial cells grown in a specific novel medium recognized the novel antigens at > 206 (associated with iron sequestration), 21.3, 7.6 and < 7.6 kDa, suggesting that the latter medium conserves the synthesis of natural bacterial cell proteins in vitro. In vivo growth of the pathogen induced the synthesis of more toxic ECPs in comparison with in vitro growth and an inverse correlation between total protein concentration in the ECPs and toxicity per unit of protein was observed. Substrate-polyacrylamide electrophoresis revealed the presence of in vivo synthesized ECPs of the pathogen (proteases) at 175, 132, < 79 and 48.3 kDa. Histological examination of tissues isolated from fish injected with these ECPs revealed inflammatory and necrotic lesions in the spleen, liver, head kidney, intestine and heart as soon as 48 h post-introduction of the ECPs.     

Evidence of exotoxin secretion of Piscirickettsia salmonis,the causative agent of piscirickettsiosis

Piscirickettsia salmonis is the aetiological agent of piscirickettsiosis, a disease which affects a variety of teleost species and that is particularly severe in salmonid fish. Bacterial‐free supernatants, obtained from cultures of three isolates of Piscirickettsia salmonis, were inoculated in Atlantic salmon, Salmo salar L., and in three continuous cell lines in an effort to determine the presence of secretion of extracellular products (ECPs) by this microorganism. Although steatosis was found in some liver samples, no mortalities or clinical signs occurred in the inoculated fish. Clear cytotoxicity was observed after inoculation in the cell lines CHSE‐214 and ASK, derived from salmonid tissues, but not in MDBK, which is of mammalian origin. The degree of cytotoxicity of the ECPs was different among the P. salmonis isolates tested. The isolate that evidenced the highest cytotoxicity in its ECPs exhibited only an intermediate virulence level after challenging fish with bacterial suspensions of the three P. salmonis isolates. Almost complete inhibition of the cytotoxic activity of ECPs was seen after proteinase K treatment, indicating their peptidic nature, and a total preclusion of the cytotoxicity was shown after their incubation at 50 °C for 30 min. Results show that P. salmonis can produce ECPs and at least some of them are thermolabile exotoxins that probably play a role in the pathogenesis of piscirickettsiosis.     

New perspective to control of tenacibaculosis in sea bass Dicentrarchus labrax L

This work was carried out to investigate the effects of injection of Tenacibaculum maritimum formalin‐killed cells (FKC), extracellular products (ECPs) and crude lipopolysaccharide (LPS) as well as 1% feed supplements of oil extracts of Echinacea purpurea and Origanum vulgare on sea bass immunity improvement to the favour of T. maritimum experimental infection control after 4 weeks of the experiment. Tenacibaculum maritimum isolated from naturally infected sea bass showed brown to yellowish‐brown lesions (sores) on gills, skin and/or fins and identified by different biochemical methods and polymerase chain reaction technique. Immune parameters namely, total protein, globulin and lysozyme activity, as well as the relative level of protection were improved by T. maritimum (FKC), (LPS), (ECPs), O. vulgare and E. purpurea, respectively compared with control. Histopathological examination of T. maritimum naturally infected sea bass indicated many pathological changes in gill, skin and musculatures. Present study could be concluded that application of T. maritimum (FKC), (LPS), (ECPs), O. vulgare or E. purpurea improved sea bass immunity to the favour of disease resistance against T. maritimum. Further investigations on the combination between the previous control methods and the vaccine application methods will be needed.     

The effect of iron limitation growth conditions on the cell and extracellular components of the fish pathogen Pasteurella piscicida

The effect of iron limitation, using the iron-chelating agent 2,2 dipyridyl, on the electrophoretic profiles of outer membrane proteins (OMPs) and extracellular products (ECPs) from 21 Pasteurella piscicida strains isolated from Europe and Japan was investigated. In addition, the effect of iron-limited and iron-surplus growth conditions on caseinase activity in culture supernatants of the pathogen was examined. The majority of P. piscicida strains, from Greece, Italy and France, cultured under iron-limited conditions, produced four novel OMPs (63 and three at and above 200 kDa). In contrast, iron-regulated outer membrane proteins were not induced in Japanese strains. Electrophoretic analysis of the ECPs from the pathogen grown under iron surplus and iron limitation revealed a large range of products and additional high molecular mass (MM) bands were evident under iron-limited conditions. When culture supernatants were analysed for their activity, most of the bacteria tested showed elevated activities under iron limited conditions. Finally, neither hydroxamate nor phenolate type siderophores could be detected with any of the chemical assays used.     

Vibrio species isolated from diseased farmed sole,Solea senegalensis (Kaup), and evaluation of the potential virulence role of their extracellular products

Bacteria isolated from an outbreak with moderate mortalities in farmed sole, Solea senegalensis (Kaup), in the south of Spain were identified as Vibrio harveyi and V. parahaemolyticus. Only bacterial strains showing swarming were virulent in sole and caused mortalities in experimentally inoculated fish. However, the signs of the disease were only reproduced with V. harveyi. The intramuscular inoculation of the extracellular products (ECPs) of both species produced mortalities in inoculated fish and the appearance of surface ulcers in the case of V. harveyi. However, the inoculation of sublethal doses of ECPs to fish showed a protective effect against V. harveyi.     

Investigation of media formulations promoting differential antigen expression by Photobacterium damsela ssp. piscicida and recognition by sea bass,Dicentrarchus labrax (L.), immune sera

Photobacterium damsela ssp. piscicida (Phdp) isolates were grown in various bacteriological media, in eukaryotic cell culture media and in the presence of fish cells (resembling some aspects of in vivo growth environments). Bacterial cells, extracellular products (ECPs) and crude capsular polysaccharide were isolated and analysed by electrophoresis and Western blot using sea bass sera. Growth in bacteriological media conserved the synthesis of cell and extracellular components when these were compared with those prepared under near-in vivo growth conditions. In fact, synthesis of a larger range of cell components was induced after growth in bacteriological media. Certain media based on yeast extract and peptones from various sources and a specific salt formulation induced the synthesis of novel cell components at approximately 21.3 and 14 kDa. These antigens were recognized by sea bass sera collected after natural pasteurellosis outbreaks and other sea bass sera raised against live or inactivated Phdp cells. The ECPs of the pathogen were not good immunogens in their soluble form despite various treatments prior to immunization. The results are discussed with respect to vaccine development.