异硫氰酸荧光素标记鳗弧菌条件优化及应用条件对荧光信号的影响

为了实现对目标细菌的有效示踪,实验以异硫氰酸荧光素(FITC)为标记物,分别设置FITC标记浓度0.2、2、20、50、100、200μg/mL,标记时间1、15、30、60、120、240 min,研究了FITC标记浓度与标记时间对鳗弧菌标记效果的影响,同时对在不同温度(25、4、-20、-70℃)及不同时间(24、48、96、192 h)存储条件下鳗弧菌标记荧光的稳定性进行了比较,对标记鳗弧菌经甲醛灭活后的荧光变化进行了观察,研究了不同光照条件(0、150、1 500、25 000 lx)对标记鳗弧菌的荧光衰减影响,以及在-20和-70℃条件下反复冻融对标记鳗弧菌荧光强度的影响。结果表明,FITC的最适标记浓度范围为20~100μg/mL,最适标记时间范围60~120 min,储藏在-20和-70℃条件下,标记后的鳗弧菌荧光信号比储藏在4和25℃条件下稳定,标记后的鳗弧菌经甲醛灭活后的荧光信号稳定性优于对照组,不同强度自然光照对标记鳗弧菌的荧光衰减效果不明显,标记细菌经3次冻融,发现在-70℃条件下冻存的细菌荧光信号稳定性保持良好。本研究结果为FITC用于标记海洋细菌提供了实用的技术参数。     

鱼肠道弧菌胶体金快速检测试纸研制

鱼肠道弧菌(Vibrio ichthyoenteri)可引起多种养殖鱼类发病死亡, 给鱼类养殖业带来严重经济损失。为解决养殖过程中鱼肠道弧菌的现场快速检测问题, 本研究研制了鱼肠道弧菌胶体金快速检测试纸。通过制备兔抗鱼肠道弧菌多克隆抗体, 间接ELISA分析发现其与鱼肠道弧菌的外膜蛋白、鞭毛蛋白、胞外产物及全菌破碎蛋白发生阳性免疫反应, 与副溶血弧菌(Vibrio parahaemolyticus)、鳗弧菌(Vibrio anguillarum)、溶藻弧菌(Vibrio alginolyticus)、哈维氏弧菌(Vibrio harveyi)的4种抗原蛋白发生程度不等的免疫交叉反应。制备胶体金标记鱼肠道弧菌抗体, 确定了鱼肠道弧菌4种抗原蛋白的划线浓度, 以4种抗原蛋白作为4条检测线, 羊抗兔IgG作为质控线, 基于竞争法免疫层析技术研制出鱼肠道弧菌快速检测试纸。对鱼肠道弧菌、副溶血弧菌、鳗弧菌、溶藻弧菌和哈维氏弧菌的检测结果显示, 该试纸可以准确鉴别出鱼肠道弧菌, 并能判别其他病原菌的交叉反应。该试纸对鱼肠道弧菌的最低检测限为5×10⁵ CFU/mL, 检测耗时为10 min。对患病牙鲆组织的检测结果显示, 该试纸与ELISA结果一致, 表明具有较好的检测准确性。本研究为水产养殖现场的鱼肠道弧菌快速、准确检测提供了有力工具。     

鳗弧菌多克隆抗体的制备及其特性分析

<正>鳗弧菌是隶属弧菌科、弧菌属的革兰氏阴性菌。该病原最早分离自欧洲鳗鲡,可导致多种海水养殖鱼类的出血性败血症。鳗弧菌病发病迅速、死亡率高、流行范围广,会让渔业生产造成严重的经济损失。多克隆抗体具有制备简单、耗时短、免疫学研究用途广等优点。因此,本研究制备鳗弧菌特异性多克隆抗体,以期为鳗弧菌免疫保护性抗原的筛选和快速检测方法的建立打下物质基础。     

海水养殖动物致病性(Pathogenicity)鳗弧菌(Vibro anguillarum)的研究综述

鳗弧菌（Vibrio anguillarum）广泛存在于沿岸海水、沉积物和海洋动物体中。是海水鱼、虾、贝类的一种常见的细菌性病原。随着海水养殖业的日益发展．致病性鳗弧菌对养殖动物的危害也越来越严重。主要对鳗弧菌的生物学性状、检测技术、鳗弧菌病症状和病理变化及海水养殖动物鳗弧菌病的防治作了简单回顾，并对鳗弧菌病的诊断、防治方法及其研究方向进行了探讨和展望。     

花鲈弧菌病病原菌（哈维氏弧菌）的分离与鉴定

1999年4－5月，山东省青岛市胶南海区网箱养殖场花鲈（Lateolabrax japonicus)幼鱼发生暴发性传染病，死亡率达50％。从具有明显症状的病鱼的病灶组织分离到1株优势菌SF－1，经人工感染和从人工感染发病的花鲈再分离的SF－3菌株的再感染试验结果表明，所分离的菌株为此次花钙烂层病的致病菌，经形态、生理生化等64项特征指标鉴定，SF－1和SF－3均为哈维氏弧菌（Vibrio harveyi)。药敏试验结果表明，头孢噻肟，头孢三嗪，头孢孟多，呋喃妥因，氯霉素,氧哌嗪青霉素，磺胺类，复方磺胺、三甲氧苄氨嘧啶，多粘菌素E等10种药物对该菌株有明显的抑制作用。     

豚鼠气单胞菌胶体金免疫层析试纸条的研制

采用淋巴细胞杂交瘤技术制备抗豚鼠气单胞菌单克隆抗体分泌细胞株，获取抗豚鼠气单胞菌单克隆抗体，柠檬酸三钠还原氯金酸制备胶体金颗粒，选择直径20 nm的胶体金颗粒，标记抗豚鼠气单胞菌单克隆抗体并制备胶体金垫。将胶体金垫与喷涂有抗豚鼠气单胞菌兔多克隆抗体和羊抗鼠抗体的硝酸纤维素膜及样品吸收垫等组装成免疫层析试纸条，建立豚鼠气单胞菌的快速检测方法。用灭活细菌与血清混合的模拟样本测定试纸条的特异性、灵敏度，结果显示，试纸条对嗜水气单胞菌、温和气单胞菌、鳗弧菌、溶藻弧菌、荧光假单胞菌、副溶血弧菌等13种常见病原菌没有交叉反应，与豚鼠气单胞菌显示特异性反应，检测灵敏度为1×10⁶ CFU/mL，结果显示时间小于5 min。研制的豚鼠气单胞菌胶体金免疫层析检测试纸条具有快速、简便、特异性高、适用于基层临床生产推广应用等优点。     

3株O3血清型鳗弧菌灭活疫苗的免疫原性和免疫保护效果

鳗弧菌(Vibrio anguillarum)O3血清型菌株是感染鱼类的重要病原菌,本文研究了3株O3血清型鳗弧菌(SMP1、SMP3和SMP4)灭活疫苗的免疫原性和免疫保护。首先在牙鲆(Paralichthys olivaceus)体内对3株鳗弧菌进行复壮;检测复壮后的菌株毒力,检测的3株鳗弧菌对蓝蔓龙(Trichogaser trichopterus)的半数致死量(LD50)分别为105.1 CFU/ml(SMP1)、104.7 CFU/ml(SMP3)和105.4 CFU/ml(SMP4);制备了3株菌的甲醛灭活疫苗,注射免疫牙鲆,免疫后第7天可检测到牙鲆的血清特异性抗体产生,免疫后第28天的血清特异性抗体效价为1:1280(SMP1)、1:640(SMP3)和1:905(SMP4),提供的免疫保护率(Relative percent survival rate,RPS)为94.4%(SMP1)、100%(SMP3)和73.7%(SMP4)。研究表明,3株致病性O3血清型鳗弧菌菌株具有良好的免疫原性,其中SMP3为最适疫苗候选株。本研究为鳗弧菌O3血清型疫苗的开发应用奠定了基础。     

5株鲆鲽鱼类病原菌多克隆抗体的制备与应用

本研究制备了鳗弧菌Vibrio anguillarum、杀鲑气单胞菌Aeromonas salmonicida、副溶血弧菌V. parahaemolyticus、哈维氏弧菌V. harveyi和腐败希瓦氏菌Shewanella putrefaciens 5株鲆鲽鱼类病原菌的兔抗血清，建立了5种菌的间接ELISA检测方法，并将该检测方法用于鱼类细菌分离物病原检测。人工感染结果显示，5株菌对大菱鲆的半数致死量(LD50)在102~107CFU/fish；制备的兔抗血清效价分别为1∶2 048 000、1∶16 000、1∶16 000、1∶1 024 000、1∶128 000；交叉反应结果显示，鳗弧菌、副溶血弧菌、哈维氏弧菌3株弧菌与抗血清相互之间存在交叉反应；抗血清特异检测灵敏度分别为104、108、107、105、106cell/ml；对13株海水鱼类细菌分离物进行检测，有1株腐败希瓦氏菌阳性，两株哈维氏弧菌阳性；1株副溶血弧菌和哈维氏弧菌均为阳性，该结果与16S rDNA序列的分子分析方法一致。     

鳗弧菌和溶藻弧菌二联疫苗对大菱鲆的免疫效果

将鳗弧菌（Vibrio anguillarum）和溶藻弧菌（V.alginolyticus）分别用0.3%福尔马林灭活,制备成鳗弧菌和溶藻弧菌2种单苗,并将2种菌等量配制成二联疫苗,单次腹腔注射免疫大菱鲆（Scophtalmus maximus）.通过检测免疫后替代途径补体活力、溶菌酶活力、抗体凝集效价的变化并分别进行攻毒实验比较疫苗的免疫效果.结果显示,3种疫苗对所测定的各免疫指标都有一定的促进作用,但二联疫苗后期效果低于2种单苗.二联疫苗对鳗弧菌和溶藻弧菌的免疫保护率分别为83.52%和83.24%,而鳗弧菌单苗对鳗弧菌攻毒、溶藻弧菌单苗对溶藻弧菌攻毒的免疫保护率分别为80.37%和74.89%.可以认为2株菌具有制备二联疫苗的可能性.[中国水产科学,2006,13（3）：397-402]     

养殖刺参“腐皮综合征”致病菌——灿烂弧菌的原位杂交检测方法的建立与应用

以养殖刺参“腐皮综合征”的重要致病菌——灿烂弧菌Vibrio splendidus的16S~23S间隔区序列，设计特异性引物，用PCR方法制备地高辛(DIG)标记探针，建立了原位杂交技术检测感染刺参体内灿烂弧菌的技术方法，并利用该方法对人工感染刺参和健康刺参各组织进行检测。结果显示，感染刺参的体壁结缔组织、肌肉组织、肠粘膜上皮、辐水管等组织的原位杂交检测呈阳性，而与健康刺参组织无交叉反应。在感染组织中，阳性信号（显色）强弱清晰，能准确反映出灿烂弧菌的侵染部位及感染程度，这为探明灿烂弧菌的感染途径、感染病程等致病机理研究奠定了基础，也为养殖刺参疾病预防和健康管理提供了技术支撑。     

Salmonid whirling disease: myxosporean and actinosporean stages cross-react in direct fluorescent antibody test

Abstract. Serologic relatedness of the two life stages of the salmonid whirling disease parasite Myxosoma cerebralis Hofer, 1903 — myxosporean spores from fish cartilage and actinosporean triactinomyxon spores from aquatic tubificids — were investigated. When the direct fluorescent antibody technique was used, anti-triactinomyxon and anti- M. cerebralis rabbit sera conjugated with fluorescein isothiocyanate cross-reacted with the respective heterologous life stage. Both stages showed similar locations of specific fluorescence with conjugates of either homologous or heterologous serum. Thus, serology supports the relatedness of the myxosporean M. cerebralis and the actinosporean triactinomyxon stages.     

Challenge studies of European stocks of redfin perch, Perca fluviatilis L., and rainbow trout, Oncorhynchus mykiss (Walbaum), with epizootic haematopoietic necrosis virus

A challenge model for comparison of the virulence of epizootic haematopoietic necrosis virus (EHNV) to European stocks of redfin perch, Perca fluviatilis L., and rainbow trout, Oncorhynchus mykiss (Walbaum), was tested. The model investigated intraperitoneal (IP), bath and cohabitation routes at 10, 15 and 20 °C for 5–6 g fish and 15 °C for 20 g perch. In the IP challenges of perch, significant mortality occurred at 15 °C and 20 °C. In challenge trials for rainbow trout, significant mortalities were observed in IP and bath challenges at 20 °C. The mortality observed in IP challenged 20 g perch was not significantly different from that recorded for 6 g fish challenged IP. No significant mortality was observed in any other treatment groups. Re-isolation of ranavirus was confirmed by IFAT and was consistently associated with dead or moribund fish in the trial groups challenged with EHNV. The findings indicate that EHNV does not pose a high risk for wild perch and trout populations in Europe by natural exposure. Mortality appears to be primarily a function of environmental factors, with temperature playing an important role, and not just the presence of the virus in the fish.     

Further studies of an epizootic of perch Perca fluviatilis L., of uncertain aetiology

Abstract. The geographical distribution of an epizootic of perch Perca fluviatilis L., characterized by severe epidermal lesions and marked fish kills, is described. A wide variety of fungal and bacterial infections were found in diseased fish but the evidence suggests that they were not the primary cause of this condition. The primary pathogenic agent (assuming that it exists) or predisposing environmental factor has yet to be discovered. The severity of this disease is illustrated by monitoring the course of the epizootic in the perch population in Windermere. All age groups of fish were affected during the course of one year although the fish at spawning time appeared to be particularly vulnerable. There was a dramatic increase in the perch mortality in Windermere in 1976 and it is estimated that over 98% of the adult population (over 1 million fish) died in that year.     

显微注射技术在制备鱼类嵌合体和转基因海水鱼上的应用

以鱼类胚胎细胞和胚胎干细胞为核供体进行细胞移植构建鱼类嵌合体研究方面，现有的成功报道均采用显微注射方法；在转基因海水鱼类研究中，显微注射也是最为常用的技术，本实验室在花鲈胚胎干细胞嵌合体构建和外源基因向花鲈胚胎的转移研究中取得的结果也充分证实，显微注射技术是开展海水鱼类细胞移植和转基因研究的首选技术。     

Occurrence and pathogenicity of Yersinia ruckeri at fish farms in northern and central Finland

Abstract. Salmonid fish at fish farms in northern and central Finland and perch, Perca fluviatilis L., roach, Rutilus rutilus (L.), and whitefish, Coregonus sp., from four lakes in central Finland were studied between 1985 and 1990 for the occurrence of Yersinia ruckeri. The bacteria were found in fish from both areas, but in most cases, only single diseased salmon, Salmo salar L., brown trout, S. trutta L., rainbow trout, Oncorhynchus mykiss (Walbaum), whitefish and perch were encountered and were always connected with stress conditions. One clinical outbreak occured in salmon fingerlings in northern Finland, and the fish were successfully treated with trimethoprim-sulpha. Monthly monitoring of lake fish revealed two symptomless carrier perch in two lakes. Outwith the main study a moribund perch with yersiniosis was found in a polluted lake, and for the first time in Finland, a rainbow trout was also found to have contracted yersiniosis in a small private pond. Sorbitol-positive and negative isolates have been found to occur in both moribund and carrier farmed fish, indicating that the sorbitol test is not essential when evaluating the pathogenicity of Y. ruckeri.     

饥饿及恢复投饵过程中花鲈肌肉组成及非特异免疫水平的变化

探讨了饥饿及恢复投饵过程中花鲈（Lateolabrax japonicus）（196±38g）肌肉组成分及鱼体非特异免疫水平的变化。实验设S0组（对照组，持续正常投喂），S5组（饥饿5d后恢复正常投喂），S10组（饥饿10d后恢复正常投喂），S15组（饥饿15d后恢复正常投喂）。S0组每隔5d取样，S5，S10和S15组分别在结束饥饿及恢复投饵后每隔5d取样测定。结果显示：饥饿程度对花鲈肌肉组成有显著影响。与对照组相比，S10组饥饿结束时肌肉的总脂含量显著降低；S15组饥饿结束时肌肉的总脂含量和粗蛋白含量都显著降，同时显著增加了肌肉的水分含量。表明饥饿过程中花鲈先动用肌肉脂肪，后动用肌肉蛋白。在恢复投饵过程中，S10和S15组总脂含量表现出先降后升的规律，而S15组蛋白表现为逐步回升的趋势。表明花鲈在饥饿后恢复投饵的过程中，先恢复肌肉蛋白含量，后恢复肌肉脂肪含量。饥饿15d时花鲈血清蛋白浓度显著降低。与对照组相比，S5组血清、脾脏和头肾溶菌酶活性均无显著变化；S10组头肾溶菌酶活性在恢复投饵10d时补偿性升高。S15组在饥饿结束时显著降低了花鲈头肾溶菌酶活性，但在恢复投饵5d时恢复到对照组水平。花鲈白细胞的吞噬活性也受饥饿程度及恢复投饵的影响。饥饿会降低花鲈血液白细胞的吞噬活性。在恢复投饵过程中,饥饿（5d）后恢复投饵会引起白细胞吞噬百分率和吞噬指数的补偿性升高。表明饥饿及恢复投饵也影响花鲈的非特异免疫水平。     

Development of Microsatellite Markers in Sea Perch,Lateolabrax Japonicus,from Codominant Amplified Fragment Length Polymorphism Bands

A fast and cost‐effective protocol to develop candidate microsatellite markers from sea perch, Lateolabrax japonicus, was described here. Ten suites of codominant bands that contained seven microsatellites were discovered in this marine fish, in which no microsatellite development was reported previously. All the seven microsatellites were found to be polymorphic among tested 20 individuals of sea perch. Five of the above seven loci conformed to Hardy–Weinberg equilibrium (HWE) as determined by using the Markov‐Chain method implemented, the other two loci significantly deviated from HWE, both of them showed a large heterozygote excess (H_E). Out of 21 possible pair‐wise comparisons among the seven loci applied to sea perch, none showed significant linkage disequilibrium (LD) (P > 0.008). Five additional fish species assessed for cross‐species amplification revealed that some of the loci appear to be applicable in close‐related species.     

不同因子对花鲈胚胎干细胞增殖的影响

胚胎干细胞(ES细胞)是从动物早期发育胚胎中分离出来的具有发育全能性的一种未分化细胞系。维持花鲈胚胎干细胞(LJES1)的体外生长、增殖及未分化状态需要在培养基中添加一些生长因子。实验通过配制省略1种或多种因子的培养基PESM0～10，计数LJES1细胞在各培养基中增殖的数量，确定各种生长因子对LJES1细胞的增殖作用。同时，对LIF因子和bFGF因子的作用进行了重点的研究，发现LIF因子对早期的LJES1细胞增殖几乎没有作用，其主要作用是维持LJES1细胞的未分化状态，但对晚期LJES1细胞的增殖有一定的作用；bFGF因子对LJES1细胞有强烈的刺激增殖的作用；鲈鱼胚胎抽提液(PEE)以及鲈鱼血清(FS)也促进了LJES1细胞的增殖，2-巯基乙醇(2-ME)具有还原血清中的含硫化合物、防止过氧化物对LJES1细胞的损害及促进贴壁的作用，因而也促进了LJES1细胞的增殖。     

Evaluation of immunological parameters in farmed gilthead sea bream, Sparus aurata L., before and during outbreaks of 'winter syndrome'

In recent years, 'winter syndrome' has caused severe economic losses in many marine farms in southern Europe. This study compared the activity of the immune system in healthy, asymptomatic and diseased gilthead sea bream. Serum protein and immunoglobulin content were evaluated as well as serum complement activity from these groups of fish. Differential leucocyte counts were also determined. Immunological assays were performed to determine intra- and extracellular 'respiratory burst' activity as non-specific immune response parameters. Diseased fish showed a significant reduction in haemolytic activity (90%), and in serum proteins and immunoglobulins in comparison with the other groups. A significant increase in the lymphocyte percentage (50%) and a decrease in the granulocyte percentage (over 70%) was found in asymptomatic fish. Respiratory burst activity was reduced in both the clinical and preclinical stages of the disease, compared with the controls. These observations confirm a severe immunodeficiency in diseased fish but also the presence of a cellular immune dysfunction in fish without clinical signs, before the onset of the disease.     

鲤鱼棘头虫病的病理及预防

本文报导了病鱼的组织(肠、肝、胰、肾)病理变化,及血液(血清的钠、钾、氯化物、肌酐、尿素氮、谷草转氨酶总活力及谷草转氨酶同工酶。白细胞血式)病理变化,从而阐明了引起病鱼慢性死亡,而累计死亡率又高达60％的机理。查明了病原(崇明长棘吻虫)的中间寄主是模糊裸腹溞,找到了用生石灰或漂白粉清塘、在发病地区用鱼种池培育的鲤鱼鱼种的有效预防方法。