Protective immune responses of recombinant outer membrane proteins OmpF and OmpK of Aeromonas hydrophila in European eel (Anguilla anguilla)

Outer membrane proteins (Omps) of Gram‐negative bacteria have been proven to be efficient subunit vaccines against bacteriosis. In this study, OmpF and OmpK of Aeromonas hydrophila were expressed, and their immune protective effects in European eel (Anguilla anguilla) were evaluated. The genomic DNA of A. hydrophila 322A was used as a template, and two kinds of prokaryotic expression plasmids, pET‐32a‐OmpF and pET‐32a‐OmpK, were constructed. Recombinant OmpF protein (r‐OmpF) and r‐OmpK were purified and were proven to have antigenicity by Western‐blot analysis. r‐OmpF and r‐OmpK were used as immunogens to immunize European eel by intraperitoneal injection. The mRNA expression of 6 immune‐related genes (IgM, IL‐10, IRF3, IRF7, LysG4 and HexB) in the liver tissues of eels at 1 hr, 3 hr, 6 hr, 12 hr, 24 hr, 72 hr and 10 days postimmunization was analysed by real‐time PCR. At 30 dpi, the serum antibody response was measured by ELISA. Fish were attacked at 15 dpi by live 322A to assess the protective immunity of r‐OmpF and r‐OmpK. All the six tested genes responded to r‐OmpF or r‐OmpK vaccination at varying degrees. The serum antibody titre of r‐OmpF‐ and r‐OmpK‐immunized groups was 1:1,600 and 1:3,200 respectively. In addition, r‐OmpF gave 35.5% of the relative immune protection rate to European eels, while r‐OmpK gave 70.0%. By analysing the protective immunity and the regulatory role in the immune‐related gene expression of the two recombinant proteins that were studied, it was found that r‐OmpK was a potential vaccine candidate against A. hydrophila.     

澳洲长鳍鳗染色体组型的初步研究

以澳洲长鳍鳗(Anguilla reinhardtii)头肾组织为材料,采用腹腔注射植物血细胞凝集素(PHA)、秋水仙素和空气干燥法制备染色体标本,进行染色体组型分析.结果显示:澳洲长鳍鳗染色体为38条,核型公式为2n=14m+6sm+18t,NF=58;未发现随体、次级缢痕及异形染色体.     

同位素稀释液质联用法测定鱼体中氯丙嗪的残留

建立了高效液相色谱-串联质谱(HPLC/MS/MS)分析鱼体中氯丙嗪残留的方法.样品用乙腈提取,MCX固相萃取柱净化,氨化甲醇洗脱.在正离子模式下以电喷雾串联质谱仪进行测定,采用内标法定量,氘代氯丙嗪为内标物.以欧洲鳗鲡、鳜鱼和大口黑鲈为研究对象,在1.0、5.0、25.0μg/kg添加水平下,方法平均回收率为90.1...     

几种环保型饲料添加剂对欧洲鳗鲡消化酶活性的影响

选取健康均匀的欧洲鳗苗1000尾,分为5组,每组2个重复,分别在基础料中添加4种环保型饲料添加剂,分析了4种环保型饲料添加剂对欧洲鳗鲡消化酶活性的影响。结果表明,1号饲料添加剂对于提高欧洲鳗鲡消化酶整体效果最好,对中草药原料的配伍进行选择优化,适当添加半胱胺,能显著提高欧洲鳗鲡幼鱼消化酶活性。     

Migratory history of the giant mottled eel (Anguilla marmorata) in the Bonin Islands of Japan

Chino N, Arai T. Migratory history of the giant mottled eel ( Anguilla marmorata ) in the Bonin Islands of Japan.
Ecology of Freshwater Fish 2010: 19: 19–25. © 2009 John Wiley & Sons A/S
Abstract –  In order to elucidate the migratory history and habitat use of the giant mottled eel Anguilla marmorata, in the Bonin Islands of Japan, strontium (Sr) and calcium (Ca) concentrations in otoliths were examined by X-ray electron microprobe analysis. The X-ray intensity map and line analyses showed three fluctuation patterns: (i) freshwater residence, (ii) continuous residence in brackish water, and (iii) residence in freshwater after recruitment, while returning to brackish water. Based on these results and previous studies, the migratory behaviours of A. marmorata appear to differ in each habitat in response to inter- and intra-specific competition. The results suggest that A. marmorata has a flexible pattern of migration, with an ability to adapt to various habitats and salinities.     

日本鳗鲡腐皮病病原菌的分离及鉴定

从患腐皮病的日本鳗鲡(Anguilla japonica)体表溃烂处分离到1株病原菌(322A),对其进行了人工感染实验和生理生化分析,测定了该菌株的16S rRNA基因序列和促旋酶(gyrase)B亚单位gyrB基因序列,并分别构建系统发育树。结果显示:感染实验证实菌株322A具有致病性;生理生化分析鉴定该菌株属于气单胞菌属(Aero-monassp.);16S rRNA基因分析显示,该菌株与气单胞菌属细菌的同源性均在99%~100%,构建的系统树显示,菌株322A与嗜水气单胞菌(A.hydrophila(FJ462702))亲缘关系最近;gyrB基因分析表明,该菌株与A.hydrophila种内序列的相似性为96%~98%,种间序列的相似性为94%~95%,构建的系统树结果显示,该菌株与A.hydrophila(FJ608553、FJ608552、AF208259)聚为一个分支。综合上述实验结果,菌株322A可鉴定为嗜水气单胞菌(A.hydrophila)。     

Differences in head shape of the European eel, Anguilla anguilla (L.)

Differences in eel, Anguilla anguilla (L.), head shape were the result of greater increases in head width, rather than changes in head length. The ratio of head width:total length (HW:TL) increased significantly ( t -test, P < 0.05) from the glass-yellow eel stage. Cultured yellow eels were exclusively narrow headed (mean HW:TL = 0.027, range = 0.023–0.032), while wild yellow eels displayed an array of head shapes (mean HW:TL = 0.033, range = 0.023–0.046). Therefore, broad heads (HW:TL 0.033) occurred only among wild yellow eels sampled and may have resulted from diet. Cultured yellow eels consumed only small pellet material. Of wild yellow eels stomachs containing food, 78% of broad-headed eels consumed large and/or hard-bodied organisms (e.g. beetles, fish, molluscs and Notonecta sp.), while 83% of narrow-headed eels consumed exclusively small/soft-bodied prey (e.g. amphipods and chironomids).     

日本鳗鲡胶原蛋白和小清蛋白的过敏原性

以日本鳗鲡皮与肌肉组织为研究对象,采用碱溶、酸溶、盐析、冻干等方法纯化得到胶原蛋白,采用加热、饱和硫酸铵分级盐析、DEAE-Sepharose阴离子交换层析等方法纯化得到两种亚型的小清蛋白(PV-Ⅰ和PV-Ⅱ),纯化的目标蛋白经动物特异性抗体的免疫印迹实验确证。酶联免疫吸附测定和免疫印迹分析结果显示,纯化的胶原蛋白和小清蛋白分别与鱼类过敏患者阳性血清发生特异性反应,且二者之间无免疫交叉反应。体外模拟胃液消化实验和SDS-PAGE分析结果显示,胶原蛋白和小清蛋白均具有较高的消化稳定性。结果提示,日本鳗胶原蛋白和小清蛋白具有较高的消化稳定性和免疫原性,二者可引发不同患者的IgE介导特异性超敏反应。     

人工诱导日本鳗鲡成熟过程中血清及性腺内类固醇激素含量的变化

为分析日本鳗鲡(Anguilla japonica)经过多次注射鲤鱼脑垂体和人绒毛膜促性腺激素后,血清及性腺组织中性类固醇激素的含量及变化,采用超高效液相色谱-串联质谱联用(UPLC-MS/MS)对血清内6种类固醇激素[睾酮(Testosterone,T)、17α-羟基孕酮(17α-hydroxyprogesterone,17α-OHP)、17α,20β-二羟基-4-孕烯-3-酮(17α,20β-dihydroxy-4-pregnen-3-one,DHP)、雌酮(estrone,E_1)、雌二醇(estradiol-17β,E_2)、雌三醇(estriol,E_3)]以及性腺中4种类固醇激素(T、DHP、E_2、E_3)进行研究。结果表明,血清及性腺组织样品平均回收率分别为86.9%~98%、75.6%~88.1%;方法检出限(S/N10)分别为0.1 ng/g、0.1 ng/m L。对样品进行测定,结果表明,随着日本鳗鲡性腺发育,实验组日本鳗鲡血清及性腺中类固醇激素的含量呈逐步增加的趋势,其中,实验组日本鳗鲡血清中除E_3(难产时含量最高)外,其他5种类固醇激素均在产卵时含量最高;实验组日本鳗鲡性腺内E_2及DHP在难产时含量最高。对照组日本鳗鲡血清内E_1、E_3以及性腺内E_2、E_3在整个实验周期均未检测到。应用本方法对日本鳗鲡血清及性腺内类固醇激素进行定量分析,具有灵敏、高回收率、准确、稳定等特点,研究结果不仅对日本鳗鲡性腺发育过程中性类固醇激素的研究有了初步的认识,更对其他硬骨鱼类性腺发育过程中体内激素变化的研究具有重要参考意义。     

Natural dietary additive yellow loess as potential antibiotic replacer in Japanese eel,Anguilla japonica: Effects on growth,immune responses,serological characteristics and disease resistance against Edwardsiella tarda

The present experiment was conducted to evaluate the efficacy of an additive derived from the nature as an alternative of dietary antibiotic in Japanese eel, Anguilla japonica. Six experimental diets were formulated to contain no antibiotics or additive (yellow loess/YL) (control/CON), three graded levels of yellow loess at 5 (YL₅), 10 (YL₁₀) and 20 g/kg (YL₂₀), oxytetracycline at 5 (OTC) and amoxicillin at 10 g/kg amoxicillin (AMX) of diet. Weight gain (WG) and specific growth rate (SGR) from fish fed CON or YL₅ diets were significantly lower than those of fish fed YL₂₀ or OTC diets. Among non‐specific enzyme, lysozyme activity of fish fed YL₂₀, OTC or AMX was detected to be significantly higher than that from fish fed CON or YL₅ diets, whereas superoxide dismutase (SOD) activity of the fish fed CON was significantly lower than that for fish fed other experimental diets. Challenge test with bacteria, Edwardsiella tarda, showed improved disease resistance among the fish fed different levels of natural additive without any statistical difference from those fed antibiotics (OTC and/or AMX) supplemented diets. Therefore, these results demonstrated the potential of natural feed additive, yellow loess to replace oxytetracycline and/or amoxicillin in Japanese eel, A. japonica.