Yersinia ruckeri produces four iron-regulated outer membrane proteins but does not produce detectable siderophores

Abstract. The expression of iron-regulated outer membrane proteins (OMPs) was examined in 36 isolates of Yersinia ruckeri selected to represent a range of biotypes, serotypes and OMP-types. Iron-restricted growth was achieved in tryptone soya broth by chelation with 200 μm α,α'-dipyridyl, although similar results could be achieved with 25 μm transferrin or 280 μM ethylenediaminedihydroxyphenylacetic acid (EDDA). During growth under iron-restricted conditions, all 36 isolates produced four additional outer membrane proteins of molecular weights 72, 69·5, 68 and 66 kDa. Expression of these proteins was repressed by the addition of 100 μm FeCl₃ to the growth medium, confirming that the proteins were iron-regulated. The facts that all of the isolates examined possessed the same four iron-regulated OMPs, and that the plasmid content of different serotypes is known to vary indicated that production of these proteins is probably chromosomally mediated and not plasmid-mediated. Production of siderophores was not detected in low iron media and suggested a direct iron-uptake mechanism for this organism.     

Bacterial pathogens in rainbow trout, Oncorhynchus mykiss (Walbaum), reared at Danish freshwater farms

During a 2-year period, bacterial fish pathogens were monitored on five rainbow trout, Oncorhynchus mykiss (Walbaum), freshwater farms in Denmark. A total of 1206 fish were examined and 361 bacterial isolates were identified phenotypically. Enteric redmouth disease, furunculosis and rainbow trout fry syndrome/coldwater disease were recorded. Infections caused by Flavobacterium psychrophilum occurred most frequently, but only one outbreak of enteric redmouth disease caused by Yersinia ruckeri serotype O1 and one of furunculosis caused by Aeromonas salmonicida were recorded during the monitoring period. Flavobacterium psychrophilum was isolated on all farms, both during disease outbreaks and from fish without any signs of disease. Serological investigations of F. psychrophilum showed that serotype Th was the dominant serotype found. The serotypes Th and Fd were involved in disease outbreaks of fry and larger fish. All isolates of F. psychrophilum showed proteolytic activities; however, a few isolates, belonging to serotype FpT did not degrade elastin and were not associated with mortality. Increasing resistance problems to oxytetracycline were demonstrated. More than half of the F. psychrophilum isolates showed resistance to oxolinic acid and oxytetracycline. No antibiotic resistant isolates were found among Y. ruckeri and A. salmonicida .     

Molecular characterization of Portuguese strains of Yersinia ruckeri isolated from fish culture systems

A total of 23 Portuguese strains of Yersinia ruckeri , the causative agent of enteric redmouth disease (ERM), were comparatively studied by means of lipopolysaccharide (LPS) and outer membrane protein (OMP) analysis, plasmid profiling and ribotyping in order to investigate the heterogeneity among isolates and the usefulness of these methods as epidemiological markers. Only two LPS profiles were observed among the isolates studied, corresponding with the serotypes O1 and O3 of Y. ruckeri . A higher heterogeneity was detected analysing the OMP, seven different patterns being observed. Although some isolates carried different small plasmids, all the serotype O1 isolates showed a plasmid band of 50 MDa and all the serotype O3 strains shared in common a extrachromosomic DNA band of 30 MDa. The analysis of the ribopatterns obtained using three different enzymes, separated the strains into 10 ribotypes, indicating genetic heterogeneity among the isolates. The heterogeneity was greater within serotype O1 isolates, six ribotypes being detected, than within serotype O3 in which only three ribotypes were found. Although OMP analysis and ribotyping can be useful for the differentiation of strains on the basis of farm and/or season of isolation, we consider that ribotyping is the best candidate for epidemiological studies because it is easier to perform and offers a slightly better discriminative power.     

Antigenic differences of the scuticociliate Miamiensis avidus from Japan

In Japan and Korea, outbreaks of scuticociliatosis have frequently occurred in Japanese flounder, Paralichthys olivaceus . Morphological observations and small subunit rRNA gene sequences have shown that the causative agent of scuticociliatosis in the flounder is Miamiensis avidus (syn. Philasterides dicentrarchi ). In this study, we elucidated the antigenic differences between six Japanese M. avidus isolates as an initial step toward developing an effective vaccine against the disease. Four Japanese flounder isolates (IyoI, Nakajima, JF05To and Mie0301 isolates), one spotted knifejaw, Oplegnathus punctatus, isolate (SK05Kyo), and one ridged-eye flounder, Pleuronichthys cornutus , isolate (RF05To) were subjected to serological analysis. Antisera against IyoI, SK05Kyo, Nakajima and Mie0301 isolates were raised in rabbits and used for immobilization assays and Western blotting. Immobilization assays showed that the six isolates could be divided into three groups, tentatively designated serotype I for IyoI, JF05To, RF05To, SK05Kyo, serotype II for Nakajima and serotype III for Mie0301. Western blotting results supported these three serotypes, with marked similarities in the banding profiles of IyoI, JF05To, RF05To and SK05Kyo isolates, which were distinct from the Nakajima and Mie0301 isolates. Three isolates, IyoI, Nakajima and Mie0301 that were selected as representatives of each serotype, were highly pathogenic to Japanese flounder by experimental infection. Based on these findings, we propose that there are at least three M. avidus serotypes in Japan.     

Prevalence and different characteristics of two serotypes of Streptococcus parauberis isolated from the farmed olive flounder, Paralichthys olivaceus (Temminck and Schlegel), in Korea

The prevalence of two serotypes of Streptococcus parauberis isolated from the olive flounder, Paralichthys olivaceus, was evaluated in a total of 29 isolates between 2003 and 2010 in Korea. Streptococcus parauberis isolates were divided into two serologically distinct types (serotype 1 and serotype 2), except for one strain (S1091), using an agglutination assay with rabbit antiserum, and serotype 1 was identified as the dominant type (24 of 29 isolates) in this study. To identify the characteristics of the two serotypes of S. parauberis, we conducted a biochemical test using the API 20 Strep kit, a transmission electron microscopy (TEM) assay, sequence analysis of 16S‐23S rRNA intergenic spacer region (ISR) and a pathogenicity test. In TEM, both serotypes possessed polysaccharide capsule layers around the cell surface when bacterial cells were treated with a homologous serotype of rabbit antiserum. However, we were unable to discriminate serotype‐specific biochemical characteristics and genetic characteristics of 16S‐23S rRNA ISR between the two serotypes. In the pathogenicity test, the serotype 1 strains induced significantly higher mortality than the serotype 2 strains in olive flounder when experimentally inoculated via the intraperitoneal route.     

虹鳟源嗜冷黄杆菌的分离鉴定及致病性研究

为确定患病虹鳟的病原,本实验从患病鱼溃烂肌肉中分离到2株细菌,分别命名为CH06和CH07,经回归感染证实分离菌株为导致此次虹鳟患病的病原菌,并进一步对其形态特征、理化特性、分子特征、血清型及耐药性进行分析。结果显示,CH06和CH07株在TYES琼脂平板上呈煎蛋状外观,产黄色素,氧化酶和过氧化氢酶呈阳性,能水解明胶和酪蛋白,不能水解淀粉,不能利用果糖、半乳糖和七叶苷等。16S rRNA比对结果显示,CH06和CH07株与嗜冷黄杆菌模式株NBRC 15942的同源性分别为99.35%和99.42%。综合菌株理化和分子特性确定CH06和CH07株为嗜冷黄杆菌。利用多重PCR方法鉴定CH06和CH07株的血清型均为1型(Fd型);多位点序列分型(MLST)分析表明,CH06和CH07株的基因型分别为ST-12和ST-78型,且均属于CC-ST10克隆型。人工感染结果显示,CH06和CH07株对虹鳟幼鱼具有较高致病性,其半致死浓度(LD₅₀)分别为7.1×10⁵和1.1×10⁵ CFU/mL,攻毒剂量与临床病症出现时间呈反比,从人工感染实验鱼的肌肉、脾脏等组织中可重新分离到嗜冷黄杆菌。组织病理变化显示,病鱼肝细胞肿胀,空泡变性,部分肝细胞溶解坏死,细胞核溶解消失;脾脏充血、出血,淋巴细胞减少,红细胞和含铁血黄素增多;肌纤维间隙增宽、断裂、弯曲不齐,部分肌细胞肌浆溶解呈蜂窝状。CH06和CH07株对10种抗菌药物的耐药谱略有不同,均对氨苄西林和甲氧苄啶-磺胺甲噁唑敏感;CH06株对恩诺沙星、氟苯尼考等耐药,而CH07株对恩诺沙星和氟苯尼考中度敏感。本研究首次报道了我国虹鳟源嗜冷黄杆菌的分离鉴定及生物学特性,以期为虹鳟细菌性冷水病的防控提供科学依据。     

Protection of turbot, Scophthalmus maximus (L.), and rainbow trout, Oncorhynchus mykiss (Richardson), against vibriosis using two different vaccines

Abstact. The potency of a whole-cell bacterin (WCB) and a toxoid enriched whole-cell vaccine (WCEB) administered intraperitoncally into rainbow trout, Oncorhynchus mykiss (Richardson), were compared. The most effective vaccine was further evaluated by bathing turbot, Scophthalmus maximus (L.). These vaccines were composed of three strains of V. anguillarun , of the serotypes 01 and 02. Both vaccines conferred the highest protection against strains of serotype 01 within 4 weeks. With the toxoid enriched vaccine giving the best results (77 RPS). When trout were revaccinated after 7 weeks with this vaccine, good protection was achieved against strains of serotypes 01 and 02. Interestingly, when the WCEB was administered by bath to turbot, acceptable levels of protection against strains of both serotypes were obtained after 4 weeks of immunization.     

南海北部陆坡金鼻眶灯鱼秋季摄食节律

中层鱼是深海食物网的重要组成部分,其摄食节律的研究对探讨以中层鱼为核心的能量流动与物质循环具有重要意义。本研究利用2014年10月南海北部陆坡海域拖网调查数据,对中层鱼优势种金鼻眶灯鱼(Diaphus chrysorhynchus)秋季的摄食节律进行了研究。结果表明:(1)金鼻眶灯鱼取样体长范围为28~110 mm,集中于50~59 mm体长组,各时间段摄食等级与体长间均不存在正相关;(2)金鼻眶灯鱼在18:00左右上浮摄食,次日05:30左右达到摄食高峰,为连续摄食,5个取样时间段平均摄食等级范围为1.50~2.57,呈增长趋势;(3)差异性分析表明,金鼻眶灯鱼傍晚与夜间或清晨间摄食等级差异性显著,表现在18:30~19:30时间段与01:00~02:00、04:45~05:50或06:30~07:30三个时间段间(P=0.041;P=0.000;P=0.007),另03:30~04:30与04:45~05:50时间段间摄食等级也存在极显著差异(P=0.005);(4)金鼻眶灯鱼各时间段摄食等级频率呈波浪形变化,4级频率在06:30~07:30时间段达到最高峰;0级频率在黄昏18:30~19:30时最高,01:00~02:00降为0,03:30~04:30上升到黄昏时的一半,此后连续下降。总体而言,金鼻眶灯鱼傍晚至次日清晨连续摄食,连续排遗,摄食集中于清晨。综上所述,金鼻眶灯鱼是一种昼夜垂直迁移、夜间连续摄食与排遗的浮游动物食性鱼类,其在南海陆坡水域能量流与物质流中起着承上启下的作用。     

A serotypic survey and cross-protection test of North American field isolates of Yersinia ruckeri

Abstract. A serological study of 23 North American strains of Yersinia ruckeri showed that they were comprised essentially of one serotype–the classical 'Hager-man' serotype. Cross-protection tests showed that rainbow trout could be protected from challenge with various strains. Continuous surveillance of enteric redmouth disease and further work including DNA binding studies is necessary to determine the precise significance of the other related serotypes.     

Chemiluminescent responses of striped bass, Morone saxatilis (Walbaum), phagocytes to strains of Yersinia ruckeri

Abstract. Seventeen strains of Yersinia ruckeri were examined for plasmid DNA by means of agarose gel electrophoresis of partially cleared lysates. All 10 serotype I strains examined contained a 70 Megadalton (Mdal) plasmid, whereas only one of the 7 serotype II strains examined contained a plasmid of about 2–3 Mdal. Yersinia ruckeri exhibited distinctive colonial morphology and serological reactivities which suggested a possible correlation with plasmid content. The luminol-enhanced Chemiluminescent (CL) responses of striped bass, Morone saxatilis (Walbaum), phagocytes to different strains of Y. ruckeri were also studied. Some variability was observed; however, the greatest phagocyte CL responses were elicited by serotype II strains without the plasmid while serotype I strains harbouring the plasmid consistently elicited weak CL responses from phagocytes.     

Antigenic analysis of reference strains and Norwegian field strains of aquatic birnaviruses by the use of six monoclonal antibodies produced against the infectious pancreatic necrosis virus N1 strain

Abstract. Six monoclonal antibodies (MAbs) produced against the infectious pancreatic necrosis virus (IPNV) N1 strain were used in a dot-blot assay to examine reference strains of the nine proposed serotypes and a representative selection of 81 Norwegian aquatic birnavirus isolates. These isolates had earlier been serotyped by use of a panel of 11 MAbs produced against other strains of IPNV. Correlations between the reaction patterns of the two panels of MAbs were found. All reference strains and field isolates shared two epitopes, one on VP2 and one on VP3. Seventy-seven of the field isolates reacted identically with the N1 strain (positive with all six MAbs). The Sp type strain was positive with five of the MAbs and was different from all the field strains. The other reference strains (WB, VR299, Ab, Ja, Te, He, C1, C2 and C3) were positive with two to four of the MAbs. Together with previously published data, these findings indicate that most Norwegian isolates are closely related to, or identical with, the N1 strain and belong to the Sp serotype. No correlation between the health status of Atlantic salmon and antigenicity of the isolates was found. Testing of the reference strains in ELISA revealed some discrepancies with the dot-blot results.     

Specific immunoglobulins in serum from Atlantic salmon, Salmo salar L., immunized with Vibrio salmonicida and infectious pancreatic necrosis virus

Abstract. Atlantic salmon, Salmo salar L., responded to intraperitoneal injection of formalin killed Vibrio salmonicida or live infectious pancreatic necrosis virus ( ipnv ) by producing specific antibodies. The antibody titre varied significantly within the group tested. Western blot analysis demonstrated that high-titre antisera recognized two major bacterial antigens with molecular weights of 12–15 kD and 22–27 kD. In addition, a few narrow bands with higher molecular weights were observed. An antiserum raised against IPNV recognised two major antigens corresponding to the structural proteins of the virus. E lisa and Western blot analysis showed that the immune serum raised against Vibrio salmonicida reacted slightly with Vibrio anguillarum , whereas no reaction to Yersinia ruckeri or Aeromonas salmonicida was detected. Indirect elisa and an elisa competition assay revealed that the immune serum raised against the N1 serotype was specific for this serotype of ipnv . The results demonstrate that Atlantic salmon has a humoral immune system capable of producing antibodies which discriminate between related bacterial antigens and between different serotypes of a virus.     

草鱼D-loop多态性与幼苗生长性状的关联分析

为了探索草鱼(Ctenopharyngodon idella)线粒体DNA(mitochondrial DNA,mt DNA)多态性对生长性状的影响,鉴于mt DNA的母性遗传特征,本研究基于2011年繁殖用的20尾母本的D-loop序列信息,与通过亲子鉴定获得的853尾40日龄子代的体长、体质量进行关联分析。结果显示,草鱼6种D-loop单倍型对生长性状表型差异具有极显著影响(P0.01);其中,单倍型为Hap16的子代的体长最大,并显著大于单倍型为Hap4的子代的体长(P0.05);单倍型为Hap18和Hap16的子代的体质量较大,依次大于其他单倍型子代的体质量,并显著大于单倍型为Hap4的子代的体质量(P0.05)。此外,草鱼D-loop序列各变异位点基因型对生长性状的影响水平不同;其中,Site01、Site06和Site07等3个位点对体长的差异存在显著影响(P0.05),Site06和Site07等2个位点对体质量的差异存在显著影响(P0.05)。研究表明,草鱼D-loop序列变异对子代生长性状具有显著影响,推测在草鱼生长性状改良的选育进程中,可以利用mt DNA多态性信息进行辅助选择。     

Comparative studies of Danish Flavobacterium psychrophilum isolates: ribotypes, plasmid profiles, serotypes and virulence

Ribotyping and plasmid profiling were carried out on 299 Danish Flavobacterium psychrophilum isolates from farmed rainbow trout, Oncorhynchus mykiss (Walbaum). The isolates had been characterized biochemically and serologically in another study. The isolates were very homogeneous, 254 isolates had the same ribotype A (restriction enzyme Eco RI) and 284 isolates harboured one 3.3 kb plasmid. Seventy-five per cent of the F. psychrophilum isolates had ribotype A, one 3.3 kb plasmid and belonged to either serotype Th or Fd. Virulence studies with representatives of the dominant groups classified such isolates as virulent, and an extra small or large plasmid did not change the virulence level. A relationship between the serotypes Fd and Th, certain ribotypes, and virulence was found. The isolates belonging to serotype FpT and to ribotype B were less virulent. Only a few isolates with other ribotypes were found and these were also less virulent. Isolates that did not have the 3.3 kb plasmid were also less virulent. The virulent isolates all harboured the 3.3 kb plasmid; however, there was no clear correlation between virulence and plasmid content as a 3.3 kb plasmid was also found in less virulent isolates.     

Development and validation of real-time PCR for the detection of Yersinia ruckeri

Yersiniosis (enteric red mouth disease) is a contagious bacterial disease caused by Yersinia ruckeri, which primarily affects salmonids. A real-time PCR assay using a molecular beacon has been developed and validated to improve the detection of the causative biotypes of Y. ruckeri. The assay, which targets the glnA (glutamine synthetase) gene, proved to have 100% analytical specificity and analytical sensitivities of 5 fg and 3 × 10(3) CFU g(-1) for DNA and seeded kidney tissue, respectively. The assay was highly repeatable with low % CV for intra- and inter-run experiments, and the optimized parameters transferred easily between different real-time PCR platforms. Following analytical validation, diagnostic specificity was determined using New Zealand farmed Chinook salmon (n = 750) from 10 farms during 2007/08. The real-time PCR was run in parallel with the bacterial culture detection method, and all fish tested were found to be negative by both methods for Y. ruckeri, resulting in 100% diagnostic specificity (95% confidence interval). The molecular beacon real-time PCR system is specific, sensitive, reproducible and a rapid method for the detection of Y. ruckeri and has the potential to be used for routine diagnostic testing, health certification and active surveillance programmes.     

甲壳素发酵液在彭泽鲫幼鱼养殖上应用效果

为研究甲壳素发酵液(Chitin fermentation liquid,CFL)在彭泽鲫(Carassius auratus variety pengze)幼鱼上效果,通过在室外养殖池水体中定期泼洒CFL,比较不同用量CFL(0.01%和0.02%)以及0.02%CFL和商品壳聚糖(Chitosan,C)联合应用效果,每个处理3次重复。饲养初始彭泽鲫体重为35 g左右,培养2个月后测定幼鱼生长性能和水体指标。结果显示:0.01%CFL、0.02%CFL和0.02%CFL+C三个处理彭泽鲫总体重与养殖初期比较增重均达到20%以上,增重效果分别比对照高101.5%、110.4%和172.7%。0.01%CFL和0.02%CFL处理的彭泽鲫消化系统中脂肪酶、蛋白酶和淀粉酶比对照高22.03%以上,但两个浓度之间差异不显著。CFL处理降低养殖水体中亚硝态氮、总氮和总磷含量,0.01%CFL处理分别比对照低29.4%、15.2%和77.8%,其它两个处理与0.01%CFL差异不显著。CFL处理降低了感染嗜水气单胞菌后彭泽鲫的死亡率,提高了保护效果。结果表明,甲壳素发酵液明显提高彭泽鲫抗病能力,促进鱼体生长,改善水质。     

主养草鱼池塘中氨氧化古菌的多样性和种群分布

为了解淡水养殖池塘环境中氨氧化古菌的多样性和种群分布,本研究以荆州地区风生水起生态农场的主养草鱼(Ctenopharyngodon idellus)池塘为研究对象,采集不同养殖区的典型的沉积物样品和水样,利用AOA氨单加氧酶(amoA)基因通用引物(amoAF/amoAR)扩增amoA基因,构建amoA基因克隆文库。从每个克隆文库中随机挑选阳性克隆子进行测序、比对,利用MEGA和MOTHUR软件构建amoA基因系统发育树和可操作分类单元(OTU)分析。结果显示:主养草鱼池塘水体和沉积物中AOA存在着差异,且沉积物中AOA种类数和多样性都更为丰富。从三个采样区域的沉积物和水体共325个AOA amoA基因阳性克隆中得到24个OTU(基于3%差异度),其中水体克隆文库中得到13个OTU,沉积物克隆文库中得到18个OTU。OTU01、OTU02、OTU05、OTU07、OTU11、OTU13、OTU16是沉积物和水体中共有的OTU,并且OTU01、OTU02是养殖池塘中的优势类群,分别占克隆子的47.38%和21.84%。系统发育分析表明,AOA主要隶属于Nitrososphaera sister group、Nitrososphaera cluster、Nitrosopumilus cluster,并且OTU01、OTU02均属于Nitrososphaera sister group。主养草鱼池塘沉积物和水体中的AOA存在差异,且在沉积物中更为丰富,主要类群为Nitrososphaera sister。     

Cellular components of probiotics control Yersinia ruckeri infection in rainbow trout, Oncorhynchus mykiss (Walbaum)

Subcellular components of the probiotics Aeromonas sobria GC2 and Bacillus subtilis JB-1, when administered to rainbow trout, Oncorhynchus mykiss , conferred protection against a new biogroup of Yersinia ruckeri . Thus, intraperitoneal or intramuscular injection of rainbow trout with cell wall proteins (CWPs), outer membrane proteins (OMPs), lipopolysaccharides (LPS), whole cell proteins (WCPs) and live cells followed by challenge on day 8 with Y. ruckeri led to 80–100% survival compared with 10% survival in the controls. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of WCPs and OMPs from GC2 had 10 and 5 variable protein bands in comparison to 11 and 5 bands in the WCPs and CWPs from JB-1. Proteomic analyses were employed following SDS-PAGE to categorize one dominant protein of 104.7 kDa from the CWPs of JB-1 and equated it with ' Bacillus spp. endoglucanase' with a Mascot score >69. These results point to the potential of using cellular components of probiotics for protection of fish against bacterial diseases.     

红头丽体鱼×红魔丽体鱼杂交子一代胚胎发育及仔鱼形态学观察

对红头丽体鱼[Cichlasoma synspilum（♀）]×红魔丽体鱼[C. citrinellum（♂）]杂交子一代（F1）（俗称“血鹦鹉”）的胚胎及仔鱼形态发育进行观察,描述了各发育时期的发育时序和形态特征。平均受精率为（91.33±3.06）％,平均孵化率为（91.67±2.08）％;血鹦鹉的受精卵呈椭圆球形,黏性,有浅黄、白灰和红褐3种颜色,无油球,平均卵长径为（1.89±0.04）mm;发育过程可分为6个阶段：受精卵阶段、卵裂阶段、原肠期、神经胚期、器官形成期和孵出期,并进一步分为28个发育分期;在水温（30±0.5）℃下历时约52 h 36 min 完成孵化。初孵仔鱼全长（3.71±0.05）mm,卵黄长径为（1.74±0.09）mm,短径为（1.25±0.07）mm,于第3天开始摄食轮虫,孵出后第5天卵黄囊完全消失。第13天全长（11.86±1.02）mm,各鳍条基本形成,进入稚鱼期。     

凡纳滨对虾全同胞家系的建立及生长比较

全同胞家系是遗传分析的重要材料。采用自然交配法建立了62个凡纳滨对虾第一代全同胞家系,对其中17个家系进行了比较研究。结果表明,各家系的孵化率、出苗率与雌性亲本的大小相关性不显著。17个家系中有7个家系显示生长优势,其大小顺序依次是A02＞A11＞A01＞A17＞A06＞A08＞A16;7个家系在40周内的生长速度较17个家系的平均值高23.2%。在所有家系中,A01、A02、A11等3个家系的体重显著大于其它家系（P＜0.05）,其平均生长速度较所有家系平均生长速度分别快25.5%、31.6%和31.0%。这3个家系的整齐度良好,在养殖中期的个体变异系数分别为11.58%、9.95%和8.48%,后期均小于5%,显示出优良的经济性状,为进一步选择培育凡纳滨对虾的快速生长新品系奠定了基础。