发病鲆鲽类分离菌株的16S rRNA基因测序分析

细菌性疾病是中国海水养殖鲆鲽类的主要病害,为全面了解病原菌种类,本研究对1999~2012年从山东、江苏、河北、天津等沿海地区养殖场发病鲆鲽鱼类中分离得到的124株优势菌株进行了16S rRNA基因测序和系统发育学分析。将基因序列与GenBank核酸序列数据库进行相似度比对分析,结果显示,有83株与弧菌属(Vibriosp.)细菌相似度最高,11株与气单胞菌属(Aeromonas sp.)细菌相似度最高,4株与爱德华氏菌属(Edwardsiella sp.)细菌相似度最高,26株为其他15种属的细菌。根据系统发育学分析结果,进一步将66株菌鉴定为16个种,优势种为溶藻弧菌(V.alginolyticus)、哈氏弧菌(V.harveyi)、鳗弧菌(V.anguillarum)、杀鲑气单胞菌(A.salmonicida)和迟缓爱德华氏菌(E.tarda)。选择其中的9株鳗弧菌和4株迟缓爱德华氏菌进行人工感染实验,结果显示,其中7株鳗弧菌和3株迟缓爱德华氏菌对大菱鲆(Scophthalmus maximus)有较强的致病性。研究结果可为阐明中国海水养殖鲆鲽类的流行病发生历史、病原种类、病原监测及疾病控制提供重要参考。     

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.     

牙鲆迟钝爱德华氏菌血清型及荧光抗体检验

以迟钝爱德华氏菌的代表菌株(HC010907-1)为免疫原,制备免疫血清,对分离于牙鲆鱼的130株迟钝爱德华氏菌进行了血清型检定,结果表明:供试的130株迟钝爱德华氏菌均为同种血清型;同时以此免疫血清为第一抗体,以标准羊抗兔IgG荧光抗体为第二抗体,进行了荧光抗体技术检验迟钝爱德华氏菌的可行性试验,表明亦具有较强的特异性。     

Analysis of exotoxins produced by atypical isolates of Aeromonas salmonicida,by enzymatic and serological methods

In this study, exotoxins produced by 62 Aeromonas salmonicida strains and the bacterium Haemophilus piscium were analysed. Enzymatic assays, zymograms and serological detection were used to monitor secretion by bacterial strains of the previously described exotoxins P1, GCAT and AsaP1 and also the extracellular P2 metallo-gelatinase and a serine caseinase, which is different from the P1 protease and has not yet been characterized. Based on the results, the strains were divided into five groups. One comprised the type strains for A. salmonicida ssp. masoucida, H. piscium and 36% of the atypical isolates, and another, a type strain for A. salmonicida ssp. smithia together with 14% of the atypical isolates. A second type strain of A. salmonicida ssp. smithia was grouped with 8% of the atypical isolates. The largest group contained the type strains for A. salmonicida ssp. achromogenes and 38% of the atypical isolates. The type strains for A. salmonicida ssp. salmonicida were in the last group with all the four typical strains and 4% of the atypical isolates. The combination of zymogram and serological detection used is recommended as the most reliable method for characterizing A. salmonicida strains according to their exotoxin secretion.     

大菱鲆迟缓爱德华氏菌福尔马林灭活疫苗研究

以大菱鲆为免疫对象,采用0.5福尔马林灭活的方法,将迟缓爱德华氏菌制成全菌疫苗,验证了其具有可靠的安全性。以浸泡和注射两种免疫接种方法对养殖大菱鲆14d内进行2次免疫,第二次免疫后第10天,对免疫鱼和各自的对照鱼进行爱德华氏菌的人工感染试验,获得注射接种疫苗的大菱鲆对腹水病的免疫保护率为70,浸泡接种疫苗的大菱鲆对腹水病的免疫保护率为35。表明注射和浸泡接种迟缓爱德华氏菌全菌疫苗都能使大菱鲆对腹水病产生免疫效果,并且注射免役效果优于浸泡免疫。     

The macrophage chemotactic activity of Edwardsiella tarda extracellular products

The chemoattractant capabilities of Edwardsiella tarda extracellular products (ECP) were investigated from two isolates, the virulent FL6-60 parent and less virulent RET-04 mutant. Chemotaxis and chemokinesis were assayed in vitro using blind well chambers with peritoneal macrophages obtained from Nile tilapia, Oreochromis niloticus, 5 days following squalene injection. Non-purified ECP derived from both isolates stimulated predominantly chemokinetic migration of macrophages. Additionally, the ECP were semi-purified by high pressure liquid chromatography. The FL6-60 parent ECP yielded higher molecular weight components than did the ECP from the RET-04 mutant. The chemotactic activity of the macrophages for both the FL6-60 parent and RET-04 mutant semi-purified ECP was increased over the non-purified ECP and overall migration was primarily chemotactic. Exposure to ECP derived from virulent and less virulent E. tarda isolates promoted chemokinetic movement of macrophages that may be involved in inflammatory responses of Nile tilapia to E. tarda infection.     

Edwardsiellosis in farmed turbot,Scophthalmus maximus (L.), associated with an unusual variant of Edwardsiella tarda: a clinical,aetiological and histopathological study

During 2005 and 2010, a survey of edwardsiellosis on eight turbot, Scophthalmus maximus (L.), farms was conducted in China. This report presents the detailed results of the study on this disease. Diseased turbot displayed two distinct types of gross signs: black discoloration of the dorsal skin on the posterior portion of the body; and red cutaneous foci on the ventral side. Internally, the most pronounced clinical signs in all fish examined were enlarged kidneys. The causal agent of the disease was finally proved to be one species of bacterium that was identified as Edwardsiella tarda by physiological and biochemical tests, API 32E and 16S ribosomal RNA sequence analysis. It is noteworthy that unlike the commonly described E. tarda strains, the isolates in this study were non‐motile strains without flagella. A histopathological study revealed that E. tarda infection was systemic in turbot and that kidney showed the most significant pathological changes, including acute focal necrosis, an influx of macrophages and formation of granuloma. The most common histopathological characteristics of this disease are the proliferation of macrophage in various organs and formation of granuloma. In addition, this article also gave background information on the disease and presented the results of virulence tests with the E. tarda strain identified in this study.     

Experimental infection of turbot, Psetta maxima (L.), with strains of viral haemorrhagic septicaemia virus isolated from wild and farmed marine fish

The susceptibility of turbot, Psetta maxima, to infection with two strains of viral haemorrhagic septicaemia virus (VHSV) obtained from wild Greenland halibut, Reinhardtius hippoglossoides, and from farmed turbot was examined. A marine VHSV strain known to be highly pathogenic for turbot was also utilized for comparative purposes. Fish were infected by intra-peritoneal (i.p.), immersion or cohabitation, and maintained at two different temperatures (8 and 15 degrees C). Infection trials showed that the three VHSV isolates were pathogenic for turbot fingerlings by i.p. injection at both temperatures, with high levels of mortality. Virus was recovered from most pools of dead fish i.p. challenged, but not from surviving fish. Although clinical signs were not induced following waterborne exposure, viral growth was obtained from some pools of surviving fish challenged by immersion with strain GH40 from Greenland halibut, which indicates that the virus can survive in sea water and infect other fish via horizontal transmission. Furthermore, although low, the clinical signs and mortality observed in fish cohabitating with turbot challenged with strain GH40 confirms horizontal transmission and indicates that the passage through fish increases the virulence of this strain for turbot. These findings indicate that Greenland halibut, as other wild fish, may play an important role in the epizootiology of VHSV and suggest a potential risk for the turbot farming industry.     

维生素E及脂肪源对大菱鲆非特异性免疫的影响

在大菱鲆饲料中分别添加鲤鱼鱼油及花生油作脂肪源,每种脂肪中添加两个水平的维生素E（8mg／kg和300mg／kg维生素E醋酸酯）,饲养大菱鲆84d,探讨其对大菱鲆非特异性免疫的影响。结果发现,（1）以鲤鱼鱼油为脂肪源的添加高剂量的维生素E能提高大菱鲆血清补体活性,而以花生油为脂肪源的饲料添加高剂量的维生素E不影响大菱鲆血清补体活性;（2）以鲤鱼鱼油和花生油为脂肪源的饲料添加高剂量的维生素E不影响大菱鲆中性粒细胞的吞噬率及白细胞总数;（3）以鲤鱼鱼油为脂肪源的饲料添加高剂量的维生素E能提高大菱鲆的溶茵酶活性,但随着时间推移其影响减弱;（4）饲喂n-3HUFA含量高的饲料,对大菱鲆腹水病的抵抗能力高于n-3HUFA含量低的饲料。     

Comparison of Edwardsiella ictaluri isolates from different hosts and geographic origins

The intraspecific variability of E. ictaluri isolates from different origins was investigated. Isolates were recovered from farm‐raised catfish (Ictalurus punctatus) in Mississippi, USA, tilapia (Oreochromis niloticus) cultured in the Western Hemisphere and zebrafish (Danio rerio) propagated in Florida, USA. These isolates were phenotypically homologous and antimicrobial profiles were largely similar. Genetically, isolates possessed differences that could be exploited by repetitive‐sequence‐mediated PCR and gyrB sequence, which identified three distinct E. ictaluri genotypes: one associated with catfish, one from tilapia and a third from zebrafish. Plasmid profiles were also group specific and correlated with rep‐PCR and gyrB sequences. The catfish isolates possessed profiles typical of those described for E. ictaluri isolates; however, plasmids from the zebrafish and tilapia isolates differed in both composition and arrangement. Furthermore, some zebrafish and tilapia isolates were PCR negative for several E. ictaluri virulence factors. Isolates were serologically heterogenous, as serum from a channel catfish exposed to a catfish isolate had reduced antibody activity to tilapia and zebrafish isolates. This work identifies three genetically distinct strains of E. ictaluri from different origins using rep‐PCR, 16S, gyrB and plasmid sequencing, in addition to antimicrobial and serological profiling.     

The development of size variation in Dover sole,Solea solea and turbot,Scophthalmus maximus: genetic variability between different geographical and among year class farmed strains

Growth and variation in size of turbot and sole larvae originating from different genetic sources were compared at different stocking densities, including genetically based information that might lead to a better knowledge of heterogeneity in cultivated populations. There was no effect of density on growth rate in either species. However, between the two sole batches there was an earlier metamorphosis of faster growing fish from Norway. In sole groups after metamorphosis, variation was stable and therefore no development of size variation was observed. There was no density effect on size variation within turbot batches before, or after metamorphosis. Spreading rate and growth rate were positively correlated in sole larvae indicating that growth of the smaller fish was not equal to the larger ones, but size variation followed a different pattern in turbot. No evidence for either overall significant heterozygosity excess, or deficiency was evidenced in the farmed strains of either species . However, significant differences between farmed populations in terms of genetic variability (θ) were reported, which was rather attributed to within differentiation than between strains. One could safely argue that different strains of Dover sole originated from different genetic sources (factorial analysis). Moreover, when the analysis was extended to individual loci to assess whether specific loci had any consistently larger, or smaller effect on heterozygosity, locus specific significant trends were discerned in turbot. A positive Heterozygosity Fitness (growth rate) Correlation (HFC) suggests that heterozygotes reported enhanced growth rates compared with homozygous ones in flatfish larvae, thus special precautions should be taken into account whenever different genetic structure patterns emerge, because of various flatfish broodstock geographical origin and year class samples.     

Antigenic relationships among rotaviruses isolated from fish

Abstract. An antigenic comparison of five rotaviruses isolated from fish in Spain, the USA and Canada was carried out using neutralization, immunodot and ELISA tests. The Spanish isolate (turbot rotavirus) was serologically different from the other four rotaviruses, by neutralization and immunodot. By these two techniques, among the four American strains the two isolates from Canada (smelt rolavirus and Atlantic salmon rotavirus) were closely related antigenically, and the two strains from USA (striped bass rotavirus and the Hetrick & Baya rotavirus, isolated from salmon) seemed to be the same scrotype. These results were supported by those obtained in the comparison of the RNA patterns of the five viruses. However, the antigenie relationships among these rotaviruses were very different when employing the ELISA technique.     

大菱鲆饲料转化率相关微卫星标记的筛选

饲料转化率(FCR)是大菱鲆重要的经济性状，通过选择育种提高饲料转化率，能够有效地降低大菱鲆的养殖成本，进而推动产业的发展。微卫星标记是鱼类分子标记辅助选育中常用的分子标记，为了筛选出与大菱鲆饲料转化率相关的微卫星标记，提高育种效率，实验以300尾大菱鲆幼鱼为研究对象，通过特制的网箱养殖系统，测定个体饲料转化率，分别选取饲料转化率最高和最低的30个样本作为高饲料转化率组(H组)和低饲料转化率组(L组)。利用40对大菱鲆微卫星引物，对H组和L组的DNA混池进行PCR扩增，统计两组个体PCR产物的基因型，筛选两池之间出现差异等位基因片段的位点，通过进一步的群体验证和家系验证，分析微卫星位点与大菱鲆饲料转化率的相关性。结果显示，微卫星位点YSKr148在238 bp的等位基因片段与大菱鲆饲料转化率存在极显著正相关，相关系数达到0.359，家系验证中该位点的阳性组的饲料转化率显著高于阴性组。研究表明，大菱鲆微卫星位点YSKr148与饲料转化率性状显著相关，可以用于该性状的分子标记辅助选育。本研究首次获得了与大菱鲆饲料转化率性状显著相关的分子标记，为研究该性状的遗传基础以及相关分子机制提供了依据...     

Effect of dietary inulin and oligosaccharides as prebiotics for weaning turbot, Psetta maxima (Linnaeus, C. 1758)

Preliminary experiments were undertaken to investigate the effect of dietary inulin (Raftiline ST), oligofructose (Raftilose P95) and lactosucrose on the growth and intestinal bacteria of the marine carnivorous turbot, Psetta maxima. Turbot larvae were weaned on compound diets containing 2% Rafiline ST, 2% Raftilose P95 or 2% lactosucrose; 2% cellulose was the carbon source in the control group. The final mean weight of the group weaned with Raftilose P95 was significantly higher than those observed with the other diets. The bacterial load was highly variable in weaning turbot, especially with respect to the putative Vibrio spp. growing on TCBS agar which, in general, seemed to be dominant. Of the total load of bacterial isolates from turbot weaned on oligofructose, 14% consisted of a strain of Bacillus spp. This strain could use Raftilose P95 as a single source of carbon, and it might play a role in the beneficial effect of oligofructose on turbot growth, since Bacillus spp. have been documented as probiotics in fish.     

柱状嗜纤维菌血清型的研究

采用从各种患病淡水鱼类上分离到的柱状嗜纤维菌（Ｃｙｔｏｐｈａｇａｃｏｌｕｍｎａｒｉｓ）４０个菌株,通过交叉凝集和琼脂内扩散反应,研究了Ｃ．ｃｏｌｕｍｎａｒｉｓ的血清型。结果表明：根据不同菌株的热稳定性具有明显差别的现象,首次证明了从我国各地收集的Ｃｃｏｌｕｍｎａｒｉｓ菌存在３个不同的血清型。     

抗迟缓爱德华菌单克隆抗体的应用

检测了抗迟缓爱德华菌单抗对牙鲆的保护性。从１０株自制单抗中筛选出一株对牙鲆具有较强保护性的单抗３Ｆ７。与仅以该菌感染的对照组相比,该抗体可显著提高牙鲆感染迟缓爱德华菌后的存活率。在以每条鱼０．１ｍＬ（１０＾９ＣＦＵ）菌量做攻击保护时,其保护率可达８０％。另外,利用所制单克隆抗体以免疫组织组织化学ＳＡＢＣ法检查了迟缓爱德华菌经腹腔感染牙鲆鱼后细菌的侵染途径,结果显示牙鲆对该菌易感的器官为肝、肾、脾等     

An assessment of the antigenic homogeneity of Edwardsiella ictaluri using monoclonal antibody

Abstract. Monoclonal antibodies were made against the reference strain of Edwardsiella ictaluri (ATCC 3320). Antibody produced by one of seven anti- E. ictaluri hybridomas reacted positively by the immunofluorescent antibody technique against 17 other E. ictaluri isolates. All hybridoma antibodies failed to react with six other bacterial species pathogenic to fish including E. tarda . Ouchterlony tests indicated that four anti- E. ictaluri clones produced only one kind of immunoglobulin. Electrophoresis of 14 different E. ictaluri isolates indicated identical protein bands at 36 and 60 kilodaltons (KD) in all isolates except an isolate from Thailand. Using the immunoblot method, channel catfish anti- E. ictaluri serum reacted with protein bands at 34 and 60 KD, which indicates that this molecular weight protein in the bacterium may be the dominant immunoprotein.     

Lancefield group C Streptococcus dysgalactiae infection responsible for fish mortalities in Japan

A Lancefield serological group C Streptococcus sp. was isolated from cultured amberjack, Seriola dumerili Risso, and yellowtail, Seriola quinqueradiata Temminck and Schlegel, immunized with Lactococcus garvieae commercial vaccines in Japan. The isolated bacteria were Gram-positive cocci, auto-aggregating in saline, morphologically long chains in growth medium, catalase negative and alpha-haemolytic on blood agar. An almost complete gene sequence of the 16S rDNA of two isolates was determined and compared with that of bacterial strains in the database. The isolates were identified as Streptococcus dysgalactiae based on the results of the 16S rDNA sequence, the bacteriological properties and the Lancefield serological grouping. Oligonucleotide primers specifically designed for the 16S-23S rDNA intergenic spacer region of S. dysgalactiae amplified a gene from all the fish isolates, as well as the type strains alpha-haemolytic S. dysgalactiae subsp. dysgalactiae ATCC430738 and beta-haemolytic S. dysgalactiae subsp. equisimilis ATCC35666, but not those of S. equi ATCC33398, Lactococcus garvieae ATCC43921 and L. garvieae KG9408. The severe necrotic lesions of the caudal peduncle seen in experimentally infected fish were similar to those seen in naturally infected fish.