Interactions of microorganisms isolated from gilthead sea bream, Sparus aurata L., on Vibrio harveyi, a pathogen of farmed Senegalese sole, Solea senegalensis (Kaup)

Four bacterial isolates from farmed gilthead sea bream, Sparus aurata, included in a previous study as members of the Vibrionaceae and Pseudomonodaceae and the genus Micrococcus, have been evaluated for their adhesive ability to skin and intestinal mucus of farmed Senegalese sole, Solea senegalensis, and their antagonistic effect on Vibrio harveyi, a pathogen of sole. These isolates showed higher adhesion to sole mucus than the pathogenic strains of V. harveyi assayed. Only two of the isolates showed antagonistic activity to V. harveyi. Interactions of the four isolates with V. harveyi in respect of adhesion to skin and intestinal mucus under exclusion, competition and displacement conditions were studied. Three isolates were able to reduce the attachment to skin and intestinal sole mucus of a pathogenic strain of V. harveyi under displacement and exclusion conditions, but not under competition conditions. The in vivo probiotic potential of isolate Pdp11 was assessed by oral administration followed by challenge with the pathogenic V. harveyi strain Lg14/00. A group of 50 Senegalese sole received a commercial diet supplemented with 10(8) cfu g(-1) of lyophilized Lg14/00 for 15 days. A second group of fish received a non-supplemented commercial diet. After challenge the mortality of the fish receiving the diet supplemented with the potential probiotic isolate was significantly lower than that in the fish receiving the non-supplemented commercial diet. This study has shown that the ability to interfere with attachment of pathogens, as well as the adhesion to host surfaces, are suitable criteria for selection of candidate probiotics for use in the culture of Senegalese sole.     

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.     

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.     

Rapid and sensitive detection of Vibrio harveyi by loop‐mediated isothermal amplification combined with lateral flow dipstick targeted to vhhP2 gene

Vibrio harveyi is a causative agent of the Vibriosis or luminescent bacterial disease in worldwide aquaculture industry. A reliable assay for identification of V. harveyi infection is important to prevent the bacterial spread. In this study, biotinylated loop‐mediated isothermal amplification (LAMP) amplicons were produced by a set of four designed primers that recognized specifically the V. harveyi vhhP2 gene, encoding a putative outer membrane protein with unknown function, followed by hybridization with an fluorescein isothiocyanate (FITC)‐labelled probe and lateral flow dipstick (LFD) detection. A novel set of PCR primer was also designed specifically to vhhP2 gene and appear to be a species‐specific tool for V. harveyi detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65°C. The LAMP‐LFD and PCR methods accurately identified 22 isolates of V. harveyi but did not detect 16 non‐harveyi Vibrio isolates, and 34 non‐Vibrio bacterial isolates. The sensitivity of LAMP‐LFD for V. harveyi detection in pure culture was 1.1 × 10² CFU mL^?1 or equivalent to 0.6 CFU per reaction, while that of PCR was 6 CFU per reaction. For spiked shrimp sample, the sensitivity of LAMP was 1.8 × 10³ CFU g^?1 or equivalent to 5 CFU per reaction, while that of PCR was 50 CFU per reaction. In conclusion, the established LAMP‐LFD methods provided a valuable tool for rapid identification of V. harveyi and can be used to distinguish V. harveyi from V. campbellii.     

Purification and characterization of lipovitellin from Pacific saury Cololabis saira

ABSTRACT:   Lipovitellin (Lv), the major yolk protein derived from vitellogenin (Vg), was purified from vitellogenic ovaries of Pacific saury Cololabis saira using hydroxylapatite column chromatography followed by gel filtration. The apparent native mass of purified Lv was approximately 420 kDa, while the tertiary structure of Lv revealed by sodium dodecylsulfate–polyacrylamide gel electrophoresis was typical of teleost Lvs, consisting of a heavy chain (∼99 kDa) and a light chain (∼34 kDa). Western blot analysis using rabbit antiserum raised against Pacific saury Lv revealed a specific reaction with a polypeptide (∼194 kDa) that is present in serum from female Pacific saury but not in male serum, suggesting the approximately 194-kDa polypeptide to be the Vg monomer. This study describes the first step toward the development of specific immunoassays for Pacific saury Vg, which will be an effective tool for monitoring the reproductive development of this species.     

大蒜对印度明对虾黑鳃病原耐药菌株哈维氏弧菌抗菌活性的研究

本研究针对大蒜提取物对印度明对虾(Fenneropenaeus indicus)黑腮病病原多重耐药哈维氏弧菌(Vibrio harveyi)体外和体内的抗菌活性进行了评价。先利用市售的几种抗生素记录致病性哈维氏弧菌的抗菌谱。然后,利用琼脂扩散法来研究大蒜对哈维氏弧菌的抗菌活性。将哈维氏弧菌接种在琼脂平板上,对每种大蒜提取物(鲜榨大蒜提取物—FSGE,冻干大蒜提取物—FDGE以及用甲醇提取的大蒜提取物—MGE)设计三种不同的浓度。结果表明,鲜榨大蒜提取物对哈维氏弧菌的抑菌活性明显优于冻干大蒜提取物和用甲醇提取的大蒜提取物。本研究分别在对虾基础饲料中添加0%(对照)、0.5%和1%浓度梯度的大蒜来评价大蒜治疗感染哈维氏弧菌的对虾的效果。结果表明,综合大蒜提取物的短期和长期防治(1%含量)的效果分析,对虾累积死亡率降低了75%。本研究探讨了鲜榨大蒜提取物替代抗生素在印度明对虾饲养防病上的优点。     

矛尾复鰕虎鱼病原哈氏弧菌的鉴定及特异性检测方法的建立

取体表出血、肌肉溃疡的虾蟹养殖塘混养大量死亡的矛尾复鰕虎鱼的深层溃烂组织及肝脏进行细菌分离,2种被检组织中均分离到2种优势生长菌落;人工感染试验表明,其中的一株分离菌(S090801)浓度为106～108 cfu/ml可引起矛尾复鰕虎鱼全部死亡,该菌的形态特征、生理生化特性及基于16SrRNA和gyrB 2种基因的系统发育学分析确定为弧菌属的哈氏弧菌。同时基于gyrB基因序列设计1对特异性引物,建立了一种哈氏弧菌快速、敏感的PCR检测方法,扩增目的片段大小为363bp,该方法检测哈氏弧菌模板DNA最低质量浓度为0.046875ng/μl,可用于哈氏弧菌引起的水产动物疾病的快速诊断及分子流行病学的调查研究。     

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.     

哈维氏弧菌外膜蛋白OmpK基因的克隆及原核表达

根据哈维氏弧菌外膜蛋白OmpK的基因序列设计一对引物,应用聚合酶链式反应（PCR）方法,从分离自患病大黄鱼的哈维氏弧菌基因组中扩增获得一段约800bp的序列。将其克隆到pGEM-Teasy载体,测序结果证明该序列是哈维氏弧菌外膜蛋白OmpK基因。用PCR方法去除其信号肽序列,定向克隆到原核表达载体pGEX-4T-2构建重组表达质粒pGEX-4T-OmpK。IPTG诱导后能够在大肠杆菌BL21中高效表达分子量约为53kD的GST-OmpK融合蛋白。用纯化后的融合蛋白免疫新西兰兔获得了高效价的抗血清。Western-blotting分析表明,它与从哈维氏弧菌中提取的约27kD的外膜蛋白能够发生特异反应,提示外膜蛋白OmpK可能是哈维氏弧菌的重要保护性抗原之一。     

Enhancement of resistance to bacterial infection in rainbow trout, Oncorhynchus mykiss (Walbaum), by oral administration of bovine lactoferrin

Abstract Bovine lactoferrin was evaluated for its ability to enhance resistance of rainbow trout, Oncorhynchus mykiss (Walbaum), to infection with Vibrio anguillarum. Oral administration of lactoferrin (100 mg kg⁻¹) to fish daily for 3 days prior to an intraperitoneal challenge with V. anguillarum resulted in increased survival rates, and enhanced resistance against Streptococcus sp., although to a lesser extent. In lactoferrin-treated fish, an increase in phagocytic and chemiluminescent (CL) activities of pronephros cells against V. anguillarum was observed. The phagocytic and CL activities of cells against Streptococcus sp. were also significantly increased. However, no in vitro bactericidal activities of lactoferrin against V. anguillarum or Streptococcus sp. were observed. This suggests that the lactoferrin enhanced the resistance of the rainbow trout against bacterial infection through the activation of phagocytes.     

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.     

Use of probiotics to enhance growth,stimulate immunity and confer disease resistance to Aeromonas salmonicida in rainbow trout (Oncorhynchus mykiss)

An 8‐week feeding trial was conducted to evaluate the effects of dietary probiotics on growth, non‐specific immune responses and disease resistance in juvenile rainbow trout, Oncorhynchus mykiss. Fish averaging 5.8 ± 0.8 g (mean ± SD) were fed one of the five experimental diets; one control (Cont), and four other diets were prepared by supplementing single probiotics 1 (Bacillus subtilis; SP₁, 0.5%), single probiotics 2 (Bacillus licheniformis; SP₂, 0.5%), multi‐probiotics (B. subtilis + B. licheniformis; MP, 0.5%) and oxytetracycline (OTC) at 5 g OTC kg^?1 diet. After 8 weeks of the feeding trial, weight gain and specific growth rate of fish fed SP₁, SP₂ and OTC diets were significantly higher than those of fish fed Cont diet (P < 0.05). Superoxide dismutase (SOD) and lysozyme activities of fish fed SP₁, SP₂ and MP diets were significantly higher than those of fish fed Cont diet (P < 0.05). There were no significant differences in SOD and lysozyme activities among fish fed SP₁, SP₂, MP and OTC diets. In challenge test with Aeromonas salmonicida for 15 days, fish fed SP₁, SP₂ and MP diets showed significantly higher cumulative survival rate than those of fish fed Cont diet (P < 0.05). However, there were no significant differences in cumulative survival rate among fish fed SP₁, SP₂, MP and OTC diets. Although there was a little advantage in fish fed MP diet in terms of non‐specific immune responses, single or multi‐probiotics are equally effective statistically. These results indicate that single or multi‐probiotics had equal beneficial effects as an antibiotic replacer on growth performance, non‐specific immune responses and disease resistance in juvenile rainbow trout.     

致病性哈维氏弧菌溶血素基因克隆及其检测

从山东沿海的发病鲈鱼（Lateolabrax japonicus）分离到1株致病性哈维氏弧菌（Vibrio harveyi），从该菌的染色体DNA扩增出一条长约1．4kb的特异性片段。DNA序列分析表明，该克隆片段含有完整的1254bp溶血素基因，该溶血素基因与哈维氏弧菌VIB645的溶血素基因VhhA和VhhB的相似性分别为99．0％和98．5％；与副溶血弧菌（V．parahaemolyticus）热稳定性溶血素基因（TDH）的相似性为74．5％；与拟态弧菌（V．mimicus）、创伤弧菌（V．vulnificus）、霍乱弧菌（V．chderae）磷脂酶基因的序列相似性分别为57．4％、59．2％、53．0％；与最小弧菌、创伤弧菌、霍乱弧菌、霍氏弧菌（V．hollisae）、河流弧菌（V．tguvialis）、鳗弧菌（V．anguillarum）的溶血素基因的相似性仅为19．9％～24．8％。根据溶血素基因的保守区段，设计了1对特异引物。分析表明，该引物能特异检测哈维氏弧菌。其可检测的DNA最小量为0．001ng。用该方法对55株不同来源的患病鱼分离的疑似病原菌进行检测，结果检出8株哈维氏弧菌，检出率为14．55％，从健康动物中检出数占所检弧菌数的6．25％，海洋环境为10．53％，海水养殖环境为26．53％，表明哈维氏弧菌在不同的海水环境及健康海洋动物中普遍存在，在养殖水体中的数量高于其他海水环境。     

哈氏弧菌外膜蛋白与溶藻弧菌脂多糖偶联疫苗的效果研究

采用十二烷基肌氨酸钠法提取哈氏弧菌(Vibrio harveyi)SpGY020601株的外膜蛋白(OMPC),采用酚水法提取溶藻弧菌(Vibrio alginolyticus)EpGS021001株的脂多糖(LPS).通过碳化二亚铵(EDC)介导的缩合反应,将哈氏弧菌的OMPC与溶藻弧菌的LPS偶联.OMPC-LPS偶联物、未偶联的哈氏弧菌OMPC、溶藻弧菌LPS、哈氏弧菌OMPC和溶藻弧菌LPS的简单混合物以及生理盐水按相同程序免疫卵形鲳鲹(Trachinotus cvatus).检测血清溶菌酶活性、血清抗体微量凝集反应结果显示,卵型鲳鲹经OMPC-LPS偶联物、OMPC、LPS以及二者的简单混合物免疫后,各免疫组间血清溶菌酶活性没有显著差异(P＞0.05),但均显著高于生理盐水对照组(P＜0.05);OMPC-LPS偶联物免疫组的血清抗溶藻弧菌EpGS021001抗体效价较单纯溶藻弧菌LPS免疫组、简单混合物免疫组出现得早,抗体滴度高;与此类似,OMPC-LPS偶联组中抗哈氏弧菌SpGY020601的血清抗体效价较单纯哈氏弧菌OMPC组、简单混合组出现得早,抗体滴度高,且持续时间长;对EpGS021001、SpGY020601的攻击,OMPC-LPS偶联物免疫组的保护率分别为85%和95%,高于单纯溶藻弧菌LPS免疫组的70%、单纯哈氏弧菌OMPC免疫组的75%,也高于简单混合物免疫组的80%和82.5%.     

哈维弧菌vhh基因缺失株的构建及其相关生物学特性研究

哈维弧菌(Vibrio harveyi)是水生动物的重要病原,为研究哈维弧菌溶血素基因vhh缺失后对其生物学特性的影响,该研究利用同源重组技术构建了V.harveyi 345的vhh基因缺失突变株,并比较了野生株和突变株的生物学特性变化。结果显示,vhh基因的缺失不影响菌株的生长、胞外蛋白酶分泌、过氧化氢(H2O2)和铜离子(Cu2+)的压力感应、铁的吸收利用、15种抗生素抗性和生物膜形成等生物学特性,但会导致菌株游动和涌动显著增强;另发现,vhh基因虽然在野生菌株内高表达,对绵羊红细胞却未表现出溶血活性。结果表明该基因负调控着菌株的运动能力。该研究为认识哈维弧菌vhh基因功能研究提供新的资料。     

Immunohistochemical evaluation of experimental Vagococcus salmoninarum infection in rainbow trout (Oncorhynchus mykiss,Walbaum 1792)

The aim of this study was to investigate the pathogenesis and histopathological and immunohistochemical findings in rainbow trout (Oncorhynchus mykiss) following experimental vagococcosis. For this purpose, 60 rainbow trout were used. The experimental study used the pathogen Vagococcus salmoninarum. The fish were intraperitoneally (IP) administered with an inoculate containing 0.1 mL of the bacteria, resulting in a dose of 1.2 × 10⁹ cfu mL^?1 per fish. For histopathological observations, tissue samples were taken from fish that died during the experiment and fish that survived until the end of the trial (60th day). All the tissue samples were immunohistochemically stained by the avidin–biotin–peroxidase complex and immunofluorescence methods using polyclonal antibody to detect V. salmoninarum antigens. In immunoperoxidase staining, positive reactions to bacterial antigens were most commonly seen in the kidney, heart and liver. In the immunofluorescence analysis, the distribution of antigens in the tissue and organs was similar to that observed with the immunoperoxidase staining. The results reveal an important correlation between histochemical and immunohistochemical staining in demonstrating the distribution of V. salmoninarum antigens in the affected tissues.     

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.     

虹鳟IgM重链恒定区的表达及兔抗血清的制备

为了能够成功表达虹鳟IgM,本研究利用生物信息学软件对虹鳟IgM的亲水性及抗原性进行了分析,根据GenBank收录的虹鳟IgM重链恒定区,参照生物信息学分析结果,设计用于扩增截短的IgM基因的引物,以虹鳟头肾RNA提取物为模板,利用RT-PCR方法扩增虹鳟截短的IgM重链恒定区部分基因片段,连接原核表达载体pET-27b,利用大肠杆菌Rosetta进行表达。SDS-PAGE及HPLC结果显示,纯化后截短的IgM大小约为47.7 ku,且纯度达到90%。利用其制备兔抗血清后ELISA分析结果显示,所制备的兔抗血清与本研究所表达的截短IgM蛋白的反应效价为1∶40 000,与虹鳟血清提取的全长IgM反应效价为1∶20 000并且呈现出抗原计量依赖性。研究表明,重组IgM蛋白与虹鳟血清中的天然IgM重链恒定区具有近似的结构,利用其所制备的兔抗血清能够与虹鳟鱼体中的天然IgM发生特异性反应。     

对虾养殖池中一株弧菌拮抗菌的分离鉴定

从对虾养殖池中分离出1株编号为2013082515(简称菌株15)的菌株,以鳗弧菌(Vibrio anguillarum)、哈维氏弧菌(Vibrio harveyi)和副溶血弧菌(Vibrio parahaemolyticus)为指示菌,分析了菌株15对指示菌的抑菌效果及最低抑菌浓度,同时,分析了菌株15对其他7株弧菌的抑菌效果.结果显示,菌株15对3株指示菌均具有抑菌效果,对鳗弧菌、哈维氏弧菌及副溶血弧菌的最低抑菌浓度分别为2.50×104、2.50×105、2.50×105 CFU/ml;对其他弧菌也具有一定的抑菌效果.采用注射及浸泡感染方法分析了菌株15对对虾的生物安全性,结果显示,在高浓度时,菌株15对对虾具有潜在毒性.分别用细菌全细胞脂肪酸气相色谱法和16S rDNA序列分析比对法对该菌进行分类鉴定,表明菌株15为一株假交替单胞菌(Pseudoalteromonas sp.).