Interference of Listonella anguillarum with potential probiotic microorganisms isolated from farmed gilthead seabream (Sparus aurata, L.)

Four isolates obtained from gilthead seabream have been tested for their adhesion to the skin, gill and intestinal mucus of gilthead seabream, and for their ability to interfere with Listonella anguillarum, an important pathogen of farmed gilthead seabream. The ability to adhere to mucus was higher than 7% for all isolates. Three isolates showed an antagonistic effect against some of the pathogenic strains tested. They were assayed to interfere with the attachment of L. anguillarum to the mucus of gilthead seabream. Only two isolates significantly reduced the adhesion of L. anguillarum to all of the mucus assayed under exclusion, competition and displacement conditions. According to the criteria applied, the isolate Pdp11 was selected and its in vivo probiotic potential was assessed by oral administration followed by challenge with the pathogen L. anguillarum. For the feeding trial, a group of 50 gilthead seabreams received a commercial diet supplemented with lyophilized 10⁸ CFU g^?1 of this isolate for 15 days. An other group of similar characteristics received the non‐supplemented commercial diet. After the challenge, the mortality of the fish receiving the diet supplemented with the potential probiotic was significantly lower than that observed in the groups of fish receiving the non‐supplemented commercial diet.     

Prevalence of a fish pathogen, Listonella anguillarum, in the intestinal tract of fish collected off the coast of Japan

This study evaluates the influence of temperature manipulation on circulating levels of sex steroids and gonadal maturation in F1 generation Senegalese sole Solea senegalensis breeders kept under either continuous light (LL) or natural photoperiod (NP). Under LL, progressive water temperature reduction during early winter [treatment MT (modified temperature)] significantly elevated [compared with exposure to LL and constant temperature (CT)] plasma steroid levels in females (January–February) and males (January) and proportion of females initiating maturation (January), suggesting a modulatory role of decreasing temperature in the early phases of sole gonadal recrudescence. Under NP, treatment MT did not stimulate female gonadal maturation despite the significantly higher plasma testosterone levels (February) compared with exposure to NP and CT, which may indicate that females at early‐intermediate phases of ovarian development, the predominant stages of maturation at early winter, were less responsive to the treatment. Conversely, treatment MT significantly enhanced male androgen production from January onwards, together with an increase in proportions of running individuals (March), indicating that males were also responsive to this treatment at mid‐late phases of testicular development. Our data further support the strong influence of water temperature on the overall control of gonadal maturation in Senegalese sole.     

环介导等温扩增技术与横向流动试纸条法快速检测鳗利斯顿氏菌的研究

采用环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP)、横向流动试纸条技术(Lateral flow dipstick,LFD),建立一种鳗利斯顿氏菌快速检测方法。针对鳗利斯顿氏菌金属蛋白酶基因设计一套特异性引物及异硫氰酸荧光素(Fluorescein isothiocyanate,FITC)标记的探针,结合生物素标记的环介导等温扩增技术扩增反应和横向流动试纸条技术对鳗利斯顿氏菌进行检测;比较LAMP-AGE、LAMP-LFD和PCR-AGE的灵敏度;选取4株鳗利斯顿氏菌和6株非鳗利斯顿氏菌验证LAMP-LFD特异性。试验结果表明,应用LAMP-LFD,能在30 min内完成鳗利斯顿氏菌的检测;LAMP-LFD检测的灵敏度为7.7 cfu/ml,LAMP-AGE和PCR-AGE检测的灵敏度均为77 cfu/ml;4株鳗利斯顿氏菌均呈阳性反应,其他6株非鳗利斯顿氏菌均为阴性。应用LAMP-LFD检测鳗利斯顿氏菌特异性强、灵敏度高、并且操作安全、简便、快捷。     

Occurrence of Listonella anguillarum in seed production environments of Japanese flounder Paralichthys olivaceus (Temminck et Schlegel)

The present study was undertaken to investigate the distribution of Listonella anguillarum in the rearing water, fish and diets (rotifers) of Japanese flounder (Paralichthys olivaceus). A total of 793 isolates were obtained from the seed production environment of Japanese flounder and 175 out of them were identified as L. anguillarum by biochemical characterization, polymerase chain reaction (PCR) detection for VAH1 haemolysin gene and phylogenetic analysis of 16S ribosomal deoxyribonucleic acids (rDNA) sequences. These results strongly suggested that L. anguillarum is rapidly and accurately identified by the combination of incubation on thiosulphate–citrate–bile salt–sucrose agar at 35°C overnight and PCR detection for the VAH1 haemolysin gene. All flounder specimens and all rotifer samples harboured L. anguillarum at high densities of 6.9 × 10³–6.3 × 10⁵ colony forming units (CFU) g^?1 and 1.5 × 10⁴–2.3 × 10⁶ CFU g^?1, respectively, while as low as 5.0 × 10⁰–2.0 × 10¹ CFU mL^?1 of L. aguillarum were detected in only two of 11 seawater samples, even though no vibriosis occurred in larval and juvenile flounder of tanks. This fact strongly suggests that L. anguillarum is an inhabitant in the seed production environments of Japanese flounder.     

鲤病原鳗利斯顿氏菌的分离鉴定及生物学特性研究

从患病鲤(Cyprinus carpio L.)体内分离到一株优势生长菌,人工感染试验证明该菌对鲤有较强的致病性。对分离菌进行了形态特征、理化特性等生物学性状检验;测定了分离菌的16S rRNA和gyrB基因的部分序列,并与相关细菌16S rRNA和gyrB基因序列进行比对后,构建了基于两种基因的系统发生树。结果显示:分离菌所扩增的16S rRNA和gyrB基因序列与GenBank数据库中鳗利斯顿氏菌的16S rRNA和gyrB基因序列相似性均在97%以上,其16S rRNA基因序列长度为1449 bp(GenBank登录号:FJ824662),gyrB基因序列长度为1202 bp(Gen-Bank登录号:GQ452957);胞外酶活性及溶血活性检测表明分离菌具有淀粉酶、蛋白酶、明胶酶、卵磷脂酶活性,但不具有脂酶活性;在含7%家兔脱纤血液营养琼脂培养基上,呈β型溶血,分离菌均具有金属蛋白酶基因及溶血素基因。根据分离菌的表型特征及分子特征,判定分离菌为利斯顿氏菌属(Listonella MacDonell and Colwell1986)的鳗利斯顿氏菌(Listonella anguillarum)。分离菌的耐药谱测定结果显示,对供试49种抗菌药物中的青霉素G等12种药物耐药,对克林霉素等5种药物敏感,对恩诺沙星等32种药物高度敏感。     

Growth parameters,innate immune response and resistance to Listonella (Vibrio) anguillarum of Dicentrarchus labrax fed carvacrol supplemented diets

The research was aimed to assess the effect of dietary carvacrol (0.025% and 0.05%) on sea bass (Dicentrarchus labrax) growth, immune response and resistance to Listonella anguillarum. Fish (69.2 ± 0.22 g) were fed the experimental diets for 9 weeks. Dietary carvacrol did not negatively affect fish survival, growth performance, feed intake and feed conversion ratio (P > 0.05) nor carcass yield and viscerosomatic, hepatosomatic and mesenteric fat index (P > 0.05). Serum and head kidney leucocytes were collected after 1, 4 and 8 weeks of feeding. Carvacrol significantly reduced serum proteins, immunoglobulins and lysozyme activity (P < 0.01) and moderately increased phagocytosis and pinocytosis of head kidney macrophages. The release of reactive oxygen species by leucocytes was reduced in carvacrol‐fed fish, even if significantly (P < 0.05) only in those fed 0.05% carvacrol for 1 week. Dietary carvacrol did not significantly affect the aspecific immune response, although a potential antioxidant activity might be speculated. Moreover, feeding carvacrol provided an appreciable resistance to a challenge with L. anguillarum, when a bacterial dose lower than the Lethal Dose₅₀ was used. Cumulative mortality in fish fed 0.025% carvacrol was significantly lower than that of untreated controls (75% Relative Per cent Survival).     

Surface-exposed expression of Edwardsiella tarda EseB in live attenuated Vibrio anguillarum based on novel surface display systems

Live, attenuated Vibrio anguillarum strains can serve as vectors for the delivery of heterologous antigens for development of multivalent recombinant vaccines. Based on the outer membrane anchoring elements of V. anguillarum , we have previously constructed several efficient surface display systems Lpp-Omporf1, Lpp-OmpU, Lpp-Omp26La, Wza-Omporf1, Wza-OmpU and Wza-Omp26La. In this study, with these constructed surface display systems, a putative antigen protein EseB from pathogenic Edwardsiella tarda was successfully expressed on the surface of an attenuated V. anguillarum strain to get multivalent vaccine candidates. Further immune protection evaluation in zebra fish ( Danio rerio ) demonstrated that the V. anguillarum EseB-display strain AV/pW-26La-B could trigger full protection against V. anguillarum infection and early protection against E. tarda infection in the immunized fish. These results suggest that surface display of heterologous protective antigens in attenuated V. anguillarum could be used as a tool to develop potential V. anguillarum vector vaccine.     

不同环境条件对溶藻弧菌粘附大黄鱼肠粘液的影响

在低盐度5~6和水温25~29℃条件下,分别投喂1种对照饲料和7种添加二甲基-β-丙酸噻亭(DMPT)(添加量分别为100、2003、00、400、5006、00和700mg.kg-1)的试验饲料,饲养初始体重约每尾0.77g的凡纳滨对虾56d,观察DMPT对凡纳滨对虾的生长、蜕壳和渗透压调节的影响。实验结果表明,添加DMPT的各组凡纳滨对虾增重率随着DMPT添加量的增加,呈现先升后降的趋势,在400mg.kg-1凡纳滨对虾增重率达到最高,显著高于对照组;与对照组相比,各试验组凡纳滨对虾的饲料系数均不同程度降低,500mg.kg-1DMPT组凡纳滨对虾的饲料系数降低达到显著水平。添加DMPT对凡纳滨对虾的蜕壳次数无显著影响。除100mg.kg-1DMPT组外,DMPT组凡纳滨对虾的渗透压均明显低于对照组,其中,添加300和500mg.kg-1组凡纳滨对虾的渗透压降低呈现显著性差异。凡纳滨对虾肌肉粗蛋白和粗脂肪含量随着DMPT水平增加呈现出先上升后下降的趋势。粗蛋白含量在DMPT添加量为400mg.kg-1时达到最高,粗脂肪含量在DMPT添加量为600mg.kg-1时达到最高,均显著高于对照组。以增重率为判定指标,DMPT在凡纳滨对虾饲料中的适宜添加量为383mg.kg-1。     

不同环境条件对溶藻弧菌粘附大黄鱼肠粘液的影响

研究了不同环境条件下溶藻弧菌对大黄鱼肠粘液的粘附作用。采用细菌计数法测定溶藻弧菌的粘附作用。粘附的细菌经SYBR GreenⅠ染色后在荧光显微镜下观察,用数码相机拍照后在电脑上计数。试验结果表明,溶藻弧菌对大黄鱼肠粘液的粘附量随菌浓度的升高而升高,并在1—1．5h内趋于饱和;粘附作用在15—30℃、pH偏酸时较强;盐度在5—35范围内对前肠粘液的粘附作用影响不明显,后肠粘液的粘附作用在此范围内随盐度增大而加强,在盐度为0时,溶藻弧菌对前、后肠粘液都无粘附作用;56℃热处理5min及60℃处理1h均能大幅减弱溶藻弧菌对两种肠粘液的粘附作用,表明溶藻弧菌表面的某些热敏结构在粘附作用中起着重要作用。根据以上结果,可以认为溶藻弧菌能够很好地粘附于大黄鱼肠粘液层,其粘附作用受温度、盐度、pH值等环境因子影响很大,溶藻弧菌表面的某些热敏结构可能在粘附过程中起着重要作用。研究结果表明,溶藻弧菌对大黄鱼肠粘液的粘附作用是可控制的,这对于鱼类养殖疾病的控制有一定的参考价值。     

溶血相关基因缺失降低鳗弧菌感染花鲈的致病性

为阐明毒力因子溶血素对鳗弧菌感染花鲈过程中发挥的作用,实验以花鲈为研究模型,采用临床剖检、组织病理学观察、分子生物学检测、转录组分析等检测方法开展实验。通过对花鲈幼鱼分别注射5.0×105 CFU鳗弧菌野生株和缺失株△vah1-vah4-rtxA,评估了溶血相关基因缺失对花鲈的感染能力、花鲈各组织的病理变化情况以及免疫响应的影响。结果显示,花鲈感染野生株后的LD50为2.103×105 CFU/mL,感染缺失株△vah1-vah4-rtxA后LD50为1.837×106 CFU/mL,溶血相关基因缺失使鳗弧菌毒性降低8.74倍;鳗弧菌主要定殖在花鲈的肠道和鳃中,溶血相关基因缺失降低鳗弧菌在花鲈体内的定殖能力,同时降低对肠道黏膜空泡化、鳃坏死的损伤程度;转录组分析表明vah1、vah4、rtxA缺失后,头肾组织的差异表达基因显著富集到造血细胞谱系、抗原处理和呈递、细胞粘附分子、肠道免疫网络的IgA生产等免疫通路。这些结果表明,溶血相关基因缺失降低了鳗弧菌感染花鲈的致病能力。研究结果有助于进一步了解鳗弧菌的致病机制,并为开发花鲈抗弧菌免疫增强剂或减毒疫苗提供依据。     

几株益生菌对鲤前肠黏液的体外黏附特性

采用体外固定鲤前肠黏液,结合细菌同位素示踪的方法,对来源于鲤肠道的2株肠球菌和1株柠檬酸杆菌以及养殖水体的2株芽孢杆菌的细菌表面凝集素和黏液蛋白受体的化学组成、对病原菌附着鲤前肠黏液的影响等进行了研究。结果表明,5株菌经高碘酸钠和蛋白水解酶修饰后,高碘酸钠能极显著降低芽孢杆菌、肠球菌和柠檬酸杆菌的黏附率(P<0.01),而蛋白水解酶对多数菌株的影响不显著(P>0.05),推测细菌表面的凝集素主要为具糖蛋白性质的物质。黏液蛋白经蛋白水解酶处理后,部分菌株的附着数量显著降低(P<0.05),但经高碘酸钠处理后,5株菌的黏附率均显著上升(P<0.05),提示黏液蛋白上存在的5株细菌的特异性受体物质可能为蛋白质。益生菌通过排斥、竞争和取代作用能够显著降低部分病原菌的黏附率。取代作用对病原菌黏附的抑制效果最好,但具有菌株特异性。     

Evaluation of the AQUARAPID-Va, AQUAEIA-Va and dot-blot assays for the detection of Vibrio anguillarum in fish tissues

The comparative accuracy of the serological assays AQUARAPID-Va, AQUAEIA-Va (BIONOR AS), and dot-blot for a rapid diagnosis of vibriosis in fish was evaluated. Twenty-one Vibrio anguillarum strains, representative of pathogenic and environmental serotypes, and 13 strains of other fish pathogenic bacteria were used to assess the sensitivity and specificity of the detection methods. The serological assays tested detected all the strains of V. anguillarum serotypes O1 and O2. The dot-blot assay was the most specific and sensitive method, detecting almost all isolates from serotypes O1, O2 and O3, with an average sensitivity of 1 x 10(6) bacteria g(-1) of fish tissue. The AQUARAPID-Va and the AQUAEIA-Va systems were able to detect 5 x 10(6) and 5 x 10(7) bacteria g(-1) of fish tissue, respectively. The simplicity, effectiveness and speed of the AQUARAPID-Va system confirmed this method as the most suitable serological test for the detection of V. anguillarum in field analysis and small-scale laboratory studies.     

鳗弧菌L-18株的限铁条件和铁调节外膜蛋白的免疫效果研究

利用Western-blot技术,对鳗弧菌L-18株的OMPs和IROMPs的免疫反应性进行了分析,并通过免疫牙鲆比较二者免疫保护力的差异,以期从IROMPs方向探索开发鳗弧菌新型鱼用疫苗,并为研究其免疫原理提供重要的科学依据。结果表明,培养基中加入不同浓度的联铁剂,鳗弧菌L-18株的生长和铁载体的表达出现规律性变化,其中加入100~130μmol·L-1 2,2’-联吡啶为最适合限铁条件,此时铁载体的表达量最高且生长受抑制程度较小。在此条件下,菌株表达出74.3ku和77.4ku的IROMPs,其中77.4ku蛋白具有免疫反应性,为重要抗原。用全菌灭活疫苗、OMPs、IROMPs分别腹腔注射免疫牙鲆7周后攻毒, IROMPs疫苗的相对免疫保护力达到75.9%,远高于OMPs疫苗的51.8%。相对于OMPs,IROMPs的抗体效价显著提高,接近全菌疫苗组的水平,而血清杀菌活力没有显著变化。 关键词：鳗弧菌;铁调节外膜蛋白;牙鲆     

黏红酵母表面黏附蛋白及肠黏液受体的初步研究

为获得对大菱鲆肠道具有较强黏附能力的益生菌株,了解其黏附机制,采用体外黏液黏附模型及Western-blot技术,对黏红酵母等4株益生菌的大菱鲆肠黏液黏附性能及黏红酵母表面粘附蛋白和肠黏液受体进行了初步研究.结果表明,4株益生菌均可黏附于大菱鲆肠黏液;但各菌株的黏附能力存在较大差异,其中黏红酵母黏附能力最强,鼠李糖乳杆菌次之,假丝酵母和枯草芽孢杆菌相对较低.黏红酵母表面蛋白预先与肠黏液孵育2h后,显著抑制了黏红酵母与肠黏液的结合,其黏附百分率下降了83.6%;但上述处理对其他3株益生菌的黏附没有明显影响.Western-blot显示黏红酵母表面蛋白中分子量为38.5ku和28.6ku的两个蛋白参与对肠黏液的黏附过程,大菱鲆肠黏液中分子量为27.3ku和22.3ku两个蛋白可与黏红酵母表面蛋白特异性结合.糖原染色显示上述4个蛋白均为糖蛋白.     

两种弧菌对菲律宾蛤仔代谢途径影响的比较研究

采用基于核磁共振波谱技术的代谢组学技术,探索菲律宾蛤仔在受到鳗弧菌和灿烂弧菌感染的代谢物变化特征,构建菲律宾蛤仔对弧菌感染后的代谢网络调控图谱,并比较两种弧菌毒性效应的差异。试验结果表明,3种代谢物葡萄糖、谷氨酸、苏氨酸在两种弧菌感染时均发生了变化,表征鳗弧菌感染的代谢物为牛磺酸、精氨酸、酪氨酸、苯丙氨酸、赖氨酸、亮氨酸、异亮氨酸、缬氨酸;而表征灿烂弧菌污染的代谢物为甜菜碱、二甲基甘氨酸、胆碱、谷氨酸、亚牛磺酸。     

鳗弧菌L-18株的限铁条件和铁调节外膜蛋白的免疫效果

利用Western-blot技术,对鳗弧菌L-18株的OMPs和IROMPs的免疫反应性进行了分析,并通过免疫牙鲆比较二者免疫保护力的差异,以期从IROMPs方向探索开发鳗弧菌新型鱼用疫苗,并为研究其免疫原理提供重要的科学依据。结果表明,培养基中加入不同浓度的联铁剂,鳗弧菌L-18株的生长和铁载体的表达出现规律性变化,其中加入100~130μmol.L-12,2′-联吡啶为最适合限铁条件,此时铁载体的表达量最高且生长受抑制程度较小。在此条件下,菌株表达出74.3 ku和77.4 ku的IROMPs,其中77.4 ku蛋白具有免疫反应性,为重要抗原。用全菌灭活疫苗、OMPs、IROMPs分别腹腔注射免疫牙鲆7周后攻毒,IROMPs疫苗的相对免疫保护力达到75.9%,远高于OMPs疫苗的51.8%。相对于OMPs,IROMPs的抗体效价显著提高,接近全菌疫苗组的水平,而血清杀菌活力没有显著变化。     

Chemotaxis towards, adhesion to, and growth in carp gut mucus of two Aeromonas hydrophila strains with different pathogenicity for common carp, Cyprinus carpio L

Characteristics that promote bacterial colonization of the intestinal mucosal surface were examined in two strains of the common fish pathogen Aeromonas hydrophila, with different pathogenicity. The characteristics examined were chemotactic activity towards mucus, bacterial adherence to mucus and growth in mucus. Intestinal gut mucus of healthy common carp was used. The results indicate that chemotaxis is not necessary for a bacterium to become pathogenic, but it may be a necessary parameter for a bacterium to be an obligate pathogen. Adhesion also seems to be a factor influencing pathogenicity. The results suggest that higher adhesion to mucus and subsequent growth is associated with differences in pathogenicity.     

Application of in situ detection techniques to determine the systemic condition of lymphocystis disease virus infection in cultured gilt-head seabream, Sparus aurata L

Immunohistochemistry (IHC) and in situ hybridization (ISH) techniques have been used for the detection of lymphocystis disease virus (LCDV) in formalin-fixed, paraffin-embedded tissues from gilt-head seabream, Sparus aurata L. Diseased and recovered fish from the same population were analysed. IHC was performed with a polyclonal antibody against a 60-kDa viral protein. A specific digoxigenin-labelled probe, obtained by PCR amplification of a 270-bp fragment of the gene coding the LCDV major capsid protein, was used for ISH. LCDV was detected in skin dermis and gill lamellae, as well as in several internal organs such as the intestine, liver, spleen and kidney using both techniques. Fibroblasts, hepatocytes and macrophages seem to be target cells for virus replication. The presence of lymphocystis cells in the dermis of the skin and caudal fin, and necrotic changes in the epithelium of proximal renal tubules were the only histological alterations observed in fish showing signs of the disease.     

Application of the rpoS gene for the detection of Vibrio anguillarum in flounder and prawn by polymerase chain reaction

Vibrio anguillarum , an opportunistic fish pathogen, is the main species responsible for vibriosis, a disease that affects feral and farmed fish and shellfish, and causes considerable economic losses in marine aquaculture. In this study, we used polymerase chain reaction (PCR) to detect V. anguillarum . PCR specificity was evaluated by amplifying the rpoS gene, a general stress regulator, in six strains of V. anguillarum and 36 other bacterial species. PCR amplified a species-specific fragment (689 bp) from V. anguillarum . Furthermore, the PCR assay was sensitive enough to detect rpoS expression from 3 pg of genomic DNA , or from six colony-forming units (CFU) mL⁻¹ of cultured V. anguillarum . However, the assay was less sensitive when genomic DNA from the infected flounder and prawn was used (limit of detection, 50 ng and 10 ng g⁻¹ tissue, respectively). These data demonstrate that PCR amplification of the rpoS gene is a sensitive and species-specific method to detect V. anguillarum in practical situations.     

载铜蒙脱石对嗜水气单胞菌粘附尼罗罗非鱼上皮细胞的影响

采用尼罗罗非鱼上皮细胞培养模型,观察嗜水气单胞菌对鳃上皮、皮肤上皮、肠上皮细胞的粘附率,研究载铜蒙脱石对细菌粘附的阻断作用及其对细菌粘附引起鱼上皮细胞膜生物特性变化的影响。结果显示：嗜水气单胞菌与鱼上皮细胞均有不同程度的粘附作用,粘附率大小顺序为鳃上皮〉皮肤上皮〉肠上皮细胞。载铜蒙脱石均显著降低了嗜水气单胞菌对鳃、皮肤和肠上皮细胞的粘附率（P〈0．05）,而且对不同上皮细胞的粘附阻断率无显著差异。嗜水气单胞菌粘附肠上皮细胞后细胞膜生物特性发生了显著变化,与正常对照组相比,嗜水气单胞菌粘附鱼上皮细胞后,胞浆游离钙和细胞膜磷脂酶A2活性显著上升。载铜蒙脱石可显著降低由于嗜水气单胞菌粘附鱼上皮细胞引起的胞浆游离钙和细胞膜磷脂酶A2活性的升高。结果表明,载铜蒙脱石可有效阻断病原菌粘附,从而防治细菌感染和细菌移位。