柱状黄杆菌胞外多糖对草鱼的免疫促进作用

将柱状黄杆菌胞外多糖（EPS）溶液经腹腔注射免疫草鱼，以注射灭菌生理盐水作对照。免疫一周后，分别提取受免鱼与对照鱼肝脏、脾脏、体肾和头肾4种组织中的总RNA，并通过RTPCR半定量的方法检测不同组织中C反应蛋白（CRP）、白介素1（IL-1）、主要组织 相容性复合体I（MHC-I）、免疫球蛋白M（IgM）、干扰素（IFN）等5种免疫基因的表达情况。结果显示，CRP在受免鱼肝脏中的表达显著上升；MHC I在受免鱼脾脏、体肾中的表达显著下降；IgM在受免鱼4种组织中的表达皆显著上升；IL-1在受免鱼4种组织中的表达与对照组没有显著差异；无论受免组还是对照组都只能检测到微弱的IFN表达，且两组之间没有显著差异。结果表明，柱状黄杆菌EPS对草鱼的先天免疫能力以及特异性体液免疫能力具有促进作用。     

柱状黄杆菌间接ELISA快速检测方法的研究

应用间接酶联免疫吸附试验(Indirect-ELISA)技术,建立了快速检测柱状黄杆菌的方法。结果显示:根据棋盘试验,确定最佳抗原包被浓度和免疫血清最佳工作浓度分别为1×106cfu/mL和1∶5000,酶标抗体最佳工作浓度为1∶10000。在该条件下,所建立的间接ELISA能检测出5×104cfu/mL的柱状黄杆菌,且与爱德华氏菌、哈维氏菌弧菌、嗜水气单胞菌、副溶血弧菌等常见鱼类致病菌无交叉反应。结果表明,所建立的间接ELISA可不经细菌培养而直接检测人工感染后草鱼鳃组织中的柱状黄杆菌,该方法对柱状黄杆菌的检测具有快速、敏感、特异、实用的优点。     

柱状黄杆菌胶原蛋白酶的克隆与表达

利用PCR方法从柱状黄杆菌(Flavobacterium.columnare)G4株中克隆了1个胶原蛋白酶基因(GenBank登录号EF501979).同源性比对发现该基因与柱状黄杆菌的另外1种胶原酶基因(GenBank登录号EAZ95511.1)最为相近,有84%的一致性和93%的相似性.该属的另外2种细菌,约氏黄杆菌(Flavobacterium.johnsoniae)和嗜冷黄杆菌(F.psychrophilum)都有与该胶原酶基因相似的基因,其相似性分别为92%和83%.该基因经KpnI和SalI酶切后连接到表达载体pET-32a上,转人大肠杆菌BL21(DE3)plysS内进行表达,经SDS-PAGE电泳后显示重组融合蛋白在44 kD处有明显表达带,与预期分子量大小一致,且主要以不溶的包涵体形式存在,变性条件下利用His·Bind树脂成功纯化了融合蛋白,将其免疫家兔,获得兔抗柱状黄杆菌胶原蛋白酶抗体,Western blotting表明该胶原酶在柱状黄杆菌的胞内和胞外都存在.这些结果为进一步研究这种胶原蛋白酶的功能及柱状黄杆菌的致病机理奠定了基础.     

MiR-155靶向踝蛋白调控柱状黄杆菌胞外多糖诱导的EPC细胞凋亡

柱状黄杆菌胞外多糖 (FC-EPS) 孵育能显著提高鲤上皮瘤细胞 (EPC) 中 miR-155 的表达,导致细胞发生凋亡。为研究 miR-155 在 FC-EPS 诱导的细胞凋亡中的作用机制,本文采用 RNAi 技术,通过过表达 miR-155 和敲降踝蛋白 (talin-1),皆能抑制 FC-EPS 诱导的细胞凋亡,并鉴定出 talin-1 为 miR-155 的靶蛋白。在 FC-EPS 诱导细胞凋亡的过程中,踝蛋白水平显著升高,在凋亡发生阶段检测到凋亡执行蛋白 caspase-3 的活化,同时检测到两条踝蛋白剪切异构体,大小约为200 kDa 和 250 kDa;将构建的鲤 talin-1 真核表达质粒转染 EPC 细胞,过表达的 talin-1 延迟且延长了细胞凋亡的进程,并检测到上述两条踝蛋白异构体。然而,当以 FC-EPS 转染 EPC 细胞时,talin-1 先下降再恢复到正常水平,细胞不发生凋亡。因此,在 FC-EPS 诱导细胞凋亡的过程中,细胞通过上调 miR-155 来调控靶基因 talin-1 mRNA 和蛋白转录的变化,以及产生 talin-1 蛋白异构体来抑制细胞发生凋亡。本文为有效防治柱状黄杆菌疾病提供新思路。     

柱状黄杆菌常规PCR检测体系的建立

根据NCBI公布的柱状黄杆菌硫酸软骨素裂解酶的基因序列,设计并选取一对能够快速而准确地检测柱状黄杆菌的PCR引物,建立PCR快速检测体系,同时测试该体系的特异性和灵敏度,并对患病的鱼组织进行检测。研究结果表明,使用设计的引物能够扩增出与预计大小相符合的138bp的特异性片段,具有较好的检测特异性,对靶标DNA的检测灵敏度为pg级,对柱状黄杆菌的检测灵敏度为25cfu/20μl。样品的检测结果与实际发病情况一致,表明本研究成功建立了柱状黄杆菌常规PCR检测体系,该体系可以用于柱状黄杆菌的诊断和样品的检测。     

枯草芽孢杆菌表达柱状黄杆菌lip基因口服疫苗对草鱼免疫保护效果的研究

枯草芽孢杆菌(Bacillus subtilis)是一种水产益生菌,最近也被用作口服疫苗的投递载体。本研究利用前期筛选到的柱状黄杆菌(Flavobacterium columnare)保护性抗原基因lip,与pBE2R载体连接后,以质粒的方式转入枯草芽孢杆菌WB800中,构建了重组菌株WB800 (pBE2R-lip)。聚丙酰胺电泳和免疫印迹实验表明,枯草芽孢杆菌WB800 (pBE2R-lip)能表达lip蛋白,其分子量约为32 kDa。利用枯草芽孢杆菌WB800 (pBE2R-lip)口服免疫草鱼(Ctenopharyngodon idellus)后,通过酶联免疫吸附实验,在受免草鱼血清中检测到特异性抗体效价的上升,在人工感染实验中,受免草鱼的免疫保护率为52.4%。本研究表明,利用枯草芽孢杆菌能有效表达柱状黄杆菌的lip基因,以其作为口服疫苗能引起草鱼的免疫应答并提供免疫保护效果。     

鳜源柱状黄杆菌的分离鉴定及其对10种中草药的敏感性研究

为探索养殖鳜细菌性疾病的防治方法,从湖北省某养殖场患烂鳃病鳜鱼体内分离出一株柱状黄杆菌。经形态学观察、16S rRNA基因序列测定,确定其为柱状黄杆菌。为确定10种中草药对鳜源柱状黄杆菌的抑菌效果,分别采用10种中草药的水提物进行抑菌试验。试验结果表明,诃子、乌梅、连翘、五倍子有较好的抑菌效果,其中诃子抑菌效果最好;五味子、秦皮、鱼腥草、牡丹皮、青蒿、穿心莲的抑菌效果较差。本研究可为开发中草药防治鳜柱形病提供一定的理论依据。     

柱状黄杆菌乙酰辅酶A合成酶基因及其上游调控序列分析

柱状黄杆菌(Flavobacterium columnare)是世界范围内危害淡水鱼类的柱形病的病原。目前对该病原菌遗传操作系统的研究进展较慢,而寻找高效稳定的启动子来调控外源基因在细菌体内的表达,有可能促进该细菌遗传操作系统的构建。本研究获得了柱状黄杆菌乙酰辅酶A合成酶基因(acetyl-coenzyme A synthetase gene,acs)的编码序列及其上游调控序列,该基因全长2 323 bp,编码635个氨基酸。通过序列分析,发现在该基因起始密码子ATG的上游存在核糖体结合位点(ribosome biding site,RBS)序列TAAAA,和启动子–7和–33的保守基序TATTTTCG和TTG。将acs的上游调控序列(promoter sequence,Pacs)置于氯霉素抗性基因(chloramphenicol acetyltransferase,cat)的上游并导入柱状黄杆菌G4株后,cat基因得以表达,并使宿主细胞产生稳定的氯霉素抗性。通过5′RACE技术,确定了外源的cat基因和内源的acs基因的转录起始位点都是位于起始密码子上游46 bp处的T。通过删减分析调控序列Pacs,发现起...     

短小芽孢杆菌X93及胞外产物抑菌活性的研究

从健康养殖大黄鱼肠道中筛选出对病原弧菌有拮抗作用的潜在益生菌株X93,利用细菌快速鉴定系统和16S rDNA方法进行细菌种类鉴定;通过点种法对其抗菌谱进行测定;采用平板扩散法,研究了培养时间、温度、盐度、蛋白酶K和Fe3+等因素对其胞外产物抑菌活性的影响。结果显示:该菌株为短小芽孢杆菌(GenBank accession No.HM137033);对20株病原指示菌中的13株均产生不同程度的抑制作用,抗菌谱较广;菌株培养48 h后的胞外产物抑菌作用最强;在pH为7时,抑菌活性最大;达到最大抑菌活性的温度和盐度分别为28 ℃、5;蛋白酶K和不同浓度的FeCl3均会降低其抑菌活性。该菌对病原弧菌、迟缓爱德华菌和嗜水气单胞菌等水产上常见主要致病菌有良好的拮抗效果,且具有抑菌作用的胞外产物对温度、盐度和Fe3+有一定的适应范围,适宜作为益生菌株,有望在实际养殖环境中得到应用。     

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.     

Development and characterization of rifampicin-resistant mutants from high virulent strains of Flavobacterium columnare

Flavobacterium columnare is divided into three genetic groups or genomovars, genomovar II being highly virulent for channel catfish. A modified live vaccine is currently available to prevent columnaris disease under the licensed name Aquavac‐Col^®. The strain of F. columnare used to generate the avirulent rifampicin‐resistant mutant used in Aquavac‐Col^® belonged to genomovar I, the less virulent group towards channel catfish. In this study, we describe the generation and characterization of rifampicin‐resistant mutants from genomovar II strains. A total of 13 new mutants were obtained, and eight of them (two from each parent strain) were genetically and phenotypically characterized. Highly conserved regions within the ribosomal operons were identical between parent and mutant strains. Genetic differences between mutants and their parent strains were revealed by amplified fragment length polymorphism (AFLP). Genetic changes were distinctive among different mutants. Analysis of the lipopolysaccharide (LPS) showed that while some mutants lacked a few molecular bands of the LPS, some exhibited the same LPS profiles as their parent strains. Comparison between immunogenic proteins from mutants and parents was carried out by immunoblot analysis and further confirmed the uniqueness of individual mutants. A complete set of rifampicin‐resistant mutants with different genetic and immunogenic properties from the highly virulent genomovar II has been created. These mutants may have the potential of becoming vaccine candidates against columnaris disease.     

Comparison of lipopolysaccharide and protein profiles between Flavobacterium columnare strains from different genomovars

Lipopolysaccharide (LPS) and total protein profiles from four Flavobacterium columnare isolates were compar. These strains belonged to genetically different groups and/or presented distinct virulence properties. Flavobacterium columnare isolates ALG-00-530 and ARS-1 are highly virulent strains that belong to different genomovars while F. columnare FC-RR is an attenuated mutant used as a live vaccine against F. columnare. Strain ALG-03-063 is included in the same genomovar group as FC-RR and presents a similar genomic fingerprint. Electrophoresis of LPS showed qualitative differences among the four strains. Further analysis of LPS by immunoblotting revealed that the avirulent mutant lacks the higher molecular bands in the LPS. Total protein analysis displayed by immunoblotting showed differences between the strains analysed although common bands were present in all the isolates. FC-RR lacked two distinct common bands (34 and 33 kDa) shared by the other three isolates. Based on the difference of LPS and total protein profiles, it is possible to discriminate the attenuated mutant FC-RR from other F. columnare strains.     

Morphological and genetic characteristics of Flavobacterium columnare isolates: correlations with virulence in fish

Variability in pathogenicity of Flavobacterium columnare makes disease treatment difficult because there is currently no way to easily recognize those strains that warrant aggressive treatments. In order to identify suitable virulence markers, 17 isolates of F. columnare were cultured from six different fish species. The DNA from all isolates was analysed using random amplified polymorphic DNA (RAPD). Bootstrap analysis of the RAPD data produced a tree with three major groups supported by bootstrap scores of 80-100%. Virulence of the isolates was determined by bath exposure of channel catfish, Ictaluruspunctatus (Rafinesque), and golden shiners, Notemigonus crysoleucas (Mitchill), to broth cultures of F. columnare. In channel catfish, 13 of 17 isolates produced 100% mortality within 48 h post-exposure. All isolates of cyprinid fish origin clustered in a single RAPD group. At least two of the four isolates that were not virulent in channel catfish were of cyprinid fish origin. There was a wide variation in cell morphology between isolates with lengths of cells or cell chains ranging from 3 to 11 microm, even under identical culture conditions. Most of the shorter or single cell isolates fell into a single RAPD group. No clear association was identified between virulence and any other characteristic, including RAPD group.     

Isolation and characterization of strains of Flavobacterium columnare from Brazil

Flavobacterium columnare is an important pathogen of freshwater fish, implicated in skin and gill disease, often causing high mortality. An outbreak of skin disease in fingerling and adult Nile tilapia, Oreochromis niloticus (L.), cultivated in a recirculation system, was investigated. Four strains were isolated and characterized by biochemical reactions, enzyme production, fatty acid profile and analysis of the 16S-23S rDNA intergenic spacer region. All strains were identified as F. columnare. Experimental infection assays with one of these strains (BZ-5-02) were conducted and pathogenicity (by intramuscular route) was demonstrated in Nile tilapia and channel catfish, Ictalurus punctatus (Rafinesque). This is the first report of characterization of Brazilian strains of F. columnare.     

Chondroitin AC lyase activity is related to virulence of fish pathogenic Flavobacterium columnare

The virulence of eight Flavobacterium columnare strains was studied to find correlations between several virulence-related factors and virulence. Virulence was tested in vivo using rainbow trout, Oncorhynchus mykiss (Walbaum). Suggested virulence-related factors such as production of the degradative enzyme chondroitin lyase, plasmid occurrence and adhesion capability were studied in vitro. Infection with the four most virulent strains resulted in 95-100% mortality within 114 h. Chondroitin lyase activity was found to be significantly related to the virulence of the strains at 25 degrees C and it was also shown to be temperature-dependent, being higher at 25 degrees C than at 20 degrees C. Virulence was not plasmid associated. The adhesion capability of the strains in vitro varied substantially when tested on crude mucus-coated slides and no statistical relationship between adhesion and virulence was found using this method.     

Lack of association between Flavobacterium columnare genomovar and virulence in hybrid tilapia Oreochromis niloticus (L.) × Oreochromis aureus (Steindachner)

Columnaris disease can be problematic in tilapia (Oreochromis spp.) production. An understanding of the pathogenesis and virulence of Flavobacterium columnare is needed to develop prevention strategies. The objective of this study was to determine the virulence of genetically defined isolates of F. columnare in sex‐reversed hybrid tilapia, Oreochromis niloticus (L.) × O. aureus (Steindachner). A series of immersion challenge trials were performed using isolates of the five established genomovars of F. columnare: I, II, II‐B, III and I/II. The mean per cent mortality of fish challenged with genomovar I, II and III isolates ranged from 0 to 100, 3.3–78 and 3.3–75%, respectively. The mean per cent mortality of fish challenged with genomovar II‐B ranged from 35 to 96.7%, and the only genomovar I/II isolate tested caused no mortality. Contrary to previous work in other fish species, there did not appear to be an association between F. columnare genomovar and virulence in tilapia. The challenge model used resulted in acute mortality. An alternative challenge model was tested by cohabitating healthy fish with dead fish infected with F. columnare. This method resulted in rapid appearance of clinical signs and mortality, suggesting the potential for F. columnare to increase in virulence upon growth on/in a fish host.     

Difference in genes between a high virulence strain G4 and a low virulence strain G18 of Flavobacterium columnare by using suppression subtractive hybridization

Flavobacterium columnare is the causative agent of columnaris disease. Different genetic groups of F. columnare show to some extent different degrees of virulence. To identify genetic differences between the high virulence strain G₄ and the low virulence strain G₁₈ of F. columnare, suppression subtractive hybridization was used. A total of 46 genes were identified from the virulent strain G₄, 35 of which showed some degree of homology with known proteins and can be classified into 11 categories: DNA replication or recombination proteins, inorganic ion transport proteins, outer membrane proteins, enterotoxin, binding proteins, YD repeat proteins, transposase, chaperon, signal transduction‐related proteins, regulatory proteins, metabolism‐related proteins. Several putative virulence factors identified in other bacteria could also be identified in the virulent strain G₄, such as ferrous iron transport protein, TonB‐dependent receptor, transposases, as well as ABC transporter permease protein. The flanking region of a putative transposase ISFclI was analysed, and a putative Rhs element was located at the downstream of the putative transposase. The analysis of isfclI gene in 24 strains of F. columnare isolated in China revealed that 11 strains have isfclI, and all the strains from Zhaoqing, Anhui and Qingjiang have isfclI.     

Influence of native catfish mucus on Flavobacterium columnare growth and proteolytic activity

Flavobacterium columnare causes columnaris disease of farmed and wild freshwater fish. Skin mucus is an important factor in early stages of columnaris pathogenesis, albeit little studied. Our objectives were to (a) characterize the terminal glycosylation pattern (TGP) of catfish mucus, (b) determine the growth of F. columnare in formulated water (FW)‐containing channel catfish (Ictalurus punctatus) or hybrid catfish (Ictalurus punctatus X Ictalurus furcatus) mucus and (c) examine extracellular protease activity of two F. columnare isolates differing in virulence. The TGP of catfish mucus by lectin binding was as follows: alpha‐D‐mannose/alpha‐D‐glucose >N‐acetyl‐beta‐D‐glucosamine >N‐acetyl‐beta‐D‐glucosamine/N‐acetylneuraminic acid >N‐acetyl‐D‐galactosamine >alpha‐D‐galactose/N‐acetyl‐alpha‐D‐galactosamine >beta‐D‐galactose = alpha‐L‐fucose. Virulence studies demonstrated isolate AL‐02‐36 was highly virulent in channel catfish fry (0.1 g) with cumulative mortality of 90%‐100% versus 60% for isolate ALG‐00‐530 at equivalent doses (~3 × 10⁶ CFU/ml); a similar result was observed in larger (0.7 g) catfish. In multiple experiments, F. columnare replicated (2‐3 logs) and survived (28 days) in formulated water‐containing catfish mucus. Highly virulent isolate AL‐02‐36 possessed at least 2.5‐ to fivefold higher protease activity following growth in mucus than the less virulent ALG‐00‐530. Flavobacterium columnare utilized catfish mucus as a nutrient source and mucus presence modulated extracellular protease production.     

Reduction of in vitro growth in Flavobacterium columnare and Saprolegnia parasitica by products containing peracetic acid

Commercial products containing peracetic acid (PAA) are strong disinfectants with a wide spectrum of antimicrobial activity and have been suggested as potential therapeutic agents in aquaculture. The aim of this study was to compare the in vitro reduction of growth on two fish pathogens, Flavobacterium columnare and Saprolegnia parasitica, by seven commercial PAA‐containing products. Flavobacterium columnare was exposed to 1, 2, 4, 6, 8 and 10 mg L^?1 PAA and S. parasitica was exposed to 0.5, 1, 2, 4, 6, 8 and 10 mg L^?1 PAA in petri dishes for 24 h incubation. The reduction of growth was measured in comparison to a PAA‐free control. A reduction of the growth was observed for both pathogens with increasing PAA concentration. Hydrogen peroxide (H₂O₂) possibly has a role in the effectiveness of the products, since products with lower PAA concentrations had a higher concentration of H₂O₂. The commercial products with a low concentration of PAA and a low PAA:H₂O₂‐ratio were generally more effective against pathogens. The practical application of the products with low PAA concentration should be prioritized.