Immunogenicity and protective role of antigenic regions from five outer membrane proteins of Flavobacterium columnare in grass carp Ctenopharyngodon idella

Flavobacterium columnare causes columnaris disease in freshwater fi sh. In the present study, the antigenic regions of fi ve outer membrane proteins(OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, were bioinformatically analyzed, fused together, and then expressed as a recombinant fusion protein in Escherichia coli. The expressed protein of 95.6 k Da, as estimated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was consistent with the molecular weight deduced from the amino acid sequence. The purifi ed recombinant protein was used to vaccinate the grass carp, C tenopharyngodon idella. Following vaccination of the fi sh their Ig M antibody levels were examined, as was the expression of I g M, Ig D and Ig Z immunoglobulin genes and other genes such as MHC Iα and MHC I I β, which are also involved in adaptive immunity. Interleukin genes( IL), including I L- 1β, IL- 8 and I L- 10, and type I and type II interferon(I FN) genes were also examined. At 3 and 4 weeks post-vaccination(wpv), signifi cant increases in Ig M antibody levels were observed in the fi sh vaccinated with the recombinant fusion protein, and an increase in the expression levels of I g M, Ig D and Ig Z genes was also detected following the vaccinations, thus indicating that an adaptive immune response was induced by the vaccinations. Early increases in the expression levels of IL and IFN genes were also observed in the vaccinated fi sh. At four wpv, the fi sh were challenged with F. column a re, and the vaccinated fi sh showed a good level of protection against this pathogen, with 39% relative percent survival(RPS) compared with the control group. It can be concluded, therefore, that the fi ve OMPs, in the form of a recombinant fusion protein vaccine, induced an immune response in fi sh and protection against F. columnare.

Involvement of two glycoside hydrolase family 19 members in colony morphotype and virulence in Flavobacterium columnare

Flavobacterium columnare is the pathogenic agent of columnaris disease in aquaculture. Using a recently developed gene deletion strategy, two genes that encode the Glyco hydro_19 domain （GH19 domain） containing proteins, ghd-1 and ghd-2, were deleted separately and together from the F. columnare G4 wild type strain. Surprisingly, the single-, Aghd-1 and Aghd-2, and double-gene mutants, Aghd-1 Aghd-2, all had rhizoid and non-rhizoid colony morphotypes, which we named Aghd-1, Aghd-2, Aghd-1 Aghd-2, and NAghd-1, NAghd-2, and NAghd-1 Aghd-2. However, chitin utilization was not detected in either these mutants or in the wild type. Instead, skimmed milk degradation was observed for the mutants and the wild type; the non-rhizoid strain NAghd-2 exhibited higher degradation activity as revealed by the larger transparent circle on the skimmed milk plate. Using zebrafish as the model organism, we found that non-rhizoid mutants had higher LDs0 values and were less virulent because zebrafish infected with these survived longer. Transcriptome analysis between the non-rhizoid and rhizoid colony morphotypes of each mutant, i.e., NAghd-1 versus （vs） Aghd-1, NAghd-2 vs Aghd-2, and NAghd-1 Aghd-2 vs Aghd-1 Aghd-2, revealed a large number of differentially expressed genes, among which 39 genes were common in three of the pairs compared. Although most of these genes encode hypothetical proteins, a few molecules such as phage tail protein, rhs element Vgr protein, thiol-activated cytolysin, and TonB-dependent outer membrane receptor precursor, expression of which was down-regulated in non-rhizoid mutants but up-regulated in rhizoid mutants, may play a role F. columnare virulence.

Construction of two selectable markers for integrative/conjugative plasmids in Flavobacterium columnare

Flavobacterium columnare, the etiological agent of colunmaris disease, is one of the most important and widespread bacterial pathogens of freshwater fish. In this study, we constructed two artificial selectable markers （chloramphenicol and spectinomycin resistance） for gene transfer in F. columnare. These two new artificial selectable markers, which were created by placing the chloramphenicol or spectinomycin resistance gene under the control of the native acs regulatory region of F. columnare, were functional in both F. columnare and Escherichia coli. The integrative/conjugative plasmids constructed by using these markers were introduced into F. columnare G4 via electroporation or conjugation. The integrated plasmid DNA was confirmed by Southern blotting and PCR analysis. These two markers can be employed in future investigations into gene deletion and the pathogenicity of virulence factors in F. columnare.

Effects of Supplementation of Dries Bio-Floc as Immuno-Stimulants on Survival Rate of Tilapia Infected with Flavobacterium columnare, VETSV01

Objective： The aim of the research was to investigate efficacy of dried bio-floc supplemented with immuno-stimulants as beta-glucan and nucleotide on mortality rate and relative percent survival （RPS） of tilapia infected with Flavobacterium columnare, VETSV01. Materials and Methods： A research was performed in 1-inch long tilapia stimulated its immunity by infection with Flavobacterium columnare, VETSV01. Pathogenesis of 0.2 mL Flavobacterium columnare, VETSV01 in the range of 10^2-10^8 mL-1 fish^-1 was injected into its intraperitoneal injection and intramuscular injection and suitable concentrations were screened to obtain for further experiment in a late restage. Based on data of minimal concentration, 50% mortality rate was caused and it was found the concentration of 108 mL^-1 fish^-1 caused 100% mortality rate and no lethal rate was found in other concentrations. Afterwards, efficacy of bio-floc sediment on survival rate of Tilapia infected with Flavobacterium columnare, VETSV01 was examined. Completely randomized design was employed and experiments were divided into 4 treatment： （1） contxol-commercial feed only, （2） commercial feed attached with dried bio-floc, （3） commercial feed attached with bio-floc and beta-glucan and （4） commercial feed attached with bio-floc and nucleotide. Experiment design was performed as 3 replicates with 10 fishes/replicates in 24 inch glass aquarium. Analysis of difference was performed and determination of mean differences of mortality rate mad relative percent survival was done through Duncan＇s Multiple Range Test （p 〈 0.05）. Results and Discussions： It was demonstrated feeding tilapia with commercial feed in combination with dried bio-floc with or without supplementation of 2 immuno-stimulants could lead to 100% survival rate and 100% relative percent survival rate and it was statistically significant different （p 〈 0.05） compared to control group. Bio-floc product supplemented with beta glucan or nucleotide could effectively stimulate immunity against Flavobacterium columnare, VETSV01 and such high effective performance is viewed as a promising potential product to be further developed and practically employed for sake of aquaculture industry in the near future.

Antibacterial compounds from Rutaceae with activities against Flavobacterium columnare and Streptococcus iniae

From the ethyl acetate extract of Murraya koenegii (Rutaceae) leaves, isomahanine (1) and mahanine (2) were isolated that showed antibacterial activity towards Flavobacterium columnare and Streptococcus iniae which caused columnaris disease and streptococcosis respectively. Isomahanine was found to have the strongest activity against F. columnare (isolate ALM-00-173) and S. iniae (isolate LA94-426) based on 24-h 50% inhibition concentration (IC50) and minimum inhibition concentration (MIC). Although compound (7), a nicotinamide isolated from Amyris texana had the lowest MIC (2.8 ± 0 mg/L) of any of the test compounds against F. columnare, the 24-h IC50 of 14.8 ± 0.6 mg/L was higher than that of isomahanine and subsequently the 24-h IC50 RDC values for (7) were almost a magnitude of order higher than those obtained for isomahanine. Isomahanine also had the strongest activity against S. iniae, with a 24-h IC50 of 1.3 ± 0.1 mg/L and MIC of 3.5 ± 0 mg/L, respectively.

Antibacterial Activity of Acylglucinol Derivatives against Flavobacterium columnare

Columnaris (caused by Flavobacterium columnare) is one of the most common bacterial diseases affecting the pond-raised channel catfish (Ictalurus punctatus) in the southeastern United States of America resulting in annual losses of millions of dollars. As part of our continuing effort to discover environmentally benign compounds for the control of columnaris disease, acyl derivatives of phloroglucinol were synthesized and tested against F. columnare using a rapid bioassay. Among the analogs that were tested, diacyl analogs showed very high antibacterial activity against F. columnare in the laboratory bioassay. Diisovaleryl and diisobutyryl analogs were found to have the strongest activity against F. columnare (isolate ALM-00-173) based on 24-h 50% inhibitory concentration (IC50) and minimum inhibitory concentration (MIC). Diisovaleryl and diisobutyryl analogs had IC50 values 0.82 mg/L and 0.80 mg/L, respectively, whereas the drug control florfenicol had an IC50 value of 0.81 mg/L. Diisovaleryl and diisobutyryl analogs also had 24-h relative-to- drug-control IC50 values around 1.0 indicating activities similar to florfenicol, which is included in medicated feed and is one of the current management approach for columnaris.

Immunogenic proteins and their vaccine development potential evaluation in outer membrane proteins(OMPs)of Flavobacterium columnare

Flavobacterium columnare is a Gram-negative bacterium that causes columnaris disease in freshwater fish worldwide.Many studies have focused on the identification of protective antigens to aid in the development of novel vaccines against the disease.In this study,an immunoblotting approach was employed to identify immunogenic outer membrane proteins(OMPs)from F.columnare in two-dimensional electrophoresis(2-DE)map gels using antibacterial sera obtained from grass carp(Ctenopharyngodon idella),and anti-grass carp-recombinant Ig(rIg)monoclonal antibodies.Five unique immunogenic proteins,including the gliding motility lipoprotein GldJ(GldJ),hypothetical protein FCOL_13420(Fco1),lipoprotein(Lip),F0F1 ATP synthase subunit beta(F0f1)and outer membrane efflux protein precursor(Omep),were characterized.Over-expression of these proteins in Escherichia coli DE3,and their immunogenicity and protective efficacy were evaluated in grass carp.The relative percent survival(RPS)of the groups immunized separately with recombinant GldJ,Lip and Omep was 72%,64%and 68%,respectively when compared to control fish.Up-regulation of immuno-related genes and specific antibodies were detected in immunized fish and sera of immunized fish inhibited the growth of F.columnare.The results suggest that GldJ,Lip and Omep are major protective antigens and may be considered as novel candidates in the development of vaccines against columnaris disease in fish.

MiR-155靶向talin-1基因调控柱状黄杆菌胞外多糖诱导的EPC细胞凋亡

为研究miR-155在柱状黄杆菌胞外多糖(FC-EPS)诱导的细胞凋亡中的作用机制,本实验采用RNA干扰(RNAi)技术,通过过表达miR-155和敲降踝蛋白基因(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2条踝蛋白剪切异构体,大小分别约为200 ku和250 ku;将构建的鲤Talin-1真核表达质粒转染鲤上皮瘤细胞(EPC),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2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。本研究为有效防治柱状黄杆菌疾病提供新思路。

笋壳鱼源柱状黄杆菌的分离鉴定及致病性和药物敏感性

【目的】明确2022年5月广东省珠海市某养殖场笋壳鱼烂鳃病的病原,为防控笋壳鱼相关病害提供参考依据。【方法】从患病笋壳鱼的腐烂鳃丝、肾脏和体表病灶等部位分离细菌,并纯化优势菌落,通过PCR方法扩增分离菌株的16S rRNA序列并进行测序分析,同时构建该基因的系统发育进化树,鉴定患病笋壳鱼的病原菌。选取代表菌株Om2202进行毒力基因检测和斑马鱼人工感染试验。测定中草药和消毒剂对分离菌株Om2202的抑菌和杀菌活性,分析菌株Om2202的致病性和药物敏感性。【结果】从患病鱼鳃部和肾脏分离到的优势菌落呈细长的弯曲或直杆状,菌体表面未见鞭毛和菌毛,有荚膜,有胞外分泌物;通过特异性PCR检测和16S rRNA序列的系统发育进化树分析,结合菌落形态分析结果,确定分离菌株Om2202为柱状黄杆菌(Flavobacterium columnare)。毒力基因检测结果显示,菌株Om2202携带gldD、hly、cslA、ompA和chiA等11种毒力相关基因。斑马鱼致病性试验结果显示,菌株Om2202对斑马鱼具有较强致病性,其LC50为7.50×106 CFU/mL。药物敏感性试验结果显示,Om2202菌株对芦荟大黄素、大黄素和苯扎溴铵等药物敏感,对连翘精油、二氧化氯、蛋氨酸碘和过氧化钙等药物不敏感。【结论】引起笋壳鱼烂鳃的主要致病菌为柱状黄杆菌,属于广泛流行的基因Ⅱ型菌株,且携带大量的毒力相关基因,对斑马鱼具有较强的致病性,可用芦荟大黄素、大黄素和苯扎溴铵等药物进行防控。

柱状黄杆菌弱毒株和车轮虫混合感染致金鱼烂鳃病的病原分析

【目的】确诊北京地区某养殖场金鱼发生烂鳃病的病原。【方法】采集具有临床症状的濒死金鱼,制作鳃组织的水浸片进行寄生虫和真菌观察,取肝脏、肾脏、脾脏、鳃组织开展金鱼造血器官坏死病病毒(GFHNV)检测。同时,从鳃上分离病原菌,分析病原菌的形态特征、理化特性、分类地位及药物敏感性等特性;采取鳃组织划痕浸泡和未划痕浸泡方式开展病原菌的人工感染试验。【结果】自然发病鱼鳃丝上有少量车轮虫寄生,未观察到真菌菌丝,GFHNV核酸检测呈阴性。从鳃组织分离到大量假根状菌落,该菌株为革兰染色阴性长杆菌,菌体大小(0.5~0.7)μm×(4~8)μm,氧化酶、过氧化氢酶阳性,降解明胶和硫酸软骨素,产生硫化氢,不发酵葡萄糖等糖醇类,基于16S rDNA序列构建的进化树与柱状黄杆菌聚为一枝,确认分离菌为柱状黄杆菌(Flavobacterium columnare),基因型Ⅰ型。划痕浸泡的各组金鱼均出现死亡,症状与自然发病鱼相似,划痕对照组和未划痕浸泡组均未出现死亡,经计算该菌对金鱼的半数致死量(LD 50)为8.8×10^(6) CFU,为弱毒株。该菌对恩诺沙星、盐酸多西环素和磺胺类药物敏感,而对试验的其他抗菌药物不敏感。【结论】本研究分离的柱状黄杆菌弱毒株可造成鳃组织破损金鱼的死亡,结合实践生产中许多金鱼感染少量车轮虫造成鳃组织破损但未出现发病死亡的病例,确定这起慢性、低死亡率的金鱼烂鳃病是由车轮虫和柱状黄杆菌弱毒株混合感染所致,而继发感染的柱状黄杆菌弱毒株是造成金鱼死亡的主要原因。

鱼胆草多糖的提取及其对柱状黄杆菌抑制活性研究

通过正交试验优化鱼胆草多糖提取工艺,考察鱼胆草多糖对柱状黄杆菌抑制活性。鱼胆草多糖最佳提取工艺为提取温度为80℃,提取时间60 min,料液比为1∶50,醇沉体积分数为75%时提取效果最佳。鱼胆草多糖对柱状黄杆菌表现出较好的抑制清除效果,MIC为12.5 mg/mL。该工艺简单稳定,鱼胆草多糖抑制柱状黄杆菌明显,为该资源的进一步开发提供了研究基础。

草鱼的一种急性细菌性传染病病原的分离鉴定及致病性研究

草鱼(Ctenopharyngodon idellus)的细菌性疾病是草鱼人工养殖过程中经常出现的疾病。文章对河南一草鱼养殖场2009年8月出现的暴发性细菌性传染病进行了研究。该渔场饲养的草鱼成鱼和鱼苗出现了体表溃烂、局部出血,肠系膜充血、出血,腹腔积水等症状,并大量死亡。从濒死草鱼的内脏中分离到3株细菌,经形态学、生理生化特性及16S rDNA序列分析,鉴定为霍乱弧菌(Vibrio cholerae)、嗜水气单胞菌(Aeromonas hydrophila)和柱状黄杆菌(Flavobacterium columnare)。其中,从患病鱼苗体内分离到霍乱弧菌;从成鱼分离到嗜水气单胞菌和柱状黄杆菌。回归试验证明3株细菌均能使草鱼致死,其中霍乱弧菌和嗜水气单胞菌对草鱼的半数致死量(LD50)分别是0.15×104 cfu/g和0.96×103 cfu/g。从草鱼中分离到致病性霍乱弧菌是首次报道。药敏试验表明,在所试的17种抗生素中,霍乱弧菌仅对利福平、左旋氧氟沙星、链霉素、奥复星、庆大霉素等5种抗生素敏感;嗜水气单胞菌仅对菌必治、奥复星、庆大霉素等3种抗生素敏感;而柱状黄杆菌则对其中的氨苄青霉素、利福平、左旋氧氟沙星等10种抗生素均敏感。

我国淡水鱼类柱形病病原菌柱状黄杆菌的遗传多样性

为认识我国淡水鱼类烂鳃病的病原以及柱形病在我国的发生情况,实验从发生烂鳃病的病鱼中分离细菌性病原,经过生理生化特性分析以及是否在含托普霉素的Shieh培养基中生长并形成黄色假根状菌落,是否产生降解明胶和硫酸软骨素的酶类等特性的鉴定,并结合16SrDNA序列分析,证实柱状黄杆菌(Flavobacterium columnare)是所分离的烂鳃病的病原。同时,研究也证实20世纪曾经命名为烂鳃(Gill-rot)病病原的鱼害黏球菌(Myxococcus piscicola Lu,Nie & Ko,1975)是柱状黄杆菌的同物异名。利用分离到的16株柱状黄杆菌的16SrDNA序列,以及已经发表的柱状黄杆菌的相关序列,构建了系统发育树,发现柱状黄杆菌的菌株聚成3枝,与柱状黄杆菌的三种基因组型(Genomovar)相对应。其中当时命名为鱼害黏球菌的强毒株G4与分别分离自日本和美国的两株聚为一枝。另外两枝包括的菌株较多,它们中的一些菌株来源于相同的鱼类宿主,如鲤形目的种类;但是,这两枝也包括一些特有的株,如从欧洲和美国的鲑形目鱼类上分离的柱状黄杆菌聚为一枝,这一枝还包括我国曾经命名为鱼害黏球菌的G18弱毒株。从我国隶属于鲈形目的鳜鱼和鲟形目的中华鲟上分离到的柱状黄杆菌则聚为另外一枝。作者认为对不同基因组型菌株的致病性和致病机理的研究将可能从根本上认识鱼类柱形病的流行规律。

剑尾鱼烂鳃、烂尾病病菌的分离鉴定

从有明显烂尾、烂鳃症状的剑尾鱼(Xiphophorus hellerii)体表病灶处、鳃及肝脏分离出优势菌株(1201F、1201H),分别采用肌肉注射和腹腔注射方式进行人工感染实验,证实菌株1201F和1201H均能够独立引起养殖剑尾鱼发病死亡,其对剑尾鱼的半数致死量分别为2.99×107cfu/尾、1.19×106cfu/尾。经形态学观察、生理生化特性分析以及16S rDNA基因测序确定菌株1201F为柱状黄杆菌(Flavobacterium cloumnare),菌株1201H为维氏气单胞菌(Aeromonas veronii)。其中菌株1201F与柱状黄杆菌(AB078047)的亲缘关系最近,置信度为90%;菌株1201H与维氏气单胞菌的亲缘关系最近,与维氏气单胞菌(JQ795750)聚为一个分支,置信度高达93%。对24种药物的敏感实验证实菌株1201F和菌株1201H同时敏感的抗生素有阿奇霉素、诺氟沙星、恩诺沙星、左氟沙星、环丙沙星。

福尔马林灭活柱状黄杆菌对草鱼免疫相关基因表达的影响

在本研究中,将福尔马林灭活的柱状黄杆菌菌苗(FKG4)经腹腔注射免疫草鱼,以注射灭菌PBS作对照,分别在免疫后1、7、15和28d,提取受免鱼和对照鱼肝脏、脾脏和头肾3种组织中的总RNA并反转录成cDNA,利用Real-time PCR方法对不同组织中C-反应蛋白(CRP)、主要组织相容性复合体I(MHCⅠ)、肿瘤坏死因子(TNFα)、白介素1(IL-1β)、白介素8(IL-8)、Ⅰ型干扰素(IFNⅠ)等6种免疫相关基因的表达进行定量分析。结果发现CRP在受免鱼肝脏中的表达于免疫后1、7d显著高于对照鱼,而在脾脏与头肾中的表达未出现显著差异;MHCⅠ在受免鱼3种组织中的表达于免疫后1、7、15d都显著高于对照鱼;TNFα在3种组织中的表达水平都较低,但都于免疫后1、7d显著高于对照鱼;IL-1β在3种组织中的表达于免疫后1d都显著高于对照鱼,但只有在头肾中的表达在免疫后7d时仍保持显著差异;IL-8在3种组织中的表达都只是于免疫后1d显著高于对照鱼;TNFα、IL-1β及IL-8等3种炎症因子的基因表达在免疫后15d都恢复到对照鱼的水平;而IFNⅠ在3种组织中的表达与对照组之间未出现显著差异。结果表明,FKG4注射免疫可以显著提高草鱼3种组织中与抗菌免疫相关的基因表达,从而增强鱼体抵抗细菌性病原的免疫力。

斑点叉尾鮰烂鳃病病原柱状黄杆菌的分离及鉴定

[目的]对从斑点叉尾鮰烂鳃病中分离的病原菌进行分类学地位鉴定。[方法]采用人工感染确定其病原性,常规生理生化鉴定和16SrRNA基因序列分析方法进行分析。[结果]该菌株为革兰氏阴性杆菌,滑行运动,无鞭毛,菌体大小为(0.3～0.5)μm×(5～10)μm,过氧化氢酶反应阳性,不能发酵和利用葡萄糖等多糖,不水解酪氨酸,不产生吲哚。所测16SrRNA基因序列经BLAST分析表明,与GeneBank上登录的柱状黄杆菌的16SrRNA序列同源性最高,其相似性达98.2%。基于16SrRNA基因序列构建的系统进化树表明,菌株GHS061212与柱状黄杆菌(AY841899)聚为1个分支,且置信度较高,为71.0%。[结论]菌株GHS061212被鉴定为柱状黄杆菌,这是国内首次报道斑点叉尾鮰烂鳃病病原为柱状黄杆菌。

柱状黄杆菌部分生物学特性的研究

采用革兰氏染色法、比色法和平板扩散法对柱状黄杆菌Flavobacterium columnare的部分生物学特性进行了研究。结果表明:用肉汤培养柱状黄杆菌24 h时,其形态多样,如半圆形、U型、V型或S型等;培养48 h时,柱状黄杆菌中开始出现颗粒状老龄培养物;培养72 h时,有部分菌体发生的"分节"现象,而老龄培养物也增多。用肉汤培养时,该菌的最佳生长条件分别是NaCl浓度为2.0 g/L,pH为7.4,温度为25～28℃。在最佳培养条件下,该菌的胞外产物具有蛋白酶、脂酶和卵磷脂酶活性,然而却检测不到淀粉酶、明胶酶和脲酶活性。

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。

大鲵柱状黄杆菌SYBR GreenⅠ荧光定量PCR诊断试剂盒的研制

根据Gen Bank中柱状黄杆菌16S r RNA序列,设计1对特异性引物,PCR扩增获得16S r RNA基因片段,并克隆到p MD-18T载体上作为阳性标准品。通过对SYBR Green I荧光定量PCR反应条件的优化,建立了黄杆菌的SYBR Green I荧光定量PCR诊断方法 ,以此为基础研制试剂盒。试剂盒扩增产物的熔解曲线分析只出现1个单特异峰,无引物二聚体,对嗜水气单胞菌、荧光假单胞菌、迟缓爱德华、弗氏柠檬酸杆菌均无阳性信号扩增,重复性好,灵敏度可达12拷贝/μL。结果表明,研制的柱状黄杆菌SYBR Green I实时荧光定量PCR试剂盒具有特异、灵敏、快速、重复性好等特征,适合于大鲵临床样品的检测。

斑鳠烂鳃病病原菌的研究

从池塘患烂鳃病的斑鳠(Mystus guttatus)亲鱼病灶中分离出一株毒力较强的致病菌-Mg2,经形态学观察、生理生化特性和16SrRNA基因序列分析鉴定,其主要特征为:菌体细长,无鞭毛和荚膜,革兰氏阴性,大小0.5×(6.5—11)μm,菌落黄色,边缘不整齐呈假树根状。氧化酶、过氧化氢酶阳性,分解酪素和明胶,硝酸盐还原阳性;不分解纤维素、几丁质、酪氨酸、七叶灵和淀粉,吲哚和葡萄糖产气阴性。对恩诺沙星、诺氟沙星、萘啶酸、红霉素、洁霉素敏感,菌株的16SrRNA基因序列分析结果表明:Mg2菌株与柱状黄杆菌聚类,基因序列的同源性在97.5%以上,综合生理生化、分子生物学两方面的分类鉴定结果,Mg2菌株应归类鉴定为柱状黄杆菌(FlavobacteriumColumnare)。