Potential use of a Yersinia ruckeri O1 auxotrophic aroA mutant as a live attenuated vaccine

The aroA gene of Yersinia ruckeri, which encodes 5-enolpyruvylshikimate 3-phosphate synthase, was insertionally inactivated with a DNA fragment containing a kanamycin resistance determinant and reintroduced by allelic exchange into the chromosome of Y. ruckeri 21102 O1 by means of the suicide vector pIVET8. The Y. ruckeri aroA::Kan(r) mutant was highly attenuated when inoculated intraperitoneally into rainbow trout, with a 50% lethal dose of >5 x 10(7) CFU. The mutants were not recoverable from the internal organs 48 h post-inoculation or later. The vaccination of rainbow trout with the AroA mutant as a live vaccine conferred significant protection (relative percentage survival = 90%) against the pathogenic wild-type strain of Y. ruckeri.     

鲁氏耶尔森菌invF基因无痕缺失突变株的构建及生物学特性

鲁氏耶尔森菌是一种具有广泛致病性的条件致病肠杆菌。三型分泌系统(T3SS)是该菌的重要毒力系统,其中invF基因是T3SS功能表达的重要调控因子。为探讨invF和T3SS对Y.ruckeri致病作用的影响,本研究构建了Y.ruckeri SC09株invF基因的无痕缺失株,并对其生物学特性进行研究。通过融合PCR方法,将invF基因的上、下游片段A、C融合,构建同源臂AC;将获得的同源臂AC连接入自杀质粒pLP12,构建pLP12-invF同源重组载体;pLP12-invF电转化进入供体菌株大肠杆菌β2163,并利用接合转移方法转入受体菌株Y.ruckeri SC09,利用抗生素正向筛选和vmt反向筛选分别对插入突变株和缺失突变株进行筛选,并利用PCR技术和序列测定对Y.ruckeri invF缺失株进行鉴定;对突变株和野生株进行菌体菌落形态观察、生化特性鉴定和生长曲线测定。结果显示,融合PCR、AC片段经氯霉素抗性正向筛选和vmt反向筛选,PCR鉴定和测序鉴定后,成功获得了Y.ruckeri SC09 invF基因的无痕缺失突变株,突变株和野生株菌体菌落形态和生化特性基本一致,突变株菌落较野生株小,各个时期突变株的生长浓度较野生株低。研究表明,采用自杀质粒pLP12和大肠杆菌β2163接合转移系统,利用抗生素正向筛选和vmt反向筛选技术,在对其基本生物学特性无显著影响的情况下,可简捷高效地获得invF基因的无痕缺失突变株。     

Development and validation of real-time PCR for the detection of Yersinia ruckeri

Yersiniosis (enteric red mouth disease) is a contagious bacterial disease caused by Yersinia ruckeri, which primarily affects salmonids. A real-time PCR assay using a molecular beacon has been developed and validated to improve the detection of the causative biotypes of Y. ruckeri. The assay, which targets the glnA (glutamine synthetase) gene, proved to have 100% analytical specificity and analytical sensitivities of 5 fg and 3 × 10(3) CFU g(-1) for DNA and seeded kidney tissue, respectively. The assay was highly repeatable with low % CV for intra- and inter-run experiments, and the optimized parameters transferred easily between different real-time PCR platforms. Following analytical validation, diagnostic specificity was determined using New Zealand farmed Chinook salmon (n = 750) from 10 farms during 2007/08. The real-time PCR was run in parallel with the bacterial culture detection method, and all fish tested were found to be negative by both methods for Y. ruckeri, resulting in 100% diagnostic specificity (95% confidence interval). The molecular beacon real-time PCR system is specific, sensitive, reproducible and a rapid method for the detection of Y. ruckeri and has the potential to be used for routine diagnostic testing, health certification and active surveillance programmes.     

鲁氏耶尔森菌qPCR快速检测方法的建立和应用

鲁氏耶尔森菌(Yersinia ruckeri)是导致虹鳟(Oncorhynchus mykiss)肠炎红嘴病的病原。本研究以鲁氏耶尔森菌毒力因子rup A基因为靶标设计1对特异性引物,以含有rup A基因部分序列的重组质粒为标准品构建标准曲线,优化建立检测鲁氏耶尔森菌的SYBR Green I实时荧光定量PCR检测方法,并对腹腔注射鲁氏耶尔森菌菌悬液的虹鳟肝、肾、脾、血液样品进行检测。结果显示,设计的引物具有良好的种间特异性,标准曲线线性方程为y=–3.3766x+40.012(R~2=0.9958);最低检测限为57 copy/μL,较常规PCR的灵敏度高出约100倍。应用检测结果表明,本方法可准确检测被鲁氏耶尔森菌感染的虹鳟样品。研究表明,所建立的qPCR方法具有特异、灵敏、快速、定量的优点,可用于快速诊断虹鳟肠炎红嘴病早期病症及定量检测鲁氏耶尔森菌。     

Cloning and expression of the gene encoding an extracellular alkaline serine protease from Vibrio alginolyticus strain HY9901, the causative agent of vibriosis in Lutjanus erythopterus (Bloch)

A 750-bp internal fragment of the alkaline serine protease gene (asp) from the Vibrio alginolyticus strain HY9901 was amplified by polymerase chain reaction (PCR). The flanking sequences of the 5'- and 3'- ends of the asp gene were characterized by reverse and nested PCR. Sequence analysis showed that the asp gene contained an 1893-bp ORF encoding 630 amino acids. The deduced amino acid sequence of the ASP (alkaline serine protease) precursor showed significant homology with several bacterial alkaline serine proteases. Expression of the asp gene in Escherichia coli and activity tests of the ASP indicated that the N-signal peptide of the ASP precursor was essential to autocatalyse and fold correctly the enzyme to obtain activity. The purified ASP was lethal for Lutjanus erythopterus with an LD(50) of 0.25 microg protein g(-1) body weight.     

基于lolB基因的SYBR GreenⅠ实时定量PCR检测病原霍乱弧菌方法的建立

基于霍乱弧菌lolB基因保守序列设计1对特异性引物,建立了SYBR GreenⅠ实时定量PCR检测霍乱弧菌的方法。常规PCR检测仅霍乱弧菌扩增出大小为519bp的目的片段,其他3种病原弧菌均为阴性;SYBR GreenⅠ实时定量PCR,在Tm为86.5～87℃,扩增产物的熔解曲线只出现1个单特异峰,无引物二聚体,表明该引物具有较好的特异性。SYBR GreenⅠ实时定量PCR扩增曲线反映了PCR的指数增长阶段和平台阶段;所制作的标准曲线在2.59×108～2.59×100拷贝数之间有较好的线性关系,相关系数为0.993,能对霍乱弧菌进行准确的定量分析。该方法检测时间从核酸抽提到结果分析仅需4～5h,较传统方法敏感、操作简单、耗时短,是霍乱弧菌引起的水产动物疾病的快速诊断及流行病调查的有效方法。     

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.     

The use of a real-time PCR primer/probe set to observe infectivity of Yersinia ruckeri in Chinook salmon, Oncorhynchus tshawytscha (Walbaum), and steelhead trout, Oncorhynchus mykiss (Walbaum)

Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM), a common pathogen affecting aquaculture facilities and implicated in large losses of cultured fish. Fisheries scientists continue to gain a greater understanding of the disease and the pathogen by investigating methods of identification and pre- and post-infection treatment. In this study, a real-time PCR probe set for Y. ruckeri was developed to detect daily changes in the bacterial load during pathogen challenges. Two species of fish, Chinook salmon, Oncorhynchus tshawytscha, and steelhead trout, Oncorhynchus mykiss, were exposed to two strains of Y. ruckeri (Hag and SC) during bath challenges. A subset of fish was killed daily for 14 days, and the kidney tissue was biopsied to enumerate copies of pathogen DNA per gram of tissue. While Chinook exposed to either the Hag or SC strains exhibited similar pathogen loads, those exposed to the Hag strain displayed higher mortality (～66%) than fish exposed to the SC strain (～24% mortality). Steelhead exposed to the Hag strain exhibited a greater pathogen load and higher mortality (～42%) than those exposed to the SC strain (<1% mortality). Steelhead challenged with either strain showed lower pathogen loads than Chinook. The study illustrates the efficacy of the probe set to enumerate Y. ruckeri bacterial growth in the kidneys of fish. Also, strains of Y. ruckeri display species-specific growth patterns that result in differential mortality and pathogen load.     

一种简便、快速检测鸡毒支原体的PCR方法

鸡毒支原体(MG)是造成鸡慢性呼吸道疾病的主要病原菌。通常采用血清学方法诊断该病,由于存在非特异性交叉反应,这给临床诊断带来困难。为解决MG快速诊断问题,该研究根据已发表的鸡毒支原体种特异性序列FMG-2合成一对引物(MG1,MG2),用PCR方法检测MG。结果表明,该PCR方法对MG能特异性扩增732bp目的片断,而对照菌(大肠杆菌、金黄色葡萄球菌等)却不能扩增出目的片段。PCR产物经测序,其序列与Genbank序列同源性为98.86%。用PCR方法检测60份感染样本,其阳性检出率为80%,而用传统的分离培养方法检出率仅为50%。     

基于toxR基因病原哈氏弧菌PCR检测方法的建立

基于toxR基因序列设计检测哈氏弧菌的1对特异性引物,通过PCR反应条件优化、PCR反应特异性及敏感性检测,建立一种哈氏弧菌的特异性分子检测方法.试验结果表明,该引物可使哈氏弧菌扩增出大小为390 bp的toxR基因片段,3种水产动物病原弧菌未扩增出任何条带,敏感性检测结果为该PCR反应最低能检测出0.375 ng/μ...     

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.     

嗜水气单胞菌外膜蛋白基因ompTS的克隆与序列分析

根据已发表的外膜蛋白基因ompⅡ的核苷酸序列设计引物，从分离 自患红底板病的中华鳖的嗜水气单胞菌中扩增得到了ompTS基因，对ompTS 基因进行序列分析，发现其与ompⅡ基因的核苷酸序列有83.5%的同源性。ompTS基因最长的开放阅读框（ORF）为1068nt，编码由355个氨基酸组成，分子量为38.9kDa的蛋白质OmpTS，其氨基酸序列的前20个氨基酸残基可能组成信号肽。由ompTS基因的编码氨基酸序列与其它细菌外膜蛋白的氨基酸序列的比较结果，进一步证实细菌外膜蛋白氨基酸序列的N端存在高度保守区。根据序列分析结果推测，ompTS基因很可能是一个新的基因，编码38.9kDa的嗜水气单胞菌外膜蛋白OmpTS，该蛋白质在膜中极有可能形成孔道，具备与孔蛋白相似的性质。     

林蛙红腿病病原菌基因的克隆及序列分析

在长白山地区某林蛙养殖场送检的红腿病病例中分离病原体,鉴定为嗜水气单胞菌,根据已发表的嗜水气单胞菌(溶血素基因AF443388)序列设计了一对特异性引物,通过聚合酶链式反应(PCR)扩增出一条约952 bp的基因片段,将该基因克隆到pMD-18T载体上,通过核苷酸序列测定,结果表明此序列与已发表的其他AH溶血素基因序列具有很高的同源性.为进一步对该病的分子流行病学调查,以及准确诊断和治疗提供依据.     

Development of a Penaeus monodon-type baculovirus (MBV) DNA probe by polymerase chain reaction and sequence analysis

Abstract. Penaeus monodon -type baculovirus (MBV) was isolated and purified from the hepatopancreases of MBV-infected Penaeus monodon Fabricius. MBV DNA was extracted and used as a template in a polymerase chain reaction (PCR). The primers were chosen from conserved regions of the polyhedrin gene of Autographa californica nuclear polyhedrosis virus (AcNPV). One DNA fragment (674 base pairs) was amplified after PCR. There was a 65% homology between the predicted amino acid sequence of this PCR product with that of the polyhedrin polypeptide of AcNPV. Nucleotide sequence analysis indicated that the amplified DNA is the open reading frame of the MBV polyhedrin gene. This 674 bp DNA fragment was subsequently used as a probe in a dot blot analysis. The probe was able to hybridize with the DNA extracted from the purified MBV and from the MBV-infected P. monodon , but not from the MBV uninfected P. monodon.     

Immunosuppression in rainbow trout, Salmo gairdneri Richardson, caused by the haemoflagellate Cryptobia salmositica Katz, 1951

Abstract. An experimental Cryptobia salmositica infection in rainbow trout, Salmo gairdneri Richardson, produced suppression of the humoral response against sheep red blood cells as measured by direct haemagglutination. Two-month and 5-month infections produced equal suppression. The parasite also produced suppression of the humoral response against a bacterial pathogen, Yersinia ruckeri . Anti- Y. ritckeri titres were significantly lower in most fish infected with C. salmositica than in non-infected fish. Immunosuppression became evident when C. salmositica first appeared in the blood (first 2 weeks of infection), Immunosuppression was confirmed by challenge with Y. ruckeri . Mortality at challenge occurred in 64·3% to 83·3% of the fish already infected with C. salmositica at the time of initial Y. ruckeri exposure. There was no mortality at challenge if fish were not infected with C. salmositica at initial bacterial exposure, nor in those concurrently infected with both pathogens. Antigenic competition may have caused the immunosuppression.     

温和气单胞菌PCR快速诊断方法的建立

根据NCBI公布的温和气单胞菌(Aeromonas sobria)丝氨酸蛋白酶的基因序列,设计并选取一对能够快速而准确地检测温和气单胞菌的PCR引物,建立PCR快速检测体系,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131 bp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1.0 pg/20μL,对温和气单胞菌的检测灵敏度为50 cfu/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温和气单胞菌常规PCR检测体系,该体系可以用于温和气单胞菌的诊断和样品的检测。     

Development of a selective-differential medium for the isolation of Yersinia ruckeri and its application in epidemiological studies

Abstract. The development of a selective-differential medium (ROD) for the isolation of Yersinia ruckeri from carrier and clinically infected rainbow trout, Oncorhynchus mykiss (Walbaum), is described. This medium was a useful aid for the detection of Y. ruckeri in faecal material and allowed the epidemiology of enteric redmouth disease to be studied in greater detail. Regular use of ROD at two fish farms in southern England has demonstrated that isolation of Y. ruckeri from faeces was possible under field conditions up to 4–6 weeks before acute clinical kidney infection occurred.     

基于vhhP2基因的SYBR Green I实时定量PCR检测哈维氏弧菌方法的建立

根据哈维氏弧菌(Vibro harveyi)溶血素基因vhhP2的保守序列设计特异性引物, 建立了SYBR Green I实时定量PCR检测哈维氏弧菌的方法。构建含vhhP2基因的重组质粒作为标准品, 进行SYBR Green I实时定量PCR, 在Tm为60℃时, 扩增产物的熔解曲线仅有一个特异峰, 扩增所得标准曲线为y= –3.331x+37.48, 相关系数为0.998, 扩增效率为1, 最低可检测到7个拷贝。实验结果表明该检测技术具有较高的特异性、敏感性和重复性, 对哈维氏弧菌病的快速诊断和流行病学调查有重要意义。

     

一个奥利亚罗非鱼雌性特异性SCAR标记的建立

以奥利亚罗非鱼Oreochromis aureus为实验材料,通过对800多条随机引物的筛选,获得了1个奥利亚罗非鱼雌性特异性的、长度为1 488 bp的RAPD标记片段RAPD71699-1488,经琼脂糖凝胶电泳后回收、克隆、测序,并根据测序结果设计PCR特异引物,再经PCR条件优化,成功地将该RAPD标记片段转化为实验结果稳定、操作简便的SCAR标记,即SCAROaF1488。采用双重PCR技术,以mtDNA 16S rRNA基因片段为PCR扩增阳性对照,对该标记的有效性在两个群体共200个个体(雌、雄各100个)中进行验证。结果显示,标记检测结果与表型性别的符合度为100%。SCA-ROaF1 488标记的获得为奥利亚罗非鱼遗传性别鉴定及标记辅助选择提供了有效工具,为深入研究鱼类性别相关基因及性别决定机制提供了重要线索和新的思路。