虹鳟肠炎红嘴病病理模型的构建

为探讨鲁氏耶尔森菌侵染虹鳟的致病机制,本实验建立了鲁氏耶尔森菌感染虹鳟引起的肠炎红嘴病的病理模型,制定相应的临床症状及组织病理学评分系统,并对该模型进行研究。将43尾平均体质量约为12 g的健康虹鳟随机分成5组:3个实验组(n=30)、对照组(n=10)和哨兵组(n=3)。3个实验组分别采用2.0×106、2.0×107和2.0×108 CFU/m L的鲁氏耶尔森菌感染浓度,通过腹腔注射方式进行人工感染试验。对感染鲁氏耶尔森菌的虹鳟肠、肝脏、脾脏和肾脏组织进行镜检及临床症状、剖检病变判断,结合细菌学检测,按制定的评分系统评价各组肠炎红嘴病造模效果,确定最佳造模方案。结果显示,各攻毒组虹鳟感染后72 h均出现不同程度死亡,临床症状表现为红嘴、肛门红肿、鳍(胸鳍、腹鳍、臀鳍)等出现不同程度充血,下颌部、腹部出现出血点等。组织病理学可见肝脏、脾脏、肾脏及肠组织均有炎性细胞浸润现象出现,肝细胞、肠上皮细胞和肾小管上皮细胞等实质细胞变性、坏死,脾脏部位淋巴细胞减少、红细胞死亡堆积。综合各组得分发现,2.0×107 CFU/m L组的鲁氏耶尔森菌感染虹鳟造模效果最佳,患病虹鳟的临床症状显著且组内差异较小,病程迁延较长,便于研究。研究表明,对体质量约12 g的虹鳟幼鱼腹腔注射0.1 m L浓度为2.0×107 CFU/m L的鲁氏耶尔森菌可成功构建肠炎红嘴病病理模型。     

实时荧光定量PCR扩增特异性vapA基因检测杀鲑气单胞菌

为实现杀鲑气单胞菌早期快速准确定量检测,研究旨在建立杀鲑气单胞菌的SYBR Green Ⅰ实时荧光定量PCR(Real-time PCR)检测方法。根据GenBank中杀鲑气单胞菌毒力阵列蛋白基因(vapA)保守序列设计并合成一对特异性引物,对其特异性、灵敏度、可重复性和应用性进行评价。结果显示,研究设计的引物具有良好的种间特异性,仅对杀鲑气单胞菌及其亚种有阳性扩增,与其他细菌不发生交叉反应。构建的Real-time PCR标准曲线质粒拷贝数与循环阈值呈良好的线性关系,扩增所得标准曲线分别为y=–4.8345x+42.535,相关系数R~2为0.998,最低检测限为34拷贝/μL,较常规PCR的灵敏度高出约1000倍。应用建立的方法检测人工感染的虹鳟病样,15个被检样品呈阳性反应,与细菌常规鉴定方法结果一致。研究表明,所建立的基于实时荧光定量PCR技术的杀鲑气单胞菌检测方法快速、特异、灵敏,可用于临床诊断和疫病监测。     

迟缓爱德华氏菌SYBR Green I实时荧光定量PCR检测方法的建立及其应用

根据克隆得到的迟缓爱德华氏菌gyrB基因序列设计并合成一对特异性引物,通用性和特异性检测结果显示所设计的引物具有良好的种间特异性和种内通用性,构建含gyrB基因的重组质粒作为标准品,经过反应体系优化后建立了检测迟缓爱德华氏菌的SYBR Green I实时荧光定量PCR检测方法。结果显示,该方法线性关系良好,在Tm为63℃时,扩增产物的熔解曲线仅有一个单特异峰,扩增所得标准曲线为y=-3.32x 39.38,相关系数为0.998,扩增效率为1.00,最低能检测到60个拷贝。应用建立的方法对人工感染的大菱鲆病样进行了检测,三个被检样品均呈阳性反应,证明该方法具有较好的适用性。实验结果表明所建立的实时荧光定量PCR方法具有特异、敏感、快速、定量的优点,可用于迟缓爱德华氏菌病的快速检测。     

耶尔森氏菌对水产养殖的危害及防治

耶尔森氏菌是肠杆菌科、耶尔森氏菌属的总称，属兼性厌氧革兰氏阴性杆菌群。该属包含鼠疫耶尔森氏菌、假结核耶尔森氏菌、小肠结肠炎耶尔森氏菌等共11个种。其中鲁氏耶尔森氏菌是引起冷水性鲑鳟负类红嘴病的主要病原菌，是淡水养殖鱼类暴发性败血症的常见致病菌（气单胞菌、河弧菌、鲁氏耶尔森氏菌）之一。     

应用多聚酶链反应检测生鸡肉中沙门氏菌的研究

目的:建立快速、特异、灵敏的PCR方法以检测生鸡肉中的肠炎沙门氏菌。方法:以肠炎沙门氏菌的sefA基因作为靶序列设计一对引物,将样品增菌后用煮沸法提取细菌基因组DNA进行检测。结果:每克生鸡肉污染3.0×10°CFU肠炎沙门氏菌,经16h增菌培养后可扩增出500bp特异性性DNA带,对28份屠宰场样品的检测结果与传统方法相符合。结论:PCR法适于生鸡肉中肠炎沙门氏菌的快速、准确检测。     

5种食源性致病菌多重PCR检测方法的建立

本研究建立一种同时特异性地检测5种常见食源性致病菌的多重PCR方法。根据目前食品中常见病原菌副溶血弧菌Vibrio parahaemolyticus、金黄色葡萄球菌Staphylococcus aureus、单核细胞增生性李斯特菌Listeria monocytogenes、肠炎沙门氏菌Salmonella enteritidis、福氏志贺氏菌Shigella flexneri的相关毒力基因,选择具有特异性的副溶血弧菌不耐热溶血毒素基因、金黄色葡萄球菌的耐热核酸酶基因、单核细胞增生性李斯特菌编码溶血素O基因、沙门氏菌侵袭蛋白A基因及志贺氏菌侵袭性质粒抗原H基因,设计5对特异性引物进行多重PCR检测,对反应条件进行优化并评价了其特异性和灵敏度。通过对48份实际样品检测,验证了此多重PCR体系具有很好的可靠性和实用性。     

中草药添加剂对鲁氏耶尔森菌感染虹鳟血液生化指标的影响

为研究复方中草药对鲁氏耶尔森菌(Yersinia ruckeri)感染虹鳟(Oncorhynchus mykiss)后其血液生化指标的影响,在虹鳟饲料中分别添加两种复方中草药(复方1组、复方2组),持续投喂28 d。在第28天感染鲁氏耶尔森菌,检测感染后72 h的血清生化指标,并统计各组存活率。结果显示:复方中草药组对虹鳟增重无抑制作用。添加复方中草药可提高虹鳟感染鲁氏耶尔森菌后的存活率,其中复方2组的保护效果最好。感染72 h后,复方2组总蛋白、白蛋白、球蛋白、甘油三酯、总胆固醇含量和碱性磷酸酶活性均高于对照组。复方2组谷丙转氨酶、谷草转氨酶活性低于对照组,乳酸脱氢酶活性显著低于对照组。复方1组和复方2组尿酸含量显著低于对照组。复方中草药组总胆红素、尿素氮、肌酐和血糖含量与对照组相比无显著性差异。结果表明,添加复方中草药能提高鲁氏耶尔森菌感染后虹鳟的存活率,其中复方2组的保护效果最好。     

基于gyrB基因的SYBR Green I实时定量PCR检测病原灿烂弧菌

灿烂弧菌Vibrio splendidus是多数海水养殖动物的主要致病菌,对养殖业危害较大。本研究根据灿烂弧菌gyrB基因的保守序列设计特异性引物,建立了SYBR Green I实时定量PCR检测灿烂弧菌的方法。构建含gyrB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62℃时,扩增产物的熔解曲线仅有一个单特异峰,扩增所得标准曲线为y=-3.338x+37.67,相关系数为0.999,扩增效率为0.99,最低能检测到20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,对灿烂弧菌病的快速诊断和流行病学调查有重要意义。     

养殖沉积环境中气单胞菌属细菌的实时荧光定量PCR检测方法的建立

利用GyrB基因的特异性引物建立沉积环境中气单胞菌属细菌的实时荧光定量PCR检测方法。将已知浓度的气单胞菌菌液加入灭菌的沉积物样品中,作为模拟沉积物样品。通过选择和优化沉积物DNA提取方法、特异性引物、标准曲线模板,建立沉积环境中气单胞菌属细菌准确检验的实时荧光定量PCR方法,同时验证该方法的特异性、灵敏性、重复性。结果表明,采用改进的溶菌酶-SDS温和裂解法提取沉积物DNA,以扩增GyrB基因片段的IAF和IAR为特异性引物,并直接以模拟沉积物样品DNA为标准品构建标准曲线,可以建立适用于定量检测沉积环境中气单胞菌属细菌的实时荧光定量PCR方法。该方法可灵敏、特异、准确地定量检测刺参养殖池塘底泥中气单胞菌属中不同种的细菌,检出效率可达103CFU/g。统计分析显示,变异系数为0.21%-0.80%,均小于5%,表明重复性良好。     

(鱼师)鱼诺卡氏菌SYBR Green Ⅰ实时荧光定量PCR检测方法的建立与应用

根据GenBank中(鱼师)鱼诺卡氏菌16S-23S rRNA基因序列设计并合成一对特异性引物,经反应体系优化后建立了检测(鱼师)鱼诺卡氏菌的SYBR Green Ⅰ实时荧光定量PCR方法.结果显示,该方法线性关系良好,标准曲线的相关系数为0.998;溶解曲线分析显示产物为单一的特异峰；检测灵敏度可达10-6 μg/μL的DNA含量,与嗜水气单胞菌、大肠杆菌、金黄色葡萄球菌、副溶血弧菌、麦氏弧菌不发生交叉反应,具有良好的特异性.应用建立的方法在(鱼师)鱼诺卡氏菌病爆发时期,对16份鱼体组织、养殖水体、饲料等样品进行了检测,结果7份为阳性,与细菌分离、培养检查结果100％相符.既能检测发病鱼,又能检出未发病且已感染的病鱼,对病害的早期防控体现应用价值.结果表明,建立的实时荧光定量PCR方法具有特异、敏感、快速、定量等优点,可用于鱼类致病(鱼师)鱼诺卡氏菌的快速检测.     

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.     

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.     

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.     

A sensitive loop-mediated isothermal amplification (LAMP) method for detection of Renibacterium salmoninarum, causative agent of bacterial kidney disease in salmonids

Loop-mediated isothermal amplification (LAMP) is a novel technique for nucleic acid amplification with high specificity, sensitivity and rapidity and does not require expensive equipment or reagents. In the present study, we developed and evaluated a LAMP method for the rapid detection of Renibacterium salmoninarum causing the bacterial kidney disease in salmonids. This method was more sensitive than quantitative real-time polymerase chain reaction (qPCR). Using DNA template extracted from cultured R. salmoninarum , the LAMP method gave an amplification signal from template diluted to 10⁻⁸ while the limit of detection of qPCR was10⁻⁷. The LAMP method was also highly specific and did not amplify DNA purified from five other Gram-positive and -negative bacterial fish pathogens. The method also worked well using extracts of macrophages infected with R. salmoninarum and kidney material from rainbow trout, which were positive for R. salmoninarum by qPCR and crude R. salmoninarum culture. There was some evidence for inhibitors of the LAMP reaction in the kidney samples, which was overcome by diluting the sample.     

Bacterial pathogens in rainbow trout, Oncorhynchus mykiss (Walbaum), reared at Danish freshwater farms

During a 2-year period, bacterial fish pathogens were monitored on five rainbow trout, Oncorhynchus mykiss (Walbaum), freshwater farms in Denmark. A total of 1206 fish were examined and 361 bacterial isolates were identified phenotypically. Enteric redmouth disease, furunculosis and rainbow trout fry syndrome/coldwater disease were recorded. Infections caused by Flavobacterium psychrophilum occurred most frequently, but only one outbreak of enteric redmouth disease caused by Yersinia ruckeri serotype O1 and one of furunculosis caused by Aeromonas salmonicida were recorded during the monitoring period. Flavobacterium psychrophilum was isolated on all farms, both during disease outbreaks and from fish without any signs of disease. Serological investigations of F. psychrophilum showed that serotype Th was the dominant serotype found. The serotypes Th and Fd were involved in disease outbreaks of fry and larger fish. All isolates of F. psychrophilum showed proteolytic activities; however, a few isolates, belonging to serotype FpT did not degrade elastin and were not associated with mortality. Increasing resistance problems to oxytetracycline were demonstrated. More than half of the F. psychrophilum isolates showed resistance to oxolinic acid and oxytetracycline. No antibiotic resistant isolates were found among Y. ruckeri and A. salmonicida .     

Quantification of Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum), tissues by qPCR

A qPCR assay was developed for rapid and sensitive detection of Flavobacterium psychrophilum, the aetiological agent of bacterial cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide. A set of F. psychrophilum-specific primers based on 16S rRNA gene sequences was designed and validated for specific detection and quantification of DNA isolated from representative strains of F. psychrophilum. The qPCR assay exhibited a high specificity for the 16S rRNA gene of F. psychrophilum (from 4 × 10(8) down to 11 copies per reaction) but not for other Flavobacterium species or other bacteria including fish pathogens. This qPCR-based method proved to be useful in the quantification of the F. psychrophilum titre present within organs dissected out from diseased fish. As the F. psychrophilum genome contains six copies of the 16S rRNA gene, we could infer a limit of detection corresponding to two bacteria per reaction, corresponding to 800 bacteria per fish tissue sample, and therefore 20 F. psychrophilum cells mg(-1) of tissue (for sample weighing 40 mg). The qPCR assay reported here could be a useful tool for veterinary diagnostic laboratories to monitor the F. psychrophilum infection level in fish farms.     

Occurrence and pathogenicity of Yersinia ruckeri at fish farms in northern and central Finland

Abstract. Salmonid fish at fish farms in northern and central Finland and perch, Perca fluviatilis L., roach, Rutilus rutilus (L.), and whitefish, Coregonus sp., from four lakes in central Finland were studied between 1985 and 1990 for the occurrence of Yersinia ruckeri. The bacteria were found in fish from both areas, but in most cases, only single diseased salmon, Salmo salar L., brown trout, S. trutta L., rainbow trout, Oncorhynchus mykiss (Walbaum), whitefish and perch were encountered and were always connected with stress conditions. One clinical outbreak occured in salmon fingerlings in northern Finland, and the fish were successfully treated with trimethoprim-sulpha. Monthly monitoring of lake fish revealed two symptomless carrier perch in two lakes. Outwith the main study a moribund perch with yersiniosis was found in a polluted lake, and for the first time in Finland, a rainbow trout was also found to have contracted yersiniosis in a small private pond. Sorbitol-positive and negative isolates have been found to occur in both moribund and carrier farmed fish, indicating that the sorbitol test is not essential when evaluating the pathogenicity of Y. ruckeri.