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。     

大口黑鲈鲁氏耶尔森氏菌的分离鉴定、主要毒力基因及致病性研究

为探明2021年3月福建某水库养殖大口黑鲈疾病暴发的原因,实验从患病大口黑鲈肝脏、肾脏、脾脏中分离优势菌,通过人工感染实验确定病原菌,并综合16S rDNA基因序列分析、生理生化和质谱特征等技术对该病原菌进行种属鉴定,同时,进行毒力基因检测、药物敏感性实验以及组织病理学观察。结果显示,从病鱼脾脏中分离获得1株优势菌并鉴定为鲁氏耶尔森氏菌;该菌株对大口黑鲈的半致死剂量为3.8×10⁵ CFU/尾;该菌株携带yrP1、yhl A、yhl B等毒力基因,对恩诺沙星、盐酸多西环素、氟甲喹、硫酸新霉素、氟苯尼考等5种药物相对敏感。组织病理学观察发现,鲁氏耶尔森氏菌的感染造成大口黑鲈肝脏、肾脏、脾脏不同程度的损伤,表现为明显的变性、坏死及炎症细胞的浸润等。本研究首次报道了鲁氏耶尔森氏菌对养殖大口黑鲈的致病性,可为养殖大口黑鲈鲁氏耶尔森氏菌病的诊断和药物防治提供参考依据。     

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。     

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。     

斑点叉尾鮰冬季大规模死亡病原分离与鉴定

2016年1—2月湖南安化地区网箱养殖的斑点叉尾鮰暴发大规模死亡,死亡率接近80%,发病鱼体重为1.5~2.0 kg。本研究从患病濒死鱼肝、脾、肾分离到1株致病菌,对其进行常规理化特性、分子生物学、人工感染实验以及药敏性检测。结果显示,分离菌株理化特性与鲁氏耶尔森氏菌(Yersinia ruckeri)特征相符,与NCBI中登录的鲁氏耶尔森氏菌的16S rDNA基因同源性达99%以上,在系统发育树上与鲁氏耶尔森氏菌聚为一族。人工感染鱼出现与自然发病相似症状,并能够从发病鱼组织中再次分离到相同病原菌,表明鲁氏耶尔森氏菌为斑点叉尾鮰该病病原菌。该分离菌株对氟苯尼考、多西环素及新霉素等药物高度敏感,本研究为斑点叉尾鮰暴发性出血病防控提供了理论依据。     

四川彭州地区养殖虹鳟传染性造血器官坏死病病毒的分离、鉴定及病理学观察

近期,四川省成都彭州市某虹鳟养殖场暴发流行疾病,导致养殖虹鳟死亡率高达90%。现场采样观察发现患病鱼主要症状为背部发黑,鳔壁、腹膜严重出血,心包积液,空肠、空胃和显著肠炎。同时对病鱼进行细菌学与组织病理学检测,细菌学检查结果为阴性,病理学观察发现脾脏有典型凝固性坏死,肝脏组织广泛变性、坏死,肠道黏膜下层水肿,肠上皮充血及上皮细胞脱落坏死,脑膜和心外膜水肿。将病鱼的脾组织研磨过滤除菌后,腹腔注射60尾健康虹鳟,注射组均表现为急性死亡(累计死亡率达85%),试验鱼出现与自然发病鱼相同的症状而对照组无异常。取病鱼的脾脏组织研磨过滤后接种胖头鲤细胞(fathead minnow cell,FHM),细胞感染3 d后出现典型的细胞病变效应(CPE)。针对编码IHNV糖蛋白(Glycoprotein,G)基因进行逆转录-聚合酶链反应(RTPCR)检测显示,患病鱼、人工感染病鱼和病变细胞均为IHNV阳性,扩增序列与IHNV糖蛋白基因同源性为98.2%。对该病毒分离株的G基因进行系统发育分析,结果显示,该分离株与亚洲分离株聚为一簇,属于JRt基因型。     

光合细菌和枯草芽孢杆菌对养殖水质的净化作用

以光合细菌(Photosynthetic bacteria,P)和枯草芽孢杆菌(Bacillus subtilis,B)为实验菌种,研究两者最佳浓度配比的复合菌组对淡水养殖水质的净化作用。试验设置1个对照组(CK)和5个复合菌组(PB1、PB2、PB3、PB4、PB5),5个复合菌组浓度配比分别为(2.0×105+1.5×105)CFU/m L、(2.0×105+3.0×105)CFU/m L、(2.0×105+4.5×105)CFU/m L、(4.0×105+1.5×105)CFU/m L、(6.0×105+1.5×105)CFU/m L,分析各试验组的化学需氧量(COD)、氨氮(NH3-N)、溶解氧(DO)、p H等水质指标。结果显示:复合菌能够明显去除水体CODMn,PB2组去除率最高达,44.98%;能有效增加DO,PB2组增氧率最高,为27.9%;能明显去除氨氮,PB2组去除率最高达78%;并且能稳定p H值在8.6左右,5个复合菌组差异不显著(P0.01)。复合菌发挥最佳净化能力的时间约在6~8 d。结果表明,复合菌最佳浓度配比为(2.0×105+3.0×105)CFU/m L,该浓度组较对照组和其他试验组能够显著净化淡水养殖水质,有效改善养殖环境。     

大黄鱼致病嗜水气单胞菌的分离鉴定及其多克隆抗体制备

为探究引起网箱养殖大黄鱼肠炎病的病原及其特性,自患病大黄鱼肝脏组织分离到1株优势菌NDLc-P,经回归感染试验确认该菌为大黄鱼致病菌.人工感染7 d后,该菌对体质量(46.4±3.5)g的健康大黄鱼的半数致死密度为3.98×107 cf u/m L,表明该菌株对大黄鱼有一定的致病性.利用梅里埃微量多项鉴定系统对病原菌N...     

肌肉注射感染嗜冷黄杆菌虹鳟的组织嗜性与动态分布

嗜冷黄杆菌(Flavobacterium psychrophilum)是鲑鳟类细菌性冷水病(bacterial cold water disease, BCWD)的病原菌, 该病的发生和流行严重制约了鲑鳟产业的健康发展。本研究分析嗜冷黄杆菌肌肉注射感染虹鳟(Oncorhynchus mykiss)后病原菌的动态组织分布情况, 以期为 BCWD 的防控提供理论支撑。以 1.0×108 CFU/mL 浓度的嗜冷黄杆菌 CH06 株菌液肌肉注射感染实验虹鳟, 感染后 12 h、24 h、96 h 观察虹鳟临床症状及组织病变, 并利用 qPCR Taqman 探针法检测各组织病原载量。患病鱼的临床病征表现为体色发黑、游动缓慢或不动、食欲不振, 尾柄部注射处肌肉溃烂, 鳃苍白, 脾脏肿大, 伴有腹水。组织病理观察显示, 嗜冷黄杆菌感染后实验鱼注射处肌纤维断裂、溶解; 脾窦扩张, 其内充满红细胞; 肾脏组织含铁血黄素增加, 出现大量空泡变性, 肾小管上皮细胞变性、坏死, 肾间质炎性细胞浸润。qPCR 检测结果表明, 肌肉注射感染 12 h 后即可在脾脏、肝脏、肾脏、肠道、鳃、注射处肌肉、脑和尾鳍中检出嗜冷黄杆菌, 注射处肌肉病原载量最高为(5.85±2.11)×10⁵ copy/μL。感染后 24 h, 脾脏、脑中病原载量较 12 h时上升最多, 其他组织病原载量与感染 12 h 时水平一致, 注射处肌肉病原载量为(6.48±2.07)×10⁵ copy/μL, 显著高于其他组织(P＜0.05)。感染 96 h 后脾脏中的病原载量显著高于其他组织(P＜0.05), 达到(1.15±0.58)× 10⁷ copy/μL; 肝脏、肾脏、脾脏中的病原载量较 24 h 时上升最多。所有被检组织中病原菌载量随时间延长均呈上升趋势。另外, 3 个时间点注射处肌肉的病原平均载量最高, 其次为脾脏, 然后是肾脏和鳃。嗜冷黄杆菌人工感染虹鳟后, 注射处肌肉、脾脏是细菌的重要增殖场所。综上, 嗜冷黄杆菌可随着血液循环进入虹鳟各组织, 并表现出对注射处肌肉、脾脏、肾脏和鳃较强的组织嗜性, 而对肝脏、尾鳍、肠道和脑组织的嗜性相对较弱; 感染时间和病变程度与组织中的病原载量呈正相关。     

无乳链球菌感染尼罗罗非鱼的脑膜炎模型

目的：脑膜炎是罗非鱼无乳链球菌病的特征性临床症状,建立罗非鱼无乳链球菌脑膜炎模型,并制定相应的临床症状及组织病理学评分系统,对研究罗非鱼无乳链球菌病的致病机制具有十分重要的意义。方法：将健康罗非鱼43尾,随机分为4组：对照组(n=10)和试验组(n=33)。3组试验组根据不同感染剂量分为：10₆cfu组(n=11)、10₇cfu组(n=11)、10₈cfu组(n=11)。试验组分别将10₆、10₇、10₈CFU的无乳链球菌菌液腹腔注射罗非鱼复制脑膜炎模型。对感染罗非鱼的临床症状、剖检病变、脑组织(端脑、中脑、小脑、延脑)病理学进行判定,并结合细菌学检测,按照拟定的评分系统评价各组脑膜炎的造模效果,确定最佳造模方案。结果：各试验攻毒组罗非鱼感染后2天均出现不同程度死亡,未见明显神经症状。感染3天后鱼体开始出现异常活动的神经症状及突眼、角膜混浊等病理变化。病理组织学观察可见：蛛网膜下腔和脑软膜上有大量炎性渗出和染成蓝紫色微细颗粒的细菌团块,脑膜充血、水肿、疏松、增厚,有大量的中性白细胞等炎性细胞浸润;脑实质基质疏松水肿,呈海绵状;脑组织炎性细胞浸润,胶质细胞增生等软脑膜炎、脑膜脑炎症状。比较各组得分发现,10₇CFU组的无乳链球菌攻毒罗非鱼造模效果最好：临床症状显著而组内罗非鱼差异性最小,组织病理学观察患病罗非鱼病情和缓适中、利于观察研究。结论：使用10₇CFU无乳链球菌腹腔攻毒罗非鱼可在第3天成功构建脑膜炎模型,造模率为100%。     

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.     

Cellular immune response in rainbow trout Salmo gairdneri Richardson to Yersinia ruckeri O-antigen monitored by the passive haemolytic plaque assay test

Abstract. The specificity and kinetics of the immune response of rainbow trout ( Salmo gairdneri ) to single injections of an O-antigen extracted from the bacterial pathogen Yersinia ruckeri , which causes enteric redmouth in fish, were investigated by the passive haemolytic plaque assay and serum antibody quantitation. Doses ranging from 5 ng to 500 mg in 10-fold increments were injected intraperitoneally into groups of trout held at 17 × 1°5°C. The occurrence of plaque forming cells (PFC) and humoral antibody was followed for 35 days after injection. Trout gave an immune response to doses of 500 ng and above. Seven days after injection no humoral antibody was detected, but PFC were found in the spleen. The maximum PFC numbers occurred 11 days after injection. On day 21, few PFC were found, whereas serum antibody titres were highest. The antibody from immunized trout showed little or no cross-reactions with sheep red blood cells passively labelled With antigens from other fish pathogens.     

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.     

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 .     

Tribrissen® and tiamulin for control of enteric redmouth disease

Abstract. Fifteen drugs were tested for in vitro control of Yersinia ruckeri . Those drugs yielding in vitro control were subsequently tested for in vivo control of induced enteric redmouth disease in rainbow trout. Two of the drugs, Tribrissen®v and tiamulin, controlled Y. nickeri in vivo under laboratory conditions. The effective doses used in this study were 1–0 mg of Tribrissen/kg fish/day and 5–0 mg of tiamulin/kg fish/day, when the compounds were administered orally for 14 days. No toxic effects were noted at these dose levels, or from Tribrissen given at 15 times the above effective dose.     

Yersinia ruckeri—A threat not only to rainbow trout

Yersinia ruckeri (Y. ruckeri) can cause mortalities that are contributing to substantial economic losses in the rainbow trout (Oncorhynchus mykiss) aquaculture sector. Because of its most characteristic clinical signs, the disease in rainbow trout caused by this pathogen is called enteric redmouth disease. Although it is considered to affect mainly salmonids, there are reports in the available literature of isolating this bacterium from other fish species, both clinically healthy and diseased. The aim of this study was to analyse the available data concerning yersiniosis in non‐salmonid fish. The analysed data indicate that Y. ruckeri is a threat not only to rainbow trout. Some of the affected species have high commercial importance and mortalities may contribute to high economic losses. The disease symptoms may not be specific and can be different from those characteristic for enteric redmouth in trout, which may lead to misdiagnosis. Collected information indicates that infection with Y. ruckeri should be taken into account in the diagnostic procedures not only in salmonids.     

Epizootics in farm-raised channel catfish, Ictalurus punctatus (Rafinesque), caused by the enteric redmouth bacterium Yersinia ruckeri

In the winters of 1995 and 1996, unusual disease outbreaks occurred on two separate channel catfish farms in Arkansas, USA. Affected fish exhibited extraordinary haemorrhaged rings around the eyes and raised haemorrhaged areas overlying the frontal foramens. Other signs included abnormal swimming, lethargy, loss of equilibrium, and exophthalmia. Bacterial isolates from the moribund fish were identified as Yersinia ruckeri by biochemical tests, no lysis by the Hafnia-specific bacteriophage 1672, and Y. ruckeri-specific growth patterns on Shotts-Waltman media. Fingerling catfish injected intraperitoneally with the bacterial isolate at 7.8 × 10⁶ bacteria fish^?1 developed lesions characteristic of the epizootics at 13, 18, and 22 °C and a biochemically identical isolate was recovered. Fingerling rainbow trout injected with the channel catfish isolate at 1 × 10⁵ bacteria fish^?1 and held at 20 °C developed signs typical of enteric redmouth by 4 days post-inoculation and were moribund by 5 days post-inoculation. Some differences of clinical signs occurred between experimentally infected rainbow trout and channel catfish. Clinical and biochemical similarities between infections of Y. ruckeri and many warmwater pathogens in affected catfish may lead to incorrect diagnosis of ERM infections.     

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.