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。     

绿鳍马面鲀与许氏平鲉杀鲑气单胞菌病原的分离和鉴定

2018年和2019年,山东省烟台市蓬莱市一养殖场工厂化养殖的绿鳍马面鲀(Thamnaconus septentrionalis)和许氏平鲉(Sebastes schlegeli)发病死亡,主要症状为嘴部溃疡、红肿和出血。从发病鱼内脏中均可分离到大量形态一致的优势菌,分别命名为2018TS-1和2019SS-1,分离菌株经16S rRNA测序、生理生化鉴定和vapA基因分析确定为杀鲑气单胞菌杀日本鲑亚种(Aeromonas salmonicida subsp. masoucida)。人工感染结果显示,2018TS-1和2019SS-1分别能引起绿鳍马面鲀和许氏平鲉的死亡,被感染鱼呈嘴部红肿症状,与自然发病症状一致,其半数致死量分别为1.78×105和0.89×105 CFU/尾。本研究首次报道了国内工厂化养殖绿鳍马面鲀和许氏平鲉感染杀鲑气单胞菌的病例,是目前人工养殖绿鳍马面鲀的首个疾病报道,也是继大西洋鲑(Salmo salar)、大菱鲆(Scophthalmus maximus)和裸盖鱼(Anoplopoma fimbria)等品种后,在山东省海水养殖鱼类中再次发现杀鲑气单胞菌杀日本鲑亚种的感染。本研究结果丰富了杀鲑气单胞菌杀日本鲑亚种的感染宿主范围,也为绿鳍马面鲀和许氏平鲉养殖的病害防控提供依据。     

杀鲑气单胞菌一新亚种的生物学特性及系统发育学分析

从石鲽（Kareius bicoloratus L．）细菌性败血感染症的病鱼（濒死及死亡不久）中分离到相应病原菌，进行形态特征、理化特性等较系统的表观分类学指征鉴定及代表菌株DNA中G＋Ct001％的测定。同时，选择代表菌株进行16S rRNA基因的分子鉴定，测定16S rRNA基因序列、分析相关细菌相应序列的同源性，构建系统发生树。结果表明，分离鉴定的60株菌为杀鲑气单胞菌的一个新亚种（subsp．nov．），定名为杀鲑气单胞菌杀鲽亚种（Aeromonas salmonicida subsp．flounderacia subsp．nov．）。代表菌株HQ010320-1及HQ010320-5的16S rRNA基因序列与GenBank数据库中的杀鲑气单胞菌的同源性在99％和100％。     

杀鲑气单胞菌杀日本鲑亚种胞外产物毒性及免疫原性分析

用玻璃纸平板法提取了杀鲑气单胞菌杀日本鲑亚种Aeromonassal monicida masoucida的胞外产物（ECP）。毒性试验证实，ECP对剑尾鱼Xiphophorus helleri具有致死性，其半致死量（LD50）为4.72μg蛋白/g体重。SDS-PAGE表明，ECP由13条蛋白带组成。利用大鼠抗ECP血清进行的Western-blot印迹显示，组成ECP的13条蛋白带中有7条具有免疫原性，能够引发机体的免疫应答反应产生抗体，其分子量分别为88、70、42、39、36、22和15kDa。     

杀鲑气单胞菌可视化重组酶聚合酶扩增检测技术的建立

本研究根据杀鲑气单胞菌(Aeromonas salmonicida)的铁载受体(fstA)基因的保守序列设计特异性引物和探针,建立了一种基于重组酶聚合酶扩增(recombinase polymerase amplification,RPA)与侧向流试纸条(lateral flow strips,LFS)相结合的可视化快...     

杀鲑气单胞菌的实时定量PCR检测方法的建立和应用

选取杀鲑气单胞菌A层A蛋白(VapA)保守序列,利用Primer Express2.0软件设计引物和探针.以ATCC标准株10倍系列稀释,通过血球计数板计数后进行实时定量PCR,制作标准曲线.通过SDS软件获得细菌数量(X)与Ct值的关系为:Ct＝-3.1574lgX 43.6841(相关系数R2=0.992).实时定量PCR的检测限为6个细菌.建立的方法对杀鲑气单胞菌典型株和非典型株具有较好的特异性,与其他细菌没有交叉反应.杀鲑气单胞菌实时定量PCR方法的建立,对于快速口岸检疫、临床诊断和疫病监测具有重要的应用价值.     

杀鲑气单胞菌对大西洋鲑游泳行为和血细胞数量的影响

为探讨利用鱼类行为及血细胞数量变化预警杀鲑气单胞菌(Aeromonas salmonicida)病害发生的可行性,监测了生产中感染杀鲑气单胞菌的大西洋鲑(Salmo salar L.)的游泳行为,以及杀鲑气单胞菌攻毒后大西洋鲑血细胞数量的变化。实验采用同一养殖基地和同一批次的大西洋鲑,其中现场实验鱼选自生产车间健康的和感染杀鲑气单胞菌的养殖鱼,攻毒实验中处理组实验鱼每尾背肌注射100μL、浓度为3.05×107CFU/m L的菌液,对照组注射等体积灭菌生理盐水。现场实验表明,感染杀鲑气单胞菌的大西洋鲑临界游泳速度较健康鱼低26.7%(P0.05),摆尾频率与游泳速度的线性回归方程的斜率也存在显著差异(P0.05)。攻毒实验表明,从攻毒的第4天开始,处理组大西洋鲑白细胞、淋巴细胞、单核细胞和粒细胞数量较对照组均发生显著变化,其中第6天的变化最为显著,白细胞总数、粒细胞数分别降低了2.8%和43.9%(P0.05),淋巴细胞数及单核细胞数分别升高了63.3%和23.9%(P0.05),且处理组4种血细胞数随时间呈现显著的线性变化(P0.05)。研究结果表明通过监测大西洋鲑游泳行为(临界游泳速度和摆尾频率)以及血细胞相关指标的变化可快速判断其健康状况,为病害的早期预警提供依据。     

大西洋鲑杀鲑气单胞菌的分离鉴定

从皮肤溃烂的大西洋鲑(Salmon salar)肌肉、肝、肾分离到一致病性的菌株(AB080226),经人工感染实验证实其为该病的病原菌,研究了该菌的形态、生理生化特征并对其16SrDNA序列进行了在线Classifer和Blast分析。结果显示:该菌为革兰氏阴性杆菌,葡萄糖氧化发酵阳性,氧化酶检测阳性,0%NaCl生长;能利用甘露醇,不具运动性,能发酵麦芽糖,葡萄糖产酸阳性,精氨酸双水解酶阳性,苯丙氨酸脱氨酶阴性;该菌属于气单胞菌属的细菌,在系统发育树上与杀鲑气单胞菌形成一个簇群,同源性高达99.9%以上。综合形态学、生理生化特征及16SrDNA序列鉴定其为杀鲑气单胞菌(Aeromonas salmonicida)。     

2株亚东鲑源气单胞菌的鉴定及其致病性与药敏特性分析

亚东鲑(Salmo trutta fario)是西藏地区重要的冷水性经济鱼类之一。为明确亚东鲑暴发性死亡的原因, 对从患病亚东鲑体内分离得到的 2 株优势细菌 B1、A3-2 进行种类鉴定、毒力基因检测、动物回归感染、耐药基因检测和药敏试验。结果显示, 2 株优势细菌鉴定为杀鲑气单胞菌(Aeromonas salmonicida) B1 和温和气单胞菌(Aeromonas sobria) A3-2。杀鲑气单胞菌 B1 对亚东鲑具有较强的致病性, 而温和气单胞菌 A3-2 对亚东鲑未表现出致病性。杀鲑气单胞菌 B1 携带有 10 种毒力基因: 外毒素(AerA、Act 和 hly 基因)、胞外酶(gcat、ahyB 和 Lip 基因)、Ⅲ型分泌系统(aexT、aopP 和 ascF-G 基因)、鞭毛(Fla 基因); 温和气单胞菌 A3-2 携带有 5 种毒力基因: 外毒素(Act 和 Alt 基因)、胞外酶(gcat 基因)、Ⅲ型分泌系统(aexT 和 aopP 基因)。杀鲑气单胞菌 B1 对头孢曲松、阿莫西林、氟苯尼考、环丙沙星、四环素、链霉素等 21 种抗菌药物敏感, 仅对万古霉素耐药; 温和气单胞菌 A3-2 对头孢曲松、氟苯尼考、环丙沙星、吡哌酸、四环素、链霉素等 17 种抗菌药物敏感, 对青霉素、阿莫西林、磺胺异噁唑、复方新诺明、万古霉素等 6 种药物耐药。杀鲑气单胞菌 B1 含有 AmpC、gyrA 和 parC 等 3 种耐药基因; 温和气单胞菌 A3-2 含有 AmpC、gyrA、parC 和 tetE 等 4 种耐药基因, 2 种气单胞菌的耐药基因检出结果与耐药表型基本一致。杀鲑气单胞菌 B1 是引起亚东鲑暴发性死亡的重要病原菌, 本研究为亚东鲑养殖过程中杀鲑气单胞菌的感染特征、疫苗研制和疾病防控提供了基础数据。     

美洲红点鲑溃烂病的病原菌研究

对从患病美洲红点鲑(Salvelinus fontinalis)体表患病部位和肾脏分离到菌株进行了鉴定及药物筛选。结果显示:人工感染试验证实为病原菌,该菌革兰氏染色阴性,菌体呈短杆状。综合该菌形态、生理生化分析结果初步鉴定两株菌均为杀鲑气单胞菌(American salvelinus)杀鲑亚种。用引物AP1和AP2对纯化后的细菌进行PCR扩增,结果扩增出长度为550 bp的DNA片段,对扩增片段进行测序,用NCBI BLAST在GenBank中搜寻相似序列,结果与杀鲑气单胞菌各株A层蛋白部分编码基因有99%以上的序列同源性。进一步证明了鉴定结果。该菌对强力霉素、左氟沙星、氟罗沙星、庆大霉素等13种药物均敏感。     

Plasma ladderlectin concentration following sterile inflammation and Aeromonas salmonicida subsp. salmonicida infection

Plasma samples obtained from rainbow trout either experimentally infected with Aeromonas salmonicida or injected with either A. salmonicida lipopolysaccharide (LPS) or a commercial A. salmonicida vaccine (Lipogen) were analysed by enzyme immunoassay to evaluate changes in rainbow trout ladderlectin (RTLL) concentrations during the acute phase response (APR). Plasma RTLL concentrations in fish injected with A. salmonicida LPS, vaccine or live A. salmonicida varied over a 10 day period, but did not significantly increase. In contrast, fish experimentally infected with A. salmonicida exhibited a modest, but statistically significant ( P  <   0.05), decrease in RTLL concentration. These studies demonstrate that RTLL is not detectably induced during the trout APR to sterile inflammation or A. salmonicida infection, but plasma concentration of this protein may be reduced during bacterial infection.     

Improvements of virulence factor phenotypic tests for Aeromonas salmonicida subsp. salmonicida,a major fish pathogen

Aeromonas salmonicida subspecies salmonicida, a fish pathogen, expresses various virulence factors such as an A-layer, lipases and proteases during the infection process. Not all strains of this bacterium express the same virulence factors. It is important to be able to evaluate which factors are present when characterizing strains. The A-layer and secreted lipases and proteases are usually detected by agar-based tests that require long incubation (24 h and more) and may provide ambiguous results. In the present study, protocols have been optimized to determine the presence of these virulence factors using liquid tests. For A-layer detection, the optimized method stains the positive bacteria with Coomassie Brilliant Blue. The lipases are detected by a colorimetric biochemical reaction triggered by the degradation of p-nitrophenyl dodecanoate into a yellow product detectable by spectrophotometry, if the result is positive. Both of these tests show results in less than an hour. Finally, the protease activity is measured by clarification of a medium containing milk during an overnight bacterial growth. These new protocols provide opportunities for quicker characterization of A. salmonicida subsp. salmonicida strains and, particularly, provide more precise results.     

Pathogenic characterization of Aeromonas salmonicida subsp. masoucida turbot isolate from China

Aeromonas salmonicida is a gram-negative bacterium that is the causative agent of furunculosis. An A. salmonicida strain was isolated from diseased turbot (Scophthalmus maximus) with the sign of furunculosis from North China. Based on vapA gene, the strain was further classified as A. salmonicida subsp. masoucida RZ6S-1. Culturing RZ6S-1 strain at high temperature (28°C) obtained the virulence attenuated strain RZ6S. Genome sequence comparison between the two strains revealed the loss of the type IV secretion system (T4SS) and type III secretion system (T3SS) from the native plasmid pAsmB-1 and pAsmC-1 of wild-type strain RZ6S-1, respectively. Further study demonstrated that the wild-type strain RZ6S-1, but not its derivative mutant RZ6S, can stimulate apoptosis. Elevated protein level of cleaved caspase-3 was detected from epithelioma papulosum cyprinid (EPC) cells infected with wild-type strain RZ6S-1 as compared with that infected with RZ6S strain. Meanwhile, the invasion of the mutant strain RZ6S was about 17-fold higher than the wild-type strain RZ6S-1, suggesting that some protein(s) from A. salmonicida subsp. masoucida RZ6S-1 suppress its invasion. The RZ6S mutant strain was attenuated, since its LD₅₀ is over 10,000 times higher compared to the wild-type strain as revealed in the turbot infection model.     

Experimental infection of turbot, Scophthalmus maximus (L.), by Aeromonas salmonicida subsp. achromogenes and evaluation of cross protection induced by a furunculosis vaccine

Turbot was shown to be sensitive to injection challenges by Aeromonas salmonicida subsp. achromogenes (Asa). A systemic disease was induced and the bacterium was isolated from various internal organs. Histopathological changes involved haemorrhages, necrosis and degeneration in skin and muscle, haemorrhages and necrosis in kidney, degeneration in the heart muscle, and fusion of the secondary gill lamellae. A polyvalent commercial salmon vaccine, containing A. salmonicida subsp. salmonicida as one of five antigens, did not confer protection in turbot against an experimental Asa infection 13 weeks post-vaccination. Vaccination induced a significant antibody response against Asa cells but not against extracellular products of the bacterium. The results of the study indicate that Asa may be a potential threat to turbot farming and that the development of new turbot vaccines is needed.     

Construction of Aeromonas salmonicida subsp. achromogenes AsaP1‐toxoid strains and study of their ability to induce immunity in Arctic char,Salvelinus alpinus L.

The metalloendopeptidase AsaP1 is one of the major extracellular virulence factors of A. salmonicida subsp. achromogenes, expressed as a 37‐kDa pre‐pro‐peptide and processed to a 19‐kDa active peptide. The aim of this study was to construct mutant strains secreting an AsaP1‐toxoid instead of AsaP1‐wt, to study virulence of these strains and to test the potency of the AsaP1‐toxoid bacterin and the recombinant AsaP1‐toxoids to induce protective immunity in Arctic char. Two A. salmonicida mutants were constructed that secrete either AsaP1_E294A or AsaP1_Y309F. The secreted AsaP1_Y309F‐toxoid had weak caseinolytic activity and was processed to the 19‐kDa peptide, whereas the AsaP1_E294A‐toxoid was found as a 37‐kDa pre‐pro‐peptide suggesting that AsaP1 is auto‐catalytically processed. The LD₅₀ of the AsaP1_Y309F‐toxoid mutant in Arctic char was significantly higher than that of the corresponding wt strain, and LD₅₀ of the AsaP1_E294A‐toxoid mutant was comparable with that of an AsaP1‐deficient strain. Bacterin based on AsaP1_Y309F‐toxoid mutant provided significant protection, comparable with that induced by a commercial polyvalent furunculosis vaccine. Detoxification of AsaP1 is very hard, expensive and time consuming. Therefore, an AsaP1‐toxoid‐secreting mutant is more suitable than the respective wt strain for production of fish bacterins aimed to protect against atypical furunculosis.     

患病细鳞鱼杀鲑气单胞菌的分离与鉴定

从患病细鳞鱼(Brachymystax lenok)的病变组织处分离到1株致病菌,经过分离培养,生化鉴定,16 S rRNA序列分析和人工感染实验确定该病原菌为杀鲑气单胞菌无色亚种(Aeromonas salmonicida subsp.achromogenes)。采用20种药物进行药敏分析,结果显示:分离菌株对阿米卡星、哌拉西林、恩诺沙星等9种抗生素敏感;对环丙沙星、诺氟沙星等4种抗生素中度敏感;对卡那霉素、青霉素、氨苄西林等7种抗生素耐药。     

Antibody-coated gold nanoparticles immunoassay for direct detection of Aeromonas salmonicida in fish tissues

Aeromonas salmonicida is the causative agent of furunculosis, a disease that affects both salmonid and non‐salmonid fish. Detection of A. salmonicida can be labour intensive and time consuming because of the difficulties in distinguishing the bacterium from other species given the wide variety of existing biochemical profiles and the slow growth characteristics which allow other organisms to overgrow the A. salmonicida. Herein, we report the development of a specific immunoassay using gold‐conjugated polyclonal antibodies for the rapid detection of A. salmonicida in fish tissues. Monodispersible 13‐nm gold nanoparticles were coated with polyclonal antibodies specific to A. salmonicida. Reddish purple agglutination of gold particles indicated the presence of A. salmonicida in samples. Positive reactions were detected visually with the naked eye. No agglutination was observed when A. salmonicida antibody‐coated gold nanoparticles were tested with other common bacterial fish pathogens, thereby verifying the specificity of the assay. The assay could detect A. salmonicida in fish tissues down to 1 × 10⁴ CFU mL^?1, and results were obtained within 45 min. The antibody‐coated gold nanoparticles were stable for at least 2 months at 4°C. The immunoassay using antibody‐coated gold nanoparticles represents a promising tool for the rapid and specific detection of A. salmonicida in fish tissues.     

养殖裸盖鱼(Anoplopoma fimbria)疥疮病病原菌的分离与鉴定

裸盖鱼(Anoplopoma fimbria)是一种名贵的冷水经济鱼类,已在我国北方地区开展小规模的工厂化繁育和养成.目前,我国关于裸盖鱼的病害研究仍是空白.本研究对山东烟台一养殖场自然发生疥疮病的裸盖鱼进行了病原分析,从发病鱼体内分离得到形态一致的优势菌株,命名为AF-1,并对其进行了致病性检测、菌种鉴定及药物敏感性研究.人工感染实验证明,AF-1对裸盖鱼有致病性,呈现症状与自然发病状态一致;结合形态观察、革兰氏染色、生理生化特征、16S rRNA和gyrB基因序列进化树分析,将AF-1鉴定为杀鲑气单胞菌(Aeromonas salmonicida);药物敏感性实验结果显示,AF-1对青霉素、阿莫西林、头孢噻吩等13种抗生素具有耐药性,对氟苯尼考、氟甲喹等16种抗生素敏感.综上所述,本研究首次报道了我国养殖裸盖鱼感染杀鲑气单胞菌病例,为裸盖鱼养殖过程中的疾病防控和疫苗开发提供了参考.