鳜致病性嗜水气单胞菌GYK1株的鉴定、毒力及溶血性

从患溃疡病的鳜肾脏中分离出一株病原细菌编号为GYK1。此株病菌人工感染鳜、银鲫、剑尾鱼,实验鱼出现出血、肌肉坏死或溃疡症状;腹腔注射攻毒,对鳜、银鲫、剑尾鱼、小鼠的半致死剂量(LD50)分别为8.33×104、1.06×105、1.26×105、1.05×106CFU/g;菌培养液上清能溶解鳜、加洲鲈、银鲫、小鼠、兔、绵羊、人O型血等的红细胞,在绵羊血平板上为β 溶血,不同代次和保存条件的细菌溶血性稳定。细菌在电镜下的形态、生化特性和ID32E系统自动鉴定的结果均符合嗜水气单胞菌的特征;PCR扩增检测到嗜水气单胞菌特异性的毒力基因气溶素基因(aerA)209bp片段,进一步说明此株病原细菌为含有毒力基因的嗜水气单胞菌。实验结果表明,GYK1是一株毒力和溶血性均较强的嗜水气单胞菌。     

羊源杀鲑气单胞菌16S rRNA基因的克隆测序及分析

从重庆市丰都县无菌采集山羊血液,提取全血基因组,用16SrRNA基因的引物进行PCR扩增,得到2条长约1 500bp的扩增片段,将其克隆到pMD19-T载体后进行测序,并与3条豚鼠气单胞菌,3条嗜水气单胞菌,3条中间气单胞菌,4条杀鲑气单胞菌,2条温和气单胞菌,3条维氏气单胞菌和5条舒氏气单胞菌16SrRNA基因序列进行系统发育分析.结果表明:所克隆的基因片段长度均为1 507bp,GenBank登录号为JF823571和JF823572.系统发育分析发现2条序列均被聚类到杀鲑气单胞菌群,并与温和气单胞菌聚类到一个大的分支.同源性比对结果显示所获得的序列与杀鲑气单胞菌法国株(ATCC33658T)X74681和阿根廷株AF134065的同源性最高,均为99.6%,表明重庆地区存在受杀鲑气单胞菌感染的山羊.     

Molecular characterization of tetracycline- and quinolone-resistant Aeromonas salmonicida isolated in Korea

The antibiotic resistance of 16 Aeromonas (A.) salmonicida strains isolated from diseased fish and environmental samples in Korea from 2006 to 2009 were investigated in this study. Tetracycline or quinolone resistance was observed in eight and 16 of the isolates, respectively, based on the measured minimal inhibitory concentrations. Among the tetracycline-resistant strains, seven of the isolates harbored tetA gene and one isolate harbored tetE gene. Additionally, quinolone-resistance determining regions (QRDRs) consisting of the gyrA and parC genes were amplified and sequenced. Among the quinolone-resistant A. salmonicida strains, 15 harbored point mutations in the gyrA codon 83 which were responsible for the corresponding amino acid substitutions of Ser⁸³→Arg⁸³ or Ser⁸³→Asn⁸³. We detected no point mutations in other QRDRs, such as gyrA codons 87 and 92, and parC codons 80 and 84. Genetic similarity was assessed via pulsed-field gel electrophoresis, and the results indicated high clonality among the Korean antibiotic-resistant strains of A. salmonicida.     

青虾源维氏气单胞菌双重PCR检测方法的建立

根据NCBI已发表的维氏气单胞菌(Aeromonas veronii)促旋酶B亚单位基因gyrB和编码细菌RNA聚合酶σ70因子基因rpoD的保守序列设计引物,建立了一种快速检测青虾源维氏气单胞菌的双重PCR检测方法。结果显示:青虾源维氏气单胞菌gyrB和rpoD基因PCR扩增产物大小分别为815 bp、554 bp,而对嗜水气单胞菌(Aeromonas hydrophila)、弗氏柠檬酸杆菌(Citrobacter freundii)、哈维弧菌(Vibrio harveyi)、副溶血性弧菌(Vibrio parahemolyticus)、需钠弧菌(Vibrio natriegens)、非O1霍乱弧菌(non-O1 Vibrio cholerae)、阴沟肠杆菌(Enterobacter cloacae)、产气肠杆菌(Enterobacter aerogenes)扩增结果皆为阴性;灵敏性试验结果显示该方法检测到的最低菌体DNA量为1.8×10^-2 ng/μL,且10份送检样本检测结果与传统细菌分离鉴定结果相一致。结果表明,双重PCR检测方法特异性好、灵敏性高,适用于...     

单壁碳纳米管载嗜水气单胞菌外膜蛋白亚单位疫苗的研制及免疫效果评价

为了筛选细菌的疫苗候选蛋白,本实验根据课题组前期的研究,挑选了2个嗜水气单胞菌外膜蛋白(A0KLQ6和A0KH89),以A0KHR9(OmpAII)为对照,通过比较外膜蛋白与碳纳米管载外膜蛋白通过腹腔注射与浸泡免疫模式,比较和评价斑马鱼的免疫反应与免疫保护效果。结果显示,腹腔注射组和浸泡组与普通蛋白组对比,碳纳米管载蛋白能够显著提高斑马鱼免疫相关基因表达量。腹腔注射组在5μg剂量下免疫A0KHR9、A0KLQ6和A0KH89蛋白分别获得61.60%、 65.39%和84.96%相对免疫保护率(relative immune protection rate,RPS),在5μg剂量下免疫碳纳米管载蛋白SWCNTs-A0KHR9、SWCNTsA0KLQ6和SWCNTs-A0KH89分别获得68.10%、77.50%和90.43%的RPSs。浸泡组在40 mg/L剂量下免疫A0KHR9、A0KLQ6和A0KH89蛋白分别获得30.80%、25.85%和37.80%的RPSs,在40 mg/L最高剂量下免疫碳纳米管载蛋白疫苗SWCNTs-A0KHR9、SWCNTsA0KLQ6和SWCNTs-A0...     

鱼源嗜水气单胞菌多重耐药菌株整合子的分子特征

为研究嗜水气单胞菌多重耐药菌株整合子-基因盒分布及分子特征, 首先采用K-B纸片扩散法检测28株鱼源嗜水气单胞菌对18种抗生素的耐药性, 然后利用PCR方法检测菌株中Ⅰ、Ⅱ、Ⅲ型整合酶基因并对其携带基因盒序列进行分析。结果表明, 分离到的鱼源嗜水气单胞菌呈多重耐药性, 对b-内酰胺类、大环内酯类、氯霉素类和四环素类药物的耐药率超过60%, 而对氟喹诺酮类药物较敏感, 且菌株间耐药谱差异较大。53.57%的菌株Ⅰ类整合子阳性, 21.43%的菌株Ⅱ类整合子阳性, 整合子阳性菌株对多种药物的耐药率均高于阴性株, 且Ⅰ类整合子阳性株多重耐药率明显高于Ⅱ类整合子阳性株, 表明整合子系统在嗜水气单胞菌多重耐药性中发挥重要作用。Ⅰ类整合子基因盒以aadA、dfrA、catB家族为主, 分别介导氨基糖苷类、甲氧磺胺嘧啶类和氯霉素类药物耐药; 基因盒的排列以aadA2+dfrA12类型为主。此外, Ⅰ类整合子阳性的嗜水气单胞菌多重耐药性在不同个体间也存在较大差异, 提示多重耐药菌株的耐药表型与基因盒的类型无直接相关性。

     

Genetic variations in survival of rohu carp (Labeo rohita, Hamilton) after Aeromonas hydrophila infection in challenge tests

A challenge test against Aeromonas hydrophila was conducted using 2062 rohu carp (Labeo rohita) fingerlings obtained from 52 dams and 87 sires (87 full-sib families) of two year-classes (2003 and 2004). Attempts to establish a cohabitant challenge model were not successful. Therefore, fish were challenged by intraperitoneal injection with A. hydrophila in two replicate tanks per year-class, and dead fish were collected hourly. The mortality reached its peak at 16–22 h after challenge and had almost completely stopped after 58 h. The test was terminated after 382 h at which the average survival was 43.9 and 48.9% in the two 2003 year-class tanks, and 77.1 and 35.8% in the two 2004 year-class tanks. Heritability estimates for survival were obtained from sire and dam threshold models (THR) and sire and dam linear repeatability models (LINR). For both types of models the heritability estimates based on data from single tanks were not consistent. For both year-classes, data from one of the two challenged tanks demonstrated significant additive genetic variation in survival during the A. hydrophila infection, whereas the heritability estimates were not significantly different from zero for the other tank. Further, genetic correlation between survivals in the two replicate tanks in each year-class was not significantly different from zero. The differential results from the replicate tanks demonstrate that additional challenge test experiments are needed before firm conclusions can be drawn about the magnitude of additive genetic variation for survival to aeromonasis in rohu carp. A cohabitant challenge model that allows the testing of important defence mechanisms in the skin and mucous membranes of the fish might have been more appropriate. To establish a valid cohabitant challenge model for rohu carp should be given high priority.     

健康草鱼肠道气单胞菌季节变化及胞外酶调查

暴发性鱼病流行季节及非发病季节的健康草鱼肠道气单胞菌的分布调查表明,两季中气单胞菌占总菌数的比例变化不大,只是冬季前肠道的气单胞菌比例明显增高,应视为草鱼肠道的正常寄生菌落。部分胞外酶中发现了较多产木聚糖酶的气单胞菌。     

鲤竖鳞病病原及其疫苗的研究

对发生在山东境内的鲤竖鳞病进行了病原学研究,经过病原分离,人工感染实验和细菌学鉴定,确定豚鼠气单胞菌（Ａｅｒｏｍｏｎａｓｃａｕｉａｅ）是鲤竖鳞病的病原菌,在此基础上,得到了免疫原性较高和生产工艺简单的鲤竖鳞病疫苗,同时,对该疫功的安全性,免疫保护力和效价等进行了系统研究,并进行了一定范围的口服免疫应用试验。