维氏气单胞菌菌影疫苗的构建及其溶菌动力学研究

细菌菌影(Bacterial ghosts)是通过诱导PhiX 174噬菌体中裂解基因E在革兰氏阴性菌中的表达所获得的无细胞内容物的空细菌体.它保持了与活菌相同的膜蛋白成分、天然结构、细胞表面抗原等特性,是一种新型的死菌疫苗.为优化维氏气单胞菌(A.veronii)菌影制备条件,本实验将带有庆大抗性的裂解基因E转入A.veroniiATCC35624株中,通过温度变化对其进行溶菌动力学试验,每隔30 min对菌液OD600nm及其活菌数进行测定,当OD600nm稳定不再下降时,通过扫描电镜观察裂解后菌体的形态变化.结果显示,在诱导30 min后OD600nm值开始持续下降,当其下降到最低值时,活菌检测结果表明几乎无活菌存在.本研究利用菌影形成机制构建A.veronii菌影,为新型疫苗的制备提供依据.     

鳜嗜水气单胞菌GYK1株胞外产物提取、纯化及生物学活性分析

以鳜分离的嗜水气单胞菌GYK1株为代表,采用玻璃纸覆盖技术提取了8株嗜水气单胞菌和1株温和气单胞菌的胞外产物,SDS-PAGE电泳分析显示,8株嗜水气单胞菌的胞外产物均有35kDa的共同蛋白带。酶活性和溶血性分析表明,GYK1株胞外产物具有酪蛋白酶、淀粉酶、脂肪酶、明胶酶活性,但不具备脲酶活性;溶血性较强,蛋白浓度为365μg·mL-1的胞外产物对鳜血细胞的溶血价为213,对加州鲈、银鲫、小白鼠、兔、绵羊、人O型血的红细胞的溶血价在211~214之间。应用聚丙烯酰胺葡聚糖凝胶柱层析,对GYK1株的胞外产物进行了初步纯化,得到分子量为35kDa的蛋白带,该纯化产物具溶血性,但溶血性比粗胞外产物降低,对鳜和小鼠血细胞的溶血价均为25。     

一株尼罗罗非鱼致病性嗜水气单胞菌的 分离鉴定及药敏试验

从自然感染发病的尼罗罗非鱼肝脏和脑组织中分离出1 株细菌,经革兰氏染色镜检、生化鉴定和PCR 鉴定,确定为嗜水气单胞菌,命名为GD001,进一步采用药敏纸片法测定分离菌株对多种抗生素的敏感性,并用该菌 株接种健康尼罗罗非鱼,鉴定其致病力。结果发现,GD001 菌株对恩诺沙星、头孢噻吩、四环素和克莱霉素等抗生素 敏感,对链霉素轻度敏感,对卡那霉素和青霉素不敏感。从人工感染10 g 左右的健康罗非鱼发现,GD001 菌株具有很 强的致病力,试验鱼在感染后3~4 h内全部死亡,且患病症状与自然发病症状的鱼一致。     

Use of a probiotic to control lactococcosis and streptococcosis in rainbow trout, Oncorhynchus mykiss (Walbaum)

From a comparison of 125 bacterial isolates recovered from the digestive tract of rainbow trout, Oncorhynchus mykiss, and carp, Cyprinus sp., a culture was obtained which was effective at preventing clinical disease caused by Lactococcus garvieae and Streptococcus iniae when used as a feed additive. The culture, Aeromonas sobria GC2, was incorporated into the feed and fed to rainbow trout (average weight = 20 g) for 14 days at a dose equivalent to 5 x 10(7) cells g(-1) of feed. Whereas the untreated controls experienced losses of 75-100% when challenged intraperitoneally with L. garvieae and S. iniae, the probiotic-treated groups remained healthy with total mortalities of only 0-6%. Formalized and sonicated preparations of GC2 and cell-free supernatant fared less well. The mode of action reflected stimulation of innate immunity, namely an increased number of leucocytes and enhanced phagocytic and respiratory burst activity.     

养殖刺参溃疡病杀鲑气单胞菌的分离、致病性及胞外产物特性分析

从患溃疡病的养殖刺参(Apostichopus japonicus)病灶处分离出1株优势菌H1,以浸浴、创伤浸浴、体腔注射和体壁肌肉注射等方式进行感染实验,证实菌株H1为养殖刺参溃疡病病原菌,并证明该菌通过体表创伤侵入的方式感染刺参,以创伤浸浴和体壁肌肉注射感染的LD50(半数致死量)分别为2.26×107CFU/尾和1.80×107CFU/尾。经形态学观察、生理生化特性分析和mini API系统鉴定,确定菌株H1为杀鲑气单胞菌杀日本鲑亚种(Aeromonas salmonicida ma-soucida)。提取菌株H1的胞外产物(ECP)进行致病性实验,结果表明ECP可导致刺参死亡,其对刺参的LD50为5.24μg蛋白/g体质量。H1-ECP具有酪蛋白酶、明胶蛋白酶、几丁质酶和淀粉酶活性,并具有溶血素活性;对底物偶氮酪蛋白(Azocasin)作用的酶比活力可达到674.5活力单位/mg蛋白,最适作用温度为50℃;对热不稳定,70℃作用30 min时,酪蛋白酶活性降到0;100℃作用30 min,ECP对刺参的毒性消失;ECP酶活可被10 mmol/L EDTA完全抑制,可被5 mmol/L PMSF抑制98.8%,Ca2 和Mg2 可使酶活性分别提高约9%和4%。结论认为,该病原菌通过体表创伤侵入方式感染宿主刺参,菌株H1胞外产物是其对刺参致病的因子之一。     

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.     

Efficacy of furunculosis vaccines in turbot, Scophthalmus maximus (L.): evaluation of immersion, oral and injection delivery

The commercial furunculosis vaccine Aquavac Furovac 5 and an autogenous vaccine, based on the challenge strain, induced immune protection in turbot, Scophthalmus maximus (L.), as shown in challenge tests 120 days post-immunization by injection (relative percentage of survival, RPS = 72-99%). This protective effect lasted for at least 6 months post-immunization at appreciable levels (RPS = 50-52%). Neither the autogenous vaccine nor the commercial vaccine was able to induce significant levels of protection against Aeromonas salmonicida in turbot when administered by immersion. Antibody levels were high or moderate in fish vaccinated by injection with the different vaccines and very low in fish vaccinated by immersion. The field results show that delivering an oral boost after the primary vaccination by injection did not enhance protection of turbot against furunculosis and that water-based (autogenous vaccine) and oil adjuvanted (Alpha Ject 1200) vaccines administered by injection conferred similar levels of protection (RPS > 80%) in turbot.     

Characterization of the goldfish fecal microflora by the fluorescent in situ hybridization method

ABSTRACT:   Bacterial populations in goldfish feces were characterized by the fluorescent in situ hybridization (FISH) method. A total of nine different group-specific rRNA-targeted oligonucleotide probes were used. Approximately half of the microbial cells (57.8 ± 16.7%) were detected with a probe EUB338 and found to be bacteria. The microbial cells in 33–35 of the 35 samples from five specimens strongly hybridized with probes ALF1b, BET42a and GAM42a, suggesting that goldfish intestinal bacteria are mainly composed of α, β and γ-subclasses of Proteobacteria. The fact that a probe AER66 reacted with 25.6 ± 14.2% of the total microbial cells in all 35 samples, demonstrated that genus Aeromonas was the dominant species in the goldfish intestines. Genus Bacteroides including Bacteroides type A detected with a probe BAC303 was observed in 15 of 35 samples while other taxonomic groups determined with HGC69a, CF39a and P72 were detected in 6–11 of 35 samples. These results strongly suggest that Bacteroides shows the greatest daily fluctuation and interindividual variation in the intestines of goldfish. Moreover, the FISH method was proven to be useful for rapid enumeration of taxonomic groups of fish intestinal bacteria.     

鲤鱼细菌性败血症的病理学研究

报道了由嗜水气单胞菌嗜水亚种 (Aeromonashydrophilasubsp .hydrophila)感染鲤鱼引起细菌性败血症的病理学变化 ,通过对自然感染和人工感染病例的病理学观察 ,病理变化主要表现为鱼体多处出血 ,特别是体表的出血最为明显 ,上下颌、眼睛、鳃盖、鳍基充血发红 ,皮肤有瘀斑瘀点 ,并发生糜烂、溃疡 ;腹水 ,肛门红肿外突 ;心、肝、肾、肠等内脏器官充血、出血、肿大、变性、坏死、发炎以及红细胞发生溶血等 ,表现出细菌性败血症的病理特征 ,并在病理变化观察的基础上探讨了该病发生的机理。     

蛙嗜水气单胞菌的分离鉴定

从丰城某养殖场发病蛙苗中分离出两株细菌 ,经人工感染试验 ,对健康鲫鱼、小白鼠产生同蛙苗相一致的病症。经细菌形态特征、培养特性及生理生化特性等试验 ,鉴定为嗜水气单胞菌 (Aeromonashydrophila ,AH)。药敏试验表明 ,两株细菌对痢特灵、多粘菌素、阿米卡星、头孢唑啉敏感