【作者】 钟蕾；

【导师】 戴良英； 肖调义；

【作者基本信息】 湖南农业大学 ， 农业微生物学， 2011， 博士

【摘要】 三角帆蚌瘟病是我国淡水贝类养殖和育珠生产中毁灭性病害,其传染快、死亡率高,危害严重。该病主要危害2龄以上已植片三角帆蚌,发病初期病蚌分泌大量粘液,此后对外界刺激反应迟钝,闭壳肌收缩无力,斧足紧缩不伸展,剖检观察可见消化腺由墨绿色变为黄褐色,胃肠道壁轻度肿胀,性腺颜色变浅呈糜烂状。关于三角帆蚌瘟病的病原一直存在细菌与病毒两种说法,因此,至今未能在该病的致病机理和防治上取得较大突破。为了探明三角帆蚌瘟病的病原、阐明其致病机理,以及寻求有效防治该病的方法,本研究从三角帆蚌瘟病病原细菌的分离培养入手,较系统研究了细菌的致病性、生物学特性、显微超微结构、生理生化及分子鉴定、药敏试验、主要致病菌毒力基因克隆与原核表达等,取得了如下结果。1三角帆蚌细菌性瘟病病原菌的致病性测定从患病三角帆蚌的病灶处分离获得15个菌株,从中选取3个优势菌株进行人工感染试验,结果表明：SJ-1、SJ-2、SJ-3菌株单独感染和混和菌感染均能引起三角帆蚌细菌性瘟病相同症状,其中混合菌攻毒后表现为急性发病,感染总死亡率达96%,SJ-1菌株感染总死亡率达68%,SJ-2菌株感染总死亡率达46%,SJ-3菌株感染总死亡率达34%。2三角帆蚌细菌性瘟病的组织病理组织涂片、美兰染色和显微镜观察,结果表明：SJ-1菌株主要感染三角帆蚌消化腺、鳃、性腺和斧足；SJ-2菌株主要感染三角帆蚌肠、消化腺、性腺和斧足；SJ-3菌株主要感染三角帆蚌肾脏、斧足、鳃和肠。对病变细胞超微结构进行观察与分析,发现病变细胞普遍肿大,细胞膜不完整、结构模糊甚至破裂。细胞核形状异变呈不规则状,核膜不完整。细胞质电子密度降低,细胞器分散,细胞器及内含物显著减少,线粒体、内质网、溶酶体、过氧化酶体、高尔基体、中心粒、肌原纤维等细胞器和上皮细胞游离面微绒毛的超微结构均发生了明显的病理变化。3三角帆蚌细菌性瘟病病原菌的鉴定及药敏试验革兰氏染色和透射电镜观察结果表明,SJ-1菌株为革兰氏阴性极生多鞭毛杆菌,SJ-2菌株为革兰氏阴性极生单鞭毛短杆菌,SJ-3菌株为革兰氏阴性,直或稍弯、两端钝圆的杆菌,有1-3根单端鞭毛。电镜观察SJ-1菌株的菌体大小为0.5～0.6μm×1.5～1.8μm左右,SJ-2菌株的菌体大小为0.5～0.6μm×1.0～1.6μm左右,SJ-3菌株的菌体大小为O.4～0.6μm×1.0～1.3μm左右。生理特征测试结果表明,SJ-1菌株的最适生长盐度为4%；最适pH生长范围为6.0～8.0；最适生长温度为25～35℃。SJ-2菌株的最适生长盐度为3%；最适pH值为7.0；最适生长温度为28-30℃,温度低于4℃和高于45℃不生长。SJ-3菌株的最适生长盐度为3%；最适pH值生长范围为6.0-7.0；最适生长温度25～30℃,4℃以下不生长,40℃起生长基本停止。16SrDNA基因进化和系统发育树分析结果显示,SJ-1菌株(GU294302)与Stenotrophomonas属的细菌自然聚类,相似性为97%,SJ-2菌株(GU294303)与Aeromonas属的细菌自然聚类,相似性达99%。SJ-3菌株(GU294304)与Pseudomonas属的细菌自然聚类,相似性均为99%。生化鉴定分析结果显示,SJ-1菌株与嗜麦芽寡养单胞菌(Senotrophomonas maltophilia)相似率为95.5%,SJ-2菌株与维氏气单胞菌(Aeromonas veronii)相似率为99%,SJ-3菌株与铜绿假单胞菌(Pseudomonas aeruginosa)相似率为99.9%。综合细菌的形态特点、生理生化特性和16S rDNA基因进化和系统发育树分析,将SJ-1菌株鉴定为为嗜麦芽寡养单胞菌(Senotrophomonas maltophilia)、SJ-2菌株鉴定为维氏气单胞菌(Aeromonas veronii)、SJ-3菌株鉴定为铜绿假单胞菌(Pseudomonas aeruginosa)。对三角帆蚌瘟病致病菌进行药敏试验,筛选出几种病原菌高度敏感的药物为丙氟哌酸、洛美沙星、左氟沙星、氧氟沙星、诺氟沙星、加替沙星和头孢他啶/棒酸。4.嗜麦芽寡养单胞菌弹性蛋白酶Las B基因的克隆与原核表达克隆获得嗜麦芽寡养单胞菌弹性蛋白酶LasB基因完整开放阅读框全长序列为1497 bp,编码498个氨基酸,软件推测其编码蛋白相对分子量为53.57KDa；氨基酸序列分析表明该蛋白是含信号肽的非跨膜蛋白；氨基酸序列同源性比较表明该基因与其它菌种的蛋白酶基因家族成员同源性很高,序列高度保守；系统进化树分析表明该基因与多个菌株的蛋白酶基因亲缘关系接近。构建了嗜麦芽寡养单胞菌弹性蛋白酶基因的原核表达载体,并将重组表达载体转化至表达菌株BL21后,在原核表达系统中诱导表达出与预期结果相符的融合蛋白,为研究嗜麦芽寡养单胞菌与宿主的相互关系、致病机理及弹性蛋白酶的开发利用奠定了重要基础。更多还原

【Abstract】 Because of its fast propagation speed and high death rate, the mussel Hyriopsis cumingii plague is the most serious disease in the freshwater shellfish cultivation and pearl production. This kind of destructive disease often occurs in the pearl base areas, as well as in the new waters, and it exacerbates the water pollution, leads to sharp dropping in the high-quality freshwater shellfish resources. The 2 years old H. cumingii, that has been planted piece, is often harmed. During earlier stage, lots of mucus are secreted from the clam body. After that, H. cumingii become dull to outside stimulation. At the same time, the shells-muscle contraction ability become more weaker, and the axe foot does not stretch. The digestive gland is from dark green into brown, the wall of the the gastrointestinal tract is mildly swell, and the color of gonad is lighter like erosion. As the etiology of H. cumingii plague with bacterial and virus is not clear, great successes in pathogenesis and effective prevention of this disease are not achieved. In order to find out the pathogens of H. cumingii plague, to offer basic information for seeking effective measures of prevention and treatment. the isolation and culture of H. cumingii plague, the pathogenicity, biological characteristics, microstructure and ultrastructure, physiological and molecular identification, susceptibility testing, the cloning and expression of mainly virulence genes in the pathogen ect are studied., those results as following:1. The bacteria pathogenicity test for Hyriopsis cumingii PlagueThe strains (SJ-1、SJ-2、SJ-3) with high virulence which isolated from 15 strains of the diseased mussels were used to infect the healthy individuals. The artificially infection experiment proved that separate infection or mixed infection of the three strains could cause the death of the mussels and those symptoms are as same as naturely diseased mussels. Thereinto, acute symptoms appeared after the mix infection of the three strains and the total mortality was as high as 96%; the total mortality was 68% when infected by SJ-1 alone; while infected by SJ-2 alone, the rate was 46% and 34% by SJ-3.2. The histopathology of bacterial Hyriopsis cumingii PlagueThrough organization, dyed with Methylene blue and the microscope observation, the results as follows:Strains SJ-1 mainly infected digestive gland, gill, gonad and axe foot of sick mussels, strains SJ-2 mainly infected intestine, digestive gland, gonad and axe foot; and strains SJ-3 mainly infected kidney, axe foot, gill, and intestine. After the ultramicrostructure of diseased cells were observed and analysed. The Results showed: diseased cells were swelling,cell membrane were incomplete,their structure wree fuzzy and even broken. The nucleus of the diseased cells were changed into irregular shape and the nuclear membrane became incomplete. The electronic density in cytoplasm decreased and the number of organelles and cytoplasmic inclusions significantly reduced. Obvious pathologic changes were appeared in free surface of epithelial cells and other organelles such as mitochondria, ER, lysosome, peroxisome, golgi apparatus, centrosome and microcilli.3. The identification of pathogens and the susceptibility testing of Hyriopsis cumingii plagueThe results of gram’s stain and TEM observation show that, SJ-1 strain is gram-negative coli bacteria with many flagellum ends; SJ-2 strain is gram-negative short coli bacteria with a flagellum in one of the ends; SJ-3 strain is gram-negative coli bacteria, which straight or slightly curved and obtuse in two ends, with 1-3 one-port flagellum. The result of physiological and biochemical tests show that, optimal growth salinity of SJ-1 strain is 4%; optimal pH ranges from 6.0 to 8.0, optimal growth temperature is 25-35℃. Optimal growth salinity of SJ-2 strain is 3%; optimal pH is 7.0; optimal growth temperature is 28-30℃, and it will not grow when the temperature is below 4℃or above 45℃. Optimal growth salinity of SJ-3 strain is 3%, optimal pH ranges from 6.0 to 7.0; optimal growth temperature is 25-30℃, and it will stop growing when the temperature is below 4℃or above 40℃. Through the 16S rDNA genetic evolution and Phylogenetic Tree analysis, the following results were found, SJ-1 strain (GU294302) cluster naturally with the genus Stenotrophomonas clustering, similarity were 97%, SJ-2 strains (GU294303) cluster naturally with the genus Aeromonas, similarity 99%. SJ-3 strain (GU294304) cluster naturally with the genus Pseudomonas, similarity were 99%. Through the determination of the automatic biochemical analyzer,SJ-1 strain is similar to the rate of 95.5% with Stenotrophomonas maltophilia,SJ-2 strain is similar to the rate of 99% with Aeromonas veronii, and SJ-3 is similar to the rate of 99.9% with Pseudomonas. We synthesized the morphological characteristics of bacteria, physiological and biochemical characteristics and 16S rDNA gene evolution and phylogenetic tree analysis, the SJ-1 strain was identified as Stenotrophomonas maltophilia, SJ-2 strain was identified as Aeromonas veronii, SJ-1 strain was identified as Pseudomonas aeruginosa.The antimicrobial susceptibility tests of pathogens of H. cumingii plague were done, and some high-sensitivity drugs to pathogens were screened, they are ciprofloracin、lomefloxacin、levofloxacin、ofloxacin、norfloxaci、gatifloxacin、ceftazidime/clavulanic acid.4. The cloning and prokaryotic expression of elastase LasB gene from stenotrophomonas maltophiliaThe complete open reading frame (ORF) sequences Of elastase gene was cloned from Stenotrophomonas maltophilia. The full length of the ORF was 1497bp which encoded a protein of 498 amino acids with a predicted molecular mass of approximately 53.57 KDa. The amino acid sequence anlaysis indicated that the protein was a non-transmembrane protein with signal peptide. The homology comparison of amino acid proved that the homology between the LasB gene and other members of the family from other stains was very high. The sequences highly conserved. Phylogenetic tree analysis showed that the gene had close relationship with many elastase genes from strains. The prokaryotic expression vector with target gene was constructed, after transformation to express strains BL21, the fusion protein as the expectation was induced to expression. The study provides the foundation for research about the correlations between stenotrophomonas maltophilia and its host, the pathogenesis and the exploitation of elastase.更多还原