栉孔扇贝大规模死亡病原学研究

IOCAS-IR > 海洋环流与波动重点实验室

	栉孔扇贝大规模死亡病原学研究
其他题名	Etiology of mass mortality in scallop Chlamys farreri
	徐彪
学位类型	博士
导师	杨红生
	2008-06-04
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
学位专业	海洋生物学
关键词	栉孔扇贝原核生物（qdp）致病性分离培养
摘要	用平板画线法从患病栉孔扇贝（Chlamys farreri）体内分离到了一种原核生物（简称QDP）。QDP可以在改进的液体培养基MEM（含2.2%NaCl，5%小牛血清）和脑心浸液（含2.2% NaCl）中生长；菌落在显微镜下（150×）为无色、透明的小点状；革兰氏染色阴性；菌体为圆形或近似圆形。QDP在发育过程中有两种状态，一种为未成熟阶段，直径小于100nm；另一种为成熟阶段，直径变化很大，最小约60nm，最大可达4µm以上。较小的个体有拟核、核糖体和新月状的空泡，未见细胞壁；较大的个体有细胞壁，胞内大部分被空泡充满，未见拟核和核糖体。栉孔扇贝组织超簿切片电镜观查证实QDP的存在。QDP的密度随着生长发育时间的不同而有所变化，繁殖高峰期密度较大。建立了密度梯度离心结合滤膜过滤分离技术，优化人工培养条件。最适生长温度为23℃，最适生长pH值为7.4，最适生长盐度相当于细胞培养液所需的盐浓度（0.85%NaCl）。提取的QDP核酸能被RNase A 降解，且没有检测到DNA。以PCR、RT-PCR扩增其16SrRNA基因序列片段，PCR反应没有扩增出扩增子，而RT-PCR则扩增出了16S rRNA基因序列片段，经测定其序列全长度为1430bp，经与GENEBANK中的16S rRNA片段比较分析，与6种不同科的微生物的同源率最高的为95%-95.47%。采用温度梯度和病原浓度梯度回归感染实验方法，较为系统地研究了QDP的致病性。研究结果表明：QDP对栉孔扇贝有强烈的致病作用，高温（23℃以上）是其致病的必要条件，证实DQP是栉孔扇贝大规模死亡的病原体之一。
其他摘要	A strain of a prokaryote(Qingdao prokaryote, QDP) was isolated from moribund scallops Chlamys farreri. QDP could reproduce independently in minimal essential medium (MEM, adding 2.2% NaCl and 5% calf serum) or heart-brain infusion(adding 2.2% NaCl). Colorless, transparent and small dots colonies could be observed under microscope (150×) formed by QDP. QDP was Gram-negative, and round or near-round in shape. It included two kinds of particles, matured and unmatured particles. The unmatured particles were less than 100nm in diameter, and the diameter of matured particles ranged from 60nm to 4000 nm or above. The relatively small cells contained nucleoid, ribosome and vacuole, but no cell-wall has been found. The relatively big cells contained cell-wall, cytoplasmic membrane and one or several irregular transparent vacuoles inside each cell, but no nucleoid and ribosome were observed. The densities in QDP varied with self-development, and were higher at the cumulative reproduction. A method of sucrose density gradient centrifugation with filtration was created for QDP isolation from moribund scallops. The optimal temperature, pH, and NaCl were about 23 ℃, 7.4 and equal to MEM. QDP was found in the ultrathin section of lesion tissue of the scallop. The extracting nucleotide could be denatured by RNase A. DNA was not found from the extracting nucleotide. The 16S rRNA gene could be amplified by RT-PCR, but not PCR, and it was a 1430 BP stretch. The highest similarity values of 16S rRNA gene with some bacteria from 6 families were about 95-95.47%. Two experiments were conducted to methodically document pathogenicity in C. farreri, which were different pathogen concentrations at the same temperature and the same pathogen concentration at different temperatures. Results indicated that QDP, which could cause heavy infection in Farrer’s scallop, was one of the causative agent of the Farrer’s scallop mortalities, and the temperature (23 ℃ or above) was a key environmental factor.
页数	105
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/1027
专题	海洋环流与波动重点实验室
作者单位	中国科学院海洋研究所
推荐引用方式 GB/T 7714	徐彪. 栉孔扇贝大规模死亡病原学研究[D]. 海洋研究所. 中国科学院海洋研究所,2008.