【作者】 李筠；

【导师】 徐怀恕； Brian Austin；

【作者基本信息】 中国海洋大学 ， 海洋生物， 2002， 博士

【摘要】 为了探索微生物区系与养殖动物疾病的关系，进一步加强对海水养殖生物病原学的认识，更好地对疾病进行诊断，我们研究了两个育苗场不同发育期的中国对虾(P.chinensis)幼体和仔虾体内的异养菌和弧菌种群变动的动态过程。以典型特征法、BIOLOG GN法、数值分类法对菌株进行鉴定。结果表明，对虾幼体样品中所分离的细菌，大多是革兰氏阴性杆菌，经鉴定为假单胞菌属(Pseudomonas)、气单胞菌属(Aeromonas)和弧菌属(Vibrio)。弧菌属主要为溶藻胶弧菌(Vibrio alginolyticus)和哈维氏弧菌(Vibrio harvevi)。在对虾幼体发育早期，假单胞菌(Pseudomonas)和气单胞菌(Aeromonas)占优势，随着对虾幼体的发育，在以后的发育过程中，弧菌(Vibrio)成为优势，其中溶藻胶弧菌(Vibrio alginolyticus)和哈维氏弧菌(Vibrio harveyi)优势明显。且二者的消长与苗期病害的发生相关，溶藻胶弧菌总是在虾苗健康时出现或成为优势，而哈维氏弧菌成为优势时，苗期病害容易发生。从而揭示了育苗期中国对虾(Penaeus chinensis)幼体中可培养的异养细菌区系及其变化与病害发生的关系。 在上述研究基础上，采用BIOLOG-GN细菌鉴定系统，分析了来自5个国家的4个养殖对虾品种苗期及部分养成期虾体的185株弧菌(分别分离自对虾健康状态及发病期间)。结果表明，来源、种类不同的养殖对虾苗期的主要弧菌组成相似，溶藻弧菌(Vibrio alginolyticus)和哈维氏弧菌(V.harveyi)是普遍存在的种类，同一种对虾在不同地区养殖，其区系组成略有差异。通常哈维氏弧菌(V.harveyi)为对虾苗期致病菌，副溶血弧菌(V.parahaemolyticus)为成虾致病菌。在健康虾苗和发病虾苗体内都可分离到溶藻弧菌(V.alginolyticus)。因此，可以说明以中国对虾为研究对象得出的结论具有普遍意义。 为探索海洋环境中可培养细菌多样性的规律，直接比较研究了水产养殖、工业污染对海洋细菌多样性的影响。我们在1999-2000年间研究了青岛近岸代表性海区(养殖密集区、工业污染区、无污染区)不同样品中的异养菌(革兰氏阴性菌)的区系组成，对从青岛沿岸的养殖密集区、工业污染区、无污染区不同月份分离的428株革兰氏阴性细菌，用BIOLOG-GN法进行了鉴定，其中106株菌对BIOLOG-GN板的95种碳源反应结果全部为阳性或阴性，而其余的322株菌对BIOLOG-GN板的95种碳源反应结果正常。根据BIOLOG结果，对其中的322株菌进行4种比较分析：1．待测菌株、参考(或标准)菌株与BIOLOG-GN与海水养殖相关的细菌多样性的研究及方法评价数据库比较;2.根据95种碳源反应结果进行聚类分析;3.聚类分析结果与标准菌株(27株)、BIOLOG毛N鉴定结果的比较;4.根据聚类分析结果，进行细菌多样性评价。同时，也对BIOLOG一GN技术进行了评价。 以上研究表明，在近岸海水环境中，弧菌是可培养细菌中的优势种群。目前，对弧菌科多样性的研究越来越集中到病原种类上，研究中发现弧菌科中仍然有很多种群还没有被描述。AFLP方法在细菌属的多样性研究、分类学研究和系统发育学研究等方面越来越起到非常重要的作用，于是出现了AFLP方法对弧菌科菌株(其中76%来源于海水养殖环境)遗传多样性的报道，其中有一些尚未定种群(即与现存的已定名的菌株不在一群中)。对此，我们选择了不能与现有的弧菌标准菌株或参考菌株归入同一类群的部分独立聚簇，依据其165rDNA全序列与标准菌株的比较结果，确立这些菌株在弧菌系统发育树中的地位，找出亲缘关系最近的已知种的标准弧菌，并与之进行DNA一DNA杂交，进一步确立这些聚簇的分类地位，以发现弧菌新的种类。研究结果表明，聚簇Al、A3、AS、AS、A23、A51、A52与A53、A55、菌株LMG 21329、菌株LMG C21330为有待于描述的新的弧菌种类。并对其中的A23和菌株LMG 21330、LMG21329进行了表型特征的测定，包括脂肪酸组成、不同碳源的利用或发酵，以及弧菌新种的命名与描述。A23命名为巧b川。xui sP.nov.，LMGZI犯9命名为风b对。户aneiensis sp.nov.，LMG 21330命名为片b厂iogallicossp.nov.。更多还原

【Abstract】 In order to improve our understanding of disease etiology and develop better diagnostics, it is necessary to research on the microflora associated with diseased animals. Microflora of aerobic heterotrophic bacteria from larvae and postlarvae of Penaeus chinensis were studied.Bacteria isolated from the samples were predominantly Gram-negative. The generic composition, especially the composition of vibrios species were found to vary with different growth stages. Pseudomonas and Aeromonas were the most frequently isolated genera from larvae at initial stages followed by Vibrio alginolyticus and Vibrio harveyi at the following stages subsequently. The high prevalence of Vibrios, especially V. alginolyticus and V. harveyi, was isolated frequently from healthy larvae and postlarvae of P. chinensis. It was suggested that V. harveyi emerging ahead of time due to the worse quality of rearing water was one factor resulting in the epizootic occurrence.On the basis of the research above, distribution of vibrios isolated from 4 species of cultured shrimp larvae in 5 countries were assayed by Biolog GN system. The isolates were isolated mainly from health or diseased shrimp larvae. The distribution of dominant Vibrio species from different species of shrimp and different countries were similar. Vibrio alginolyticus and V. harveyi (or V. carchariae) were dominant and detected in the species of shrimp cultured in the countries in the present study. The Vibrio species isolated from specie of shrimp cultured in different country were a little different from the others. V. harveyi was related to shrimp larvae disease. V. parahaemolyticus was related to adult shrimp disease. V. alginolyticus could be found both from health and diseased shrimp both.The other research was to attempt to make direct links between aquaculture. industrial pollution and their effects on biodiversity.Therefore, 428 isolates of Gram-negative bacteria were isolated in 1999 and 2000 from marine sediment, seawater. seaweed, fish and shellfish, taken from locations in Qingdao. Sampling locations were comprised of industrially and aquaculturally affected sites and a clean, control site. In order to analyze microbial diversity, a polyphasic approach was adopted for characterization of these isolates, specifically through examination of key phenotypic traits, i.e. using Biolog GN MicroPlateTM profiles. Of the 428 Gram-negative isolates examined using Biolog GN, 322 of them were identified at least to the genus level, or were unidentified, whilst 106 of them gave no profile (i.e. had no positive tests or all test results were positive). This technique yielded complex taxonomic data, which were subjected to statisticaland cluster analyses. The application of Biolog-GN method to studies of microbial communities is discussed. Marine bacteria biodiversity adjacent to aquacultural area, industral area compared with clean sites in Qingdao was evaluated according to the results of statistical and cluster analyses by Biolog-GN.It was shown that Virbio is the dominant one among the culturable bacteria in the marine costal environment and marine aquaculture environment and plays a significant role in the environment. Vibrios are active and in high abundance in aquatic ecosystems particularly in eutrophic environments, accounting for up to 14-45 %. The family Vibrionaceae harbours many taxa not yet described. The application of AFLP (amplified fragment length polymorphisms) has been shown to have a tremendous impact in the study of diversity, taxonomy and phylogeny of several bacterial genera. Genomic diversity amongst Vibrio isolates from differrnt sources (76% of them are from marine aquaculture environment) determined by fluorescent ampplified fragment length polymorphism was reported. Almost 47% of these isolates distributed in 31 clusters indicating that many culturable taxa of the Vibrionaceae remaine as yet to be described.Some vibrios FAFLP clusters were not grouped with type strains or reference strains used in this study. The closes更多还原