【作者】 王燕；

【导师】 张晓华；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2010， 博士

【摘要】 当今世界,人口不断增长,自然资源日益匮乏,为解决陆地资源的局限性,人们的注意力开始转向海洋。海洋是地球上最大的生物栖息地,在这一环境中生存的海洋细菌资源越来越受到人们关注。有些海洋细菌在生物制药、海洋污染物的生物降解和修复、生态系统物质循环、海水养殖有益菌的筛选等方面起着关键作用,而有些海洋细菌种类给人们带来极大危害,如海水养殖动物甚至人类病原菌、造成生物污损和环境污染的海洋细菌等。为综合开发利用海洋细菌资源,充分“发挥”海洋细菌资源的“优点”,消除控制有害菌或化弊为利,为人类所用,本论文从海水养殖动物病原菌(避短)和海洋新菌资源(扬长)两个方面对海洋细菌进行了研究。工厂化养殖的发展、养殖条件的恶化以及病害防治措施的不合理采用等多种因素,使养殖环境中的微生物区系平衡遭到严重破坏,海水养殖动物病害尤其是细菌性病害频繁发生,新的海水养殖病原菌不断出现。本实验分别从患病半滑舌鳎(Cynoglossus semilaevis Gunther)和大菱鲆(Scophthalmus maximus)中分离纯化出优势菌株WY06和WY28,人工感染实验证实二者均能使宿主和模式动物斑马鱼(Danio rerio)致病,WY06对半滑舌鳎和斑马鱼的半数致死量分别为5.5×103 cfu/g (5.2×105 cfu/fish)和1.9×103 cfu/g (8.9×102 cfu/fish); WY28对大菱鲆和斑马鱼的半数致死量分别为39 cfu/g (3.3×102 cfu/fish)和2.1×104 cfu/g (6.4×103 cfu/fish)。WY06和WY28分别与美人鱼发光杆菌杀鱼亚种(Photobacterium damselae subsp. piscicida)和迟缓爱德华氏菌(Edwardsiella tarda)的16S rDNA序列相似性为99.0%和99.9%。综合二者在形态、生理生化特征及16S rDNA同源性等方面的实验结果,确定WY06为美人鱼发光杆菌杀鱼亚种,WY28为迟缓爱德华氏菌。美人鱼发光杆菌杀鱼亚种作为海水养殖动物病原菌在国内属首次报道。迟缓爱德华氏菌是多种淡、海水鱼类的主要病原菌之一,给美国、欧洲各国以及包括中国在内的多个亚洲国家的多种鱼类养殖造成了巨大的经济损失,近几年,一直影响着我国大菱鲆养殖。关于该菌的致病机理至今尚不清楚,成为研究病害防治措施的瓶颈。为寻找该菌的致病因子,本研究对不同来源的迟缓爱德华氏菌致病菌株与非致病菌株的胞外酶活性、溶血性、自动凝集现象、血凝集作用、细胞表面疏水性、血清抗性、胞外蛋白和外膜蛋白图谱、粗提脂多糖和类脂A致病性等进行了比较,发现迟缓爱德华氏菌具有种内多样性。在致病菌株的外膜蛋白和胞外产物中存在三条低分子量的蛋白条带,但不存在于非致病菌株,其功能有待于进一步确定。人工感染实验证实,只有肌肉注射致病菌株的粗提脂多糖和类脂A,才能使鱼致死,从而证实了脂多糖是迟缓爱德华氏菌的重要致病因子之一,并支持了类脂A是脂多糖生物活性中心这一论点。为寻找环境友好型爱德华氏菌病的防治措施,将迟缓爱德华氏菌WY28菌株经福尔马林灭活制成灭活疫苗,对大菱鲆进行腹腔注射免疫,免疫后第四周测得免疫鱼血清中抗迟缓爱德华氏菌的抗体效价为1：1280,第六周测得免疫鱼血清中抗迟缓爱德华氏菌的抗体效价为1：3289。低剂量迟缓爱德华氏菌攻毒试验表明,受免鱼的相对存活率明显高于对照组。由此可见,利用从病鱼体内分离的迟缓爱德华氏菌菌株制备的灭活疫苗能使大菱鲆较有效地抵御迟缓爱德华氏菌强毒株的攻击。此外,将大蒜作为口服免疫刺激剂,饲喂虹鳟鱼一周,对爱德华氏菌病的抵抗力明显提高。基于16S rDNA序列分析的研究方法显示,绝大多数海洋细菌都未获得纯培养,这就意味着海洋细菌资源还具有很大的开发潜力,还蕴藏着大量未被开发利用的海洋细菌新种,而新的海洋细菌可能产生新的生物活性物质,所以本论文对海洋细菌研究的另一方面是海洋新菌资源的开发。本研究分别从健康中国对虾(Penaeus chinensis, O’sbeck)肠道、黄海水域表层海水中分离出菌株HHS02T, WH169T和DFH11T。HHS02T菌株革兰氏染色阴性,弧形,极生单鞭毛,运动,过氧化氢酶阴性,氧化酶阳性,兼性厌氧,对弧菌抑制剂O/129敏感；WH169T菌株革兰氏染色阴性,极生单鞭毛,短杆状,过氧化氢和氧化酶阳性,严格好氧。在2216E液体培养基中28℃培养3天或更长时间,菌体自动聚集。在2216E平板上28℃培养12天,有出芽现象并有菌柄产生；DFH11T菌株革兰氏染色阴性,嗜冷,严格好氧,螺旋状,极生单鞭毛,过氧化氢酶阴性,硝酸盐还原酶阳性。16S rDNA序列比对结果显示,它们分别与Vibrio sp., Aestuariibacter sp和Oleispira sp的16S rDNA序列相似性为98.5%,95.1%和97.2%,综合菌株多位点序列分析结果、化学分类、生理生化、DNA G+C含量、DNA-DNA杂交以及其他特征,证明三者均为海洋新菌,分别命名为非典型弧菌(Vibrio atypicus),海滩凝集杆菌(Aestuariibacter aggregatus)和惰性解油螺旋菌(Oleispira lenta),保存于两个不同国家的国际承认的菌种保藏中心。更多还原

【Abstract】 In recent years, pressures on continental resources have grown with rapid increasement of population and subsequent consumption. Resources exploitation switched from continent to ocean. The marine environment is the largest habitat on the earth. Within this environment, research of marine bacteria is a growing field of interest. Marine bacteria are an important and relatively unexploied resource for novel microbial products. Some species of marine bacteria are great of benefit to human, e.g. biopharmaceuty, bioremediation, biogeochemical cycles and prevension of diseases in aquaculture (probiotics). However, some spieses of marine bacteria are harmful to human, e.g. fish and human pathogens, marine biofouling. Thus, marine bacteria resources should be exploited appropriately--taking its advantages and controlling its disadvantages. This research studied marine bacteria bilaterally, e.g. bacterial pathogens of fish and exploitation of new marine bacterial species.Development of intensive marine aquaculture, poor management practices and abuse of antibiotics, disturb the stable microbial banlance and cause diseases, especially bacterial dieases in aquculture, sometimes lead to emergence of new pathogens. In present study, two strains WY06 and WY28 were isolated form diseased tongle sole (Cynoglossus semilaevis Gunther) and turbot(Scophthalmus maximus), respectively. Pathogenicity assays revealed that WY06 was virulent to tongue sole and zebrafish (Danio rerio) by intraperitoneal injection challenge, with the LD50 being calculated as 5.5×103 cfu/g of fish (5.2×105 cfu/fish) and 1.9×103 cfu/g of fish (8.9×102 cfu/fish) respectively. WY28 was virulent to turbot and zebrafish by intraperitoneal injection challenge, with the LD50 being calculated as 39 cfu/g (3.3×102cfu/fish) and 1.9×103 cfu/g of fish 2.1×104 cfu/g (6.4×103cfu/fish) respectively. WY06 and WY28 showed 99.0% and 99.9% 16S rDNA sequences similarity to Photobacterium damselae subsp. piscicida and Edwardsiella tarda respectively. On the basis of the polyphasic taxonomic evidence presented in this study, it can be concluded that WY06 and WY28 belong to P. damselae subsp. piscicida and E. tarda respectively. P. damselae subsp. piscicida was described as fish pathogen for the first time in China.E. tarda, which had a wide range of host, caused vital economic losses in aqucuture of Ameriacan, European and Asian countries including China. In past few years, E. tarda affected the turbot cultre in China. However, the pathogenic mechanism of the organism still remained to be studied. In order to find pathogenicity related factors, caseinase, gelatinase, elastase, phospholipase, lipase, haemolytic, hydrophobic and serum resistance activities, haemagglutination, autoagglutination, siderophore and profiles of extracellular protein and outer membrane protein of virulent and avirulent strains of E. tarda were examined and compared. There are three low molecular-weight bands that are prensent in vilurent strains but not in avirulent strain. However, their identities were not verified in this study. The pathogenicity of live cells, lipopolysaccharides (LPS) and lipid A have been tested and compared with those of avirulent strains, using rainbow trout (Oncorhynchus mykiss Walbaum) as a model fish. The result revealed that LPS was one of the virulence factors of E. tarda and lipid A was a biologically active determinant of LPS as previously reported.In order to find a safe and heathy way to control fish diseases caused by E. tarda, turbots were vaccinated with formalin-killed vaccine of WY28 via intraperitoneal injection. Controls were injected with the same volumes of saline. The evident antibody titres (average was 1:1280) were detected in vaccinated fish 4 weeks after immunization. The antibody titres reached higher level (average was 1:3289) 6 weeks after immunization. Vaccinated turbots were intraperitoneal injected with different concentrations of WY28 to detect relative percent survival (RPS) of formalin-killed vaccine of WY28. The vaccinated fish showed higher RPS against bacteria than the unvaccinated fish. Therefore, the inactivated vaccine of WY28, to a large extent, could prevent turbot diseases that caused by E. tarda. Furthermore, garlic was used as immonostimulant to feed rainbow trout. Pathogenic assay revealed that fish feeded with garlic showed higher RPS against E. tarda than the control fish. Thus, garlic was a valuable candidate of disease control plants.Based on 16S rDNA sequences analysis, only 1% of marine bacteria were isolated and identified. This means most of the marine bacteria resources have not been explored. Another point of this research is to explore new species in marine environment. In this study, three strains HHS02T, WH169T and DFH11T were isolated from healthy Chinese prawn(Penaeus chinensis, O’sbeck) and superficial seawater of Yeallow Sea, respectively. Strain HHS02T comprised slightly curved rod-shaped, Gram-negative, non-endospore-forming, catalase-negative, oxidase-positive,O/129 sensitive, facultatively anaerobic cells that were motile by means of single polar flagella. Strain WH169T was a Gram-negative, non-spore-forming, catalase-and oxidase-positive, strictly aerobic, short rod-shaped bacterium with a single, polar flagellum. The cells aggregated together when incubated at 28℃for more than 3 days in marine 2216E broth. Buds and prosthecae are formed when it was grown at 20℃for 12 days on marine 2216E agar. DFH11T strain comprised Gram-negative motile, psychrophilic, nitrate reductase positive, strictly aerobic spirilli that did not produce catalase. HHS02T, WH169T and DFH11T showed 98.5%,95.1 and 97.2% 16S rDNA sequences similarity to Vibrio sp., Aestuariibacter sp. and Oleispira sp. respectively. Based on differential phenotypic properties, fatty acid profile,16S rRNA gene sequence and MLSA results, it is concluded that HHS02T, WH169T and DFH11T represents the novel species for which the name Vibrio atypicus sp. nov., Aestuariibacter aggregatus sp. nov. and Oleispira lenta sp. nov. were proposed respectively. The strains are deposited in two recognized culture collections in two different countries.更多还原