基于16S rRNA和recA香鱼鳗利斯顿氏菌的分离鉴定

为研究引起香鱼(Plecoglossus altivelis)出血溃烂症病因及致病菌系统发育地位,本研究从患病香鱼的肝脏、肾脏及体表分离到11株病原菌(编号:X0901-X0911),运用常规细菌生理生化方法鉴定致病菌所属种类;运用16SrRNA基因、recA基因序列分析方法研究致病菌的系统发育地位。细菌生理生化鉴定结果表明:致病菌为鳗利斯顿氏菌,11株细菌生理生化结果相同,均为革兰氏阴性杆菌、氧化酶阳性、接触酶阳性、吲哚阳性、精氨酸脱羧酶阳性、精氨酸双水解酶阳性、硝酸盐还原阳性、甘露醇阳性、MR测定阳性;H2S阴性、V-P测定阴性等。根据16SrRNA基因、recA基因序列分别构建弧菌科常见细菌系统进化树,结果表明:致病菌与鳗利斯顿氏菌(Listonella anguillarum)均聚为一枝,聚类结果与细菌生理生化鉴定结果相符。致病菌与鳗利斯顿氏菌16SrRNA基因、recA基因的同源性分别为99.9%、99.8%。以recA基因构建的系统进化树的拓扑学结构与16SrRNA基因建树结果相类似。此外,与16SrRNA基因相比,recA基因在不同物种之间具有更高的鉴别能力,本研究表明recA基因适合作为弧菌科常见细菌物种间进化关系研究的标记。     

杂色鲍血细胞免疫特点及免疫功能的研究

杂色鲍Haliotis diversicolor感染病毒后血细胞总浓度呈现上升趋势, 颗粒细胞与无颗粒细胞浓度与百分比均呈现不同的变化;感染鲍抗菌活力在第24h时最高, 说明病毒感染刺激病鲍血细胞产生抗菌活性, 第36h时的抗菌活力大大下降;感染鲍在第6h时, 溶菌活力大大下降, 且一直持续至36h;血细胞内的活性氧总体呈明显的上升趋势;受感染的血细胞出现细胞器空泡化和分解.说明病毒感染对杂色鲍血细胞免疫功能的影响非常明显.     

拮抗香蕉枯萎病菌海洋细菌的筛选和鉴定

从93份中国南部海域近海海泥样品中分离出1800多株海洋细菌,以对峙培养法,测定海洋细菌对香蕉枯萎菌的拮抗作用,再以琼脂划线培养法(agar streak)测定菌株的抑菌活性,结果表明:约55.6%的菌株对香蕉枯萎菌有一定的拮抗作用,其中58株菌株抑制香蕉枯萎菌生长所形成的抑菌带宽度大于10.0mm,抑菌作用较好,复筛后其中13个菌株形成的抑菌带宽在15.0mm以上。经反复转接和对峙培养,筛选出拮抗效果好、抑菌活性稳定的菌株TC-1,初步鉴定为芽孢杆菌属的细菌,TC-1对14种病原微生物都有较好的抑菌效果。     

半滑舌鳎病原鳗利斯顿氏菌表型及分子特征研究

2008年12月,江苏赣榆某渔场养殖半滑舌鳎出现大量死亡,症状主要表现为:头部、鳃盖及鳍基出血,尾鳍腐烂,腹腔膨胀并积有大量腹水,部分病鱼肠管脱出肛外。从病鱼肝脏、腹水中分离出大量优势生长的细菌,人工感染试验证明其对半滑舌鳎有较强的致病性。对3株分离菌进行了形态特征、理化特性、胞外酶及溶血素活性等表型生物学性状检验;测定了代表菌株的16S rRNA和gyrB基因序列,分析了16S rRNA和gyrB两种基因序列的同源性,并构建了系统发生树,比较了两种基因对分离菌的鉴定能力;结果表明gyrB基因用于细菌种间鉴定更具优越性;基于16S rRNA和gyrB基因的系统发育学分析表明分离菌与鳗利斯顿氏菌具有高同源性,根据分离菌的表型及分子特征,判定分离菌为利斯顿氏菌属(Listonella MacDonell and Colwell 1986)的鳗利斯顿氏菌[Listonella anguillarum (Bergeman 1909) MacDonell and Colwell 1986]。胞外酶活性及溶血活性检测表明3株分离菌均具有淀粉酶、蛋白酶、卵磷脂酶等胞外酶活性,在含7%家兔脱纤血液营养琼脂培养基上呈β型溶血,PCR检测3株供试菌均可扩增出大小约493 bp的溶血素基因和大小约248bp的金属蛋白酶基因。分离菌的耐药谱测定结果显示,对供试49种抗菌药物中的青霉素G等13种药物耐药,对羧苄青霉素等6种药物存在敏感与耐药的株间差异。     

A culture-dependent survey of thermophilic bacteria from hot springs in Xiamen area in China

Microbes are believed to play important roles in ecosystem function in many environments. The hot springs of Xiamen Island are close to the Xiamen Sea, and may have some characteristics different from those of inland hot springs. Microbes living in the hot springs of Xiamen may have new characteristics. However, little is known about microbial communities of hot springs close to the Xiamen Sea. A culturedependent survey of microbial population in the Xiamen hot springs was performed by using an approach combining total cellular protein profile identification and 16S rRNA gene sequencing. A total of 328 isolates of bacteria were obtained from liquid and sediment samples from the Xiamen hot springs, including neutrophilic thermophilic bacteria and moderately thermophilic acidophiles. Neutrophilic thermophilic bacteria, which grow at a temperature range of 55—90℃ including Rhodothermus marinus (Strain 1), Thermus thermophilus (Strain 2), Thermus thiopara (Strain 3), Geobacillus stearothermophilus (Strain 4), Geobacillus thermoleovorans (Strain 5), and Pseudomonas pseudoalcaligenes (Strain 6), were recovered by 2216E plates. Moderately thermophilic acidophiles, which can grow at temperatures above 50℃ and a pH range of 1.8—3.5 such as Alicyclobacillus acidoterrestris (Strain 8), Sulfobacillus acidophilus (Strain 9), and Sulfobacillus thermosulfidooxidans (Strain 10), were isolated on selective solid medium containing sulfur and Fe2+. Among these strains, Rhodothermus marinus, Thermus thermophilus and Geobacillus stearothermophilus are not only thermophiles, but also halophiles. One bacterium strain (Strain 6) shared 99% nucleotide sequence homology with Pseudomonas pseudoalcaligenes on the 16S rRNA gene sequence, but was quite different from Pseudomonas pseudoalcaligenes in biological characteristics, suggesting that it may represent a novel thermophilic species. Results indicated that various species of neutrophilic thermophiles and moderately thermophilic acidophiles were widely distributed in the Xiamen hot springs and that Rhodothermus marinus and Thermus thermophilus dominated the cultivable microbial community.     

4种微生态制剂对对虾育苗水体主要水质指标的影响

研究了由球形红假单胞菌(Rhodopseudomlonas sphaeroides)、噬菌蛭弧菌(Bdellovibrio bacteriovorus)及黏红酵母(Rhodotorula glutinis)制成的4种微生态制剂对中国对虾(Fenneropenaeus chinensis)育苗水质的影响.结果表明,试验组与对照组相比都能明显净化水质,试验组中以添加噬菌蛭弧菌和黏红酵母组的效果最差,添加球形红假单胞茵和噬茵蛭弧茵组的效果最好,此组水质的pH值稳定,NH4+-N明显下降,而亚硝酸盐、化学需氧量及硫化物等指标也优于其他试验组.统计分析显示,试验组与对照组相比,细菌数降低了3个数量级,添加嗜菌蛭弧菌试验组在减少异养茵(包含有害细菌)方面效果较好.同时用鳗弧菌攻毒,在5 d内,对照组的死亡率显著高于试验组(P<0.05),试验组免疫保护率为29.4%～58.5%,以添加嗜菌蛭弧菌和球形红假单胞菌组最高.建议联合使用噬菌蛭孤菌和球形红假单胞菌,混合密度初步定为2×105个/mL.     

养殖大菱鲆细菌性红体病病原菌的分离与鉴定

从患有红体病的养殖大菱鲆(Scophthalmus maximus)的肌肉中分离得到一株优势菌并记为H1.经人工注射感染证实H1即为引起养殖大菱鲆红体病的病原菌,其半致死量LD50为2.82×105CFU/mL,而低浓度(1.41×103 CFU/mL)没有造成死亡,但注射部位有脓肿现象,注射相同体积1.5%无菌生理盐水的对照组没有明显的症状,注射部位也无异常.革兰氏染色显示该菌为革兰氏阴性,菌体呈杆状,周生鞭毛.综合该菌的形态、常规生理生化特征和API32E鉴定结果,发现H1与迟钝爱德华氏菌的表性特征非常相似,相似率迭到99.9%.在分子水平上对其16SRNA基因序列的测定,同源性分析,结果表明H1与迟钝爱德华氏菌的亲缘关系最近,相似度达到99%.综合上述研究结果,将该菌株初步鉴定为迟钝爱德华氏茵(Edwardsiella tarda).     

The selection of alkaline protease-producing yeasts fi＇om marine environments and evaluation of their bioactive peptide production

A total of 400 yeast strains from seawater, sediments, saltern mud, marine fish guts, and marine algae were obtained. The protease activity of the yeast cultures was estimated, after which four strains (HN3.11, N11b, YF04C and HN4.9) capable of secreting extracellular alkaline protease were isolated. The isolated strains were identified as Aureobasidium pullulans, Yarrowia lipolytica, Issatchenkia orientalis and Cryptococcus cf. aureus. The optimal pH of the protease activity produced by strains HN3.11, YF04C, and HN4.9 was 9.0, while that of the protease produced by strain N11b was 10.0. The optimal temperature for protease activity was 45°C for strains HN3.11, N11b, and YF04C, and 50°C for strain HN4.9. After digestion of shrimp (Penaeus vannamei) protein and spirulina (Arthospira platensis) protein with the four crude alkaline proteases, the filtrate from spirulina (Arthrospira platensis) powder digested by the crude alkaline protease of strain HN3.11 was found to have the highest antioxidant activity (61.4%) and the highest angiotensin I converting enzyme (ACE)-inhibitory activities (68.4%). The other filtrates had much lower antioxidant activity and ACE-inhibitory activities.     

迟缓爱德华氏菌致病相关因子的研究进展

迟缓爱德华氏菌(Edwardsiealla tarda)是引起多种鱼类产生爱德华氏菌病(edwardsiellosis)的病原菌。随着水产养殖业的日益发展,致病性迟缓爱德华氏菌对水产养殖业的危害也越来越严重。有关迟缓爱德华氏菌的致病机理至今还没有系统、清晰的阐释,但近些年对其致病相关因子的研究已逐渐深入。文中对迟缓爱德华氏菌的生物分型、快速检测技术等进行了简要阐述,并对该病原菌的主要致病相关因子,包括黏附因子、抵抗宿主免疫机制的酶类、各种毒素、Ⅲ型和Ⅵ型分泌系统、载铁体及磷酸盐特异转运操纵子进行了详细阐述,以期为迟缓爱德华氏菌致病机理的进一步深入研究及其防治提供参考和借鉴。     

一种新的流域水沙关系模型及其在年际时间尺度的应用

根据陕西子洲试验站9个流域和山西离石试验站3个流域的观测数据,研究了黄土丘陵沟壑区次暴雨径流和产沙的关系。结果表明:在研究区,当流量超过某一临界值后,含沙量保持稳定,且次洪平均含沙量主要取决于大流量时段,故对于较大的洪水事件,其平均含沙量也趋于稳定,次暴雨和次暴雨过程两个时间尺度的水沙关系表现出很好的相似性。因此可用正比关系式来拟合较大洪水次暴雨径流深和产沙模数之间的关系。由于极端事件对研究区水土流失的重要性,使得该模型有很好的实用性。模型在年际时间尺度上的应用结果表明,对仅在次暴雨期间产流的地区,该模型可以很好地预测年产沙量,而对于常年流水的流域,在丰水年根据汛期径流量该模型也有较好的计算精度。