不同毒力溶藻弧菌脂多糖的多态性研究

提取了10株不同毒力的溶藻弧菌脂多糖,其中HN08811、HN08155、HN08813、HN08335、HN07006为毒力菌株,HN08805、HN08307、HN08156、HN08801和TG06003为非毒力菌株.分别取10μg进行SDS-PAGE电泳.结果表明,不同毒力溶藻孤菌脂多糖的电泳条带存在较丰富的多态性,在类脂质A区段(分离胶的上半部分),不同菌株间图谱呈片状堆积,多态性难以分辨;在抗原多糖O-特异性侧链条带区段(分离胶的下半部分),除HN07006外,毒力菌株的条带清晰,数目在7～9条之间,所有非毒力菌株产生细而密集的可辨条带.本文为一类遗传型的溶藻弧菌毒力菌株的快速鉴别提供了一种新的分子分型指标,也为溶藻弧菌的血清型分型提供了直接理论依据.     

养殖环境及贝源溶藻弧菌MLST分型及其毒力基因、耐药性分析

溶藻弧菌(Vibrio alginolyticus)是近年来贝类病害中最常见的细菌性病原之一，对贝类养殖产业的健康发展构成严重威胁。本研究旨在分析不同养殖环境水体和贝类组织中，溶藻弧菌的基因变异、毒力基因、耐药性及其分布规律。对12株溶藻弧菌分离株开展多位点序列分型(multilocus sequence typing, MLST)、毒力因子以及菌株耐药性分析，结果显示，12株溶藻弧菌的序列型(sequence typing, ST)分型互不相同，7株为PubMLST数据库已经收录的ST型，5株因管家基因的等位基因位点变化而形成新的ST型，贝类养殖环境中的溶藻弧菌具有较高的遗传多样性。12株溶藻弧菌都携带tlh、fur和collagenase三种毒力基因，但均未检测到tdh、trh、toxR和tcpA毒力基因。溶藻弧菌携带毒力因子的种类和数量受地区分布等因素的影响。不同来源的溶藻弧菌均具有多重耐药特征，对青霉素和氨苄西林产生抗性。本研究表明，贝类养殖环境中的溶藻弧菌具有种群复杂、遗传多样性高的特点；不同来源菌株在毒力基因携带和耐药性方面存在较大差异。本研究通过探究不同区域内不同来源的溶藻弧菌遗传变异及耐药性差异，对贝源溶藻弧菌的有效防控提供一定理论参考。     

基于核酸适配体的PCR法检测溶藻弧菌及其灭活菌

溶藻弧菌(Vibrio alginolyticus)分布广,数量多,发病率高,是水产养殖中常见的条件致病菌,而对溶藻弧菌进行快速准确的识别鉴定是其病害防治的前提和基础。核酸适配体,因为具有较高的亲和特异性,在微生物的识别鉴定方面展现出了巨大的优势。本文利用核酸适配体和适配体筛选产物,通过结合、洗涤、加热分离、PCR扩增以及电泳检测等步骤,对溶藻弧菌进行了检测鉴定。结果表明,适配体和筛选产物都能对溶藻弧菌及其灭活菌进行较好的识别鉴定,适配体筛选产物对溶藻弧菌的检测下限为10~3cfu/mL,而对其灭活菌的检测下限为10~2cfu/mL,适配体对溶藻弧菌及其灭活菌的检测下限都可达到10 cfu/mL。该方法对溶藻弧菌有较好的亲和特异性,并能较好地区分溶藻弧菌与哈维氏弧菌等水产常见病原菌,在水产病害的检测中显示了较好的应用前景。     

溶藻弧菌单克隆抗体的制备及应用

小鼠骨髓瘤细胞系SP2/0与经溶藻弧菌(1.1587)免疫的BALB3/C雌鼠脾细胞在PEG条件下融合,有45.8%的培养孔有杂交瘤细胞生长,其培养上清液抗体阳性率为77.4%.经反复有限稀释法克隆杂交瘤细胞,获得7株抗溶藻弧菌的单克隆抗体杂交瘤细胞株,分别为AC1-C9,AC1-C11,BB4-D4,AD1-A3,AD1-F3,AD2-E7,AD2-F7.取AD1-F3扩大培养,注射小鼠,制备了抗溶藻弧菌的单克隆抗体腹水,滴度为11000.该腹水与其他3株溶藻弧菌菌株有强交叉反应,与其他13株标准菌株无交叉反应.利用制备的单抗腹水,建立了检测溶藻弧菌的单抗-ELISA技术.该反应系统可应用于溶藻弧菌的快速诊断,反应时间为6-7h,检测灵敏度为104cells·-1.并利用该检测方法进行了11份待测菌样本溶藻弧菌的检测.     

水产品中溶藻弧菌PCR-变性高效液相色谱检测方法的建立

采用DHPLC 技术检测溶藻弧菌特异性PCR扩增产物,优化分析条件,探讨其灵敏度特异性,并与传统检测方法进行了比较.试验结果表明,该方法有很好的特异性,灵敏度较高.得到溶藻弧菌PCR-DHPLC特征峰型图,样本检测的峰型图可用于定性或定量监测样本中溶藻弧菌的动态变化.建立的水产品中溶藻弧菌PCR-DHPLC方法具有广泛而良好的适用性.     

养殖大黄鱼病原弧菌多重PCR检测技术的建立和应用

溶藻弧菌(Vibrio alginolyticus)、副溶血弧菌(vibrio parahaemolyticus)和哈维氏弧菌(Vibrio harveyi)是浙江省养殖大黄鱼(Pseudnosciaena crocea)弧菌病的主要致病菌.本研究选择针对溶藻弧菌和副溶血弧菌的胶原酶基因,哈维氏弧菌的部分ToxR基因的特异性,优化设计了3对特异性引物,通过进行多重PCR反应体系优化,多重PCR产物的测序鉴定与特异性和敏感性实验,建立了一种检测致病性弧菌的多重PCR检测方法.经过琼脂糖凝胶电泳后的条带分析判断,可以在一个PCR管中同时成功地检测这3种病原细菌,含溶藻弧菌、哈维氏弧菌和副溶血弧菌3种致病弧菌核酸的阳性对照样品分别扩增出大小为737 hp、382 bp和271 bp的预期产物,其灵敏度是102～102 CFU/mL.将该方法应用于检测人工感染后的养殖大黄鱼病鱼肝脏和肾脏,结果在6份组织样本中,5份检出原始感染菌株,与API 20E鉴定结果相符;对弧菌病流行季节采集的未发病的16份养殖大黄鱼组织样本和16份水体样本进行抽检,结果在1份大黄鱼组织样本中检出哈维氏弧菌,7份水体样本中检出这3种弧菌中的1种或2种,鉴定结果与API 20E鉴定结果符合率为93.75%.说明该方法不仅可以检测发病鱼,还可以检测无病症带菌大黄鱼以及带菌水样,且说明海洋水体中存在着大黄鱼弧菌病的致病菌.结果说明,多重PCR检测方法具有较高的敏感性与特异性,可以缩短检测时间,降低检测成本,该方法的建立对养殖大黄鱼弧菌病的快速诊断和分子流行病学的调查具有重要意义.     

引发杂色鲍苗掉板死亡的溶藻弧菌分离鉴定及毒力分析

跟踪观察了广东汕尾鲍鱼养殖场的培苗过程,并从变白病鲍苗中分离到1株优势菌(1号菌株).2次不同时间、不同地点的回归感染实验均证明其可引发鲍苗死亡掉板,API条带分析表明其为溶藻弧菌(Vibrio alginolyticus);胞外毒力因子分析揭示该菌株可分泌蛋白酶、脂肪酶、明胶酶以及拥有明显的溶血能力.结果证明了溶藻弧菌可引发我国南方杂色鲍苗掉板死亡.     

豚鼠气单胞菌快速检测间接ELISA法的建立

应用从患败血症的鳗鲡中分离、纯化出的豚鼠气单胞菌(Aeromonas caviae)菌株AB40511NA1,制备了兔抗血清,建立了快速检测豚鼠气单胞菌的间接ELISA方法.研究结果显示:AB40511NA1兔抗血清的效价为1:16000.该法对AB40511NA1最低检测量为6×104 cfu·mL-1;与水产养殖动物主要致病菌嗜水气单胞菌(A. hydrophila)、温和气单胞菌(A. sobria)、鳗弧菌(V. anguillarum)、溶藻弧菌(V.alginolyticus)、副溶血弧菌(V. parahaemolyticus)及非O1-群霍乱弧菌(Non-O1 Vibrio cholerae)均无交叉反应,特异性强;能快速、准确、方便地检测出豚鼠气单胞菌.     

溶藻弧菌外膜蛋白OmpK基因表达和间接ELISA检测方法的初步建立

溶藻弧菌是中国南部水产养殖业中弧菌病的最主要病原菌,数量居海洋类弧菌之首,其快速检测具有重要意义。以溶藻弧菌国内标准株ATCC17749基因组DNA为模板,PCR扩增出外膜蛋白OmpK基因,将其克隆入表达载体pET-28a中,获得重组质粒pET-28a-OmpK,限制性内切酶结合琼脂糖凝胶电泳分析和序列测定结果表明,该序列开放读码框正确且与已发表的OmpK结构编码序列完全一致。将重组质粒pET-28a-OmpK转化大肠杆菌BL21 pLys E,高效表达重组蛋白,将其纯化后免疫小鼠所得的高免血清能外膜蛋白OmpK特异性结合,表明体外表达的重组蛋白OmpK具有良好的免疫原性和反应原性。利用该抗溶藻弧菌外膜蛋白OmpK的抗血清建立了间接ELISA检测方法,检测灵敏度为10⁴ CFU/mL,能特异性检测出溶藻弧菌,与其他菌株无交叉反应。     

一株大黄鱼致病性溶藻弧菌的分离鉴定与毒力相关基因分析

2018年,宁德蕉城大湾渔排大黄鱼出现体表和鳍条溃疡、出血,鳃缺损等症状。为鉴定该菌株并评估其致病性,本研究从患病大黄鱼鳃部分离得到一株优势菌株,生理生化特征显示,该菌株可在TCBS培养基上生长,菌落颜色为黄色,为革兰阴性短杆菌,具有嗜盐性,生理生化指标与标准株17700一致。结合生理生化指标与16S rDNA分析,确定该菌为溶藻弧菌,命名为Val180620。攻毒结果显示,Val180620菌株对大黄鱼具有较强的致病力,在水温25℃腹腔注射感染时,对规格(20±2)g大黄鱼的LD_(50)为2.38×10~6 CFU。毒力相关基因分析结果表明,Val180620菌株携带有CollagenaseVA、ToxR、FlaA和TRH等毒力相关基因。此外,本研究未从菌株Val180620中检测出UreR基因,但菌株Val180620仍表现出较强的致病性,说明UreR不是决定溶藻弧菌毒力的主要因子。     

六边形开口方形人工鱼礁阻力系数数值模拟与模型试验比较研究

为了对4种不同开口大小的六边形开口方形人工鱼礁在4种迎流角度下数值模拟和水槽模型试验的阻力系数进行对比验证，分别利用水槽模型和数值模拟试验方法对4种不同开口比（γ_ty=0.0625，0.14，0.25，0.39）六边形开口方形人工鱼礁在4种迎流角度（θ=0°，15°，30°，45°）状态下的阻力进行测定，并计算两种方法的阻力系数。结果表明：（1）在数值模拟和水槽模型试验中，人工鱼礁模型阻力均随着开口比的增大而减小；礁体迎流角度的变化可改变礁体阻力，且在4种迎流角度下，人工鱼礁阻力在θ=30°时最大。（2）人工鱼礁数值模拟与水槽模型试验中，当θ为15°、30°和45°时，阻力系数均随着开口比的增加而增加，具有明显的线性关系，且阻力系数在迎流角度θ=30°时最大。（3）数值模拟与模型试验阻力的相对误差在0.12%~17.18%，平均误差7.43%；礁体阻力系数的相对误差在0.03%~14.64%，平均误差5.26%。阻力及阻力系数误差均在20%以下。水槽模型试验与数值模拟阻力和阻力系数相关系数R分别为0.99和0.80，P<0.001，具有极强的相关性。因此，利用数值模拟精细化研究人工鱼礁水动力性能是可行的。     

Trophic relationships between a native and a nonnative predator in a system of natural lakes

Abstract – Bull trout, a species of char listed as threatened under the US Endangered Species Act, have been displaced from portions of their historic range following the introduction of nonnative lake trout. It has been suggested that competitive exclusion as a result of trophic overlap between bull trout and lake trout may be the causal mechanism associated with displacement of bull trout. This study used stable isotope data to evaluate trophic relationships among native bull trout, nonnative lake trout and other fishes in seven lakes in Glacier National Park (GNP), Montana. Bull trout and lake trout had greater δ¹⁵N values relative to other fishes among lakes (δ¹⁵N ≥ 3.0‰). Lake trout had greater δ¹⁵N values relative to bull trout (δ¹⁵N = +1.0‰). Bull trout had greater δ¹³C values relative to lake trout in six of the seven lakes examined. Although both bull trout and lake trout had greater δ¹⁵N values relative to other fishes within lakes in GNP, differences in δ¹⁵N and δ¹³C between bull trout and lake trout suggest that they are consuming different prey species or similar prey species in different proportions. Therefore, displacement of bull trout as a direct result of complete overlap in food resource use is not anticipated unless diet shifts occur or food resources become limiting. Additionally, future studies should evaluate food habits to identify important prey species and sources of partial dietary overlap between bull trout and lake trout.     

Characterization of a cDNA encoding a homolog of actin-related protein 4 from a marine red alga Porphyra yezoensis

ABSTRACT:   A cDNA ( PyARP4 ) containing an open reading frame for a protein of 573 amino acids was identified in the marine red alga Porphyra yezoensis . The conceptual PyARP4 protein exhibits significant similarity to actin-related protein (ARP) 4 in the terrestrial plant Arabidopsis . Comparison of the deduced amino acid sequence showed moderate sequence identity (30%) to a conventional actin in P. yezoensis , as seen in comparisons between ARP and conventional actins of other organisms. A putative bipartite nuclear localization signal and an actin motif were found within the PyARP4 amino acid sequence. In a phylogenetic analysis, the PyARP4 was found to cluster with the ARP4 of other organisms. The expression level of PyARP4 did not change significantly among four developmental stages of life cycle and was lower than that of a conventional actin. This cDNA therefore may serve as a useful internal standard in gene expression analyses of differentially expressed genes in P. yezoensis .     

山区水库网箱养殖黄颡鱼试验

网箱面积30 m2,底部设饲料托盘。黄颡鱼规格46.3 g/尾,先用动物性饵料驯食1个月,后投喂粗蛋白质量分数38%颗粒饵料。加强管理,药物预防鱼病。黄颡鱼成活率87%,平均规格206 g/尾,平均单产19.6 kg/m2,利润284.8元/m2。     

Habitat characteristics affecting occurrence of a fluvial species in a watershed altered by a large reservoir

Flooding river valleys following construction of dams restrict fluvial environments to reaches that were formerly headwaters. Whether remaining habitat is suitable for all life stages of fluvial species is poorly understood. A fluvial species, Arctic grayling Thymallus arcticus, showed a dramatic decline following flooding of the Upper Peace River and the formation of the Williston Reservoir. We related landscape and field site‐specific features with occurrence of juvenile Arctic grayling using an information theoretic approach. For the landscape model, an association was identified between stream order and Arctic grayling occurrence although stream order alone was a poor predictor. A positive association between juvenile Arctic grayling occurrence and distance from the Williston reservoir and stream order, as well as a negative association with water temperature and temperature variance, was deemed important for the field site model. Both modelling approaches indicated size of stream system to be an important influence on occurrence of juvenile grayling in the Williston watershed. River length required for suitable river habitat for salmonids has not previously been identified, but should be factored into future management plans when evaluating the impact of proposed hydroelectric dams and subsequent flooding of river systems.     

The isoelectric point, a key to understanding a variety of biochemical problems: a minireview

We address the importance of the isoelectric point (IEP) of proteins and membrane components such as phospholipids for our understanding and interpretation of isoforms and opposite charge interactions in the formation of complexes. Five examples drawn from the literature are newly approached from the IEP point of view to clarify general principles.     

The potential exceptional role of a small Baltic boulder reef as a solitary habitat in a sea of mud

1. In this study, non‐destructive seafloor imaging techniques were employed to assess the benthic community structure of a recently discovered boulder reef within the central Arkona Basin of the Baltic Sea.
2. Data indicate that geographical isolation, water column stratification and temporary oxygen deficiency create an exceptional reef habitat in a sea of mud.
3. The reef forms a saline refuge for the deepest and easternmost known population of the plumose sea anemones (Metridium dianthus Ellis, 1768) in the Baltic Sea.
4. Limited connectivity to other reefs and dominance of the aggressively colonizing and planktivorous M. dianthus hamper the recruitment of other species and lead to an overall low invertebrate richness.
5. Large boulders attract ecologically and economically important fish like Baltic cod (Gadus morhua Linnaeus, 1758) that find shelter in the otherwise heavily fished Arkona Basin.
6. Despite this very first exploration being only a snapshot of the observed community, findings imply a potential conservation importance of this Germany's deepest known natural habitat type 1170 (‘reefs’).

     

Rapid start-up of a biological filter in a closed aquaculture system

Seeding of new biological filters with nitrifying bacteria is effective in reducing activation time of the new filters. In a series of tests, seeded filters were activated in an average of 10.7 days, compared with 22.2 days for the unseeded controls. The shorter activation period results from the reduced time required for the nitrifying organisms to increase in numbers to their environmental equilibrium in the system tested.