鲶爱德华氏菌间接酶联免疫快速检测法的建立

用鲶爱德华氏菌兔抗血清作为一抗,碱性磷酸酶(AP)标记的羊抗兔IgG作为酶标二抗,建立黄颡鱼"红头病"病原菌—鲶爱德华氏菌的间接酶联免疫(ELISA)快速检测法,并优化检测条件。抗原最佳包被浓度为107/mL,一抗工作的最佳稀释度为1∶211,病原菌的检测灵敏度为105/mL,交叉反应实验证明该方法特异性强,与迟钝爱德华氏菌、弧菌等13种标准菌株无交叉。应用上述技术对人工感染发病鱼中分离的优势菌进行检测,结果表明阳性检出率为80%;对自然发病黄颡鱼体内分离获得的20株优势菌检测结果表明,12株菌为鲶爱德华氏菌。     

斑点叉尾鮰保科爱德华氏菌的分离鉴定和组织病理观察

对患病斑点叉尾鮰的肝脏病灶进行细菌分离培养,经API20E细菌生化快速试剂条鉴定为保科爱德华氏菌(Edwardsiella hoshinae)。人工感染试验可再现自然条件下鱼病的症状,表现出很强的致病性。病变组织切片观察发现,病变最初是部分肝细胞发生颗粒变性及水样变性,颗粒变性的肝细胞肿大。严重时,细胞周界不清晰,肝细胞内的小空泡连成大空泡,核悬浮在肝细胞的中央,细胞核肿大,并出现部分肝细胞的核浓缩、破裂、溶解消失。保科爱德华氏菌对斑点叉尾鮰具有显著致病性,可引起肝脏发生严重病变。     

溶藻弧菌免疫胶体金快速检测试纸条的制备

采用溶藻弧菌ATCC17749菌体作为抗原免疫新西兰大白兔,制备兔抗溶藻弧菌多克隆抗体;利用柠檬酸钠还原氯金酸法制备胶体金及胶体金-抗体结合物,并将其喷涂在玻璃纤维膜上冷冻干燥;在硝酸纤维素膜上包被兔抗溶藻弧菌多克隆抗体和羊抗兔IgG分别作为检测线和控制线;将处理后的玻璃纤维素膜、硝酸纤维膜、样品垫及吸水纸组装成双抗夹心试纸条,并对试纸条灵敏度、特异性、稳定性进行评价。结果表明,制备的兔抗溶藻弧菌多克隆抗体的效价可达1∶512 000;胶体金溶胶的最佳制备条件为:加入1.8 mL 10 mg/mL的柠檬酸三钠,500 W微波炉中火加热10 min;试纸条检测线和质控线的抗体最佳包被量分别为8.0 mg/mL和1.0 mg/mL;试纸条检测灵敏度为1×106cfu/mL,特异性试验显示,试纸条与哈氏弧菌、副溶血弧菌、霍乱弧菌、拟态弧菌、美人鱼弧菌、弗氏弧菌、河流弧菌、创伤弧菌等8株菌无交叉反应;稳定性试验表明,试纸在4℃干燥条件下能稳定保存5个月以上。制备的溶藻弧菌多克隆检测试纸条可灵敏、较特异地检测源自病鱼的溶藻弧菌,整个检测过程可在10 min内完成,适用于溶藻弧菌的快速检测。     

温度对鳜皮肤黏膜抗体产生的影响

应用间接酶联免疫吸附试验(ELISA)技术测定不同水温下鳜经嗜水气单胞菌GYK1株全菌灭活疫苗浸泡免疫后皮肤黏液中抗体,以研究抗体动态变化,揭示温度对抗体产生的影响。结果表明:27.0~29.8℃水温(下称"较高温")下,鳜皮肤黏膜抗体滴度于第7天达到峰值211,而水温介于19.8~23.9℃(下称"较低温")时,抗体滴度于第11天达到峰值26,说明温度较高时,抗体形成快,峰值出现时间早,并且抗体滴度明显升高。另取不同温度下经上述疫苗浸泡免疫的鳜皮肤黏液,采用硫酸铵盐析、离心、浓缩等生物化学技术粗提鳜皮肤黏液中的免疫球蛋白,SDS-PAGE检测发现,较高水温下,高渗免疫组(加10 mg/mL食盐)鳜皮肤黏液蛋白中目标条带(72×103和29×103)深度较其他3个实验组(同温度下全菌组、较低水温下高渗及全菌组)有所增加,ELISA检测该组抗体效价为211。     

演艺海豚混合感染大肠埃希氏菌和变形杆菌的检验分析

解剖了一条出现腹泻等症状、经治疗无效死亡的海豚 ,发现肺、肝、脾、胃等均有病变 ,从病变组织器官中分离到 7株细菌 ,通过菌株的形态、染色特征、生化特性、血清学鉴定 ,发现其中 5株菌株为大肠艾希氏菌 O8血清型 ,另 2株分别为普通变形杆菌和奇异变形杆菌。动物试验表明 ,大肠埃希氏菌和变形杆菌是海豚发病的致病菌。 7个菌株均对菌必治和先锋噻肟高敏 ,对氯霉素、链霉素、痢特灵等中敏 ,对青霉素 G、氨苄青霉素等耐药     

广东省罗非鱼主养区无乳链球菌的分离、鉴定与致病性

从广东省5个罗非鱼主养区患暴发性的罗非鱼中分离到15株菌,挑选其中的5株(不同地域、不同组织分离的菌株)进行人工感染试验,表现出自然发病的症状,确定此5株分离菌为导致罗非鱼暴发病的主要病原。通过形态学观察和生理生化实验,并结合16SrRNA基因序列分析,5株分离菌株均为无乳链球菌(Streptococcus agalactiae)。5株分离菌在形态学和生理生化实验结果上保持一致,16SrRNA基因序列同源性达98.6%～99.8%,但在致病性和对药物的敏感性上却呈现一定的差异。另外10株分离菌株通过生理生化实验鉴定也为无乳链球菌。研究表明,广东省罗非鱼流行性暴发病的病原菌主要为无乳链球菌。     

眼斑拟石首鱼烂尾病病原菌的分离鉴定及灭活疫苗的免疫效果

从患烂尾病眼斑拟石首鱼(Sciaenops ocellatus)的溃疡处分离到一株优势菌So GZ1401,回归感染试验证实该菌是引起烂尾病的病原菌,对眼斑拟石首鱼的半致死量(LD50)为8.6×103 CFU·g~(-1);结合其形态学和生理生化特征及HSP60测序分析,鉴定为哈维氏弧菌(Vibrio harveyi);利用杯碟法研究其胞外产物性质,结果表明:其胞外产物具有淀粉酶、明胶酶、酪蛋白酶、卵磷脂酶、脂酶活性以及溶血活性,但无脲酶活性。药敏试验表明,该菌株对氟哌酸、恩诺沙星、氟苯尼考和复方新诺明等抗菌药有较高的敏感性。制备哈维氏弧菌灭活疫苗,采用注射和浸泡2种途径免疫眼斑拟石首鱼,测定受免疫鱼的血清抗体效价和疫苗对眼斑拟石首鱼的免疫保护率。结果显示,免疫组血清中的抗体效价水平均高于对照组,差异有统计学意义(P0.05),注射组和浸泡组的抗体效价最高值分别达718.4和128,哈维氏弧菌攻毒后免疫保护率分别为75.0%~86.7%和46.4%~56.7%。哈维氏弧菌灭活疫苗对眼斑拟石首鱼具有较好的免疫保护性。     

近江牡蛎肠道细菌及其产酶能力

从近江牡蛎肠道中分离出21株菌,其中, 9株为革兰阳性菌, 12株革兰阴性菌。为寻找能分泌多种酶的有益微生物,研究了它们蛋白酶、脂肪酶、淀粉酶、纤维素酶的产酶能力。结果表明,有61 .9%的菌株能分泌蛋白酶或淀粉酶(各有13株菌);有52. 4%的菌株能分泌脂肪酶(11株菌);有33. 3%的菌株能产纤维素酶(7株菌)。在这21株菌中,产4种酶的有5株菌,产3种酶的3株菌,产两种酶的5株菌,产1种酶的3株菌,不产酶的仅有5株菌。不产酶的菌株仅占细菌总数的23. 8%,而各种产酶菌则高达76. 2%,由此表明细菌在近江牡蛎对食饵消化过程的重要性。     

广东水稻纹枯病菌RAPD基因型与致病型关系

分析了广东水稻纹枯病菌(Rhizoctonia solani AG-1IA)致病型多样性和RAPD多态性之间的相互关系。结果表明,供试水稻纹枯病菌表现出较为丰富的致病型多样性(H=3.485)和遗传多样性,55个菌株接种9个水稻品种表现出39种致病类型。11条RAPD随机引物共扩增出91条谱带,其中多态性条带86条,多态率为94.51%。扩增出的22个选择性RAPD位点中,每个菌株都存在特有的RAPD分子指纹图谱。55个菌株在毒力数据、RAPD数据以及两者结合的数据聚类分析UPGMA树形图上形成几个不同的聚类类群,其中一个类群包括了大部分的菌株。毒力数据与RAPD分子数据的Jacarrd相似性距阵间的相关性系数比较低(r=0.0137),说明致病型与RAPD基因型性之间为相互独立的关系。     

环境胁迫对栉孔扇贝(Chlamys farreri)血浆蛋白及其抗菌活性的影响

采集采自山东某养殖场的栉孔扇贝(Chlamys farreri)健康贝和病贝的血淋巴,测定其血浆蛋白浓度、血浆蛋白图谱和相对分子质量,以及血浆抗菌活性、凝集素活性等几种重要的体液免疫因素,研究环境胁迫对血浆蛋白及其抗菌活性的影响。结果表明:栉孔扇贝(C.farreri)血浆蛋白质量浓度约1 260~1 650μg/mL;血浆蛋白SDS-PAGE图谱显示3条蛋白带,相对分子质量分别为34 000、25 000和14 000;健康贝血浆抗菌活性指数BI为0.34,病贝BI为0.21;凝集素活力水平为1.602~2.204。氨氮胁迫下血浆蛋白浓度下降约780μg/mL,凝集素活力下降为1.301;高温胁迫下血浆蛋白质量浓度下降约为900μg/mL,凝集素活力为1.903;高盐胁迫下血浆蛋白浓度下降约为990μg/mL,凝集素活力为2.204。     

Equality testing for soil grid unit resolutions to polygon unit scales with DNDC modeling of regional SOC pools

Matching soil grid unit resolutions with polygon unit map scales is important to minimize the uncertainty of regional soil organic carbon(SOC) pool simulation due to their strong influences on the modeling.A series of soil grid units at varying cell sizes was derived from soil polygon units at six map scales,namely,1:50 000(C5),1:200 000(D2),1:500 000(P5),1:1 000 000(N1),1:4 000 000(N4) and 1:14 000 000(N14),in the Taihu Region of China.Both soil unit formats were used for regional SOC pool simulation with a De Nitrification-DeC omposition(DNDC) process-based model,which spans the time period from 1982 to 2000 at the six map scales.Four indices,namely,soil type number(STN),area(AREA),average SOC density(ASOCD) and total SOC stocks(SOCS) of surface paddy soils that were simulated by the DNDC,were distinguished from all these soil polygon and grid units.Subjecting to the four index values(IV) from the parent polygon units,the variations in an index value(VIV,%) from the grid units were used to assess its dataset accuracy and redundancy,which reflects the uncertainty in the simulation of SOC pools.Optimal soil grid unit resolutions were generated and suggested for the DNDC simulation of regional SOC pools,matching their respective soil polygon unit map scales.With these optimal raster resolutions,the soil grid units datasets can have the same accuracy as their parent polygon units datasets without any redundancy,when VIV 1% was assumed to be a criterion for all four indices.A quadratic curve regression model,namely,y = – 0.80 × 10~(–6)x~2 + 0.0228 x + 0.0211(R~2 = 0.9994,P 0.05),and a power function model R? = 10.394?~(0.2153)(R~2 = 0.9759,P 0.05) were revealed,which describe the relationship between the optimal soil grid unit resolution(y,km) and soil polygon unit map scale(1:10 000x),the ratio(R?,%) of the optimal soil grid size to average polygon patch size(?,km~2) and the ?,with the highest R~2 among different mathematical regressions,respectively.This knowledge may facilitate the grid partitioning of regions during the investigation and simulation of SOC pool dynamics at a certain map scale,and be referenced to other landscape polygon patches' mesh partition.     

Immunogenicity and protective role of antigenic regions from five outer membrane proteins of <Emphasis Type="Italic">Flavobacterium columnare</Emphasis> in grass carp <Emphasis Type="Italic">Ctenopharyngodon idella</Emphasis>

Flavobacterium columnare causes columnaris disease in freshwater fish. In the present study, the antigenic regions of five outer membrane proteins (OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, were bioinformatically analyzed, fused together, and then expressed as a recombinant fusion protein in Escherichia coli. The expressed protein of 95.6 kDa, as estimated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was consistent with the molecular weight deduced from the amino acid sequence. The purified recombinant protein was used to vaccinate the grass carp, Ctenopharyngodon idella. Following vaccination of the fish their IgM antibody levels were examined, as was the expression of IgM, IgD and IgZ immunoglobulin genes and other genes such as MHC Iα and MHC IIβ, which are also involved in adaptive immunity. Interleukin genes (IL), including IL-1β, IL-8 and IL-10, and type I and type II interferon (IFN) genes were also examined. At 3 and 4 weeks post-vaccination (wpv), significant increases in IgM antibody levels were observed in the fish vaccinated with the recombinant fusion protein, and an increase in the expression levels of IgM, IgD and IgZ genes was also detected following the vaccinations, thus indicating that an adaptive immune response was induced by the vaccinations. Early increases in the expression levels of IL and IFN genes were also observed in the vaccinated fish. At four wpv, the fish were challenged with F. columnare, and the vaccinated fish showed a good level of protection against this pathogen, with 39% relative percent survival (RPS) compared with the control group. It can be concluded, therefore, that the five OMPs, in the form of a recombinant fusion protein vaccine, induced an immune response in fish and protection against F. columnare.     

The Hydrolyzation of Collagen by Fucoidan Oligosaccharide''s Complex with CeⅣ

Fucoidan is such a polysaccharide that its hydroxies are easy to combine with lanthanons ion (CeⅣ) to form complex. This work obtained the complexes of three fucoidan oligosaccharides with different molecular weights F1( ＞5 000), F2(1 000-5 000) and F3( ＜ 1 000) by hydrolyzing Oligosaccharide collagen with sulfuric acid. It is found that the fucoidan oligosaccharide F3 can form complex with more CeⅣ than F1 and F2. Hydrolyzing collagen with the complex was carried out to produce amino acid and peptides. All the three fucoidan oligosaccharide complexes with CeⅣ( F1, F2, F3) can catalyze by the artificial hydrolytic enzyme, and the activity of the complex of F3 is the highest.     

国产遥感传感器大气层外波段平均太阳光谱辐照度计算

目前,我国大部分遥感传感器的大气层外波段平均太阳光谱辐照度(Mean solar exoatmospheric irradiances over band b,简称ESUN_b)尚未公布,这给遥感图像表观反射率的计算带来了不便,在一定程度上影响了数据资料的应用和推广。本文基于已有官方ESUN_b值的EO1/ALI、Terra/ASTER、QuickBird等多种中、高空间分辨率传感器,对9条常见的太阳光谱曲线进行比较分析。结果表明,WRC太阳光谱曲线最合适计算中分辨率传感器的ESUN_b,而Wehrli太阳光谱曲线最合适计算高分辨率传感器的ESUN_b。基于WRC太阳光谱曲线和Wehrli太阳光谱曲线计算,得到了ZY-1 02C/PMS相机、ZY-3/TLC相机和MUX相机,以及GF-1/WFV相机和PMS相机各波段ESUN_b值,并对ESUN_b值的不确定性进行了分析。结果显示,太阳光谱选取的不同,对ESUN_b的取值会产生-1.94%至1.48%的偏差。本文使用多种卫星传感器交叉比较的方式确定了最佳的太阳光谱,给出了ZY-1 02C、ZY-3和GF-1等卫星搭载的遥感传感器各波段的ESUN_b值,为这些国产卫星遥感数据的广泛应用提供便利。此外,本文所用方法简便易行,在其他新的遥感传感器上具有推广应用价值。     

Environmental anomalies in the northeastern East China Sea during the last 3 000 years: implications for El Niño activity in the Holocene

To reconstruct the productivity changes for the last 10 500 a in the northeastern East China Sea (ECS), biogenic compounds (such as carbonate, organic carbon and opal), marine micropaleontological fossils (planktonic foraminifera, benthic foraminifera, radiolarian and silicoflagellate) and the compositional characters of benthic foraminifera fauna analyses were carried out on a sediment core DOC082 obtained from the western slope of Okinawa Trough (29°13.93′N, 128°08.53′E; 1 128 m water depth). The long-term changes of biogenic and micropaleontological proxies display some similarities through the last 10 500 a, which show three different phases: lower values are recorded during the early and middle Holocene (before about 4 000 a BP), followed by an abrupt and remarkable increase at about 4 000 a BP, the late Holocene (after about 3 000 a BP) is characterized by continuously high values. The multi-proxy data of paleoproductivity and percents of benthic foraminifera genera (Uvigerina and Bulimina) show that during the early and middle Holocene (10 500–4 000 a BP) productivity was relatively low with a sudden and distinct increase at about 4 000 a BP, and the late Holocene (3 400–0 a BP) is marked by significantly higher productivity. Also, the radiolarian-based sea surface temperature (SST) records reveal a distinct decline in SST in the late Holocene after 3 200 a BP, very different from the early and middle Holocene. For the last 3 000 a, the enhanced biological productivity and distinctly lower SST indicate a major change of oceanographic conditions in the northeastern ECS. These marine environmental anomalies are consistent with other paleoclimatic records for the late Holocene in the Chinese continent and its surrounding regions. After analyzing the mechanisms of modern productivity and SST changes in the northeastern ECS, and based on the climatic anomalies in the Chinese continent and variations in the Kuroshio Current during modern El Niño periods, we suggest that the anomalous environmental conditions in the northeastern ECS may imply intensified El Niño activity during the late Holocene.     

Vegetation and Climate Reconstruction of the Planalto in Rio Grande do Sul, South Brazil (Late Glacial and Holocene)

A 405 cm long core was drilled in the Aparados da Serra National Park (Rio Grande do Sul, Brazil) at the end of 1999, and in all, 23 samples were taken for palynological investigation. The cores′ age is estimated at 27 000 a BP based on three radiocarbon dates taken from different depths of the core. Palynological methods were used to reconglacial conditions mainly influenced by the South Pole throughout several transition stages to today's humid warm climate, mainly being influenced by the Brazil stream.     

An efficient method of noroviruses recovery from oysters and clams

Noroviruses (NoVs) are widespread causes of nonbacterial gastroenteritis. Outbreaks of NoVs caused diseases are commonly ascribed to the consumption of contaminated shellfish. The concentration and RNA extraction of NoVs are crucial steps of detecting NoVs in shellfish. This study aimed to select a simple, rapid and highly efficient recovery method of NoVs detection with real-time RT-PCR. Four methods of recovering GI.3 and GII.4 NoVs from spiked digestive tissues of oysters and clams, respectively, were compared, of them, the method involving proteinase K and PEG 8000 was found the most efficient. With this method, 9.3% and 13.1% of GI.3 and GII.4 NoVs were recovered from oysters and 9.6% and 12.3% of GI.3 and GII.4 NoVs were recovered from clams, respectively. This method was further used to detect NoVs in 84 oysters (Crassostrea gigas) and 86 clams (Ruditapes philippinarum) collected from 10 coastal cities in China from Jan. 2011 to Feb. 2012. The NoVs isolation rates were 10.47% of clams (9/86) and 7.14% of oysters (6/84). All the detected NoVs belonged to genotype GII. The NoVs recovery method selected is efficient for NoVs detection in oysters and clams.     

武陵山区植被净第一性生产力的时空格局及其与地形因子的关系

本研究旨在探讨2001-2010年武陵山区植被净第一性生产力(NPP)的时空变化格局及其与主要地形因子间的关系。利用MODIS MOD17A3数据、地形数据,以及土地覆盖等数据,基于回归分析和分级统计等方法,开展了武陵山区植被NPP的时空变化格局及其与高程和坡度2个基本地形因子间关系的研究。结果表明：(1)10年间武陵山区植被年NPP的平均值为590.72 gC·m^-2·yr^-1,森林、灌草和农田的NPP平均值分别为596.79 gC·m^-2·yr^-1、586.98 gC·m^-2·yr^-1和563.31 gC·m^-2·yr^-1;(2)2001-2010年武陵山区植被NPP总量的平均值为98.90 TgC·yr^-1,波动范围为92.79 ~ 106.99 TgC·yr^-1,从NPP年际变化的空间分布来看,武陵山区植被NPP的变化趋势整体上呈北增南减的格局;(3)分别以30 m和3°为高程和坡度的分级级差时,发现武陵山区平均植被NPP随海拔和坡度的升高有明显的先增加后缓慢下降的趋势,但在海拔高于1500 m及坡度大于50°的地区,平均植被NPP出现了较大波动,然而,在波动中均有随海拔和坡度的增加而先上升后再下降的趋势;(4)在地理区间尺度上,200~1000 m的高程段以及5~25°的坡度段内植被NPP的平均值较高,500 m以上的高程区间平均植被NPP依次递减,但50°以上的坡度区间平均植被NPP反而有所增加,这在一定程度上反映了坡度高于某一临界值后坡面侵蚀强度又有所下降的趋势。     

中国1:100万遥感地貌制图方法的试验——以南京幅(Ⅰ-50)为例

地貌是自然地域综合体的主导因素,与国民经济建设关系十分密切。本文在总结前人研究地貌及地貌制图的基础上,以LandsatETM、DEN和地貌图件为基础数据源,试验全国1:100万遥感地貌制图方法和技术(南京幅为例),即利用历史地貌图和ETM影像来更新地貌界线,制定制图流程和规范,并讨论遥感地貌制图的几个关键技术和问题,包括遥感影像地貌界线划分的比例尺确定、遥感影像上地表覆盖和地貌界线划分、地貌界线与DEM等高线关系以及遥感地貌自动化制图等,为编制全国1:100万地貌图奠定基础。     

新疆绿洲城镇对称图谱及其生态经济模式分析

天山南北的绿洲城镇以天山为轴对称,分布图案似一副向西张开的弓箭。在局部区域绿洲分布呈线形、弧形、扇形和放射形模式。绿洲经济以水土资源和矿产资源为依赖,中心城市的形成不以城市配位数和平均距离的大小为关键条件。县级中心城镇人口低于10万,平均配位数为3.50,平均距离144km;地市级中心城市人口10～60万,平均配位数4.25,平均距离450km;西部地区百万人口大城市的平均距离约2000km。绿洲人口持续增长,人口密度233人/km²,超过德国人口密度230人/km²。受控于景观带谱对称格局,生态保护政策将使绿洲转向岛国经济模式,发展经济必须跨越自然空间。工业化和城市化是绿洲发展的必然途径,以发展中小城市为主线。距离省府乌鲁木齐2000km以内的配位经济中心难以带动新疆经济跨越式发展,经济跨越式发展的动力模式,不是城市等级配位分布网络的稳定模式,而是经济差级配位模式。乌鲁木齐处于欧亚大陆新丝绸之路欧亚大陆桥中点处经济地理线形谷地位置。欧亚大陆东部与西部沿海经济中心可以作为与新疆经济合作的优先目标。