海产蟹类血卵涡鞭虫病间接荧光抗体快速检测技术

以寄生于三疣梭子蟹血淋巴中的血卵涡鞭虫虫体为抗原，制备了多克隆血清抗体。抗体经健康梭子蟹血淋巴吸附处理后，间接ELISA检测效价达7680。应用该抗体建立了血卵涡鞭虫的间接荧光抗体检测技术(IFAT)。采用常用显微镜检、PCR及 IFAT三种方法对采集的养殖青蟹、梭子蟹、及海捕梭子蟹等18个样本进行了检测。检测结果显示：常规显微镜检阳性检出率为33.3%，而IFAT及PCR检测阳性率77.8%，符合率达100%；阳性虫体被染上黄绿色荧光，而正常梭子蟹血细胞则未被染色；可检测到血卵涡鞭虫不同生活阶段的营养体、腰鞭孢子及合孢体阶段。本文为血卵涡鞭虫的流行病学调查及生活史研究提供了简便实用的方法。     

血卵涡鞭虫单克隆抗体的制备与初步应用

用血卵涡鞭虫可溶性抗原免疫BALB/c小鼠,经常规融合、间接ELISA方法筛选,将所得阳性克隆再经3次亚克隆后,共获得3株针对血卵涡鞭虫的单克隆抗体(2B₂、3G₄、4G₇),单克隆抗体亚类鉴定表明,三者为IgG类抗体。用筛选的杂交瘤细胞株制备小鼠腹水抗体,其细胞上清及腹水效价分别为5.12×10^-4和8.00×10^-4。进一步利用单克隆抗体建立间接荧光抗体方法对单抗特异性进行鉴定,阳性虫体被染上黄绿色荧光,而正常梭子蟹血淋巴则未被染色。用单克隆抗体和多克隆兔抗血清以羊抗鼠HRP-IgG为酶标抗体,建立了检测血卵涡鞭虫的双抗体夹心ELISA方法,该方法对血卵涡鞭虫阳性标本检测符合率为100%。结果表明,制备的单克隆抗体效价高、特异性好,可用于血卵涡鞭虫的早期临床诊断。     

应用PCR方法检测患“黄水病”锯缘青蟹中的血卵涡鞭虫

"黄水病"是目前锯缘青蟹养殖过程中的主要疾病之一,病蟹主要症状表现为肌肉白浊,体液呈土黄色或浊白色牛奶状。因该病流行范围广、发病率和死亡率高,给青蟹养殖业主造成巨大经济损失,严重地影响了青蟹养殖的健康发展。本研究从病蟹体液中发现大量疑似血卵涡鞭虫的寄生原虫,应用已建立的梭子蟹血卵涡鞭虫病的PCR检测方法对患"黄水病"青蟹进行检测。结果从患病青蟹组织的DNA中扩增出产物大小为585bp的特异性目的片段,经序列分析比较,与三疣梭子蟹上发现的血卵涡鞭虫的序列同源,同源性达99.7%。综合病原流行病学调查、组织病理学、电镜观察等分析结果,初步确定血卵涡鞭虫是引起养殖青蟹"黄水病"的重要病原。     

三疣梭子蟹 Portunus trituberculatus (Miers) 血卵涡鞭虫病诊断与综合防治措施

血卵涡鞭虫是导致海产甲壳类疾病的主要寄生虫病原之一，国内迄今尚未见相关报道。本文根据作者近年来对海水养殖梭子蟹血卵涡鞭虫病的研究结果．就该病在养殖梭子蟹当中的流行情况及诊断方法进行介绍，并提出了相应的综合防治措施，旨在为我国海水养殖接类血卵涡鞭虫病害的研究与防治提供参考。     

环介导恒温扩增技术检测血卵涡鞭虫

血卵涡鞭虫病是海水甲壳类的重要寄生虫病,其流行范围广、死亡率高、危害非常严重。本研究根据GenBank中已登录的血卵涡鞭虫(Hematodinium sp.)ITS1序列设计了1套引物,该引物可识别目标基因中6个不同区段。以此套引物建立了一种基于环介导恒温扩增技术(Loop-mediated isothermal amplification,LAMP)的血卵涡鞕虫病诊断方法。特异性试验结果表明,该套引物对血卵涡鞕虫检测具有较高的特异性,能有效检出血卵涡鞕虫。敏感度试验结果表明,该LAMP技术的灵敏度比常规PCR技术高4个数量级。分别运用LAMP和常规PCR技术对25份临床疑似病例进行检测,LAMP方法共检出感染病例25份,常规PCR检出感染病例23份。该技术能在65℃恒温条件下45～60min完成目的DNA的扩增,可直接通过肉眼观察反应产物中是否产生白色沉淀或经SYBRGreenI染色后通过颜色变化、扩增产物的琼脂糖凝胶电泳来定性判断结果,这将为血卵涡鞕虫病的临床诊断提供一种更加简便、快速、实用的方法。     

血卵涡鞭虫的发生及流行研究进展

血卵涡鞭虫(Hematodinium)是一类危害海水甲壳类动物的致病性寄生甲藻，自1931年以来感染超过40多种甲壳类动物，导致了一些经济类甲壳动物的大量死亡。为有助于加强对血卵涡鞭虫的防控，从季节、环境、宿主类型和传播模式方面，总结了血卵涡鞭虫流行和发生的原因。     

海产甲壳类血卵涡鞭虫病研究进展

血卵涡鞭虫是导致海产甲壳类疾病的主要寄生虫病原之一,国内迄今尚未见相关报道。本文结合作者近年来对海水养殖甲壳类病害的研究结果,较系统地综述了国外甲壳类寄生血卵涡鞭虫的研究进展,包括分类地位、生活史、传播途径、流行病学、组织病理以及诊断方法,并提出了相应的防治措施,旨在为中国海水养殖蟹类血卵涡鞭虫病害的研究与防治提供参考。     

大黄鱼苗淀粉卵涡鞭虫病的诊断及防治方法

淀粉卵涡鞭虫是大黄鱼苗种培育危害最大的寄生虫之一.本文对大黄鱼苗淀粉卵涡鞭虫病发生的临床症状和防控方法进行了研究,除常规防治方法外,利用双氧水与高锰酸钾合用也可起到杀灭虫体,减少寄生数量的效果.同时,本文分析和比较了发生寄生虫病的育苗池水与正常池水的水质理化因子,为有效诊断和防治大黄鱼苗淀粉卵涡鞭虫病提供基础材料.     

双重PCR检测携带有tl和tdh基因的副溶血弧菌毒力菌株

在常规PCR技术的基础上优化条件,建立并完善双重PCR技术,并通过检测阳性对照和待检样品,进一步确定其检测的可行性.结果表明在常规PCR方法中为阳性的样品,包括阳性对照以及待检样品,在双重PCR方法中也呈现阳性,为阴性的样品在本方法中则也呈现阴性;能在血平板中出现溶血圈的在本法中也被印证含有tdh基因.由此证实本方法确实能对tl和tdh两种基因同时进行检测,成功地证明了同时检测tl和tdh两种基因的双重PCR方法的可行性.该法不但具有常规PCR的优点,而且还能节省耗材和时间,可用于检测水产食品以及临床样品中的副溶血弧菌的毒力菌株.     

免疫荧光技术

本刊讯：免疫荧光技术是利用某些荧光紊通过化学方法与特异性抗体结合制成荧光抗体，荧光抗体与被检抗原特异性结合后．形成的免疫复合物在一定的波长光的激发下可产生荧光，借助荧光显微镜可检测或定位被检抗原。免疫荧光技术将免疫化学和血清学的高度特异性和敏感性与显微术的高度精确性相结合。在水产养殖病原的检测上已得到一定应用。[第一段]     

母猪胎衣不下的病因与防治要点

猪的胎盘属于弥散型胎盘,这种胎盘的结构特点和饲养管理的不当,常常导致母猪胎衣不下发生,给生猪的生产繁殖带来极大损失。本文针对母猪胎衣不下发生病因、综合防治进行详细阐述,旨在对预防和治疗胎衣不下能有所帮助。     

Estimation of variation of virulence of Renibacterium salmoninarum by survival analysis of experimental infection of salmonid fish

The variation of virulence of Renibacterium salmoninarum , the causative agent of bacterial kidney disease (BKD) in salmonid fish, was studied by infecting rainbow trout, Oncorhynchus mykiss (Walbaum), with two isolates (strains 325 and 932) from diseased Atlantic salmon, Salmo salar L., and one isolate (strain 4366) from an apparently healthy Atlantic salmon. Coho salmon, Oncorhynchus kisutch (Walbaum), were injected with the strain 932 to estimate difference in fish species resistance. Fish were removed by random sampling for other study purposes, a study design possible with analysis of lifetime distributions incorporating both sampling-, death- and survival-times. At the end of the experiment, the rainbow trout infected with strains 325, 932 and 4366 had a survival probability of 33%, 51% and 72%, respectively. The coho salmon infected with strain 932 had a 26% survival probability. The strain differences were significant according to the log-rank test, and the risk ratio between the strains ranged from 1·8 to 5·4. The strain from the apparently healthy fish was least virulent. The survival of the fish species was different over time. Rainbow trout were more likely to die early in the time course, but high numbers of coho died later, resulting in an overall risk of mortality of 1·4 in favour of rainbow trout. Differences in virulence may reflect changed selective pressure on R. salmoninarum when introduced from feral stocks into the environment of fish farms.     

金鳟、虹鳟苗种长途运输实例

鳟鱼发眼卵和旨种运输是养鳟生产的重要环节。发眼卵多在冬季和早春运输，苗种多在晚春和夏季运输。苗种运输大多以鱼篓充氧进行短距离汽车运输，长途运输冷水性鱼苗种的实例很少。本文总结了金鳟和虹鳟苗种长途运输方法，以期与养鳟业界共同交流提高。一、包装材料和方法内包装材料是苗种运输专用塑料袋，规格54×104cm，外包装是泡沫塑料箱，规格63.5×45.5×30．5cm，纸箱规格64×46×32．5cm。塑料袋采取双层式，内装苗种和水共12.5kg，其中苗种0.5-1．3kg，100－2000尾。起运前苗种停食1－2天。运鱼用水水温调至6℃，鱼、水入袋后袋内…     

斑节对虾虾苗活力检测方法

该研究通过肉眼观察、镜检，进行干露、饥饿、盐度突降、福尔马林等抗性试验，并采用病毒检测等方法，以期建立评估斑节对虾（Penaeus monodon）虾苗活力和质量标准。结果表明，斑节对虾健康虾苗具有趋光性、集群性，体表光洁，肌肉透亮，肠胃食物充盈等特性。测试虾苗干露时间以15min为宜，健康虾苗干露后能立即恢复活力，而病弱虾苗多出现死亡、昏迷现象；虾苗的成活率随饥饿时间的延长而降低，随福尔马林浓度升高和时间延长而降低，随盐度突降幅度增加而降低。健康虾苗能忍受100～200μL·L^-1福尔马林溶液30min，成活率近100％；在盐度20～30下虾苗的成活情况较好，而其在淡水中仅能存活1h。对虾苗进行病毒检测，可以避免养殖中因虾苗携带病毒而可能导致的病毒性疾病的暴发。     

水硬度对七彩神仙鱼幼鱼发育的影响

采用不同硬度的水对七彩神仙鱼幼鱼进行饲养。6周龄幼鱼在硬度为7.94°dH±0.30°dH时饲养84d后,比在硬度为14.71°dH±0.23°dH水中的幼鱼个体大,生长速度快。表明较高硬度的水有利于七彩神仙鱼幼鱼的生长发育。     

虎斑乌贼受精卵卵黄营养成分分析

本实验对虎斑乌贼受精卵卵黄的营养成分进行分析,旨在探讨其幼体的营养需求量,为其幼体配合饲料研制提供参考数据。随机选取大约800个虎斑乌贼受精卵的卵黄,采用国家标准方法测定其水分、灰分、粗蛋白质、粗脂肪、氨基酸、脂肪酸和矿物元素含量。结果表明:1)虎斑乌贼受精卵卵黄中粗蛋白质含量为76.33%(干重基础);总氨基酸(TAA)和必需氨基酸(EAA)含量分别为71.22%和32.38%(干重基础),EAA/TAA为45.46%,氨基酸中以谷氨酸(Glu)含量最高(9.97%),必需氨基酸中亮氨酸(Leu)含量最高(7.58%)。2)其粗脂肪含量12.71%(干重基础);共检出17种脂肪酸,包括8种饱和脂肪酸(SFA)、5种单不饱和脂肪酸(MUFA)和4种多不饱和脂肪酸(PUFA),SFA、MUFA和PUFA分别占脂肪酸总量的43.47%、7.54%和49.25%,其中以DHA含量最高,达32.80%,EPA含量为7.70%,DHA/EPA为4.26。3)检测出Na、K、Ca、Mg、Sr、Mn、Fe、Cu、Zn、Al和As 矿物元素,微量元素中富含Zn、Al和Fe,含量分别为 0.77、0.71和0.43 mg/kg(鲜重基础)。由此可见,卵黄具有高蛋白、低脂肪,富含n-3PUFA的特点;虎斑乌贼幼体饲料中蛋白质需求量参考值为76.33%;氨基酸需求量参考值,如赖氨酸(Lys)为5.49%,蛋氨酸(Met)为2.63%;脂肪的需求量参考值为12.71%,DHA为4.17%,EPA为0.98%;微量元素需求量参考值,如Zn为2.77 mg/kg,Cu为0.19 mg/kg(干重基础)。     

Characterization of protease inhibitors of seminal plasma of cyprinids

Anti-proteinase activity was demonstrated in the seminal plasma of cyprinid fish species (bream, chub, ide, dace, asp, goldfish, roach, common carp) using electrophoretic techniques combined with a detection method based on inhibition of bovine trypsin. We found species-specific protease inhibitors in the seminal plasma of cyprinids. At least three bands of protease inhibitors with different migration rates could be identified by native PAGE. Higher variability was characterized for bands with slower migration rates. Visualization of inhibitors after SDS-PAGE under non-reducing conditions allowed estimation of their molecular weights. Apparent molecular weights were within the range of 51–59 and 47–54 kDa for the bands with slower and moderate migration rates, respectively. The molecular weight of fast migration bands for roach and common carp were estimated to 23 and 30 kDa, respectively. Inhibitors of common carp seminal plasma differed in their affinity toward serine proteases. Three inhibitors in common carp seminal plasma could be visualized using cod and bovine trypsin, but only two inhibitors (of high molecular weight) were recognized with chymotrypsin. There were differences in anti-proteinase activity and seminal plasma protein concentration in relation to the origin of common carp seminal plasma (breeding lines) and time of milt collection (spawning vs. post-spawning season).     

不同年龄细鳞鱼的繁育效果

研究了不同年龄组(3～8龄)细鳞鱼(Bachymystax lenok Pallas)亲鱼的繁育效果,分析其繁育主要技术,包括亲鱼培育、繁育技术、受精卵孵化、鱼苗培育等,结果发现,5～6龄和7～8龄细鳞鱼产卵量没有明显差异,产卵量、孵化率、出苗率均较高,繁殖效果明显好于3～4龄亲鱼。     

海口市近海渔业资源衰退的原因分析与对策建议

在分析海口市海洋渔业资源和海洋捕捞现状的基础上，提出了近海渔业资源衰退的6项原因和加快保护和修复资源的10项措施和建议。     

Histopathology of respiratory organs of certain air-breathing fishes of India

Air-breathing fishes have evolved bimodal respiratory mechanisms for exploitation of water (through gills and highly vascularized skin) as well as atmospheric air (through aerial respiratory organs, ABO). Mucous cells in these respiratory organs of variously stressed fishes exhibit periodic fluctuations in their density and staining properties. The main types of damage in the gills include congestion of blood capillaries (BLCs), periodic lifting and sloughing of respiratory epithelia of the secondary lamellae causing haemorrhage, extensive fusion of secondary lamellae and hyperplasia of the respiratory epithelia due to uncontrolled regeneration leading to asphyxiation, altered excretion, and death of the fish. Haemolysis has also been observed following lead exposure. The damage in the ABO of Heteropneustes fossilis includes sloughing of the epithelial cells, leading to haemorrhage causing decreased red blood corpuscles density and degeneration of the secondary gill lamellae with reduced respiratory area. Subsequent hyperplasia of the respiratory epithelia and fusion of gill lamellae increase the respiratory barrier distance. The BLCs often bulge out and protrude into the lumen, bringing blood nearer to air. The ladder-like pillar cell (PLC)-BLC components of the gill lamellae frequently collapse. Damage to the ABO of Channa striata is less severe. Often haemorrhaging due to bursting of extensively stretched BLCs causes aerial respiratory failure. Chloride cells of the ABOs also show hyperplasia. While the highly mucogenic epidermis of C. striata shows less damage, the epidermis of Clarias batrachus and H. fossilis shows severe wear and tear, sloughing, and haemorrhage. Side-by-side regeneration continues, causing altered histomorphology of the epidermis. The different gland cells also show periodic fluctuations in their density and staining. The dermis also shows severe damage with loosening of their connective tissue fibres. These fibres give stronger reactions for sulfated mucin that not only retain additional water molecules for continuance of skin breathing, but also bind the toxic ambient pollutants.