Immunohistological localization of trypsin in mucus-secreting cell layers of Atlantic salmon, Salmo salar L.

Abstract. Immunohistochcmical examination showed that trypsin was present in mucus-secreting cell layers of Atlantic salmon, such as surface epithelial cells of gills and intestine, and epidermal cells of dorsal skin. Trypsin in tissue slices was identified by an immunohisto-chemical technique which used affinity purified immunoglobulins from rabbit antisera against purified salmon pancreatic trypsin as primary antibodies. Most of the positively stained cells appeared to be granulated and secretory. The authors hypothesize that trypsin in mucus-secreting cell layers is a part of the non-specific immune defence of the fish.     

草鱼IFNa和IFNd重组蛋白表达、纯化及单克隆抗体制备

为系统研究草鱼I型干扰素的合成、分泌和免疫功能,本研究在大肠杆菌中表达并提纯了草鱼IFNa（CiIFNa）和IFNd（CiIFNd）重组蛋白。将CiIFNa和CiIFNd成熟肽分别克隆到pET-21d或pEHISTEVb表达载体上,并转化到大肠杆菌中;IPTG诱导表达得到CiIFNa和CiIFNd成熟肽的包涵体,经过盐酸胍变性、蛋白复性和浓缩后,利用AKTA分子筛层析获得了纯度较高的重组蛋白。用重组蛋白免疫小鼠,通过PEG法诱导得到杂交瘤细胞;将稳定分泌抗体的阳性细胞株的细胞悬液注射入小鼠腹腔,制备腹水抗体并进行纯化。本研究纯化了草鱼CiIFNa和CiIFNd各2株抗体,并采用SDS-PAGE、ELISA、Western blot和免疫荧光法对其进行了较全面的鉴定。研究结果表明CiIFNa和CiIFNd单克隆抗体特异性好、效价高,能够特异识别在大肠杆菌和真核细胞中表达的重组蛋白,且不存在CiIFNa和CiIFNd分子间的交叉识别。本研究制备的单克隆抗体为深入研究草鱼干扰素的细胞来源和蛋白表达规律奠定基础。     

对虾肝胰腺的微细结构

用光镜和电镜观察了对虾(Penaeus orientalis Kishnouye)肝胰腺的结构。结果为:对虾肝胰腺的结构与螫虾等甲壳类基本相同。肝胰腺由两侧对称的分枝上皮小管组成。上皮细胞分为:胚胎性、吸收、原纤维和分泌性4种。上皮的表面有整齐而紧密排列的微绒毛。上皮的基底面有电子致密度低的基板,其上有肌上皮细胞附着。     

Culture of mantle epithelial cells expressing shell matrix proteins from scallop Patinopecten yessoensis

ABSTRACT:   In molluscs, mantle epithelial cells secrete organic matrix proteins to form shells. In this study, we established a culture of mantle epithelial cells by using the mantle pallial layer of scallops. We aimed to identify the mantle epithelial cells expressing scallop shell matrix proteins and establish a culture system of epithelial cells. After the mantle pallial layer was carefully isolated from the mantle tissue, explant culture was performed at 4°C. Most cells that migrated from the explant tissue were round cells. Most of the adhered cells retained round morphology, while some of the cells adhered to the dish and showed morphology similar to that of epithelial-like and fibroblast-like cells. When the cultured cells were immunostained with a polyclonal antibody against the shell matrix protein, the antibody recognized many of the adhered cells. An estimation of the number of epithelial cells revealed that approximately 70% of the adhered cells were epithelial cells. This is the first report to describe epithelial cells in cultured mantle cells, which express shell matrix proteins. This culture system may be a useful method for characterization of the mantle epithelial cells.     

三角帆蚌珍珠囊细胞的分泌活动

本文对三角帆蚌珍珠囊细胞的分泌活动进行了亚显微结构的研究。发现珍珠囊表皮细胞能积极地进行物质合成 ,这些物质主要是蛋白质、硫酸化粘多糖和中性粘多糖 ,并将这些物质以微绒毛分泌、块状物分泌、细胞间隙分泌和大颗粒分泌等多种方式分泌到珍珠囊腔 ;而位于珍珠囊表皮细胞之间的、来源于外套膜结缔组织的腺细胞则含有丰富的中性粘多糖 ,并通过开口式分泌的方式释放到珍珠囊腔 ,同珍珠囊表皮细胞分泌的物质一起 ,共同参与珍珠结晶层的形成 ;珍珠囊细胞分泌的多样性与珍珠组成的复杂有关 ;珍珠囊细胞的分泌活动具有节律性和区段性的特点 ,这种区段性和节律性的分泌与珍珠多层结晶纤层的形成相适应。     

3种硬骨鱼类消化道肥大细胞的组织化学与免疫组化

类胰蛋白酶己被作为人类和某些哺乳动物组织中肥大细胞的标志。为检测鱼类肥大细胞胞浆中是否含有类胰蛋白酶,采用小鼠抗人类肥大细胞类胰蛋白酶单克隆抗体AA1通过Elivision^TM plus免疫组化染色技术,对尼罗罗非鱼、日本鳗鲡和欧洲鳗鲡消化道组织以及人胃癌组织石蜡切片中的肥大细胞进行染色,同时应用AB/SO和改良甲苯胺蓝法进行组织化学染色。结果表明,应用AB/SO和改良甲苯胺蓝染色都能较好显示尼罗罗非鱼肥大细胞, AB/SO染色法对日本鳗鲡和欧洲鳗鲡效果较差,改良甲苯胺蓝染色在上述两种鱼中未能检出肥大细胞;采用小鼠抗人类肥大细胞类胰蛋白酶单克隆抗体AA1通过Elivision^TMplus免疫组化染色技术,首次证实了尼罗罗非鱼肥大细胞胞浆中类胰蛋白酶的存在,但类胰蛋白酶阳性细胞数量很少,主要分布于尼罗罗非鱼消化道各段的黏膜上皮细胞基部和黏膜固有层,而日本鳗鲡与欧洲鳗鲡消化道均未检出类胰蛋白酶阳性细胞。说明不同鱼类肥大细胞胞浆颗粒中生物化学组分存在差别。人胃癌间质中可见多量类胰蛋白酶阳性细胞。     

Expression patterns of acidic and basic fibroblast growth factor in loach fish embryos

Heparin-binding fractions were taken from the heparin sepharose columns on which extracts of loach fish (Misgurnus anguillicaudatus) embryos from blastula, gastrula, 4–8 and 12–16 somites stages were applied. These heparin-binding fractions, except the fraction derived from 12–16 somite embryos, showed potent mitogenic activity on fibroblast-like cells derived from caudal fin blastema of goldfish. Western blot analysis of these heparin-binding fractions was carried out using monoclonal antibodies against human acidic and basic fibroblast growth factors (FGF-1 and-2). An immunoreactive FGF-1 band at 16 kD was detected in the heparin-binding fraction derived from embryos in each stage of blastula, gastrula and 4–8 somites. An immunoreactive FGF-2 band at 17 kDa was detected exclusively in the heparin-binding fraction derived from 4–8 somite embryos. In the heparin-binding fraction derived from 12–16 somite embryos neither immunoreactive FGF-1 nor-2 member band was detectable.     

Identification of overwintering vegetative cells of the bivalve-killing dinoflagellate <Emphasis Type="Italic">Heterocapsa circularisquama</Emphasis> in Uranouchi Inlet,Kochi Prefecture,Japan

ABSTRACT:   Red tides of Heterocapsa circularisquama have led to serious damage of bivalve aquacultures in western coastal areas of Japan. To understand the whole picture regarding the ecology of this species, it is essential to clarify its overwintering mechanisms. In this study, the population dynamics of H. circularisquama were investigated from February 2004 to November 2005, and overwintering cells were identified for the first time in water columns of Uranouchi Inlet, Kochi Prefecture, Japan. Heterocapsa circularisquama cells were detected by the indirect fluorescent antibody technique using monoclonal antibodies that specifically recognize and react to this species. Vegetative cells were almost always detected from the first observation in February 2004 to November 2005 with temperatures of 10.5–30.6°C. During the period from winter to spring, this species survived in areas with a temperature higher than 10°C. The overwintering cells of H. circularisquama were isolated in March 2004, and identification was made via observation of the morphology and body scales of the cultured cells. These overwintering cells were identified as H. circularisquama and reacted to the monoclonal antibody. These results indicate that H. circularisquama can overwinter and survive throughout the year in a vegetative cell state in Uranouchi Inlet.     

Development of monoclonal antibodies to PK'X', the causative agent of proliferative kidney disease

Abstract. Two monoclonal antibody probes were produced against PK'X' cells. The parasites were partially purified by filtration and centrifugation of kidney tissue from rainbow trout with proliferative kidney disease and used as antigen for immunization of mice. The resulting monoclonal antibodies reacted with PK'X' cell antigens in enzyme-linked immunosorbent assay and immunohistochemistry tests. One antibody (Mab 12) appeared to be specific for PK'X'in kidney tissue, while the other (Mab 18) cross-reacted with host cell antigens in the kidney tubules. These probes are invaluable tools for the investigation of parasite surface antigens and life cycle studies.     

Use of monoclonal antibodies in the indirect fluorescent antibody technique (IFA) for the diagnosis of Edwardsiella ictaluri*

Abstract. Forty-five channel catfish, Ictalurus pitnctaius (Rafinesque), fingerlings were inoculated with Edwardsiella ictaluri to determine the usefulness of monoclonal antibodies for indirect fluorescent antibody techniques for confirming clinical diagnosis of enteric septicaemia of catfish. Bacteriological and indirect fluorescent antibody results were compared statistically and found to correlate in 90·3% of the cases. A method for incorporating monoclonals for indirect fluorescent antibody into the speciation of Edwardsiella ictaluri and the use of monoclonal antibodies in diagnosis is described.     

Do fathead minnows,Pimephales promelas Rafinesque,alter their club cell investment in responses to variable risk of infection from Saprolegnia?

Fish in the Superorder Ostariophysi possess large epidermal club cells that release chemical cues warning nearby conspecifics of danger. Despite the long‐held assumption that such club cells evolved under the selective force of predation, recent studies demonstrated that predation has no effect on club cell investment. Rather, club cells have an immune function and cell production may be stimulated by skin‐penetrating pathogens and parasites. The current work investigates whether fathead minnows, Pimephales promelas, alter their club cell characteristics based on variation in infection risk. In a 2 × 3 design, we exposed minnows to infective cysts of two oomycete species (Saprolegnia ferax and S. parasitica) at three different concentrations (2, 20 or 200 cysts L^?1). Club cell characteristics (number and size) were quantified 12 days after exposure. Saprolegnia parasitica is thought to be more pathogenic than S. ferax, hence we predicted greater club cell investment and a larger turnover rate of cells by minnows exposed to S. parasitica than S. ferax. We also predicted that minnows exposed to higher numbers of cysts should invest more in club cells and have a higher turnover rate of cells. We found no difference in club cell density or size between fish exposed to the two Saprolegnia species; however, fish exposed to high concentrations of pathogens had smaller club cells than those exposed to low concentrations, indicating a higher rate of turnover of cells in the epidermis.     

二倍体泥鳅与大鳞副泥鳅及杂交F1精子结构与活力

二倍体泥鳅与大鳞副泥鳅杂交能够顺利获得杂交后代,其正反交核型都为（2n=49）,但杂交后代精巢表现出明显的发育迟缓现象,杂交泥鳅自交与回交实验表明,其雄性不可育。为了解二倍体泥鳅和大鳞副泥鳅杂交F₁雄性不育的原因,实验通过计算机辅助精子活力分析系统（CASA）、光学显微镜、扫描电镜、透射电镜、流式细胞技术分别对二倍体泥鳅（DD）、大鳞副泥鳅（PP）、其正反杂交F₁（DP）和（PD） 4种泥鳅精子的形态结构与活力进行了分析。结果显示,激活30 s时,DD和PP的精子运动率分别为76.50%±0.70%和75.17%±8.60%,极显著高于DP （3.65%±1.75%）和PD （2.68%±0.63%）,且杂交泥鳅的精子的平均运动速度（VAP）、平均曲线速度（VCL）、平均直线运动速度（VSL）等运动参数也极显著低于DD和PP,说明杂交泥鳅精子的活力极其低下。应用流式细胞技术对4种泥鳅精巢内细胞进行倍性鉴定,发现DD和PP精巢内大多为1N精子,而DP和PD精巢内除了正常单倍体精子外,还有大量的2N和4N以及少量的8N细胞。通过扫描电镜和透射电镜...     

铜、锌对泥鳅细胞DNA损伤及金属硫蛋白表达的影响

采用0、100、200、400、800μmol/L的硫酸铜和0、200、400、800、1600μmol/L的氯化锌分别处理泥鳅鳍细胞系,培养24 h后,用单细胞凝胶电泳检测其DNA损伤情况,同时对金属硫蛋白基因的表达进行研究。试验结果表明,随着两种重金属浓度的增加,泥鳅鳍细胞的DNA损伤程度呈现明显的剂量依赖性。当硫酸铜浓度≥200μmol/L、氯化锌浓度≥400μmol/L时,细胞的拖尾率、彗尾DNA比例、彗星尾长、彗星尾距和Olive尾矩均显著增大(P<0.05)。泥鳅细胞金属硫蛋白基因的表达量随着硫酸铜浓度的升高呈现出短暂稳定后显著下降的趋势,而随着氯化锌浓度的增大呈现出显著上升再显著下降的趋势,表明氯化锌浓度≤400μmol/L时可显著诱导泥鳅鳍细胞系金属硫蛋白的转录表达。综合来看,与锌相比,泥鳅鳍细胞对铜的解毒能力和耐受力更差。因此,铜污染对泥鳅细胞的遗传毒性更加明显。本研究将为探讨重金属对生物的毒性效应及生物学监测提供理论依据。     

中间球海胆体腔细胞损失后的恢复规律及恢复期中轴器观察

本研究通过人工抽离中间球海胆(Strongylocentrotus intermedius)体质量10%的体腔液诱导体腔细胞的修复更新,分组测定6、12、18和24 h后海胆围脏腔内的体腔细胞密度,分析体腔细胞的修复规律;同时,取各时间点海胆的中轴器进行组织学观察,并利用鼠抗人Ki-67(细胞增殖抗原)单克隆抗体检测中轴器内的细胞增殖信号。结果显示,人工抽离体腔液后6 h,体腔细胞密度与初始密度相比明显偏低;12 h后逐步升高,密度略低于初始密度,恢复度为(92.78±29.40)%;18 h后已明显高于初始密度,恢复度为(137.08±32.40)%,显著高于6 h和12 h的恢复度(P<0.05),随后,体腔细胞密度逐渐降低。海胆损失体腔液后,中轴器表现为内部组织消减、空腔化,外壁上皮细胞层崩解、脱离,凸起结构空泡化等变化,18 h后内部疏松组织略有增加,24 h后中央腔内出现明显的新生组织。利用Ki-67单克隆抗体检测发现,正常海胆中轴器内具有一定的细胞增殖信号,该信号在损失体腔细胞18 h后明显增强。结果表明,损失体腔细胞后,中间球海胆可启动快速恢复机制,而海胆的中轴器发生明显的组织结构变化,组织内的细胞增殖活动也明显增强。研究结果可为海胆体腔细胞的造血组织与发生机制研究提供依据。     

Susceptibility of betanodavirus in a newly established vertebra-derived cell line from Mosquitofish (Gambusia affinis)

In the present study, a new cell line from the vertebra of mosquitofish Gambusia affinis was successfully established and characterized. The cell line is named as bone Gambusia affinis (BGA) and subcultured for more than 55 passages in Leibovitz's/L15 medium supplemented with 15% FBS at 28°C. The cell line has a modal chromosome number of 48. Molecular characterization of the partial sequence of the coi gene confirmed the origin of the BGA cell line from mosquitofish. These cells exhibited epithelial morphology confirmed by the cytokeratin marker. The BGA cells showed mineralization of their extracellular matrix when stained with alizarin red and von Kossa stain. BGA cells were found to be susceptible to RGNNV and SJNNV strains of betanodavirus (NNV) showing cytopathic effect with multiple vacuolations in the cells. The RT-PCR confirmed the betanodavirus infections in BGA cells. The SEM micrograph showed the morphological changes observed in the cell during virus infection. The in vivo challenge experiment also showed the viral replicating efficiency in the Gambusia affinis with increasing viral titre. Thus, our present results show that the BGA cell line is a useful tool for isolating betanodavirus and could be used to investigate bone cell differentiation and extracellular matrix mineralization.     

The production and characterization of monoclonal antibodies against Photobacterium damselae ssp. piscicida and initial observations using immunohistochemistry

Five monoclonal antibodies (MAbs: F2B1, 1E4, 13B10, 4D4 and F3G12) were produced against lysed Photobacterium damselae ssp. piscicida ( Ph. d . ssp. piscicida ). The MAbs recognized three antigens of differing molecular weight on the Western blot of Ph. d . ssp. piscicida . They also cross-reacted with five different species of Vibrio . An enzyme linked immunosorbent assay (ELISA) with MAbs, F3G12 and 4D4 demonstrated differences between Ph. d . ssp. piscicida and three Ph. d . ssp. damselae type strains, indicating differences in the surface antigenicity between these two groups of bacteria. Antigen retrieval in conjunction with immunohistochemistry (IHC) using MAb 13B10, revealed colonies of bacteria in the kidney, spleen and liver of sea bass, Dicentrarchus labrax , infected with pasteurellosis. A number of positive colonies were observed around the mucosal layers of the intestinal tissue, especially within the lamina propria. In addition, a number of bacterial colonies were associated with red blood cells and blood vessels of the organs examined.     

Development and characterization of a monoclonal antibody against Taura syndrome virus

We produced a panel of monoclonal antibodies (MAbs) from the fusion of Taura syndrome virus variants from Belize (TSV-BZ) immunized BALB/cJ mouse spleen cells and non-immunoglobulin secreting SP2/0 mouse myeloma cells. One antibody, 2C4, showed strong specificity and sensitivity for TSV in dot-blot immunoassay and immunohistochemistry (IHC) analysis. The MAb reacted against native TSV-BZ, TSV variants from Sinaloa, Mexico (TSV-SI) and TSV variants from Hawaii (TSV-HI) in dot-blot immunoassay. By IHC, the antibody identified the virus in a pattern similar to the digoxigenin-labelled TSV-cDNA probe for the TSV-BZ, TSV-HI and TSV-SI variants, but not for the TSV variants from Venezuela (TSV-VE) and the TSV variants from Thailand (TSV-TH). MAb 2C4 did not react against other shrimp pathogens or with normal shrimp tissue. Western blot analysis showed a strong reaction against CP2, a region of high antigenic variability amongst TSV variants. This antibody has potential diagnostic application in detection and differentiation of certain TSV biotypes.     

泥鳅混合选育F_2代生长性能分析

以泥鳅太湖群体为基础群体,采用混合选育方法进行了人工选育,对产生的混合选育F2代进行生长性能分析。结果表明：泥鳅混合选育F2代的日增重率和日增长率分别达到0.022g/d和0.036cm/d,比对照组提高了4.8%和2.9%。混合选育群体体重变异系数和体长变异系数分别比对照群体降低了9.8%和8.1%,经过一代人工选育,泥鳅生长性能进一步提高,且群体规格趋于整齐。     

3种泥鳅微卫星标记和D-Loop部分序列遗传变异分析

为进一步了解中国当前主要养殖鳅类种质资源现状,实验采用7个微卫星标记和线粒体D-Loop部分序列,对泥鳅(Misgurnus anguillicaudatus,MA)、大鳞副泥鳅(Paramisgurnus dabryanus,PD)和台湾大泥鳅(未见种属分类,TW)3种泥鳅进行群体遗传变异分析.结果显示,6个微卫星位点在3种泥鳅中均能获得有效扩增,1个微卫星标记(Mac239)只在MA中获得特异性扩增,而在PD和TW中未能获得有效扩增条带.在3种泥鳅共90尾个体的D-Loop 部分序列中发现32个单倍型,仅在PD和TW间存在1个共享单倍型.实验中共检测到65个变异位点,其中MA与PD和TW间存在29个特异性位点,而PD和TW间未检测到特异性位点.瓶颈效应和中性检验显示,TW近期可能发生有效群体数量的减少.基于微卫星标记和D-Loop部分序列的遗传变异分析显示,TW和PD间的Nei's遗传距离和K2P遗传距离最近(0.297和0.006),明显小于两者与MA间的遗传距离(1.011～1.899和0.095～0.099);分子方差分析(AMOVA)显示,3种泥鳅的遗传分化极显著(P＜0.01).群体间遗传结构和单倍型网络分析显示,3种泥鳅的遗传结构相对独立,仅在TW和PD间存在一定程度的遗传结构混杂.研究表明,泥鳅和大鳞副泥鳅在遗传结构上存在明显差异,可采用分子标记进行有效鉴别;台湾大泥鳅可能是大鳞副泥鳅的生态种群或遗传改良群体,而非有效物种.     

Comparative proteomic analysis of virulent and rifampicin-attenuated Flavobacterium psychrophilum

Flavobacterium psychrophilum is the aetiologic agent of bacterial coldwater disease and rainbow trout fry syndrome. In this study, we compared a wild‐type strain (CSF 259‐93) with a rifampicin‐resistant strain and virulence‐attenuated strain of F. psychrophilum (CSF 259‐93B.17). The attenuated strain harboured a mutation in the rpoB gene consistent with resistance to rifampicin. Two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) and mass spectrometry demonstrated an altered proteome with eight proteins characteristic for the parent strain and six that were unique to the attenuated strain. Immunoblotting with a diagnostic monoclonal antibody (FL‐43) identified a putative antigen (FP1493) that was subsequently cloned, expressed as a recombinant protein and confirmed as recognized by FL‐43. 2D‐PAGE, immunoblotting with rainbow trout, Oncorhynchus mykiss (Walbaum), convalescent antisera and mass spectrometry of bacterial whole‐cell lysates revealed several uniquely expressed immunoreactive proteins including FP1493. An FP1493 recombinant subunit vaccine was tested, but did not provide protection against challenge with the CSF259‐93 strain. While the exact mechanism responsible for altered protein synthesis and attenuation of CSF 259‐93B.17 is still unknown, the differentially expressed immunoreactive proteins are a valuable resource to develop subunit vaccines and to identify proteins that are potentially involved in disease.