Phage and MLVA Typing of Salmonella Enteritidis Isolated from Layers and Humans in Belgium from 2000–2010, A Period in which Vaccination of Laying Hens was Introduced

The aim of the study was to characterize isolates of Salmonella enterica serovar Enteritidis (S. Enteritidis) obtained from humans and layer farms in Belgium collected during 2000–2010. Three periods were compared, namely (i) before implementation of vaccination (2000–2004), (ii) during voluntary vaccination (2005–2006) and (iii) during implementation of the national control program (NCP) for Salmonella including mandatory vaccination against S. Enteritidis (2007–2010). The characteristics compared across time periods were distributions of phage type and multiple‐locus variable number tandem‐repeat assay (MLVA). While PT4 and PT21 were predominantly isolated in Belgium in layers and humans before 2007, a significant reduction of those PTs was observed in both populations in the period 2007–2010. The relative proportion of PT4b, PT21c and PT6c was found to have increased considerably in the layer population since 2007. In the human population, PT8, PT1 and the group of ‘other’ PTs were more frequently isolated compared to the previous periods. When comparing the proportion of the predominant MLVA types Q2 and U2, no significant difference was found between the layer and human population in the three periods and between periods within each category (layer and human). A significant difference in isolate distribution among MLVA clusters I and II was found between human and layer isolates recovered during Period 3 and in the human population between Period 1 and 3. Results suggest that the association between S. Enteritidis in layers and the occurrence of the pathogen in humans changed since implementation of the NCP in 2007.     

用酶联免疫吸附受体法检测鱼类生长激素的生物活性

根据激素一受体反应的原理，结合酶联免疫吸附测定法（ＥＬＩＳＡ）和放射受体测定法（ＲＲＡ）的优点，应用我们纯化的Ａ昌鱼生长激素（ｇｃＧＨ）和大鳞大马哈鱼生长激素（ｓＧＨ）及其特异抗体，采用鱼类肝细胞膜受体制剂，首次建立了测定鱼类ＧＨ生物活性的酶联免疫吸附受体测定法（ＥＬＩＳＡ－ＲＡ）。此法检测草鱼ＧＨ的灵敏度达０．０６３￣０．１２５ｕｇ／ｍｌ，检测大马哈鱼ＧＨ与草鱼肝膜受体结合的灵敏度达０．２５ｕｇ     

A rapid immunoperoxidase-based virus neutralization assay for salmonid alphavirus used for a serological survey in Northern Ireland

A modified virus neutralization (VN) assay was developed to replace an existing assay read on the presence or absence of virus-induced cytopathic effect (CPE). The modified assay used a monoclonal antibody to salmon pancreas disease virus as the first layer of an immunoperoxidase (IPX)-based immunostaining technique to detect viral growth. The IPX-based VN assay required only 3 days to perform, and the adoption of a 96-well microtitre format facilitated a high throughput of samples requiring small volumes of serum, cells and virus. When 352 sera from farmed salmon and 302 sera from farmed trout were tested by both the modified and the original CPE-based assays, overall correlations of 97.72 and 96.03% were, respectively, obtained (96.94% combined). When the modified assay was used to test 188 sera collected from wild salmonids in freshwater river systems in Northern Ireland, no positive results were recorded.     

Epidermal proteases of the Japanese eel

A fluorescent-sensitive assay was used to demonstrate the protease activity in the dorsal skin of Japanese eel (Anguilla japonica). Two distinct extracts were separately prepared from skin mucus and epidermal cell layers, with no mutual contamination. The epidermal extract was sensitive to various substrates, whereas there was no, or only marginal, susceptibility to the same substrates for the mucous extract. Optimum hydrolysis pHs of the epidermal extract was variable and below pH 7.0, and the optimum hydrolysis temperatures were between 40 and 50 °C. In addition, Tos-Phe-Ch₂Cl, chymostatin, CdCl₂, CuCl₂, HgCl₂ and ZnCl₂ inhibited protease activities to different extents. Several other reagents specifically affected the protease activities, and their induced effects were useful for the identification of epidermal proteases. The findings indicate that a proteolytic factor, exhibiting various enzymological specificities, is retained within epidermal cell layers of Japanese eel. This factor is composed of 4 distinct proteases, such as cathepsins L and B-like proteases, a serine protease and an aminopeptidase.     

Purification of chinook salmon (Oncorhynchus tshawytscha) GH for receptor study

A method for the purification of chinook Salmon (Oncorhynchus tshawytscha) GH, which retains its biological activity, is described. The biological activity was investigated with an established radioreceptor assay using liver membranes from pregnant rabbits and bovine GH as standard and labelled hormone. The enrichment of the preparation was checked with electrophoresis (SDS-PAGE). Extraction and further steps were carried out using low molarity alkaline buffer (pH 8–10, M = 100 mM). Three chromatography steps were performed (Concanavalin-A sepharose, Bio-gel P60, DEAE). Ion exchange chromatography was performed under isocratic conditions (using a 50 cm column). Two isoforms (sGH1 and sGH2) were isolated. The purification yield is 0.7% compared to lyophilized pituitaries. The molecule is homogeneous in SDS-PAGE. Contamination by prolactin, gonadotrophin and corticotrophin is negligible (< 0.5%). It could be demonstrated that the biological activity of the preparation is maintained since this preparation stimulates the growth of juvenile trout (Salmo gairdneri) and binds specifically (35%) to trout liver membranes.     

Mechanochemical properties of ordinary and dark muscle myosins from seawater fish

Myosin was isolated from two types of muscle, ordinary and dark muscles, of three species of fish living in sea water. The compositions of light chains were visualized by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the mechanochemical activity was examined by in vitro motility and ATPase assays. Ordinary muscle myosin of either species had three species of light chain, whereas dark muscle myosin had another two species of light chain judged by SDS-PAGE. Sliding velocity of ordinary muscle myosin was in the range of 4.92–6.89 μm/S, whereas that of dark muscle myosin was in the range of 3.07–4.25 μm/s. Therefore, ordinary muscle myosin showed 1.26–1.95 times higher sliding velocity than dark muscle myosin in either species. The ratios of V_max of actin-activated Mg²⁺-ATPase activity of ordinary to dark muscle myosins were correlated quite well to the ratios of sliding velocity. Activity of ordinary muscle myosin was comparable to that of mammalian fast muscle myosin, but that of dark muscle myosin was twice of that of mammalian slow muscle myosin. These results may reflect the essential role of fish dark muscle myosin always used in slow cruising.     

Aquatic birnavirus induces apoptosis through activated caspase-8 and -3 in a zebrafish cell line

In this study, the possible influence of temperature on infectious pancreatic necrosis virus (IPNV)-induced apoptosis in a zebrafish liver epithelium (ZLE) cell line was investigated. At a lower temperature (18 degrees C), there was expression of viral proteins VP2 and VP3 at 4 h post-infection (p.i.). At this time no expression was found in the high temperature group at 28 degrees C. The cell survival ratio was 52 and 18% at 24 and 48 h p.i., respectively, during IPNV infection at 18 degrees C. In addition, we assayed for apoptosis in IPNV-infected cells with terminal deoxynucleotidyl transferase (TdT)-mediated end labelling (TUNEL) of DNA at different dosages of virus. We found a ratio of apoptotic cells of 8 and 25% at 12 and 18 h p.i., respectively, in the multiplicity of infection (MOI) 1 group. The MOI 10 group had 20 and 45% apoptotic cells at 12 and 18 h, respectively. Furthermore, at 18 degrees C IPNV activated the caspase-8 and 3 from 1.5 to 2 times at 12 and 18 h p.i., respectively. Taken together, these findings suggest that successful virus replication occurs at the low temperature (18 degrees C) compared with the non-permissive temperature of 28 degrees C. Thus, IPNV replication is capable of activating caspase-8 and -3 and inducing host apoptosis.     

Development of a whole-body cortisol extraction procedure for determination of stress in golden shiners, <Emphasis Type="Italic">Notemigonus crysoleucas</Emphasis>

Baitfish such as golden shiners are subjected to stress during harvesting, grading, and transport. Their small size makes it difficult to measure the stress response with the biological indicator cortisol using conventional assay methods for plasma. This paper examines the development and validation of methods for whole-body cortisol extraction from individual baitfish. Three types of extracts were tested: (1) an ethyl ether unaltered extract (UA); (2) an extract reconstituted in phosphate buffered saline (PBS); (3) an extract that had been increased in volume by the addition of food-grade vegetable oil (VO). These extracts were evaluated using validation tests with radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA). The UA extract produced inadequate volumes of extract for multiple assays and could not be used for the determination of cortisol in a single fish. The PBS reconstitution method failed the precision recovery of serial dilutions (62.3%), linearity (R ²: 0.7864), and parallelism validation tests. The VO volume-boosting method passed all validation tests [intra-assay coefficent of variation (%CV): 16.3 for ELISA and 5.9 for RIA; inter-assay %CV: 10.3; spiked recovery: 102.0%; dilution recovery: 93.0%; linearity R ²: 0.9435; log of serial dilutions was parallel] and provided enough extract for multiple assays from an individual baitfish. Based on these results, we conclude that the VO volume-boosting method presents a means for determining cortisol from individual baitfish using either RIA or ELISA assays.     

Simple and Rapid Quality Control of Sulfated Glycans by a Fluorescence Sensor Assay—Exemplarily Developed for the Sulfated Polysaccharides from Red Algae Delesseria sanguinea

Sulfated polysaccharides (SP) from algae are of great interest due to their manifold biological activities. Obstacles to commercial (especially medical) application include considerable variability and complex chemical composition making the analysis and the quality control challenging. The aim of this study was to evaluate a simple microplate assay for screening the quality of SP. It is based on the fluorescence intensity (FI) increase of the sensor molecule Polymer-H by SP and was originally developed for direct quantification of SP. Exemplarily, 65 SP batches isolated from the red alga Delesseria sanguinea (D.s.-SP) and several other algae polysaccharides were investigated. Their FI increase in the Polymer-H assay was compared with other analytical parameters. By testing just one concentration of a D.s.-SP sample, quality deviations from the reference D.s.-SP and thus both batch-to-batch variability and stability can be detected. Further, structurally distinct SP showed to differ in their concentration-dependent FI profiles. By using corresponding reference compounds, the Polymer-H assay is therefore applicable as identification assay with high negative predictability. In conclusion, the Polymer-H assay showed to represent not only a simple method for quantification, but also for characterization identification and differentiation of SP of marine origin.     

基于量子点的赭曲霉毒素AsFLISA检测技术研究

建立了基于量子点标记技术的赭曲霉毒素A(Ochratoxin A,OTA)双抗夹心荧光免疫检测(sandwich fluorescence-linked immunosorbent assay,sFLISA)体系,包含多克隆包被抗体、生物素标记的多克隆检测抗体、量子点标记的链霉亲和素等,获得了sFLISA的最佳操作参数:包被抗体浓度2.5μg/mL,检测抗体稀释500倍,量子点标记链霉亲和素稀释100倍。该方法在OTA浓度3.125~125μg/L之间时,相对荧光强度和OTA浓度呈线性关系,回归方程为y=0.0206x+0.2018,R2=0.9924;加标回收率在90.1%~110.0%之间,变异系数均小于10%,能较好地进行赭曲霉毒素A的定量检测。