8株鳖源变形杆菌外膜蛋白的比较

从 1 2批送检病鳖体内分离出 8株细菌 ,经形态学检查、生理生化特性测定、致病性测定和血清学鉴定 ,确定 7株为普通变形杆菌 ,1株为奇异变形杆菌。进一步采用十二烷基硫酸钠 (SDS)破菌法提取 8株鳖源变形杆菌的外膜蛋白 (OMP)进行SDS PAGE电泳 ,比较分析细菌OMP型。结果显示奇异变形杆菌的OMP型由 3条相对分子质量范围为 3 0× 1 0 4～4 3× 1 0 4的主要蛋白带组成 ;7株普通变形杆菌的主要外膜蛋白相对分子质量范围为 3 0× 1 0 4～6 7× 1 0 4,分属 2个OMP型 ,其中 4个分离株属OMP1型 ,由 4条主要蛋白带组成 ;其余 3个分离株属OMP2型 ,由 7条主要蛋白带组成。表明不同种变形杆菌的OMP型差异较大 ,同种不同株变形杆菌的OMP型相似 ,但蛋白带的迁移率及颜色深浅在菌株间仍有差异。此外 ,发现相对分子质量约为 4 3× 1 0 4的一条外膜蛋白带为所有菌株所共有 ,可能是变形杆菌属特异性抗原     

几种检测弓形虫血清抗体方法的比较分析

本文对5种弓形虫血清IgG抗体检测方法进行比较评价。5种方法平行检测了80份血清,比较各检测方法的阳性检出率,敏感性,特异性,符合率及Youden指数。结果经配对2检验,ELISA-200,IHA,MAT,SPA-ELISA 4种方法在阳性检出率、平行检测结果之间无显著性差异(P<0.05)。ELISA-200敏感性、特异性和符合率均高于90%,Youden指数(0.85)显著高于其他方法(P<0.05)。IHA,MAT,SPA-ELISA 3种方法各指标处于中等水平,各有适合的检测环境。ELISA-100与其他四种方法在检测结果上差异极显著(P<0.01),特异性(38.3%)、符合率(58.75%)及Youden指数(0.383)都显著低于其他方法。     

Opsonization of yeast cells with equine iC3b, C3b, and IgG

The main opsonins in serum are antibodies and complement factor C3. The opsonization mechanisms including complement activation and deposition are important in studies of phagocytosis and of mechanisms of microbial immune evasion. The objective of the present study was to monitor the deposition of complement C3 and IgG from equine serum on yeast cells (Saccharomyces cerevisiae) using a flow cytometric immunoassay. Correlations were made between the opsonic coating and phagocytic capacity using equine blood neutrophils. In addition, the bound C3 fragments were characterized by SDS–PAGE and Western blot analyses.

Opsonic coating of yeast with equine C3 and IgG occurred rapidly with detectable levels with as little as 0.75% serum. C3 deposition was a result of complement activation and no passive adsorption was observed. When complement was inactivated, the fluorescence indicating IgG deposition increased 3–6-fold, indicating spatial competition between C3 and IgG at binding.

Opsonization with 1.5% serum led to suboptimal equine neutrophil phagocytosis of yeast cells which was dependent on complement activation by the classical pathway. With ≥6.25% serum, IgG contributed to opsonization and phagocytosis. With 50% serum and more, C3 was deposited also by the alternative pathway. Phagocytosis rates became optimal with 3% serum, and did not increase further with higher serum concentrations. The main form of C3 on the yeast cells was iC3b and the rest was C3b without any detectable breakdown products (C3c or C3dg). The equine complement components are similar in size to the human equivalents.

It may be concluded that opsonization of yeast particles leading to phagocytosis, occurs at very low serum concentrations (1.5%) and that it is dependent on activation of the classical complement pathway at this low opsonic level. This is an important finding for efficient host defense, e.g. extravascular phagocytosis at infection sites.     

Accuracy of the TurfTrax Racing Data System for determination of equine speed and position

Reasons for performing study: The speed and position data collected by TurfTrax Racing Data Limited during UK Thoroughbred racing have potential to benefit equine science and welfare. The size (the 2006 data set alone consists of 30,932 individual horse starts across 2667 races) and nature (speed and 2D position for each horse at 4 updates per second) of the data make it a unique resource for questions in equine safety, welfare, performance, and animal locomotion. Objective: To determine the accuracy of the TurfTrax tracking system in estimating the speed and position of horses during racing. Methods: Measurements from the TurfTrax wireless tracking system were compared with those of a survey‐grade global positioning system (GPS) receiver. Results: The TurfTrax system was found to give position measurements to within ± 11 and ± 64 cm in the fore‐aft and lateral directions, respectively, averaging ± 38 cm (interquartile range) and speed to within 0.15 m/s. Potential relevance: The data collected by the TurfTrax system are of sufficient accuracy to inform new diagnoses, training regimens and basic locomotor scientific studies. The position data can provide the precise distance, going, inclination, rate of turn and pack positioning through which each horse has raced. The speed profile can be used to examine the level of exertion, effect of training regimens and influence of racecourse features on performance. A first clinical application would be to analyse retrospectively these factors on occurrence of injury to compare with past training regimens, levels of exertion, and/or racecourse conditions.     

马疱疹病毒gD蛋白主要抗原域的表达及初步应用

根据GenBank马疱疹病毒1型(EHV-1)和4型(EHV-4)gD基因进行分析,选取同源性较高且抗原性强的C端序列设计一对引物进行扩增。将扩增的基因插入pET-30a的BamHⅠ和SalⅠ之间构建原核表达载体pET-gD。然后将pET-gD质粒转化至BL21(DE3)宿主菌,对培养和表达条件进行优化,实现EHV gD蛋白主要抗原区域的高效表达。将重组pET-gD蛋白进行纯化并接种长白兔制备高免血清。经免疫印迹试验鉴定,证实所得纯化表达产物具有良好的抗原性。     

Autologous conditioned serum: the comparative cytokine profiles of two commercial methods (IRAP and IRAP II) using equine blood

Reasons for performing study: Osteoarthritis (OA) is one of the most prevalent and debilitating conditions affecting the horse. Autologous conditioned serum (ACS), commercially available as IRAP and IRAP II, is a recently developed treatment for OA in which plasma is prepared from venous blood by incubation with glass beads for 24 h. This product has been shown to increase anti‐inflammatory cytokines and growth factors in human blood. However, data for equine ACS preparations are lacking. Objectives: To characterise the protein profiles produced by commercially available ACS systems in equine blood. Methods: Blood was drawn from 5 horses into 6 groups: red top vacutainer (control), IRAP and IRAP II, with and without heparin. Samples were collected 1 or 24 h post draw and analysed for IL‐1ra, IL‐10, IGF‐1, TGF‐β, TNF‐α and IL‐1β using ELISAs. Results: Twenty‐four hour IRAP and IRAP II samples contained significantly higher levels of all cytokines relative to 1 h serum controls. At 24 h, IRAP II contained significantly higher levels of IL‐1ra and IRAP contained significantly higher levels of TNF‐α, compared to 24 h controls. In addition, TGF‐β, IL‐10 and IL‐1β in IRAP and IRAP II sera were similar to 24 h serum controls. The addition of heparin significantly reduced levels of IGF‐1, TNF‐α and TGF‐β, and significantly elevated levels of IL‐1ra. Conclusions: The cytokine profile that IRAP II produced is modestly better than IRAP. Incubation of whole blood in glass tubes stimulated cytokine synthesis, although not as efficiently as IRAP II. Potential relevance: Although high levels of IL‐1ra were found in ACS, elevation of other factors suggests these cytokines play a previously understated role in clinical improvements. Because ACS has been shown to alleviate clinical symptoms of OA, the present study suggests that factors other than IL‐1ra alone might be involved in its clinical efficacy. Species‐dependent elevations of cytokines warrant further investigation and optimisation of the systems appears to be necessary based on the differences between human and equine blood.     

Comparison of 4 blood storage methods in a protocol for equine pre-operative autologous donation

OBJECTIVE: To compare viability of equine whole blood stored by 4 different methods, and to establish optimal storage protocols for an equine autologous blood donation program. STUDY DESIGN: In vitro study of stored equine whole blood. Animals- Six healthy adult horses. METHODS: Blood from each horse was collected into 4 different containers: glass bottles containing acid-citrate-dextrose solution (ACD), plastic bags containing ACD, citrate-phosphate-dextrose (CPD), and CPD with supplemental adenine (CPDA-1). Blood was stored for 5 weeks and sampled at 2-day intervals. Standard hematologic and biochemical variables were evaluated, and adenosine-5-triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) concentrations were measured and normalized to total hemoglobin content. RESULTS: Plasma hemoglobin, % hemolysis, lactate, potassium, ammonia, and lactate dehydrogenase (LDH) increased, whereas glucose concentration and pH decreased in all stored blood over 5 weeks. There was a temporal increase in hemolysis with all storage methods, but the increase was greatest in glass bottles. Lactate and ammonia were highest in CPD and CPDA-1 samples, indicating more active red blood cell (RBC) metabolism. 2,3-DPG concentrations decreased during storage, but were optimally preserved with CPDA-1. ATP concentrations were significantly higher for blood stored in CPDA-1, and were lowest in glass bottles. CONCLUSIONS: Hematologic and biochemical values measured for blood stored in CPDA-1 are suggestive of improved RBC viability compared with other storage methods. With the exception of ATP, results from stored equine blood were similar to those reported for other species. CLINICAL RELEVANCE: Commercial CPDA-1 bags appear to be the optimal storage method for equine whole blood.     

Cartilage-derived retinoic acid-sensitive protein in equine synovial fluid from healthy and diseased joints

Reasons for performing study: More sensitive and specific diagnostic methods for early detection of changes in the joint cartilage are needed. Cartilage‐derived retinoic acid‐sensitive protein (CD‐RAP) is a potential marker of cartilage synthesis and regeneration. This is the first study on equine CD‐RAP. Objectives: To evaluate the ability of a commercially available human sandwich ELISA assay to detect equine CD‐RAP in synovial fluid from healthy and diseased joints. Methods: Synovial fluid was collected from 28 horses with no signs of joint disease and from 5 with induced inflammatory arthritis. CD‐RAP concentrations were measured using a human CD‐RAP ELISA. Intra‐ and interassay imprecision of the assay were evaluated by multiple measurements on pools of equine synovial fluid. Assay inaccuracy was determined by linearity under dilution. Results: The assay showed moderate to large intra‐ and interassay variation when applied to equine synovial fluid. Equine CD‐RAP was detected in synovial fluid from healthy horses ranged at 8.2–52 ng/ml. Repeated arthrocentesis (after injection of isotonic saline), age, joint or gender did not significantly affect CD‐RAP concentrations. Twelve hours after intra‐articular injection of lipopolysaccharide, concentrations of CD‐RAP were significantly lower than after injection of isotonic saline and remained significantly lower until the end of the study at 144 h. Conclusion and potential relevance: The assay is suitable for longitudinal monitoring of CD‐RAP concentration in individual horses. Disease significantly influenced CD‐RAP levels. Similar to previous results obtained in man, CD‐RAP seems to be a marker of cartilage synthesis and/or regeneration in horses.     

Comparison of four staining methods for detection of mast cells in equine bronchoalveolar lavage fluid

Mast cells normally are present in equine bronchoalveolar lavage fluid (BALF), but usually represent <2% of all cells in healthy horses. An increased percentage of mast cells has been associated with airway hyperactivity and inflammatory airway diseases, but marked differences are reported between studies in normal and diseased horses. Because an abnormal mast cell count may be of clinical relevance, we compared the ability of a fast Romanowsky method to stain mast cell granules with that of 3 metachromatic stains: automated Romanowsky, May-Grünwald Giemsa, and toluidine blue stains. The BALF cells from 24 horses were studied. A differential cell count was performed blindly on 400 cells. The percentages of mast cells obtained were analyzed by means of repeated-measures analysis of variance and Fischer's PLSD test. The Bland and Altman method was used to assess agreement among stains. The mean percentage of mast cells in BALF was significantly lower with the fast Romanowsky than with the automated Romanowsky, May-Grünwald Giemsa, and toluidine blue stains. With the fast Romanowsky stain, the metachromatic granules of mast cells were not stained, and their identification was based on morphologic criteria. Toluidine blue staining allowed detection of the highest mean percentage of mast cells, but was inadequate for performing a differential cell count on other cell types. In conclusion, fast Romanosky stain may be inadequate for detection of mast cells in equine BALF, whereas automated Romanowsky, May-Grünwald Giemsa, and toluidine blue stains provide metachromatic staining of mast cell granules.     

人血清白蛋白载体的构建及其在山羊乳腺中的瞬时表达

根据GeneBank中人血清白蛋白(HSA)的基因序列,设计一对特异性的引物,从人肝cDNA文库中扩增出其编码序列,并加入酶切位点XhoⅠ,以pCDNA 3.1为模板,设计特异性的引物,并在上游引物加入NotⅠ,下游加入ApaⅠ和NotⅠ,扩增出筛选标记新霉素抗性基因neo,然后再以pBC1为基本骨架,在其NotⅠ、XhoⅠ位点处分别插入hsa和neo,从而得到乳腺表达载体pBC-NEO-HSA,将该质粒注射入奶山羊乳腺中进行暂时性的表达,结果显示所构建的载体能够有效地指导目的基因的表达,从而为下一步构建细胞系、克隆动物作好基础。     

Comparison of isolation and expansion techniques for equine osteogenic progenitor cells from periosteal tissue

Stem cell therapy and cell-based therapies using other progenitor cells are becoming the treatment of choice for many equine orthopedic lesions. Important criteria for obtaining autogenous equine progenitor cells in vitro for use in clinical cell-based therapy include the ability to isolate and expand cells repeatedly to high numbers (millions) required for therapy, in a clinically relevant time frame. Cells must also maintain their ability to differentiate into the tissue type of choice. The objective of this study was to compare isolation and expansion techniques for preparation of periosteal-derived osteogenic progenitor cells for use in commercial autogenous cell-based therapy. Cells were allowed to migrate spontaneously from periosteal tissue or were enzymatically released. Isolated cells were expanded using enzymatic detachment of cells and subsequent monolayer or dynamic culture techniques. Viable osteogenic progenitor cells from each group were counted at 2 weeks, and osteogenic potential determined. Cells isolated or expanded using the explant or bioreactor technique yielded cells at a much lower number per gram of tissue compared with that of enzyme digestion and monolayer expansion, but all cells were able to differentiate into the ostoblast phenotype. Osteogenic progenitor cells isolated by enzymatic release and expanded using monolayer culture reached the highest number of viable cells per gram of donor periosteal tissue while maintaining the ability to differentiate into bone forming cells in vitro. This technique would be an easy, consistent method of preparation of equine osteogenic cells for clinical cell based therapy for orthopedic conditions.     

抗东方马脑炎病毒结构蛋白E2单克隆抗体的制备及其抗原表位的鉴定

为制备抗东方马脑炎病毒(EEEV)结构蛋白E2的单克隆抗体(MAb)并鉴定其抗原表位,本研究以Bac-to-Bac真核表达系统表达EEEV E2蛋白,纯化后作为免疫原免疫BALB/c小鼠,取其脾淋巴细胞与小鼠骨髓瘤细胞SP2/0进行融合.以原核表达载体pET-30a表达并纯化的EEEV E2蛋白作为包被抗原建立间接ELISA方法筛选杂交瘤细胞,获得4株稳定分泌抗EEEV E2蛋白MAbs的杂交瘤细胞株,分别命名为6F3、6F11、7C11、8B11.Western blot与间接免疫荧光试验结果表明,获得的4株MAbs均与EEEV呈阳性反应,而与西方马脑炎病毒、乙型脑炎病毒以及登革热病毒1型～4型呈阴性反应.利用部分重叠的原核表达的短肽对E2蛋白抗原表位进行鉴定,初步确定MAb 6F11、7C11和8B11识别的抗原表位均为E-33 (321EGLEYTWGNHPPKRVW336),而MAb 6F3无短肽与其反应,推测可能为构象表位.本研究结果为建立EEEV型特异性检测方法、研究E2蛋白结构功能及该病的进一步防制奠定了基础.     

Comparison of the properties of highly purified equine chorionic gonadotropin isolated from commercial concentrates of pregnant mare serum and endometrial cups

Commercial concentrates of equine chorionic gonadotropin derived from pregnant mare serum and equine endometrial cups were further purified by sulfoethyl-Sephadex C-50 chromatography and gel filtration on columns of Sephacryl S200. Highly purified fractions were obtained from the serum concentrate and from the endometrial cups (serum eCG and cups eCG, respectively). The two preparations were very similar chemically with respect to amino terminal amino acids (phenylalanine and serine), carbohydrate content (38–45%), and amino acid content. The cups eCG had a slightly greater average mobility than serum eCG by disc gel electrophoresis (R_F = 0.37 and 0.32, respectively).Notable differences existed between the preparations in certain assay systems. The cups eCG was shown to be more potent than serum eCG by LH bioassay, LH radioimmunoassay (RIA), equine and calf testes FSH radioreceptor assays (RRA), and a RIA utilizing an antibody generated against cups eCG. In contrast, the serum and cups eCGs appeared comparably active in an equine testes LH RRA and RIA using an antibody to a serum-derived eCG. Ratios of FSH:LH activity for both preparations were consistently less than 1, but varied considerably between different assay systems. Although the preparations exhibited very little FSH activity in receptor assays utilizing equine tissues and equine gonadotropins as radioligands, considerable binding activity was measured in calf and rat testes FSH RRAs.These data confirm that variations in the nature of eCG can be reflected in chemical, biological and immunological differences. Further, the properties of eCG preparations vary depending on the source material used for isolation and the assay systems employed for evaluation.     

Comparison of refractometer and biuret methods for total protein measurement in body cavity fluids

Abstract: Most hand-held medical refractometers have internal scales that limit protein measurement to results ≥ 2.5 g/dL. Tables for conversion of refraction (r) to protein concentration for values as low as 0.1 g/dL were published in the 1960s, but their accuracy for use on body fluids has not been established. The purpose of this study was to assess the reliability of body cavity fluid protein determination by refractometry. We compared the protein concentration of 25 body cavity fluids as determined by 2 Goldberg type hand-held refractometers with results obtained by the biuret method. Published charts converting refraction (r) to protein concentration were used to determine protein concentration in samples with protein <2.5 g/dL. Higher protein values were read directly from the instruments. The range of comparison was limited to ≥ 0.6 g/dL, the lowest concentration of the biuret method's standard curve. Twenty-one peritoneal fluid, 2 pleural fluid and 2 pericardial fluid samples from 16 horses, 5 cattle, 3 dogs, 2 llamas and 1 cat were tested. The results obtained by the two refractometers were closely and linearly related to biuret results (P<.001), with slopes by linear regression analysis close to 1, and correlation coefficients >0.977. Based on this study, the range for quantification of body cavity fluid protein concentration by refractometry can be extended below 2.5 g/dL, allowing for quantitative assessment of most clinical samples.     

猪圆环病毒2型Rep蛋白基因的原核表达及间接ELISA抗体检测方法的建立

采用PCR方法扩增猪圆环病毒2型(PCV-2)Rep蛋白基因,克隆入原核表达载体pET-32a(+),转化大肠杆菌BL21构建重组表达菌,经SDS-PAGE检测目的蛋白得到成功表达,采用His标签单抗和PCV-2阳性猪血清进行Western blot鉴定表明重组蛋白具有良好的抗原性.利用纯化的重组蛋白rRep作为包被抗原,通过反应条件优化,建立了间接ELISA抗体检测方法.抗原最适包被质量浓度为1 mg/L(0.1 μg/孔),待检血清最适稀释度为1∶100,作用时间为1h,酶标抗体最适稀释度为1∶3 000.用该方法检测猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、伪狂犬病病毒(PRV)阳性血清,结果均为阴性;批内和批间重复试验变异系数分别为＜5％和＜10％,表明本方法具有较好的特异性和重复性.应用rRep-ELISA对113份血清样品进行检测,与rCap-ELISA结果比较,rRep-ELISA检测阳性率为50.44％(57/113),略低于后者的54.87％(62/113),2种方法检测符合率为88.5％(100/113),具有较好的一致性.这为PCV-2感染的流行病学调查和新型ELISA诊断试剂盒的研制奠定基础.     

Lipid-rich bovine serum albumin improves the viability and hatching ability of porcine blastocysts produced in vitro

The effects of lipid-rich bovine serum albumin (LR-BSA) on the development of porcine blastocysts producedin vitro were examined. Addition of 0.5 to 5 mg/ml LR-BSA to porcine blastocyst medium(PBM) from Day 5 (Day 0 = in vitro fertilization) significantly increased the hatching ratesof blastocysts on Day 7 and the total cell numbers in Day-7 blastocysts. When Day-5 blastocysts were culturedwith PBM alone, PBM containing LR-BSA, recombinant human serum albumin or fatty acid-free BSA, addition ofLR-BSA significantly enhanced hatching rates and the cell number in blastocysts that survived compared withother treatments. The diameter, ATP content and numbers of both inner cell mass and total cells in Day-6 andDay-7 blastocysts cultured with PBM containing LR-BSA were significantly higher than in blastocysts culturedwith PBM alone, whereas LR-BSA had no effect on mitochondrial membrane potential. The mRNA levels of enzymesinvolved in fatty acid metabolism and β-oxidation (ACSL1, ACSL3,CPT1, CPT2 and KAT) in Day-7 blastocysts weresignificantly upregulated by the addition of LR-BSA. The results indicated that LR-BSA enhanced hatchingability and quality of porcine blastocysts produced in vitro, as determined by ATP content,blastocyst diameter and expression levels of the specific genes, suggesting that the stimulatory effects ofLR-BSA arise from lipids bound to albumin.     

Specific binding of bovine, ovine, caprine and equine IgG subclasses to defined types of immunoglobulin receptors in gram-positive cocci

One hundred and twenty bacterial strains were tested for non-immune binding of radiolabelled bovine, ovine, caprine and equine immunoglobulins. Bacteria possessing previously defined IgG receptors interacted in a well defined manner with purified IgG subclass immunoglobulins. Human group C and G streptococci carrying IgG receptors type III were capable of binding all IgG subclasses in the four mammalian species studied. Protein A-containing staphylococci demonstrated a restricted specificity with binding of bovine IgG1, ovine IgG1, caprine IgG1 and IgG2 as well as equine IgG(ab). Group A streptococci which can bind human IgG did not show specific reactivity. A new type of binding unrelated to the regular Fc-mediated binding was observed with equine IgG(T).The differences in specificity for IgG subclasses suggest that structures with binding capacity to streptococcal type III Fc receptors are different from staphylococcal protein A reactive sites. Inhibition experiments performed with purified immunoglobulins showed that individual IgG subclasses differed greatly in their inhibiting capacity reflecting differences in avidity.The high avidity and the broad, unrestricted immunoglobulin G reactivity of streptococcal IgG receptor type III indicate that human group C and G streptococci may provide a valuable tool for solid phase absorption of immunoglobulins from several mammalian species.