Bovine ceroid-lipofuscinosis (Batten's disease): The major component stored is the DCCD-reactive proteolipid,subunit c,of mitochondrial ATP synthase

The ceroid-lipofuscinoses (Batten's disease) are a group of recessively inherited lysosomal storage diseases of children and animals in which there is intracellular accumulation of a fluorescent lipopigment in a wide variety of cells. Lipopigment bodies isolated from pancreas, liver, kidney and brain tissue from a heifer affected with ceroid-lipofuscinosis contained between 55 and 62% protein. A dominant component comigrated on LDS-PAGE with the major low molecular weight protein stored in ovine ceroid-lipofuscinosis. It was identified by amino acid sequence and mass spectroscopy as the full subunit c of mitochondrial ATP synthase, normally found only in the inner mitochondrial membrane, where it is estimated to account for 2–4% of the membrane protein. In pancreatic lipopigment it accounted for at least 40% of the total lipopigment mass and this storage was considered specific to the disease. No other mitochondrial proteins were found in storage bodies. These results are similar to those found in studies on the ovine and the late infantile and juvenile human forms of the disease. It is concluded that bovine ceroid-lipofuscinosis is also a proteolipid proteinosis in which subunit c of mitochondrial ATP synthase is specifically stored in lysosome derived organelles.Abbrevations DCCD dicyclohexycarbodi-imide - LDS-PAGE lithium dodecyl sulphate polyacryl-amide gel electrophoresis - PTH phenylthiohydantoin - CsCl caesium chloride     

The stability of glutathione peroxidase activity in plasma from cattle,pigs and sheep on storage in the presence and absence of glutathione

Ovine, bovine and porcine plasma glutathione peroxidase (GSH-Px) activity decreased on storage at both 4°C and 20°C. The ovine and bovine enzymes were significantly less stable than the porcine enzyme. The addition of GSH to a final concentration of 2 mmol/L to plasma samples at the commencement of storage retarded the loss of both ovine and bovine plasma GSH-Px activity. The ovine enzyme was unique in that after inactivation by storage at 4°C, incubation with GSH restored the enzyme activity. It is recommended that plasma GSH-Px should be assayed fresh, or otherwise stored at –20°C.     

Changes of platelet function and blood coagulation during short-term storage of CPDA-1-stabilised ovine blood

The objective of this study was to detect the influence of short-term storage on the haemostatic function in whole citrated ovine blood at different storage temperatures. Ovine blood was collected in a commercial transfer bag system containing CPDA-1 and stored on a wobbler at room (20-25 °C; n = 5) or refrigerator temperature (4 °C; n = 5). The following analyses were performed initially and after 1, 2, 3, 4, 5, 6, 8, 12, 24, 48 and 72 h of storage: platelet count and (spontaneous) aggregates, agonist-induced platelet aggregation with two methods (impedance aggregometry, turbidimetric method), prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen concentration and resonance thrombography.Platelet count remained stable at room temperature, whereas a significant decrease was detected after 48 h storage at 4 °C. The latter was associated with the formation of a high percentage of platelet aggregates (50-60%) after 5 h storage. Decrease in platelet aggregation was significantly more pronounced when blood was stored at 4 °C. The plasmatic coagulation tests were stable within the observation period.Results indicate that platelet count and aggregability of CPDA-1-stabilised ovine blood is better preserved at room temperature and provides adequate haemostatic function for ex vivo experiments for one working day. Functional loss and high percentage of platelets within aggregates which were observed in ovine blood stored at refrigerator temperature have to be considered in blood transfusion in sheep.     

Effect of storage on measurement of ionized calcium and acid-base variables in equine, bovine, ovine, and canine venous blood.

The stability of blood ionized calcium (Ca2+) and acid-base variables in equine, bovine, ovine, and canine venous blood samples (n = 15, in each group) stored at 4 C for 3, 6, 9, 24, or 48 hours was studied. Variables included blood Ca2+ and standard ionized calcium (Ca2+ corrected to pH 7.4) concentrations, pH, blood carbon dioxide and oxygen tensions, base excess, bicarbonate concentration, and total carbon dioxide content. Results indicate that storage of blood samples at 4 C for up to 48 hours, despite appreciable acid-base changes, is associated with less than 1.5% change in equine, bovine, and ovine blood Ca2+ concentrations. Similar changes were observed in canine blood during the first 9 hours' storage. After 24 and 48 hours' storage, clinically relevant decrease (10.5 and 15.5%) in canine blood Ca2+ concentration was measured. Therefore, Ca2+ concentration in equine, bovine, and ovine venous blood samples stored up to 48 hours, and in canine blood samples stored up to 9 hours at 4 C is of diagnostic use.     

Changes in the quality of meat (Longissimus thoracis et lumborum) from Kamieniec lambs during long‐term freezer storage

The aim of this study was to determine changes in the quality of lamb meat (Longissimus thoracis et lumborum), which was vacuum‐packaged and freezer‐stored (?26°C) for 6 and 12 months. The experiment was performed on 12 male lambs of the Kamieniec Longwool breed, raised to 106 days of age. In comparison with fresh meat, thawed meat was characterized by lower ash content, higher pH, greater natural drip loss and cooking loss, and lower scores for taste intensity. Vacuum packaging and low‐temperature storage protected lamb meat against oxidative changes, and alleviated the adverse effects of oxidation on the color, aroma and taste of meat. It can be concluded that freezer storage (?26°C) of vacuum‐packaged meat can help meet consumer demand for lamb meat products in periods when fresh meat is unavailable. However, it should be noted that long‐term frozen storage induces undesirable changes in meat quality, including a decrease in water‐holding capacity and taste intensity.     

羊泰勒虫吉林株的鉴定及系统进化分析

为了解吉林省延边地区流行的羊泰勒虫种类,采用血涂片镜检和PCR方法对羊泰勒虫进行鉴定和遗传学分析。血涂片镜检观察发现,血涂片红细胞中存在圆形虫体。对羊泰勒虫MPSP基因生物信息学分析显示,MPSP编码的蛋白包含3个N-糖基化位点,14个潜在抗原表位,含有信号肽结构。建立系统进化树显示,分离到的3个虫株与Theileria luwenshunli(GQ281044)同源性为100%,亲缘关系较近,分离的羊泰勒虫为吕氏泰勒虫。由该试验结果可以得出,吉林省延边地区流行的羊泰勒虫为吕氏泰勒虫。     

Clinical and Clinicopathologic Characteristics of Ovine GM-1 Gangliosidosis

Ovine GM-1 gangliosidosis is an inherited lysosomal storage disease. Nine lambs affected with the disease were studied to characterize clinical signs and to determine if there were any pathognomonic clinicopathologic abnormalities. Evaluation included physical, ophthalmic, and neurologic examinations, complete blood counts, serum enzyme and electrolyte analyses, urinalyses, cerebrospinal fluid analyses, blood gas analyses, roentgenograms, electromyograms, and electrocardiograms. Two affected lambs had clinicopathologic tests performed before and after the onset of clinical signs. The only consistent abnormalities recognized were nonspecific signs referable to the central nervous system; predominantly ataxia, conscious proprioceptive deficit most severe in the hind limbs, blindness, and recumbency. Lambs continued to eat and drink, though at diminished levels and with loss of body condition. It was concluded that there are no pathognomonic clinicopathologic abnormalities associated with ovine GM-1 gangliosidosis, and antemortem diagnosis requires enzyme assay of leukocytes or cultured fibroblasts, or lectin histochemistry of tissues obtained by biopsy. Lysosomal storage diseases should be considered among the differential diagnoses in young animals presenting with early neonatal death or with nonspecific neurological signs, in concert with an absence of diagnostic clinicopathologic findings.     

Olive Leaf Water Extract Protects Chicken Breast Sausages Against Quality Deterioration Induced by Frozen Storage

This study aimed to determine the effect of olive leaf water extract (OEx) on the physical properties of chicken breast sausage (CBS) and the preventive effect of OEx against lipid oxidation in CBS during frozen storage. CBSs, to which 0.1 and 0.5% (w/w) OEx were added to minced meat, were stored frozen at −20°C for 60 days. The thawing weight loss of control CBS without OEx increased with the frozen storage period, while OEx-CBSs did not change, from 15 to 60 days in storage. The water-holding capacity, breaking strength, elasticity, and viscosity of control CBS decreased upon frozen storage, while those of OEx-CBSs did not change. The observation of CBSs using scanning electron microscopy showed that OEx-CBSs that were stored frozen, unlike control CBS, maintained a structure similar to their unfrozen counterparts. These results indicate that OEx confers resistance to CBS upon freezing. Furthermore, the application of OEx to CBS suppressed lipid oxidation, decrease in pH and discoloration induced by frozen storage. Thus, this natural OEx is useful in improving the physical and chemical qualities of frozen processed poultry foods.     

Epidemiology of ovine Campylobacter infection determined by numerical analysis of electrophoretic protein profiles

The relationship of 50 Campylobacter strains isolated from aborted ovine foetuses, and the faeces of sheep, cattle and chickens were determined by numerical analysis of electrophoretic (SDS-PAGE) protein profiles. Comparison of protein patterns by numerical methods revealed differences between C. fetus ssp. fetus, C. jejuni, and C. coli strains as well as heterogeneity among isolates from different outbreaks. Isolates from each farm produced a distinct cluster and flocks from different locations were found to be infected with relatively different strains. In most cases, protein patterns of ovine foetal isolates were very similar to those of ovine faecal isolates. Ovine isolates of C. fetus ssp. fetus, C. jejuni and C. coli gave similar protein patterns to the corresponding Campylobacter species isolated from cattle or chicken, on the same farm. Thus, it was concluded that certain protein types of ovine Campylobacter strains were more likely associated with local areas, and Campylobacter strains causing ovine abortions are distributed in the environment more widely than assumed to date.     

Proteomic profiling of ovine serum by SELDI-TOF MS: optimisation, reproducibility and feasibility of biomarker discovery using routinely collected samples

The diagnosis of infectious diseases in animals may be enhanced by study of the serum proteome in which myriad components are influenced by physiological and pathological processes. Surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF MS) has the capacity to detect known and unknown immunologically relevant molecules in the serum proteome. Optimum combinations of ProteinChip array surfaces, energy absorbing molecules, sample dilutions and instrument settings were determined for spectral generation from whole ovine sera. The coefficient of variation for within and between chip mass/charge and peak intensity were <0.03% and <23%, respectively. There were minor alterations in spectra associated with storage of chips or machine drift. Clotting times of 30 min to 3h did not greatly alter protein spectra although storage of sera at -20 degrees C led to alterations. However, routinely collected serum samples stored at -20 degrees C were useful for identification of biomarkers associated with vaccination with a bacterial antigen. This information will inform future studies on serum proteome profiling in livestock, but independent assessments are recommended for each species.     

Effect of storage time on the urine protein: creatinine ratio in alkaline ovine urine

The urine protein:creatinine (UPC) ratio is considered the reference method to assess proteinuria. Its diagnostic value in ovine medicine needs further elucidation. In population monitoring and/or for research purposes, it is convenient to collect many samples simultaneously and store them for later analysis. However, analyte stability data are required to ensure reliable results. We used 15 of 90 urine samples collected from sheep to assess the effect of storage time on the UPC ratio. After centrifugation, the supernatant of each sample was divided into 6 aliquots. Urine protein and creatinine concentrations were determined immediately in one aliquot using the pyrogallol red and a modified Jaffè method, respectively. The other aliquots were stored at −18°C. Based on the absence of active sediment, alkaline urine pH, and UPC ratio ≥0.2, we included 15 samples in our study. The UPC ratio was determined in the stored aliquots 2, 7, 14, 21, and 60 d after collection. The data were analyzed with univariate ANOVA. No significant difference was observed in the urinary concentrations of protein, creatinine, and the UPC ratio (0.8 ± 0.84 in conventional units and 0.09 ± 0.095 in SI units) among different times (p > 0.05). The UPC ratio remained stable for 2 mo in ovine urine samples stored at −18°C.     

重组绵羊pEGFP-PRNP质粒构建及真核细胞转染

构建了携带绵羊朊蛋白基因（PRNP）的重组真核转染质粒,通过脂质体转染试剂将其转染至神经胶质瘤细胞（C6）,以期为进一步研究绵羊朊蛋白的生理功能和从细胞水平研究朊蛋白疾病的发病机制奠定基础。采用PCR扩增目的基因序列,纯化后将其克隆到带有增强型绿色荧光蛋白（EGFP）报告基因的真核表达载体pEGFP-N1中,对重组质粒pEGFP-PRNP做酶切鉴定。而后采用阳离子脂质体转染法将重组质粒转染到C6细胞,荧光显微镜观察。经鉴定,绵羊PRNP基因已克隆到真核表达载体pEGFP-N1中,成功地构建了重组pEGFP-PRNP质粒,并可稳定地在C6细胞中表达。本研究为外源朊蛋白在细胞中表达提供了平台,为进一步在细胞水平研究朊病毒疾病奠定了基础。     

Effects of Pasteurella haemolytica cytotoxin on ovine peripheral blood leucocytes and lymphocytes obtained from gastric lymph

Ovine peripheral blood leucocytes were separated on discontinuous Percoll density gradients into fractions rich in lymphocytes (PBLy) and polymorphonuclear leucocytes. Supernatant fluid from a dialysis sac culture of Pasteurella haemolytica biotype A serotype 1 (A1) was cytotoxic to these leucocytes in vitro. PBLy retained viability after storage in liquid nitrogen and could be employed in cytotoxicity assays. However, sheep cannulated via the common gastric lymph duct were an excellent source of large numbers of homogeneous lymphoid cells (GLy) which also stored well in liquid nitrogen. As both freshly collected and stored GLy were killed by culture supernatant fluid GLy offer advantages as target cells for further characterisation of the extracellular cytotoxin produced by P. haemolytica. From the results obtained, it is considered that all ovine leucocytes are susceptible to P. haemolytica cytotoxin.     

超声波处理对牛乳浓缩蛋白水溶性的影响

经贮藏后的牛乳浓缩蛋白(milk protein concentrate,MPC)的水溶性会下降,而MPC的水溶性直接影响其应用价值.本实验利用超声波技术处理新鲜和贮藏后的MPC,研究不同超声波处理条件对贮藏后的MPC水溶性的影响.结果表明:超声处理(50、300、600 W分别处理5 min; 300 W分别处理3、5、10 min)贮藏后的MPC85,其水溶性显著下降;而超声处理(300 W或600 W分别处理3、5、10 min)新鲜MPC70后在50℃条件下贮藏30 d,其水溶性下降较缓慢且300 W处理10 min的水溶性下降得最慢.因此,在MPC生产过程中进行超声处理,可以在一定程度上减缓MPC贮藏过程中水溶性下降程度.     

Distribution of the Shadoo protein in the ovine brain assessed by immunohistochemistry

Shadow of prion protein is a gene potentially involved in the pathogenesis of prion diseases. However, the Shadoo protein encoded by this gene has not yet been studied in sheep, an important species in prion matters. Therefore, we developed a polyclonal antibody against ovine Shadoo and assessed the presence and distribution of this protein in the ovine brain by immunohistochemistry. The strongest staining level was found in the cerebellum (especially in the Purkinje cells) and in the pons, but cerebrum, hippocampus, pituitary gland, medulla oblongata, thalamus and hypothalamus were also immunopositive. Remarkably, a typical granular pattern was seen in most of the tested brain tissues, which might indicate that Shadoo is primarily expressed at synapses. The results of this study and the availability of an ovine anti-Shadoo antibody can contribute to future research on the function of Shadoo and on its potential involvement in prion diseases.     

Effect of storage temperatures and duration on quality of prepared chicken breast with paprika oleoresin

The objective of this study was to investigate the effect of storage temperatures and time on discoloration and oxidation of prepared chicken breast with paprika oleoresin. Freshly prepared chicken breast containing paprika oleoresin was stored at ?2, ?10, ?18°C, and an oscillating temperature between ?10 and ?18°C (?10/?18°C). A significant decrease in redness was detected at ?2, ?10, and ?10/?18°C. The lowest TBARS values and carbonyl contents were observed in the samples stored at ?18°C for 5 weeks. Also, the values of sulfhydryl groups gradually decreased with the increase in storage temperatures and duration. The results suggest a positive correlation between the loss of redness and oxidation in all samples. The findings indicated that the discoloration and oxidation of prepared chicken breast added with paprika oleoresin were inhibited significantly when stored at ?18°C.     

Analysis of cerebrospinal fluid from field cases of some common ovine neurological diseases.

Analysis of cerebrospinal fluid (CSF) samples from normal sheep and from cases of some common neurological diseases revealed a significant increase (P less than 0.05) in the group mean CSF protein concentration for meningitis, listeriosis and spinal abscess but not for scrapie, spinal injury, ovine pregnancy toxaemia or polioencephalomalacia. The CSF white blood cell count (WBC) was significantly increased (P less than 0.05) in the meningitis group and in those cases of listeriosis which failed to respond to antibiotic therapy. All cases of bacterial infection of the central nervous system (CNS) could be identified by the combined interpretation of the protein concentration and the differential WBC count. It is concluded that CSF analysis is useful clinically in differentiating traumatic from infective spinal lesions and toxic or metabolic lesions from bacterial meningitis in sheep.     

Comparison of methods for assay of fecal proteolytic activity

Fecal proteolytic activity (FPA) in ten normal dogs was readily detected either calorimetrically using azocasein substrate or by radial enzyme diffusion into agar gels containing casein substrate. Daily FPA ranged from 17 to 207 azocasein units/g (ACUIG) or 4 to 18 mm of casein hydrolysis, while mean 3-day FPA ranged from 58 to 10 1 ACUIG or 7 to 15 mm. Studies of proteolytic activity remaining after treatment of fecal extracts with a specific trypsin inhibitor indicated that trypsin accounted for 0% to 71% of proteolytic activity. Proteolytic activity decreased steadily in fecal specimens stored at room temperature or above, but there was only slight loss in activity during storage for up to 5 days at 4 degrees C. Proteolytic activity was unaffected by repeated freezing and thawing and samples could be stored for long periods at -2 degrees C without noticeable loss of activity. It is concluded that assays of FPA using either azoprotein substrate or radial enzyme diffusion into agar gels containing casein substrate allow evaluation of exocrine pancreatic function in dogs, provided that several samples are tested. These methods are suitable for application in a variety of species.     

Effect of storage on oxygen dissociation of canine blood.

Oxygen dissociation curve and adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (DPG) contents in red blood cells (RBC) were determined in canine blood stored in acid citrate dextrose (ACD) and citrate phosphate dextrose (CPD) solutions. The oxygen-unloading ability decreased, as shown by the left shift of oxygen dissociation curve during storage, and the shift correlated with decreasing DPG but not decreasing ATP concentrations. After 2 weeks of storage in ACD solution, oxygen dissociation curves were shifted significantly to the left. For blood stored in CPD solution, 4 weeks was required before the shift was significant. It was concluded that canine blood collected and stored in CPD solution is more efficient than that stored in ACD solution in delivering oxygen to the tissues.     

Preservation of Skin by Refrigeration for Autogenous Grafting in the Horse

Eighteen stored split thickness meshed skin grafts were applied to surgically created lesions on the metacarpal and metatarsal regions of six horses. Donor skin was harvested from the sternal region, meshed and stored at 4 degrees C in a cell culture medium containing 10% serum. Stored grafts were applied to the wounds at 1, 2, and 3 week intervals. Acceptance of the grafts stored for 1 week was generally poor (1 of 6 grafts), whereas that of the 2 and 3 week old grafts was generally excellent (10 of 12 grafts). Poor acceptance of the 1 week old grafts was attributed to streptococcal infection of the recipient wounds. Using the storage medium and grafting technique described, excellent acceptance can be expected after graft storage of up to 3 weeks.