猪流行性腹泻病毒S蛋白原核表达及其单克隆抗体制备

本研究以猪流行性腹泻病毒(PEDV)全基因组为模板,利用RT-PCR方法扩增PEDV S基因的亲水区,将其克隆至pMAl-c2E原核表达载体,获得重组质粒pMAl-c2E-PEDV-S。经EcoRⅠ/SalⅠ双酶切测序鉴定后,转化BL21菌,经IPTG诱导,成功表达了约74 kDa的重组蛋白MBP-PEDV-S,亲和层析法纯化重组蛋白。将重组蛋白免疫3只健康的6～8周龄雌性BALB/c小鼠,将小鼠脾脏B淋巴细胞与SP2/0瘤细胞融合,采用间接ELISA方法筛选阳性杂交瘤细胞株,获得了4株能稳定分泌抗PEDV S蛋白抗体的杂交瘤细胞株1B11、1B10、1C8、3L3,其腹水效价都高于1∶100 000。获得的4株单克隆抗体与PEDV经IFA和Western blot检测,结果显示均具有良好的反应性。而且3株单克隆抗体重链为IgG2a,1株重链为IgG2b;3株轻链为Kappa,1株轻链为Lambda。本研究表达了PEDV S蛋白并制备了单克隆抗体,为研究S蛋白的结构和功能提供了条件,为揭示PEDV致病机制奠定了基础。     

Paleocirculation of the deep north atlantic: 150,000-year record of benthic foraminifera and oxygen-18

Benthic foraminiferal faunas in a piston core from 3331 meters at 44 degrees N on the Mid-Atlantic Ridge show striking variations in the relative abundance of species. Uvigerina peregrina, which is broadly distributed today in the South Atlantic and in the Pacific in water that has been long isolated from the surface, is absent in the North and Equatorial Atlantic at depths occupied by highly oxygenated North Atlantic deep water. This species dominated the fauna at this site for much of the past 150,000 years. It is suggested that North Atlantic deepwater production was much reduced or eliminated at times of Uvigerina peregrina abundance, as a result of cooling and stratification of the Norwegian Sea surface, coincident with the times of the southward migration of the polar front in the North Atlantic.     

The aetiology of spinal deformity in Atlantic salmon, Salmo salar L.: influence of genetic factors on the frequency and severity in freshwater stages

A radiographic study was carried out on 2-year groups of pre-smolt parr to detect the comparative frequency of vertebral change in a range of defined pedigreed families of high growth rate of Atlantic salmon, Salmo salar L., reared under identical conditions. The observed frequency of deformity was related to the observed deformity level in the seawater parental generation, and to a potentially uncontrolled environmental determinant, i.e. stripping date. High and low parental deformity groups of families were found to produce offspring that had very similar levels of radiographically detectable spinal deformities in the pre-smolt parr, suggesting no genetic link. With regard to stripping date, the overall incidence was nearly half that seen in the preceding year (8.7% cf 16.6%). However, a non-significant trend to fewer radiographic spinal deformities was seen in the later stripping period.     

Development of a functional scoring system in dogs with acute spinal cord injuries

OBJECTIVES: To develop and compare the reliability of 2 methods of scoring pelvic limb gait in dogs recovering from thoracolumbar spinal cord injuries and to use this scoring system to determine the rate and level of functional recovery of dogs with acute thoracolumbar intervertebral disk herniations. ANIMALS: 46 dogs with spinal cord injuries resulting from intervertebral disk herniations. PROCEDURE: Dogs' gaits were videotaped at different time intervals after injury. In phase 1 of the study, the stages of recovery of pelvic limb function were identified, and a numeric scoring system was devised to reflect that recovery. In phase 2, pelvic limb gait was scored by different observers, using a numeric and a visual analog scale. Intra- and interobserver coefficients of variability of both methods were compared. In phase 3, pelvic limb function was scored, using the numeric scale at various intervals after acute thoracolumbar disk herniations. RESULTS: The numeric scale was significantly more reliable than the visual analog scale when both intra- and interobserver coefficients of variability were evaluated. Dogs that were paraplegic with no deep pain sensation recovered at different rates during the first 3 months, whereas dogs that were paraplegic with deep pain sensation typically recovered within 1 month of injury. CONCLUSIONS: Pelvic limb gait of dogs recovering from thoracolumbar spinal cord injuries can be reliably quantified, using a numeric scale. This scale will facilitate the performance of clinical trials aimed at improving the outcome of acute spinal cord injuries.     

Zinc and copper status in ewes supplemented with sulfate- and amino acid-complexed forms of zinc and copper

Thirty 6-yr-old Targhee ewes were randomly allotted to one of five supplemental treatments to evaluate supplementation effects on liver and fecal Zn and Cu concentrations and serum alkaline phosphatase activity: 1) control, 2) Zn complex, 3) Zn and Cu (ZnCu) complex, 4) Zn sulfate, and 5) ZnCu sulfates. Supplements were administered daily in gelatin capsules for 56 d. Liver biopsies and serum samples were collected every 14 d starting on d 0. Supplemental Zn and Cu levels were formulated to provide 90 mg/kg Zn and 10 mg/kg Cu, respectively, on a daily dry matter intake basis. Form (complex vs sulfate) x type (Zn vs ZnCu) interactions were not detected (P > 0.35). Therefore, contrast statements were used to make the following treatment comparisons: 1) control vs supplement, 2) Zn vs ZnCu, and 3) complex vs sulfate. Ewe BW at the end of the study (P = 0.09) and ewe BW change from beginning to end of the study (P = 0.07) were greater for supplemented than control ewes. Body weight and BW change did not differ between sulfate and complex (P > 0.39) or Zn- and ZnCu- (P > 0.40) supplemented ewes. Liver Cu concentrations did not differ (P = 0.41) between control and supplemented ewes. Liver Cu concentrations were higher (P < 0.10) for ewes supplemented with ZnCu than Zn and complex than sulfate forms of supplement. Liver Zn concentration tended (P = 0.13) to be higher in ZnCu than Zn-supplemented ewes. Liver and fecal Zn concentration were higher (P < 0.06) in ewes fed complex than sulfate supplements. However, serum alkaline phosphatase activity tended (P = 0.12) to be greater in ewes fed sulfate than complex supplements. Supplementing mature ewes with complexed minerals resulted in higher concentrations of Zn and Cu in the liver. In addition, supplemental Cu tended to increase concentrations of Zn in the livers of ewes; however, high levels of supplemental Zn did not negatively impact liver Cu concentrations.     

Evaluation of glycoprotein Ib expression on feline platelets

OBJECTIVE: To determine whether platelets obtained from cats expressed glycoprotein Ib (GPIb). SAMPLE POPULATION: Platelets obtained from 11 specific-pathogen-free cats. PROCEDURE: Platelets were analyzed by use of immunofluorescence microscopy, flow cytometry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western immunoblot analysis, and immunoprecipitation. RESULTS: Immunofluorescence microscopy and flow cytometry revealed the protein on the surface of feline platelets. Biochemical studies (western immunoblot analysis and immunoprecipitation) revealed a 140-kd membrane glycoprotein. Additional biochemical studies revealed that feline GPIb was sensitive to proteolysis, because platelet cytoskeletons prepared with low concentrations of a calpain inhibitor (ie, leupeptin; 100 microg/ml) had substantial proteolysis, and there was an association of protein fragments with the actin cytoskeleton. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of these results indicate that feline platelets express a 140-kd membrane protein that is recognized by monoclonal antibodies developed against GPIb. Application of standardized ELISA to quantitate glycocalicin, the water-soluble fragment of GPIb, may provide important information on the production of microvesicles, increased platelet turnover, and abnormal proteolysis.     

The generation and persistence of genetic variation in foot-and-mouth disease virus

Genetic variation in foot-and-mouth disease virus (FMDV) is of interest for at least two reasons. First, changes to the genes encoding capsid proteins results in antigenic variation, and affects vaccine efficiency and effectiveness of vaccination programs; second, genetic changes can lead to important insights into the transport of virus between countries, regions, herds, and even possibly individuals. Current estimates of RNA virus mutation rates suggest that an average of about one base mis-incorporation is likely to occur each time a single FMDV genome replicates. This should result in the introduction of every possible 1-step mutation from the progenitor genotype into the viraemia of a single infected animal many times a day. In the absence of purifying selection, a single infected animal should therefore generate a genetically very diverse population of virus.Viral-capsid sequences obtained from infected animals sampled over long-term FMDV epidemics suggest that these genetic changes accrue in a remarkably linear 'clock-like' fashion and at rates of around 1% change per year. While such a rate is generally regarded as quite high, it is actually somewhat lower than one might expect based on the rate at which viral diversity could be generated within a single animal. The difference might be explained in a variety of possible ways: (1) the mutation rate has been overestimated; (2) purifying selection is stronger than predicted; (3) only a restricted subset of excreted virus is actually infectious; (4) infected animals only excrete virus from a small partitioned subset of amplified virus, and that most of the generated viral diversity is unable to exit the animal; or (5) only a small fraction of all infected animals participate in the actual disease-transmission process.     

Dual Sarcocystis neurona and Toxoplasma gondii infection in a Northern sea otter from Washington state, USA

Dual Sarcocystis neurona and Toxoplasma gondii infection was observed in a Northern sea otter from Washington, USA. The animal was found stranded, convulsed, and died shortly thereafter. Encephalitis caused by both S. neurona and T. gondii was demonstrated in histological sections of brain. Immunohistochemical examination of sections with S. neurona specific antisera demonstrated developmental stages that divided by endopolygeny and produced numerous merozoites. PCR of brain tissue from the sea otter using primer pairs JNB33/JNB54 resulted in amplification of a 1100 bp product. This PCR product was cut in to 884 and 216 bp products by Dra I but was not cut by Hinf I indicating that it was S. neurona [J. Parasitol. 85 (1999) 221]. No PCR product was detected in the brain of a sea otter which had no lesions of encephalitis. Examination of brain sections using T. gondii specific antisera demonstrated tachyzoites and tissue cysts of T. gondii. The lesions induced by T. gondii suggested that the sea otter was suffering from reactivated toxoplasmosis. T. gondii was isolated in mice inoculated with brain tissue. A cat that was fed infected mouse brain tissue excreted T. gondii oocysts which were infective for mice. This is apparently the first report of dual S. neurona and T. gondii in a marine mammal.     

Postoperative performance of racing horses with tearing of the medial palmar intercarpal ligament

OBJECTIVE: To examine the relationship between medial palmar intercarpal ligament (MPICL) tearing and postoperative performance in racing horses. MATERIALS AND METHODS: The postoperative performance of 42 horses in which the midcarpal joint was examined arthroscopically was followed prospectively. Intra-articular variables examined were the severity of MPICL tearing, subchondral bone damage and articular cartilage damage. Using a scoring system based on the class of race and the position in the race, a mean score was calculated for up to five races before and after surgery. The preoperative score was subtracted from the postoperative score to give a net score. Statistical analysis was by a Mann Whitney U test and multiple linear regression. RESULTS: Thirty-two (76%) raced postoperatively, 23 (55%) won at least one race and 12 of 26 (46%) performed at the same or higher level. Horses with grades 2 to 4 MPICL tearing had significantly lower net scores than those with grade 1 or no tearing. The severity of subchondral bone damage was the only variable on its own that was significantly correlated with net score (r2 = 0.23, P < 0.05). The addition of the grade of MPICL tearing to bone damage significantly improved the prediction of postoperative performance (P < 0.05). The inclusion of articular cartilage damage had no effect on the prediction of postoperative performance. CONCLUSION: Tears involving more than one third of the MPICL as observed arthroscopically have a significant detrimental affect on postoperative performance of racing horses.