No effect of bovine interferon-tau for control of calf diarrhea and immunomodulation in calves

Newborn calves received a low dose of bovine interferon-tau (boIFN-tau) orally for 4 weeks and calves that had developed diarrhea received a low dose of boIFN-tau orally for 5 days. No effects of boIFN-tau were seen in the duration of the diarrhea, or in daily weight gain. Calves received a high dose of boIFN-tau subcutaneously 3 times and they were then stimulated with bovine herpesvirus type 1 vaccine. No adverse effects were observed after the administration of boIFN-tau and lymphocyte subsets from calves did not change after the stimulation. Our results suggest that boIFN-tau does not seem protecting for preventing calves from diarrhea, recovering the health of calves with diarrhea or immunomodulation, although the treatment itself is not toxic.     

Rapid purification of a 110-kilodalton hemolysin of Actinobacillus pleuropneumoniae by monoclonal antibody-affinity chromatography.

An efficient, single-step method for purification of the 110-kilodalton (kDa) hemolysin of Actinobacillus pleuropneumoniae was developed. An immunoaffinity column was made by cross-linking murine monoclonal antibody 8C2 to the 110-kDa hemolysin of A pleuropneumoniae strain J45 serotype 5 to protein A-agarose beads. Purified hemolysin with high hemolytic activity was obtained after washing the column with phosphate-buffered saline solution, and eluting the hemolysin with 50 mM diethylamine, pH 11.0. The same column was also used to purify the hemolysin from A pleuropneumoniae strain 4074 serotype 1. The purification procedure could be completed within 5 hours, and almost 50% of the total hemolytic activity and hemolysin protein was recovered in pure form.     

Expression and purification of canine granulocyte colony-stimulating factor (cG-CSF)

Canine granulocyte colony-stimulating factor (cG-CSF) with modification of cysteine at position 17 to serine was expressed in Brevibacillus choshinensis HPD31. cG-CSF secreted into the culture medium was purified by ammonium sulfate precipitation and consecutive column chromatography, using butyl sepharose and DEAE sepharose. Biological activity of the recombinant cG-CSF was 8.0 × 10⁶ U/mg protein, as determined by its stimulatory effect on NFS-60 cell proliferation. Purified cG-CSF was subcutaneously administered once a day for two successive days to dogs (1, 5, 25, or 125 μg). Neutrophil count increased the following day in all dogs except those administered the lowest dose (1 μg). No severe side effects were observed in dogs after administration of cG-CSF.     

可溶性E2蛋白作为抗原的检测猪瘟病毒血清抗体间接ELISA方法的建立

以可溶性重组E2蛋白作为抗原,建立了猪瘟病毒(CSFV)血清抗体间接ELISA诊断方法(rE2-ELISA).将猪瘟疫苗毒株E2基因主要抗原区(A-D)基因克隆到表达栽体pGEX-6p-1上,转化E.coli,降低诱导温度至20℃,获得48 000大小E2融合蛋白,部分目的蛋白以可溶性形式表达.Western blotting试验证实,E2融合蛋白可以和CSFV阳性血清发生特异性结合.亲和层析纯化后的E2融合蛋白作为抗原,建立了检测CSFV血清抗体的间接rE2-ELISA方法.该方法的特异性试验结果表明,与PRRSV、PCV2、PPV和PRV阳性血清之间不存在交叉反应;用rE2-ELISA和国外同类试剂盒(CSF-Ab-Kit)检测142份田问血清样品,2种试剂盒的阳性检测率分别为83.81%和88.73%.因此,rE2-ELISA猪瘟抗体检测试剂盒具有良好的敏感性和特异性,适合应用在大规模的CSFV血清抗体的检测工作中.     

猪排泄物中恩诺沙星和环丙沙星含量的HPLC检测方法

为了对猪排泄物中恩诺沙星(enrofloxacin,ENR)和环丙沙星(ciprofloxacin,CIP)进行定量检测,试验建立了测定猪粪尿中ENR和CIP含量的高效液相－荧光检测方法。将猪粪经乙腈－氨水超声提取后,加入三氯乙酸酸化,然后分别将经磷酸酸化后的猪尿和提取后的猪粪溶液经固相萃取小柱富集净化,取净化液进行HPLC分析。HPLC流动相为乙腈(A):柠檬酸/乙酸铵缓冲液(B),梯度洗脱:0~25 min,A 10%~40%;25~30 min,A 40%至10%,荧光检测器的激发波长278 nm,发射波长465 nm。结果表明,ENR和CIP 在尿中的最低检测限(LOD)<0.01 mg/L,在粪中的LOD<0.021 mg/kg,在尿中的最低检测限(LOQ)<0.03 mg/L,在粪中LOQ<0.056 mg/kg,猪尿中的ENR和CIP在0.01~1.0 mg/mL范围内线性关系良好,R²分别为0.9994和0.9992;猪粪中的ENR和CIP在0.02~2.0 mg/mL范围内线性关系良好,R²分别为0.9986和0.9981。ENR在猪粪和猪尿中的回收率分别为79.4%和88.5%,CIP在猪粪和猪尿中的回收率分别为75.8%和89.9%。该方法样品处理简单,检测结果准确可靠,且灵敏度较高,是值得推广的检测方法。     

Method for Determination of Enrofloxacin and Ciprofloxacin in Pig Excreta by HPLC

A HPLC-FLD method was developed for determination of enrofloxacin (ENR) and ciprofloxacin (CIP) levels in feces and urine of pig.The pig feces was ultrasonic extracted by acetonitrile-ammonia, then added trichloroacetic acid to make the extraction acidification.The pig urine was acidulated by phosphoric acid and the extraction of feces solution were enriched and purified by solid phase extraction small column, took purification liquid for HPLC analysis.Conditions of HPLC mobile phase was acetonitrile (A):citric acid/ammonium acetate buffer (B), the procedure of gradient elution was 0 to 25 min, A:10% to 40%;25 to 30 min, A:40% to 10%.The detector of fluorescence excitation wavelength was 278 nm, emission wavelength was 465 nm, chromatographic data were measured and recorded.The results showed that the LOD of ENR and CIP were lower than 0.01 mg/L in urine and 0.021 mg/kg in feces, the LOQ of ENR and CIP were lower than 0.03 mg/L in urine and 0.056 mg/kg in feces.ENR and CIP in the concentration of 0.01 to 1.0 mg/mL levels range had good linear relationship, R² were 0.9994 and 0.9992 in pig urine, respectively;ENR and CIP in the concentration of 0.02 to 2.0 mg/mL levels range had good linear relationship, R² were 0.9986 and 0.9981 in pig feces, respectively.The recovery ratio of ENR were 79.4% and 88.5%, and the recovery ratio of CIP were 75.8% and 89.9% in pig feces and urine.After get on validation, the method was easy in sample processing and testing, the results were accurate, reliable and high sensitivity, which was a worth promoting detection method.     

Accuracy of the composite somatic cell count to detect intra-mammary infection in dairy cows using latent class analysis

The somatic cell count (SCC) is considered an important indicator of intra-mammary infection (IMI). The purpose of this study was to determine the accuracy of both SCC and culture to detect IMI and their conditional dependence by means of latent class methods. This study involved 175 dairy cows from 2 herds with different udder infection prevalences. Quarter and composite milk samples were collected for SCC and bacteriological culture. Latent-class models using Bayesian methods were used to estimate test sensitivity (Se) and specificity (Sp) and population prevalence. The models ran involved only major mastitis pathogens and composite SCC (CSCC). Five thresholds between 100,000 and 300,000 cells/mL were evaluated and the receiver operating characteristics (ROC) curve analysis was performed. Fifty-five percent of the cows had CSCC ≥200,000 cells/mL and 95.4% of the cows had at least one infected quarter either with minor or major pathogens. Considering a threshold of 150,000 cells/mL, the estimated Se and Sp for the CSCC were, 0.80 (95% CrI 0.71–0.88) and 0.57 (95% CrI 0.44–0.71), respectively. The estimated culture Se and Sp were 0.83 (95% CrI 0.73–0.93) and 0.89 (95% CrI 0.74–0.98), respectively. There was no evidence of dependence between CSCC and culture. The area under curve for CSCC was 0.72. To the best of our knowledge, this is the first report of the CSCC accuracy to detect IMI for major pathogens considering the effect of culture misclassification. The estimates provided here could help to examine the performance of sampling schemes based on CSCC to manage udder health.     

吉林白鹅α干扰素在大肠杆菌中的表达及抗病毒活性检测

用PCR方法扩增了吉林白鹅α干扰素(IFN-α)成熟肽编码序列,将IFN-α片段定向插入原核表达载体pGEX-6p-1中,构建重组质粒pGEX-IFN-α,将重组质粒转入大肠杆菌BL21(DE3)感受态细胞里,在IPTG诱导下表达可溶性的融合蛋白(GST-IFN-α)。SDS-PAGE、Western-blot检测结果表明,重组吉林白鹅IFN-α融合蛋白(rGoIFN-α)的分子量大小约为43ku,能与IFN-α抗血清发生特异性结合反应。重组吉林白鹅α干扰素经谷胱甘肽Sepharose-4B亲和柱层析纯化后,在鸭胚成纤维细胞上抗鹅细小病毒的活性为3.32×105 U/mg,本试验结果表明,该表达系统能够表达重组鹅α干扰素,且表达的重组鹅α干扰素具有一定的抗病毒活性。     

Relationship between somatic cell count status and subsequent clinical mastitis in Dutch dairy cows

High composite somatic cell counts (CSCC) in dairy cows may develop into clinical mastitis (CM), suggesting that prevention or intervention of high CSCC may prevent CM later in lactation. The objective of this study was to quantify the relationship between high CSCC in dairy cows and the first subsequent case of CM in the same lactation. Farmer-diagnosed cases of CM and test day CSCC measurements during 1 year of 13,917 cows in 196 randomly selected Dutch dairy herds were available for analysis. Cows were followed in 1 lactation from the first test day postpartum until CM, drying off, culling or end of study. Cox proportional hazards models with time-varying CSCC levels were used to estimate the effect of high CSCC (≥200,000cells/ml) on the time until the first case of CM. A shared frailty effect was included to adjust for clustering of cows within herds. The proportion of cows developing CM after a CSCC measurement was 11%. Primiparae with a high CSCC had a 4-fold higher hazard for subsequent CM than primiparae with a low CSCC; multiparae with a high CSCC had a 2-fold higher hazard than multiparae with a low CSCC. Additionally, multiparae with a low CSCC had a 2-fold higher hazard for CM occurrence than primiparae with a low CSCC. Increasing the threshold for high CSCC showed that the risk for CM increased. If the last CSCC before CM was low, CSCC information of 2 preceding test days was more predictive than CSCC information from only the last test day. When the last CSCC was high, CSCC information of 2 preceding test days did not have added predictive value. This study identified that approximately 25% of first subsequent CM cases after a CSCC measurement can potentially be prevented when cows are prevented to get high CSCC or when high CSCC cows are removed from the population. This corresponded with a decrease in the proportion of lactating cows with CM after a CSCC measurement from 11% to 7%.     

猪圆环病毒2型Cap蛋白单克隆抗体的制备

本研究利用已构建的基因工程重组菌表达猪圆环病毒2型(porcine circovirus type 2,PCV2)ORF2编码的Cap蛋白,纯化后作为免疫原,免疫8周龄BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合。经间接免疫荧光试验(IFA)筛选及有限稀释法进行3次亚克隆后,本试验最终获得5株稳定分泌抗PCV2 Cap蛋白单克隆抗体的杂交瘤细胞,分别命名为3D12、4D5、4B9、4C9和4G10。其中4D5为IgG2b亚型,其余4株单克隆抗体均为IgG1亚型,轻链类型均为κ。Western blotting鉴定结果表明获得的5株单克隆抗体均不能特异性的识别47 ku重组PCV2 Cap蛋白;对病毒感染细胞进行IFA试验,结果显示5株单克隆抗体均特异性的识别病毒抗原,表明5株单克隆抗体识别的抗原表位均为构象表位。中和试验结果表明,5株单克隆抗体均有中和活性。本试验结果为进一步探索ORF2基因的结构、功能及建立快速准确的诊断方法奠定了基础。     

Preparation of Monoclonal Antibodies against Porcine Circovirus Type 2 Cap Protein

The structural protein Cap encoded by ORF2 of porcine circovirus type 2 (PCV2) was expressed in genetic engineering recombinant bacteria and used as the immunogen after purification.Five hybridoma cell lines against PCV2 Cap protein named as 3D12,4D5,4B9,4C9 and 4G10,respectively,were developed after fusion between SP2/0 myeloma cells and spleen cells of BALB/c mice immunized with purified recombinant PCV2 Cap protein.Except the heavy chain type of 4D5 was identified as IgG2b,others were identified as IgG1;The light chains were all kappa.In Western blotting assay,all the monoclonal antibodies (mAbs) couldn't specifically recognize the 47 ku recombinant PCV2 Cap protein,but showed strong specific fluorescence in PCV2 infected PK15 cells in IFA,which indicated that all the mAbs recognized comformational epitope.The neutralization test showed that all the mAbs had neutralization activity.These results laid the foundation for further study of the structure and function of PCV2 ORF2 gene,and establishment of the method for diagnosing PCV2 rapidly and exactly.     

High level expression of biologically active canine interferon-alpha subtype 4 using a baculovirus

In this study, a high amount of bioactive recombinant canine interferon-alpha subtype 4 (CaIFN-alpha4) was expressed in a baculovirus system. For easy purification, it was expressed as a CaIFN-alpha4 bearing histidine hexamer at the C-terminal region, designated CaIFN-alpha4His. CaIFN-alpha4His was detected in culture supernatants of insect cells infected with the recombinant virus using sodium dodecyl sulfate-polyarcylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue staining. The level of expression was very high, and approximately 1 mg of purified protein, with 5.0 x 10(7) units/mg, was obtained from 300 ml of culture supernatant. The purified product showed antiviral activity against Vesicular stomatitis virus on canine tumor cell line A72 and chicken embryo fibroblast cells.     

Fasciola hepatica: studies on vaccination of rats and mice with a surface antigen prepared from fluke homogenate by means of a monoclonal antibody

T1 tegumental antigen was isolated from a homogenate of eight- to 10-week-old Fasciola hepatica using a T1-specific monoclonal antibody bound to sepharose in an antibody-affinity column. Rats and mice were vaccinated with T1 antigen in Freund's complete adjuvant, and control groups received equivalent amounts of non-T1 antigen (eluted from the antibody-affinity column) or ovalbumin. On completion of the immunisation programme, serum samples were collected for ELISA and IFA testing. The animals were challenged by oral infection with F hepatica metacercariae or, for several vaccinated rats, by intraperitoneal transplantation of live adult flukes. At autopsy, worm-burden and liver damage was assessed for each animal and the condition of transplanted flukes was examined. Comparison of test and control groups of animals showed that neither T1 nor non-T1 antigens provided significant protection against challenge, although specific antibody responses against the appropriate sensitising antigen were engendered. Flukes transplanted to the peritoneal cavity of immunised rats survived without damage, although they became encased in hollow fibrous capsules of host origin. The results lend support to the pre-existing concept that glycocalyx turnover by discharge of T1 secretory bodies at the apical surface of migrating flukes provides an efficient means of protection for the parasite against host immunity.     

Purification and renaturation of membrane neuraminidase from Haemophilus parasuis

Haemophilus parasuis, which causes polyserositis, polysynovitis, meningitis, septicemia, and pneumonia in pigs, has emerged as an increasing problem in modern swine production systems. Co-factors for and the pathogenesis of H. parasuis disease are not defined. One of the potential virulence factors of H. parasuis is its neuraminidase (sialidase). While purifying the H. parasuis neuraminidase from the membrane fraction, we developed a protocol to renature enzymatic activity after enzyme preparations were resolved electrophorectically in denaturing polyacrylamide gels. The H. parasuis neuraminidase co-resolved with recombinant neuraminidase of Vibrio cholera; thus its apparent molecular mass is 82 kilodalton (kDa). The H. parasuis neuraminidase was associated with the membrane fraction and the purification protocol removed over 99% of the H. parasuis cell protein while retaining over 90% of the neuraminidase activity. Purified protein will provide another avenue to clone the neuraminidase gene that has been refractory to cloning and the protocol will be a means to purify recombinant protein.     

H9亚型禽流感病毒HA基因的重组禽痘病毒构建

本试验构建了H9亚型禽流感病毒HA基因禽痘病毒转移载体,经转染、蓝斑克隆、筛选和纯化,获得了遗传性状稳定的HA基因重组禽痘病毒。提取感染重组病毒的鸡胚成纤维细胞(CEF)的DNA进行PCR扩增,获得1.7kb的携带有外源目的基因片段。收集纯化的重组病毒CEF细胞,用H9亚型禽流感病毒多克隆血清作一抗,碱性磷酸酶标记的鸡IgG为二抗进行Western blot检测,结果表明重组痘病毒能在体外的CEF细胞表达HA糖蛋白。     

High level expression of recombinant chicken interferon-alpha using baculovirus

Bioactive recombinant chicken interferon-alpha (ChIFN-alpha) was expressed in a baculovirus system. For easy purification, it was expressed as ChIFN-alpha bearing histidine hexamer (His-tag) at C-terminal, designated ChIFN-alphaHis. The expressed proteins were detected by SDS-PAGE analysis with Coomassie brilliant blue staining as around 23 and 19 kDa bands thought to be immature and matured ChIFN-alphaHis respectively. The purified ChIFN-alphaHis with a nickel chelated column showed anti-viral activity in vitro.     

Isolation of Cowdria ruminantium by cellular affinity chromatography and detection by an enzyme-linked immunosorbent assay

The isolation of Cowdria ruminantium by means of wheat germ lectin affinity chromatography as described in this paper permits the recovery of partially purified viable organisms under mild conditions in short time. These conclusions are based upon results of analyses of column fractions by intravenous inoculation into sheep, protein determination, electronmicroscopy and enzyme-linked immunosorbent assay. The entire purification procedure could be completed in 4-5 hours using only either infected sheep tissue or nymphae as starting material.     

猪圆环病毒2型Cap蛋白的截短表达与活性检测

本研究参照已发表的PCV2基因组序列,设计合成1对特异性引物,以PCV2基因组DNA为模板,PCR扩增了长约480 bp的ORF2基因片段。将目的片段定向克隆到pGEX-6p-1原核表达载体,酶切及测序鉴定正确后,转化BL21(DE3)表达菌,经IPTG诱导得到了以包涵体形式表达的重组蛋白。采用亲和层析法在变性的条件下纯化重组蛋白,纯化的重组蛋白浓度为0.396 mg/mL。纯化蛋白经免疫印迹、间接ELISA检测证明具有良好的抗原性和特异性。     

绿色荧光蛋白基因与猪瘟兔化弱毒疫苗株E2基因在杆状病毒中的融合表达

为了研究猪瘟病毒E2糖蛋白的立体结构及生物学特性，将增强型荧光蛋白基因（EGFP）和猪瘟兔化弱毒疫苗株（HCI.V）E2基因经PCR扩增后克隆至pBlueBacHis2A质粒，与杆状病毒DNA共转染后经PCR鉴定获得了含有EGFP和HCLVE2融合基因（GFPTE2）的重组杆状病毒rBACTE2-339，并将其感染sf9细胞后在荧光显微镜下观察到了亮绿色荧光．说明融合基因已初步表达。