Immunofluorescent Studies of Bovine Hypersensitivity Pneumonitis

Antigens of Micropolyspora faeni were located by immunofluorescence in apparent association with alveolar macrophages within sections of lung from cases of bovine interstitial pneumonia. Pretreatment of frozen sections with pH 2.8 glycine-HC1 buffer was a necessary prerequisite for specific staining. Bovine immunoglobulin and bovine complement were identified in similar locations.     

Antigenic Relationships of Equine Herpesvirus Strains Demonstrated by the Plaque Reduction and Neutralization Kinetics Test

The antigenic relationships among 50 strains of equine herpesvirus (EHV) were studied by neutralization tests using antisera prepared in rabbits against four EHV reference strains: types 2 and 3, cytomegalo-like virus 82-A, and our leukocyte isolant H-40. No distinctive antigenic differences among reference strains were demonstrated in reciprocal neutralization tests but each antiserum neutralized its homologous virus more rapidly than any heterologous strain. Forty-six EHV strains isolated from peripheral blood leukocytes of apparently healthy horses were antigenically indistinguishable from each other and from the four reference strains. Their high degree of antigenic relatedness suggests that these viruses are isolants of a single, widely distributed serotype of which type 2 (LK) strain is a typical representative.     

Immunofluorescent Studies of Bovine Para-Influenza 3 Virus II. Experimentally Infected Calves

Fluorescent antibody (FA) studies of tissues from three colostrum deprived calves inoculated intranasally with the SF-4 strain of bovine para-influenza 3 (PI-3) virus indicated that these calves developed a mild upper respiratory infection but infected cells were not identified in the lower respiratory tract. Three other calves inoculated intranasally and intratracheally with PI-3 virus developed more severe clinical signs of infection and virus was identified, by FA techniques, in the upper and lower respiratory tract of all three calves and in the spleen of one calf. PI-3 virus was detected in smears of nasal epithelium from five of six calves at some time during the observation period.     

Immunofluorescent Studies of Bovine Para-Influenza 3 Virus I. Cell Cultures and Experimentally Infected Hamsters

Immunofluorescent techniques were used to follow the replication of the SF-4 strain of bovine para-influenza 3 (PI-3) virus in Madin-Darby Bovine Kidney (MDBK), secondary bovine kidney (BK), Embryonic Bovine Trachea (EBTr) cell cultures and experimentally infected hamsters. The virus replicated equally well in MDBK and secondary BK cell cultures but less successfully in EBTr cultures. Nuclear fluorescence was not observed in the cell systems studied.

Infection was limited to the epithelial lining of the upper respiratory tract, trachea and bronchi of experimentally infected hamsters. Virus was most easily identified in smears prepared from turbinate mucosa suggesting a potential diagnostic technique for use in identifying PI-3 infected animals.

     

利用牛Y染色体重复序列进行早期胚胎性别鉴定的研究

本试验利用Y染色体重复序列作为雄性特异性引物,以肿瘤坏死因子（TNF-α）为内标引物建立多重PCR体系,进行牛早期胚胎性别鉴定。共设计四对引物—Y染色体重复序列外引物和内引物,其大小分别为534bp和480bp;肿瘤坏死因子外引物和内引物大小分别为357bp和272bp。试验结果表明,优化后的多重PCR体系的灵敏度分别达到3个胚胎细胞,准确率100%,可以满足早期胚胎性别鉴定的需要。     

牛流行热病毒糖蛋白基因的原核表达与抗原性鉴定

为探索可用来开发牛流行热病毒(bovine ephemeral fever virus,BEFV)疫苗和诊断试剂的候选基因,本研究针对BEFV糖蛋白(G)基因设计了2对特异性引物,用PCR方法扩增基因片段,PCR产物经Xho Ⅰ和Nde Ⅰ双酶切后亚克隆到表达载体pET-30上,将鉴定正确的重组质粒(480-pET-30、1107-pET-30)转化大肠杆菌BL21(DE3)感受态细胞,培养阳性菌株D_{600 nm}值为0.6~1.0,37 ℃下用1.0 mmol/L IPTG诱导表达目的蛋白,将其纯化之后进行SDS-PAGE和Western blotting免疫原性分析。同时,应用间接ELISA、动物免疫试验及交叉反应试验对目的蛋白进行分析。结果表明,首次发现的1 107 bp基因片段是分段表达的,具有很好的生物活性和特异性,更适合作为开发疫苗和诊断试剂的候选基因,为今后建立BEFV的血清学诊断方法及疫苗研发提供了理论基础。     

Prokaryotic Expression and Antigenic Characterization Identification of G Gene for Bovine Ephemeral Fever Virus

In order to explore specific candidate gene of antigens and diagnostics development for the bovine ephemeral fever virus (BEFV), the target gene sequences were derived from G gene by PCR amplification using two specific primers.The PCR products were digested by Xho Ⅰ and Nde Ⅰ and cloned into pET-30 vector, and the recombinant plasmids (480-pET-30, 1107-pET-30) were transformed into E.coil BL21 (DE3) cells, the D_{600 nm} of positive strains were 0.6 to 1.0.The recombinant strains were induced by IPTG (1.0 mmol/L) at 37 ℃.The characteristics of the target proteins were analyzed using SDS-PAGE and the immunogenicity was analyzed through Western blotting.At the same time, the target proteins were used as coating antigens to do ELISA and all rabbits were inoculated with recombinant proteins.The results showed that the expression feature of 1 107 bp gene fragment of G gene for the first time was segmented in vitro as well as has nicer biological activity and specificity, and it more be suitable as a candidate gene for molecular vaccine and diagnostics development.This study provided the theoretical foundation of the establishment of diagnostics technique and vaccine for BEFV.     

南京地区犬的巴贝斯虫病病原形态学观察及免疫荧光鉴定

对我国南京地区犬的巴贝斯虫病的病原形态学进行了系统观察.结果发现,该虫在红细胞内呈多形性,其中圆点状、指环形和小杆形虫体为最常见.间接免疫荧光染色表明,该虫与抗吉氏巴贝斯虫血清可发生明显的特异性反应.结合虫体的形态特征,认为流行于南京地区犬的巴贝斯虫病病原为吉氏巴贝斯虫.     

Demonstration of an Antigenic Relationship Between Hog Cholera and Bovine Viral Diarrhea Viruses by Immunofluorescence

Specific staining of antigen within bovine embryo kidney tissue culture cells, infected with either Oregon C24V or NADL-MD bovine viral diarrhea virus, was accomplished using fluorescein-conjugated swine anti-hog cholera or bovine antiviral diarrhea globulin. Also specific staining of antigen within pig kidney tissue culture cells, infected with hog cholera virus, was accomplished using the same two types of conjugates. Specificity was confirmed by appropriate controls.     

两种方法检测奶牛结核病的比较分析

采用常规皮内变态反应(TST方法)对唐山、秦皇岛、沧州和石家庄四个市共计2 204头奶牛进行牛结核病检测,采用IFN-γ试验方法对PPD检测结果为阳性、可疑的牛以及部分阴性牛进行复检。将IFN-γ试验与TST检测结果相比较,其敏感性为78.26%,特异性为89.09%,符合率为84.33%。研究表明单独使用PPD皮内变态反应方法在牛结核病的诊断方面存在特异性差的缺点,用IFN-γ试验对TST试验阳性和可疑牛进行复检可以提高特异性,避免造成假阳性牛的误杀;该方法在临床上适合推广应用。     

牛结核病两种检测方法的比较

1迟发性变态反应1.1原理目前测定牛结核病的标准方法是迟发性变态反应(delayed typehypersensitivity,DTH)试验,即结核菌素(即PPD,为提纯的结核菌蛋白衍生物,其中包括蛋白、脂肪类、糖类以及核苷酸等多种抗原成分)试验,也是国际贸易的指定方法。结核菌在刺激机体发生免疫应答的时候,迟发性变态反应也随之产生,二者均由T细胞介导产生,但诱导产生免疫和变态反应的物质不同,所以检测机体对结核菌是否发生变态反应,即可判断是否具有免疫力,进而就可得知机体是否感染过结核菌。对牛皮内注射结核菌素,并在3天后测量注射部位肿胀程度。美国就是…     

Bovine Vibriosis: The Distribution and Specificity of Antibodies Induced by Vaccination and Infection and the Immunofluorescent Localization of the Organism in infected Heifers

Two groups of three Holstein heifers were immunized respectively with Vibrio fetus venerealis and Vibrio fetus intestinalis incorporated in Freund's complete adjuvant. Both serum and vaginal mucus agglutination titers increased following immunization. Vaginal mucus samples were more frequently positive when the homologous cells were used as antigen in the agglutination test.

Ten non-immunized heifers were inoculated with another strain of V. fetus venerealis and slaughtered at periods of 30 to 40 and 60 to 70 days post-inoculation (DPI). Agglutinating antibodies were present in the vaginal mucus of some infected individuals by five weeks post-inoculation. In the course of the experiment 11 vaginal mucus samples were obtained which agglutinated heated cells of the infecting strain; one aggglutinated whole cells. Precipitins toward homologous antigens could not be demonstrated in vaginal mucus but four of six samples tested precipitated a heat stable extract from an intestinal strain of the same O-serotype. Bacterial antigen was detected by immunofluorescence on the surface, as well as within and beneath the epithelium at all levels of the reproductive tract regardless of time of slaughter. Lesions in infected animals consisted of focal and diffuse lymphocytosis, plasmacytosis, and epithelial vacuolation. Diffuse neutrophilic infiltration of the oviducts was observed.

Agglutinins appeared in the serum of each of nine heifers immunized with whole cells of same venereal strain. Group mean serum titers for whole and heated cells were 1/28,000 and 1/1,300 respectively. Vaginal mucus samples agglutinated whole cells in 48% of tests while 6.3% reacted with heated cells. Serum, but not vaginal mucus, of immunized animals precipitated soluble antigens of the immunizing strain. The immunizing strain of V. fetus did not infect the reproductive tract of any of six immunized heifers upon challenge.

     

牛传染性鼻气管炎诊断方法研究进展

牛传染性鼻气管炎(IBR)是由牛传染性鼻气管炎病毒(IBRV),即牛疱疹病毒1型(BoHV-1)所引起的以上呼吸道炎症为主的一种牛的急性、热性、接触性传染病,呈世界性流行。IBR的早期准确诊断,对该病的防控具有不可忽视的作用。目前,IBR的诊断方法主要包括病原学诊断和血清学诊断方法。病原学诊断具有特异和敏感及准确等特点,而血清学诊断具有敏感、快速、方便和价廉等特点。为了实施IBR的净化和根除计划,部分国家和地区已逐渐采用IBR基因缺失疫苗,配套使用鉴别诊断方法来鉴别IBR疫苗免疫和自然感染。论文就牛传染性鼻气管炎常用诊断方法的研究进展进行综述,以期为IBR的诊断和防控提供参考。     

吖啶橙免疫荧光菌团法快速检测鱼贝类副溶血性弧菌的研究

本研究建立了适用于检测鱼贝类副溶血性弧菌的吖啶橙免疫荧光菌团培养—沉淀法.用该法可检出每毫升含10万个细菌的菌悬液,不与沙门氏菌、大肠杆菌、猪丹毒杆菌等7种杂菌发生交叉反应.采用随机抽样法对从长春市各大市场采集的351份海产鱼贝类样品进行了检测,其阳性率为46.4%,常规培养法检出率为49.8%,两者相差不显著(P>0.05).该法可在27h之内报告结果,而常规培养法需5～7d.该法具有特异性强、敏感性高、简便快速、所需仪器简单等特点,适用于大样本的检测和基层单位使用.     

Determination of Bovine Serum Low-density Lipoprotein Cholesterol using the N-geneous Method

The N-geneous method is a recently developed method for determination of low-density lipoprotein cholesterol (LDL-C) in human serum. In the present study, we attempted to adapt this method to bovine serum. The values of LDL-C obtained using the N-geneous method were highly correlated with those from the method using ultracentrifugation and heparin sepharose affinity chromatography (r = 0.934, p < 0.001). The reproducibility of this method was acceptable (intra-assay CV 4.2%, inter-assay CV 7.6%) for clinical use. Using the N-geneous method, serum LDL-C was evaluated in cows around parturition, and in cows with fatty liver induced by fasting. The concentration of LDL-C decreased significantly in cows close to parturition. A reduced concentration of LDL-C was also observed in cows with fatty liver. In both cases, the changes of LDL-C were similar to those of apolipoprotein B (apoB)-100, and the values of LDL-C were highly correlated (r = 0.876, p < 0.001) with those of apoB-100. These results suggest that the concentration of LDL-C reflects the level of apoB-100. The N-geneous method is simple and rapid, and might to be a useful tool to elucidate the clinical significance of LDL-C in bovine serum.     

两种牛结核病检测方法对广西奶牛的检测

牛结核病现行防治政策是对牛尤其是奶牛进行检疫,淘汰结核病牛[1]。现在牛结核病应用较多的检疫方法有变态反应[2]、酶联免疫试验     

A型口蹄疫病毒抗原表位与牛IgG重链恒定区的融合表达及活性鉴定

为了研制广谱高效的A型口蹄疫新型多表位疫苗,根据GenBank数据库的A型代表毒株VP1序列设计并合成了VP1结构蛋白上的主要抗原表位DNA段,即135 aa～160 aa和200 aa～213 aa,与牛IgG重链恒定区编码基因连接,将合成的2个表位基因经Xho工、EcoR Ⅰ和BamH Ⅰ酶切后依次克隆到pET-30a(+)载体上,构建重组质粒pRE2IgG,将其转化到大肠埃希菌BL21(DE3)感受态细胞,以IPTG诱导表达融合蛋白pRE2IgG,对表达产物进行SDS-PAGE电泳分析,Western blot检测.结果显示,重组蛋白获得高效表达,并以包涵体形式存在,其分子质量约为60 ku,且能与抗A型FMDV阳性血清发生特异性反应,具有良好的反应活性.