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1.
为建立区分猪种布鲁菌S2疫苗株接种奶牛与布鲁菌自然感染奶牛,BLAST比对分析羊种、牛种、猪种、犬种、沙林鼠种和绵羊种6种布鲁菌基因序列,发现repA-related基因是猪种布鲁菌与牛种及羊种布鲁菌的差异基因。设计引物PCR扩增获得repA-related基因片段,克隆并原核表达得到了布鲁菌repA-related融合蛋白,以repA-related蛋白建立间接ELISA检测方法。用repA-related蛋白间接ELISA检测猪种S2疫苗株接种动物血清为阳性,检测牛种和羊种布鲁菌自然感染动物血清为阴性。repA-related蛋白间接ELISA能从试管凝聚实验(SAT)及常规ELISA检测阳性的奶牛血清样本中,区分出S2疫苗接种牛与牛种布鲁菌感染牛。 相似文献
2.
利用SUMO-BCSP31重组大肠杆菌表达并纯化了布鲁菌BCSP31重组蛋白,并利用该蛋白研制了布鲁菌免疫胶体金检测试纸条。以BCSP31蛋白作为检测抗原,通过间接法研制胶体金试纸条检测鹿血清抗体。该试纸条与大肠杆菌、沙门菌、小肠结肠炎耶尔森菌和绿脓杆菌无交叉反应;检测阳性血清最大稀释倍数为1∶640,证明试纸条具有良好的特异性和敏感性。临床上对420只鹿进行检测,比对试纸条与虎红平板凝集试验(RBT)检测结果:阳性符合率为100%,阴性符合率为96%,Kappa值为0.73,两种方法符合率较高;比对试纸条与cELISA检测结果:阳性符合率为81%,阴性符合率为100%,Kappa值为0.88,两种方法符合率高。该试纸条简便快捷,具有较高准确性,可用于鹿布鲁菌病的现场初筛检测。 相似文献
3.
《中国动物传染病学报》2019,(5)
本研究分别以布鲁菌LPS和牛分枝杆菌MPB70蛋白作为检测抗原,建立了动物布鲁菌病和结核病双抗原夹心法胶体金抗体检测技术,并研制了同时检测动物布鲁菌病和结核病的快速抗体检测试纸条,用于动物布鲁菌病和结核病临床快速血清抗体检测。检测试纸条与其他病原无交叉反应,与牛、鹿口蹄疫血清、巴氏杆菌病血清、耶尔森氏菌病血清无交叉反应;敏感性高,临床采集的布鲁菌病和结核病阳性血清最大稀释倍数可以达到1:512;特异性好,准确率高。研制的检测试纸条与现有的检测方法具有较高符合率:与RBT检测布病结果阳性符合率为100%,阴性符合率为96%,总符合率为96%;与牛结核γ-干扰素ELISA检测结核病结果阳性符合率94%,阴性符合率为97%,总符合率为96%。试纸条检测卡在吉林省多个地区进行临床试验,具有快速、敏感、特异、操作简单、可以用于现场快速检测等优点,能够同时用于动物布鲁菌病和结核病的临床抗体检测。 相似文献
4.
为建立区分猪种布鲁菌S2疫苗株接种奶牛与布鲁菌自然感染奶牛,BLAST比对分析羊种、牛种、猪种、犬种、沙林鼠种和绵羊种6种布鲁菌基因序列,发现repA—related基因是猪种布鲁菌与牛种及羊种布鲁菌的差异基因。设计引物PCR扩增获得repA-related基因片段,克隆并原核表达得到了布鲁菌repA—related融合蛋白,以repArelated蛋白建立间接EI．IsA检测方法。用repA—related蛋白间接ELISA检测猪种s2疫苗株接种动物血清为阳性,检测牛种和羊种布鲁菌自然感染动物血清为阴性。repA—related蛋白间接EusA能从试管凝聚实验（SAT）及常规ELIsA检测阳性的奶牛血清样本中,区分出s2疫苗接种牛与牛种布鲁菌感染牛。 相似文献
5.
【目的】建立口蹄疫病毒(FMDV)感染与免疫鉴别诊断及免疫评价二联试纸稳定的生产工艺,促进其产业化生产及临床应用。【方法】本研究以胶体金免疫探针结合猪IgG,FMDV结构蛋白(SP)及非结构蛋白(NSP)的表位多肽偶联载体蛋白制备人工抗原,设置两条检测线精准拦截抗体的检测模式,以试纸条特异性及敏感性为评价指标,对胶体金免疫探针用蛋白、试纸拦截用多肽抗原、检测线位置及喷膜缓冲液、金标蛋白保存液、样品垫缓冲液以及样品稀释液等参数进行优化。采用优化后的试纸检测266份田间猪血清样品,并与口蹄疫O型抗体液相阻断ELISA检测试剂盒(LPB ELISA)和3ABC阻断ELISA抗体检测试剂盒(3ABC ELISA)检测结果进行对比,计算该试纸与商品ELISA试剂盒的符合率。【结果】经优化后,口蹄疫感染与免疫鉴别诊断及免疫评价二联试纸生产工艺参数如下：以胶体金标记金黄色葡萄球菌A蛋白(SPA)为免疫探针;选择混合多肽的形式设置拦截线,以非结构蛋白多肽(BSA-NSPs)喷涂T1线,结构蛋白多肽(BSA-SPs)喷涂T2线,以0.1 mol/L Tris-HCl为喷膜缓冲液;以ddH2 相似文献
6.
《畜牧与兽医》2017,(8):62-67
为了鉴别羊种布氏杆菌自然感染和S2疫苗(猪种)免疫羊,选用布氏杆菌S2疫苗株进行了全基因组测序,预测出的编码基因与已公布的布氏杆菌基因组数据库比对分析,找出S2特有基因GL_0002181,对GL_0002181基因进行原核表达及免疫原性验证,同时用BP26蛋白作为对照组。Western blot结果显示:以S2疫苗免疫绵羊血清作为检测抗体,重组菌BL21(pETGL_0002181)、BL21(pETBP26)与BL21(DE3)空菌比较,分别在31 ku、32 ku处出现了特异性条带,说明重组蛋白能够与S2免疫血清中的抗体发生特异性反应,具有较好的免疫原性;而重组表达菌BL21(pETGL_0002181)菌体蛋白与自然感染血清不反应,重组蛋白BP26与自然感染血清反应,说明GL_0002181基因在S2疫苗株中存在,在羊种自然流行株中不存在。结果表明:GL_0002181抗原可用于布氏杆菌S2疫苗免疫与羊种布氏杆菌自然感染鉴别诊断,为布氏杆菌病有效诊断提供理论依据。 相似文献
7.
为研制安全、有效、新型靶向化布鲁菌外膜蛋白疫苗,采用去污剂连续抽提与电洗脱法分离纯化出布鲁菌外膜蛋白,并进行α-D-甘露吡喃异硫氰酸苯酯糖基修饰、佐剂乳化.依据统计学中完全随机实验设计试验,免疫BALB/c小鼠,收集不同时间点血液样品,ELISA法检测血清中IgG和IFN-γ含量.结果显示分离纯化出37.5 kD与47.2 kD的目的蛋白并成功糖基化修饰;纳米颗粒佐剂乳化后的试验用疫苗免疫小鼠,血清IFN-γ水平呈显著升高趋势(P＜0.05);抗体水平略显优势,但差异不显著(P＞0.05).表明试验用疫苗能激发机体的细胞免疫应答,且产生了免疫记忆.本研究为研制布鲁菌新型靶向化疫苗的研发和推广提供了研究依据. 相似文献
8.
为建立一种快速诊断牛布鲁菌病的方法,以原核表达、纯化的外膜蛋白OMP22作为抗原,以带荧光标记的IgG抗体作为标记物制备免疫层析试纸条,建立一种布鲁菌病快速检测方法,并对该方法的敏感性和特异性进行验证。结果显示,制备的试纸条最低可检出1∶100稀释的牛布鲁菌标准阳性血清,且与牛结核、牛蓝舌病、牛病毒性腹泻、牛口蹄疫、牛巴氏杆菌病、牛白血病的血清无交叉反应;试纸条检测结果与商品化ELISA检测试剂盒(IDEXX)的符合率为93.5%。以上结果表明,建立的荧光标记免疫层析试纸条方法能快速检出牛血清中的布鲁菌抗体,具有良好的敏感性和特异性,是一种适合现场初筛的检测方法。 相似文献
9.
试验旨在利用胶体金免疫层析技术建立快速检测犬血清中犬细小病毒（canine parvo virus, CPV）血凝抑制（haemagglutination inhibition, HI）抗体效价的方法,用于CPV疫苗免疫效果评价。采用双抗体夹心法,以抗CPV血凝相关抗原的单克隆抗体制备CPV抗原检测试纸条;将犬血清进行不同比例系列稀释后,分别与定量CPV抗原充分反应,滴入CPV胶体金试纸条,根据试纸条检测线（test line,T线）消失时的血清最高稀释倍数判断血清中CPV抗体的HI效价;用此方法检测86份犬血清样品,并与传统血凝抑制试验方法进行分析比较。结果显示,成功制备CPV抗原检测试纸条,确定了试纸条检测犬血清CPV-HI效价的反应条件和结果判定标准。结果表明,在检测不同稀释倍数犬血清反应后的CPV抗原时,能使试纸条T线消失时的血清最高稀释倍数与HI效价具有正相关性,犬血清最高稀释倍数乘以4即为HI效价;两种方法的符合率达90.7%。本试验初步建立了胶体金试纸条检测CPV血凝抑制效价的方法,为检测CPV-HI效价提供了一种操作简单、快速的试验方法,可用于CPV疫苗免疫效果评价。 相似文献
10.
以H5N1亚型虎源流感病毒免疫Balb/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0融合,用间接ELISA试验检测细胞培养上清,获得2株阳性杂交瘤细胞克隆株,命名为3A13和1B8.分别制备腹水后进行纯化,获得了抗H5亚型虎源流感病毒血凝素的单克隆抗体,用制备的单克隆抗体结合胶体金免疫层析技术,制备了H5亚型虎源流感病毒免疫胶体金快速诊断试纸条.该试纸条对H5N1虎源流感病毒鸡胚培养毒、H5亚型禽流感病毒标准抗原,以及24份虎疑似感染H5N1亚型虎源流感病毒感染病料和小鼠模型病料等检测结果为阳性,同时对H7和H9亚型禽流感病毒标准抗原及传染性支气管炎、新城疫抗原等检测结果为阴性;与HA-HI和RT-PCR的平行对比试验表明,试纸条检测敏感度与血凝试验和血凝抑制试验的敏感性相符.本试验所研制的免疫胶体金诊断试纸条可用于H5亚型虎源流感病毒的特异诊断和流行病学调查,为开发成品化检测试纸条奠定基础. 相似文献
11.
Single-step purification and evaluation of recombinant BP26 protein for serological diagnosis of Brucella ovis infection in rams 总被引：1，自引：0，他引：1
Zygmunt MS Baucheron S Vizcaino N Bowden RA Cloeckaert A 《Veterinary microbiology》2002,87(3):213-220
To investigate the value of the BP26 protein in the serological diagnosis of ovine brucellosis caused by Brucella ovis, recombinant BP26 protein was produced in Echerichia coli and purified for use in an indirect enzyme-linked immunosorbent assay (I-ELISA). The majority of the recombinant protein was recovered from the supernatant of sonicated recombinant E. coli cells in a soluble form. This facilitated the purification of the recombinant BP26 protein which was achieved by using ion-exchange chromatography. After one step of purification, the purity of the recombinant BP26 protein was analyzed by using SDS-PAGE, Coomassie blue staining, and Western blot with a monoclonal antibody (MAb) directed against the BP26 protein. The degree of purity appeared satisfactory so that it could be directly used in I-ELISA. Although the recombinant BP26-ELISA appeared less useful than I-ELISA using the B. ovis hot saline (HS) extract as antigen, the high number of sera from B. ovis infected rams found positive (90%) in the recombinant BP26-I-ELISA indicated that the BP26 protein may be an additional suitable antigen for serological diagnosis of B. ovis infection in rams. 相似文献
12.
A dot-immunobinding assay (DIA), using as antigen a sonic extract of Brucella abortus dotted on nitrocellulose bound to a plastic strip, was employed for the detection of Brucella antibodies in 666 sheep sera. The results were compared with the complement fixation test (CFT). All the 242 sera belonging to two flocks were found to be negative by DIA. CFT was negative in 239 cases, whereas three samples showed anti-complementary activity. Of the 424 sera from the remaining three flocks, 98 were positive by both tests and six were positive in DIA, but negative in CFT. In addition, 14 of the 19 anti-complementary sera were also positive by DIA. 相似文献
13.
Brucella abortus INTA2, a novel strain 19 (Delta)bp26::luc (Delta)bmp18 double mutant lacking drug resistance markers 总被引：2，自引：0，他引：2
Campos E Cravero SL Delgui L Mora I Kahn N Arese AI Rossetti OL 《Veterinary microbiology》2002,87(1):1-13
Brucella abortus INTA2, a novel mutant strain, was constructed by inactivation of two B. abortus S19 genes: bp26 and bmp18, with the objective of obtaining a mutant strain that could be compatible with a diagnostic test and have less residual virulence than strain 19. The double mutant was constructed by replacing a large section of the bp26 coding region with the luciferase (luc) coding gene, resulting in mutant strain B. abortus M1luc, followed by partial deletion of bmp18 coding sequence. Both genes were inactivated by allelic replacement assisted by sacB counter-selection. Luciferase expression was evaluated and confirmed that it is a valid marker in the construction of mutant strains. When B. abortus INTA2 was inoculated in BALB/c mice, significantly fewer colony forming units (CFUs) were recovered from mice spleens during initial phase of infection. No splenomegaly was observed in strain INTA2-immunized mice at any time suggesting that strain INTA2 has lost some residual virulence of the parental strain. Nevertheless, similar protection levels against virulent challenge were observed in mice immunized with strains INTA2 or S19. Although strain INTA2 would still induce O-side antibodies, it does not express BP26. This would allow differentiation of INTA2-vaccinated animals from animals infected with field strains by measuring anti-BP26 antibodies, either by an agglutination test or ELISA using BP26 as antigen. Altogether these results indicate that B. abortus INTA2 might be a promising vaccine strain against brucellosis. 相似文献
14.
为了研制布鲁菌bp26-间接ELISA抗体检测试剂盒,将布鲁菌bp26基因克隆至原核表达载体pET-32a(+),重组质粒转化大肠杆菌BL21(DE3)中诱导表达,以纯化后的重组蛋白bp26包被酶标板,优化ELISA反应条件,组装试剂盒。SDS-PAGE和Western blotting分析结果表明,重组蛋白分子质量约为41 ku,经IPTG诱导后高效表达,且具有良好的免疫原性;优化试验确定重组抗原最佳包被浓度为3.6 μg/mL,血清样本的阳性临界值为0.370;特异性试验结果表明,重组蛋白与牛结核菌、副猪嗜血杆菌、链球菌等多种病原体的阳性血清均无交叉反应;敏感性试验结果表明,血清稀释至1:12800时,仍检测为阳性;该试剂盒的批内、批间变异系数均小于10%;用该试剂盒检测241份临床牛血清样本,并与试管凝集试验(SAT)检测结果比较,两者符合率为97.1%。原核表达的布鲁菌bp26具有良好的免疫原性,研制的布鲁菌bp26-间接ELISA抗体检测试剂盒敏感性、特异性和重复性较好,可为布鲁菌基因缺失标记疫苗的应用提供配套的血清学诊断方法。 相似文献
15.
为了构建能够在布鲁氏杆菌宿主细胞和宿主个体中稳定表达红色荧光蛋白的载体,试验通过PCR分别获得核糖体结合位点-红色荧光蛋白（RBS-Red）与布鲁氏菌特异DNA（BDNA）片段,利用重叠延伸PCR获得RBS-Red-BDNA重叠片段;将重叠片段插入pLB载体,利用Sma Ⅰ与Sac Ⅱ对重组pLB载体和pMC-221质粒进行双酶切后连接目的片段,连接产物转化大肠杆菌DH5α感受态细胞;提取的质粒电转布鲁氏菌16M菌株感受态细胞,将电转后的16M-pMC-Red菌株涂布于含有氯霉素抗性的布鲁氏菌培养基,倒置荧光显微镜下观察菌株颜色;16M-pMC-Red菌株侵染小鼠巨噬细胞,制成细胞爬片,激光共聚焦荧光显微镜下观察小鼠巨噬细胞,鉴定红色荧光蛋白表达情况。结果显示,利用重叠延伸PCR技术成功改造了红色荧光蛋白布鲁氏菌双启动子表达载体;将改造获得的质粒电转进布鲁氏杆菌16M菌株,菌株荧光鉴定能够稳定表达红色荧光蛋白;电转成功的16M-pMC-Red菌株侵染小鼠巨噬细胞后,可以稳定表达红色荧光蛋白。试验构建的发红光质粒pMC-Red可以与发绿光质粒pMC-221联用,为不同种株布鲁氏菌之间的联合研究奠定了基础,也为布鲁氏菌病检测提供了一个以荧光检测为指标的新策略。 相似文献
16.
P Chand J R Sadana H V Batra R N Khanna 《Zentralblatt für Veterin?rmedizin. Reihe B. Journal of veterinary medicine. Series B》1989,36(5):346-352
A dot Enzyme-linked Immunosorbent Assay (dot-ELISA), using whole cell Brucella abortus antigen dotted on the nitrocellulose membrane bound to a plastic strip (dipstick) was employed for the detection of Brucella antibodies in bovine sera. The results were compared with that of serum agglutination (SAT), Rose Bengal plate agglutination (RBPT) and Complement Fixation test (CFT). All the four tests gave negative reaction in 127 sera obtained from a brucellosis free herd. Testing of 549 sera from a chronically infected herd revealed 57 positive and 447 negative animals in all the four assays. Of the remaining 45 sera, 34 were positive in dot-ELISA. Six of these cases were independently detected by dot-ELISA while 28 showed positive reactions in combination with other tests. When serum samples from 158 aborted cases were subjected to dot-ELISA, 79 were found positive. Of these dot-ELISA positive cases, 71 gave positive reaction in SAT, 72 in RBPT and 78 in CFT. B. abortus biotype 3 was isolated from 34 of the 98 aborted fetuses examined. 相似文献
17.
狂犬病(Rabies)是一种由狂犬病病毒(RV)引起的急性人兽共患传染病,一旦发病,死亡率几乎为100％.为快速检测狂犬病病毒IgG抗体,本研究首先克隆表达并纯化狂犬病病毒G蛋白,应用G蛋白作为抗原,G蛋白单克隆抗体制备C线,鼠抗犬IgG和鼠抗猫IgG标记T线,经条件优化后,建立了一种能够用于快速检测动物狂犬病病毒血清... 相似文献
18.
A. A. ADESIYUN S. U. ABDULLAHI J. B. ADEYANJU 《The Journal of small animal practice》1986,27(1):31-37
Serum samples collected from dogs brought for routine physical examination, vaccination and other complaints at the Small Animal Clinic of Ahmadu Bello University, Zaria, Nigeria were tested for Brucella abortus and Brucella canis antibodies. Ninety-five (38-2 per cent) of 249 dogs studied were positive for B. abortus agglutinins by the Rose Bengal plate test (RBPT) but none was sero-positive by the standard agglutination test (SAT). The antibody prevalence for B. canis by the SAT was 28-6 per cent for 224 dogs tested. Exotic breeds of dogs had a prevalence of 34-9 per cent for B. canis agglutinins while 28-1 per cent of local dogs were sero-positive. Twenty-two per cent of dogs older than 2 years were sero-positive compared to a prevalence of 33-3 per cent found amongst dogs younger than 1 year. A similar B. canis infection rate was observed amongst male (29-6 per cent) and female (26-7 per cent) dogs. 相似文献
19.
Jennifer Maratea Darla R Ewalt Salvatore Frasca J Lawrence Dunn Sylvain De Guise Lech Szkudlarek David J St Aubin Richard A French 《Journal of zoo and wildlife medicine》2003,34(3):256-261
After recent isolations of Brucella sp. from pinnipeds and cetaceans, a survey was initiated to investigate the prevalence of Brucella sp. infections and serologic evidence of exposure in marine mammals stranded along the coasts of Connecticut and Rhode Island. One hundred and nineteen serum samples from four species of cetaceans and four species of pinnipeds were collected from 1985 to 2000 and tested for antibodies to Brucella sp. using the brucellosis card test, buffered acidified plate antigen test, and rivanol test. In addition, 20 of these were necropsied between 1998 and 2000, with lymphoid and visceral tissues cultured for Brucella sp. Three of 21 (14%) harbor seals (Phoca vitulina) and four of 53 (8%) harp seals (Phoca groenlandica) were seropositive. Brucella sp. was isolated from two of four (50%) harbor seals and three of nine (33%) harp seals. Of the five animals with positive cultures, two were seropositive and three seronegative. Brucella sp. was most frequently cultured from the lung and axillary, inguinal, and prescapular lymph nodes. Tissues from which Brucella sp. was isolated showed no gross or histopathologic changes. These results indicate that marine mammals stranded along the coast of southern New England can be exposed to and infected with Brucella sp. 相似文献
20.
William C Stoffregen Steven C Olsen C Jack Wheeler Betsy J Bricker Mitchell V Palmer Allen E Jensen Shirley M Halling David P Alt 《Journal of veterinary diagnostic investigation》2007,19(3):227-237
Eighty feral swine were trapped from a herd that had been documented to be seropositive for Brucella and which had been used for Brucella abortus RB51 vaccine trials on a 7,100-hectare tract of land in South Carolina. The animals were euthanized and complete necropsies were performed. Samples were taken for histopathology, Brucella culture, and Brucella serology. Brucella was cultured from 62 (77.5%) animals. Brucella suis was isolated from 55 animals (68.8%), and all isolates were biovar 1. Brucella abortus was isolated from 28 animals (35.0%), and isolates included field strain biovar 1 (21 animals; 26.3%), vaccine strain Brucella abortus S19 (8 animals, 10.0%), and vaccine strain Brucella abortus RB51 (6 animals, 7.5%). Males were significantly more likely to be culture positive than females (92.9% vs. 60.6%). Thirty-nine animals (48.8%) were seropositive. Males also had a significantly higher seropositivity rate than females (61.9% vs. 34.2%). The relative sensitivity rates were significantly higher for the standard tube test (44.6%) and fluorescence polarization assay (42.6%) than the card agglutination test (13.1%). Lesions consistent with Brucella infection were commonly found in the animals surveyed and included inflammatory lesions of the lymph nodes, liver, kidney, and male reproductive organs, which ranged from lymphoplasmacytic to pyogranulomatous with necrosis. This is the first report of an apparent enzootic Brucella abortus infection in a feral swine herd suggesting that feral swine may serve as a reservoir of infection for Brucella abortus as well as Brucella suis for domestic livestock. 相似文献