基于丝状支原体丝状亚种P35反应原性的验证及其间接ELISA方法的建立

为建立检测丝状支原体丝状亚种(Mmm)的间接ELISA方法,本研究利用生物信息学软件对Mmm的全基因组序列进行了分析,选定大小为540bp的0584基因,该基因是编码分子量为35ku的脂蛋白,并对其免疫反应原性进行研究。利用原核表达系统获得了0584基因的重组蛋白rP35,以牛传染性胸膜肺炎(CBPP)阳性血清为一抗进行western blot分析,试验结果为阴性,但Dot-blot试验结果为阳性,证明该蛋白可能是Mmm特有的一种构象依赖性免疫相关蛋白且具有反应原性。以rP35蛋白为包被抗原,通过反应条件优化初步建立了检测CBPP血清抗体的间接ELISA方法,特异性为88.0%,敏感性为89.8%,批内和批间变异系数均小于10%,与商品化试剂盒符合率为84.8%。本研究为研制CBPP血清检测试剂盒提供了一种候选蛋白。     

丝状支原体山羊亚种抗体间接血凝检测方法的建立及应用

将丝状支原体山羊亚种PG3株经超声波破碎处理后的产物致敏经戊二醛和鞣酸处理过的绵羊红细胞,建立了检测丝状支原体山羊亚种抗体的间接血凝试验方法。抗原致敏红细胞的最佳浓度是100～125μg/mL,超免血清抗体效价达1:512～1:1024。免疫山羊2周后血清抗体检出率为83.3%,对羊布氏杆菌、绵羊肺炎支原体阳性血清检测均为阴性,敏感性明显高于琼脂扩散试验。对628份田间血清进行检测,阳性率为44.9%。结果表明,该法敏感性较高、特异性强和重复性好,可用于山羊传染性胸膜肺炎免疫抗体水平的监测和流行病学调查。     

牛支原体NM001株单克隆抗体的制备与鉴定

为获得抗牛支原体NM001株单克隆抗体,并评价其特性,以牛支原体分离株NM001作为抗原免疫6周龄BALB/c小鼠,利用杂交瘤技术和间接ELISA方法筛选到2株能稳定分泌抗牛支原体的单克隆抗体细胞,命名为2C5和7G3。其细胞上清的间接ELISA抗体效价分别为6.4×10³和1.2×10⁴。经亚型测定,单抗2C5和7G3均属于IgG1类,轻链均是λ型。制备腹水并对单抗进行纯化和特性鉴定,两株单抗的间接ELISA抗体效价分别为1.02×10⁵和4.09×10⁵,且两株单抗与无乳支原体、山羊支原体山羊肺炎亚种、丝状支原体山羊亚种、绵羊肺炎支原体、牛巴氏杆菌均无交叉反应。Western Blot结果显示,2株单抗均能特异性识别牛支原体全菌蛋白中的相应蛋白。试验表明,单抗2C5和7G3能够与牛支原体发生特异性反应,从而为牛支原体血清学检测提供一定的物质基础。     

羊丝状支原体簇环介导等温扩增检测方法的建立

本试验利用环介导等温扩增技术(LAMP)建立了羊丝状支原体簇的快速检测方法,该方法以羊丝状支原体簇成员的保守性基因16S rRNA为靶序列,设计了4条特异性引物,在65 ℃等温条件下,60 min一步完成反应。在反应管中预先添加羟基萘酚蓝(HNB),阳性呈蓝色,阴性呈紫色。丝状支原体簇成员——丝状支原体山羊亚种(Mmc)、丝状支原体丝状亚种LC型(Mmm LC)、山羊支原体山羊肺炎亚种(Mccp)的LAMP检测为阳性;对其他病原菌没有交叉反应。以Mmc的核酸为模板进行灵敏度检测,LAMP的最低检出限为10 pg/μL。结果表明,本试验建立了一种特异、敏感、快速、简便的羊丝状支原体簇的LAMP方法。     

牛传染性胸膜肺炎抗体间接ELISA检测方法的建立

旨在建立一种检测牛传染性胸膜肺炎（CBPP）血清抗体的间接ELISA方法,用于该病的监测。利用生物信息学软件对CBPP的病原丝状支原体丝状亚种国内分离株Ben-1株的全基因组进行分析,选取脂蛋白rP0308作为包被抗原,通过一系列反应条件的优化,建立了基于rP0308蛋白的间接ELISA抗体检测方法,并对其性能进行评价。结果显示该方法的敏感性为92%,特异性为96%,与牛支原体、牛鼻支原体、无乳支原体、口蹄疫、牛病毒性腹泻、牛传染性鼻气管炎和牛结核分枝杆菌等阳性血清均不发生交叉反应,批内变异系数在2.41%~6.03%,批间变异系数在2.94%~6.59%,重复性良好。利用本方法和商品化的cELISA试剂盒分别对实验室保存的1 648份临床样品进行检测,该方法与商品化试剂盒的阴性符合率为89.1%,阳性符合率为79.2%,总符合率为88.7%。以上研究结果表明,本研究建立的间接ELISA方法具有良好的敏感性、特异性和重复性,具有良好的临床应用前景。     

青海地区丝状支原体山羊亚种的流行病学调查

为弄清楚丝状支原体山羊亚种在青海省地区的流行状况,笔者利用丝状支原体山羊亚种抗体检测正向间接血凝试剂盒对2007至2009年采自青海各地区的1200份山羊血清进行了检测。结果显示,平均阳性率为16.08%。不同地区山羊血清的阳性率从5%-20.9%不等。从季节上看,冬春季节发病率要高于夏秋季节。结果提示青海地区的丝状支原体山羊亚种的感染率偏高,应该加强对由丝状支原体山羊亚种的防控。     

青海地区丝状支原体山羊亚种的流行病学调查

为弄清楚丝状支原体山羊亚种在青海省地区的流行状况,试验利用丝状支原体山羊亚种抗体检测正向间接血凝试剂盒对2007至2009年采自青海各地区的1200份山羊血清进行了检测。结果显示,平均阳性率为16.08%。不同地区山羊血清的阳性率从5%~20.9%不等。从季节上看,冬春季节发病率要高于夏秋季节。结果提示青海地区的丝状支原体山羊亚种的感染率偏高,应该加强对丝状支原体山羊亚种的防控。     

牛传染性胸膜肺炎抗体间接ELISA检测方法的建立

旨在建立一种检测牛传染性胸膜肺炎(CBPP)血清抗体的间接ELISA方法,用于该病的监测。利用生物信息学软件对CBPP的病原丝状支原体丝状亚种国内分离株Ben-1株的全基因组进行分析,选取脂蛋白rP0308作为包被抗原,通过一系列反应条件的优化,建立了基于rP0308蛋白的间接ELISA抗体检测方法,并对其性能进行评价。结果显示该方法的敏感性为92%,特异性为96%,与牛支原体、牛鼻支原体、无乳支原体、口蹄疫、牛病毒性腹泻、牛传染性鼻气管炎和牛结核分枝杆菌等阳性血清均不发生交叉反应,批内变异系数在2.41%～6.03%,批间变异系数在2.94%～6.59%,重复性良好。利用本方法和商品化的cELISA试剂盒分别对实验室保存的1 648份临床样品进行检测,该方法与商品化试剂盒的阴性符合率为89.1%,阳性符合率为79.2%,总符合率为88.7%。以上研究结果表明,本研究建立的间接ELISA方法具有良好的敏感性、特异性和重复性,具有良好的临床应用前景。     

牛支原体、无乳支原体和丝状支原体丝状亚种小克隆三重PCR检测方法的建立及初步应用

本研究旨在建立一种可一次性区分牛支原体、丝状支原体丝状亚种小克隆和无乳支原体的三重PCR诊断方法,为临床诊断和流行病学调查提供可靠检测技术.根据GenBank发表的上述3种病原的基因组序列保守区域设计3对特异性引物建立三重PCR方法;确定其检测敏感性,以猪支原体、鸡支原体、无乳支原体和丝状支原体丝状亚种小克隆基因作模板检验其特异性;同时和病原分离鉴定结果对比其准确性.结果表明在优化体系和条件下能够同时得到扩增长度为448、549、375 bp 3条特异性片段,未扩增出猪、鸡支原体模板特异性片段;其敏感性(可检测到的最小模板DNA含量)为0.8 ng·μL-1;36份临床样品检测结果显示,三重PCR检测结果与分离培养鉴定方法一致,均能鉴定出牛支原体阳性病料.本研究建立的三重PCR诊断方法能够一次性鉴别3种支原体,具有高敏感性、特异性和准确性,可用于临床诊断和流行病学调查.     

一株致病性丝状支原体山羊亚种支原体的分离鉴定

通过培养纯化、形态学观察、生化试验、生长抑制试验、代谢抑制试验、人工感染试验、间接血凝试验,对疑似山羊传染性胸膜肺炎病原分离株SY2进行初步鉴定,参考国际已知支原体16S r RNA序列,选取共同保守性强的片段设计出1对引物,提取人工感染分离株SY2培养物的菌体DNA进行PCR扩增并克隆、测序。将该序列与NCBI中已知支原体序列比较,结果证明SY2株与丝状支原体山羊亚种标准株PG3同源性为99%,故确定疑似山羊传染性胸膜肺炎病原分离株SY2为丝状支原体山羊亚种。     

Contagious bovine pleuropneumonia: a rationale for the development of a mucosal sub-unit vaccine

Contagious bovine pleuropneumonia (CBPP) remains a major cattle disease in Africa with serious socio-economic consequences. Its eradication requires the development of improved vaccines. Knowledge on this disease and its causing agent, Mycoplasma mycoides subsp. mycoides biotype Small Colony (MmmSC), has been progressing significantly in the last years, opening new areas for vaccine design. Advances were achieved in the understanding of the protective immune responses to MmmSC infection and immunopathological mechanisms allowing the pathogen to escape the host immune response. Based on sequencing and genomic studies, some virulence factors and metabolic pathways were unraveled leading to the identification of potential MmmSC vaccine candidates. Based on these findings, this review presents a scientific strategy to design multi-component sub-unit vaccines for mucosal delivery as the most promising approach for efficient long-term protective vaccines to prevent CBPP.     

牛支原体新疆分离株oppD/F基因克隆及分子特征分析

试验旨在分析牛支原体新疆分离株oppD/F基因序列,并探究其序列特征及编码蛋白的结构和功能。根据GenBank中PG45菌株oppD/F基因序列（登录号：AF130119.1）设计1对引物,应用PCR技术扩增获得分离株oppD/F基因,将oppD/F基因片段克隆至pMD19-T载体中进行测序,在采用生物信息学方法分析其核苷酸序列的基础上对其编码蛋白的基本理化性质、疏水性、可溶性、信号肽、跨膜域、亚细胞定位、磷酸化位点、糖基化位点、二级结构、三级结构和功能等进行分析和预测。结果显示,牛支原体新疆分离株oppD/F基因序列全长1 617 bp,与Mb PG45株和Mb JF4278株的同源性均为100%,处于同一分支;该基因编码蛋白是由539个氨基酸残基组成,不存在信号肽及跨膜结构,是一种稳定的亲水性蛋白质;oppD/F蛋白存在1个AAA家族结构域,50个潜在的磷酸化位点和3个糖基化位点,其二级结构是混合型,其中α-螺旋所占比例最高（61.78%）,无规则卷曲次之（20.78%）。功能预测结果显示,oppD/F蛋白在信号传导、受体、结构蛋白、离子通道、免疫应答和胁迫应答等方面的几率均较高。本研究结果为进一步分析牛支原体oppD/F基因的功能提供了一定的理论依据。     

猪肺炎支原体P97蛋白单克隆抗体的制备、表位鉴定及其初步应用

为了制备能够用于阻断ELISA检测的猪肺炎支原体（Mycoplasma hyopneumoniae,Mhp）单克隆抗体（MAb）,本研究将原核表达的Mhp J株P97蛋白C末端包含R1区的肽段（P97CR1）作为免疫原免疫BALB/C雌鼠,筛选获得一株能稳定分泌抗Mhp P97蛋白MAb的杂交瘤细胞株A3。鉴定结果显示,A3 MAb是IgG1亚类,轻链为κ链。Western blot结果显示A3 MAb能够和原核表达纯化的P97CR1蛋白特异性反应,并且在流式细胞仪分析中能够识别天然的Mhp。通过逐渐截短表达分析该MAb识别的表位序列为LDDNLQ,该抗原表位在Mhp菌株中高度保守。阻断ELISA初步试验结果显示,A3 MAb与蛋白抗原的结合能够被Mhp高免阳性血清阻断。该MAb的制备为进一步建立特异性强、灵敏度高的Mhp阻断ELISA检测方法奠定了基础。     

A competitive ELISA for the specific diagnosis of contagious bovine pleuropneumonia (CBPP)

A competitive ELISA, using a specific monoclonal antibody, was designed to detect antibodies to Mycoplasma mycoides subsp. mycoides SC, the agent of contagious bovine pleuropneumonia. One monoclonal antibody was found suitable for such a test, ‘117/5', it does not cross-react with any of the other mycoplasma species tested, furthermore, its binding is inhibited by positive sera. The cutoff, 50% of inhibition, was determined using a set of negative sera from CBPP-free areas. The sensitivity was controlled with sera from artificially infected animals as well as from sera from areas where CBPP is enzootic. In both cases, cELISA compared favorably with CFT. The precocity of detection was similar but cELISA detected more positives and the positive titers seemed to persist longer than in the case of CFT. Lysis of the antigen used to coat the ELISA plates reduced the variability of fixation and improved the repeatability of the test. A field evaluation is now in progress which will determine the true sensitivity and specificity of the test and also check if antibodies are detected after vaccination.     

犊牛支原体肺炎继发牛A型多杀性巴氏杆菌感染的诊治

某奶牛场犊牛相继发生肺炎和关节炎,为确诊该牛场犊牛群发病的原因并提出防控方案,本试验剖检新生犊牛并采集病料,分别开展牛支原体及其他病原菌的分离培养、PCR鉴定及药敏分析;进行牛病毒性腹泻病毒、牛口蹄疫病毒和牛传染性鼻气管炎病毒PCR检测;制作犊牛肺脏组织病理切片并进行观察和评估。从犊牛肺脏组织分离到牛支原体和牛A型多杀性巴氏杆菌;牛病毒性腹泻病毒、牛口蹄疫病毒和牛传染性鼻气管炎病毒检测均为阴性;肺脏组织病理切片可见肺泡结构破坏、出血及大量炎性细胞浸润;药敏试验结果显示,牛支原体和牛A型多杀性巴氏杆菌分别对泰乐菌素和头孢唑啉敏感,但对青霉素、庆大霉素、林可霉素和氨苄西林均呈现耐药。该犊牛群确诊为牛支原体肺炎继发牛A型多杀性巴氏杆菌感染,采用泰乐菌素联合头孢唑啉肌肉注射,配合对症治疗和规范管理,有效控制了该场犊牛疾病。     

Evaluation of an enzyme-linked immunosorbent assay kit to detect Babesia bovis antibodies in cattle

The sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) to detect antibodies; to Babesia bovis was evaluated in 1000 sera from Holstein heifers. Five hundred of them were from cattle naturally or experimentally infected with B. bovis and 500 from uninfected heifers born and raised in a region free of the vector of cattle babesiosis. Additionally, the ELISA was evaluated and compared with an indirect immunofluorescent antibody (IFA) test in 374 heifers inoculated with different kinds of B. bovis antigens in four trials. The cross-reaction was also evaluated in 50 heifers infected with Babesia bigemina and 50 heifers infected with Anaplasma marginale. The mean percentage positivity of negative sera in relation to the ELISA strong positive sera was 8%. The seropositive/seronegative cutoff point was set as twice the mean percentage positivity of negative cattle sera ( = 16%). The sensitivity of the ELISA was 98% with a 95% confidence interval (CI) of 96–99%. The specificity was 95% (CI 93–97%). The agreement was 97% and the kappa value was 0.93. The predictive values of positive and negative results were 95% and 98% respectively. ELISA showed a similar sensitivity to that of the IFA test to detect antibodies to different B. bovis antigens. Its sensitivity ranged from 97.1% to 100% (CI 89–100%), while the sensitivity of the IFA test ranged from 92.8% to 100% (CI 83–100%). ELISA cross-reacted in 8% and 6% of the sera carrying B. bigemina and A. marginale antibodies, respectively, while the IFA showed 4% cross-reaction in each situation. The ELISA evaluated has the advantages of a proper sensitivity, objectivity and capacity to be adapted to test large number of samples in a short period of time. The results indicate that the ELISA is a suitable replacement for the IFA test to detect B. bovis antibodies in cattle sera, especially in epidemiological studies.     

Differential clustering of Mycoplasma mycoides subsp. mycoides SC strains by PCR-REA of the bgl locus

In order to develop a specific tool differentiating the African field strains of Mycoplasma mycoides subsp. mycoides SC from other potentially less virulent strains, including the vaccine strains, we have developed a PCR followed by a restriction enzyme analysis (PCR–REA). This approach also differentiates the African field strains from the Australian strains and the type strain PG1. The genomic marker detected by the PCR–REA is based on a single nucleotide change in the bgl gene that codes for 6-phospho-β-glucosidase (Bgl), an enzyme that is involved in sugar metabolism.     

抗蓝舌病病毒VP6和VP7蛋白单克隆抗体的制备及鉴定

为制备针对蓝舌病病毒(BTV)的单克隆抗体(MAb),本研究利用血清型1型BTV(BTV1)免疫BALB/c鼠,将其脾淋巴细胞与SP2/0进行融合,并用BTV1包被ELISA板,通过间接ELISA方法筛选出3株稳定分泌抗BTV1的MAb的杂交瘤细胞株(2B10、3D4和4H8)。利用表达BTV1主要蛋白的真核表达重组质粒转染BHK-21后,对所制备的杂交瘤细胞株上清进行间接免疫荧光(IFA)以及western blot鉴定,结果显示:2B10和4H8与VP7蛋白反应,而3D4与VP6蛋白反应。同时,IFA鉴定结果进一步表明,3株MAb与24个血清型的BTV均可以发生反应。本研究制备的MAb为建立BTV免疫学检测方法和相关病毒蛋白的功能研究奠定了基础。     

Clinical and serological evidence of bovine babesiosis and anaplasmosis in St. Lucia

One hundred fifty-nine Holstein calves were imported into St. Lucia from the U.S.A. An outbreak of babesiosis occurred 17 days post-arrival, and an outbreak of anaplasmosis occurred 5 months after importation. Sera obtained 3, 6 and 12 months post-importation revealed a high prevalence of IFA titres to Babesia bovis and B. bigemina 3 months after arrival and an increase in titres to Anaplasma marginale 6 months after arrival. Sera obtained arrives from native cattle from several places on the island indicated infection rates of 80, 65 and 64% with A. marginale, B. bigemina and B. bovis, respectively. The rapid card test only indicated a 25% prevalence of infection of native cattle by A. marginale. This low prevalence was probably due to deterioration of serological activity during shipment.