比较两种ELISA试剂盒在猪伪狂犬病血清抗体检测中的一致性

比较两种ELISA试剂盒检测结果的一致性,为流行病学调查和临床诊断筛选适合的商品化试剂盒。采用来自两个厂家的野毒抗体(gE)和免疫抗体(gB)ELISA检测试剂盒分别检测362份和392份猪血清,应用Kappa检验比较试验数据的一致性。结果显示:两种猪伪狂犬病抗体(g E)检测试剂盒联合检测出95份抗体阳性和256份阴性,符合率97.24%,结果一致性为极强(Kappa值0.932);两种猪伪狂犬病抗体(gB)检测试剂盒联合检测出351份抗体阳性和9份阴性,符合率91.84%,一致性为弱(Kappa值0.347)。以上结果说明,两种猪伪狂犬病抗体(gE)检测试剂盒都适用于PRV gE抗体检测,而两种猪伪狂犬病抗体(gB)检测试剂金差异较大,需慎重选择。     

青海海西州地区牛羊布鲁菌氏病监测方案的研究

目的探索适合青海省海西州地区牛、羊布鲁氏菌病的监测方案,净化布鲁氏菌病.利用琥红平板凝集试验(RBPT)、全乳环状试验(MRT)和试管凝集试验(SAT)对1200份牛血清中376份奶牛血清、677份羊血清,以及376份牛奶进行检测.结果RBPT检测1200份牛血清中9份可疑,3份阳性;376份奶牛血清中3份可疑,1份阳性;其他牛6份可疑,2份阳性;677份羊血清中3份可疑.MRT检测376份奶样中5份可疑,1份阳性.SAT重复检测的20份血清中2份为阳性,RBPT与MRT结果的符合率为100%.结论三种检测方法的联合使用适合大样本的进行布鲁氏菌病的监测,为基层布鲁氏菌病的监测方案的研究提供了理论依据.     

虎红平板凝集试验和胶体金免疫层析法诊断布鲁氏菌病效果的比较

为了比较虎红平板凝集试验(RBPT)和胶体金免疫层析法(GICA)诊断布鲁氏菌病的效果,658份牛血清和365份羊血清进行了检测,并通过试管凝集试验(SAT)对两种方法筛选到的阳性样品进行了确证。RBPT法检测出103份阳性牛血清,44份阳性羊血清;GICA法检测出129份阳性牛血清,65份阳性羊血清。通过SAT法验证结果显示牛血清有84份为阳性,羊血清有44份为阳性。GICA法的敏感性要高于RBPT法,可以作为家畜布病初筛试验的一种比较理想的检测方法。     

山东日照市养禽场（户）新城疫免疫效果监测

为了解山东省日照市养禽场(户)新城疫疫苗的免疫效果,应用血凝抑制试验方法对全市9家养禽场(户)的315份血清样品进行实验室检测。检测结果显示,新城疫抗体检测中,检测315份,免疫合格290份,抗体水平合格率达到92.1%,符合国家要求。     

牛传染性鼻气管炎病毒血清抗体检测试验及结果分析

采用SYNBIOTICS公司酶联免疫吸附试验(ELISA)检测试剂盒对来自新疆15个不同地区(包括4个养牛场)共635份牛血清进行了牛传染性鼻气管炎病毒血清抗体检测,共检出阳性血清329份,最高感染率达90%,最低感染率为5%,平均感染率为51.8%,结果显示该病已在全疆普遍存在,感染没有明确的地域分布规律。对其中的106份血清用Institute Pourquier公司ELISA试剂盒重新检测,发现两试剂盒的符合率为90.6%,Institute Pourquier公司ELISA检测试剂盒敏感性稍高。从该106份血清中再次随机抽取其中的35份用病毒中和实验(VN)检测方法进行了检测,检测结果与SYNBIOTICS公司ELISA检测试剂盒相比较,同为阳性样品24份,同为阴性样品7份,两者符合率88.6%;与Institute Pourquier公司ELISA检测试剂盒相比较,同为阳性样品24份,同为阴性样品6份,两者符合率85.7%。试验结果表明,与VN相比,ELISA检测试剂盒具有操作简便,敏感性高等优点。     

对昌平区某羊群布鲁氏杆菌感染情况的分析

布鲁氏杆菌病(Brucellosis)是由布鲁氏杆菌(Blucella)引起的对养殖业危害巨大的传染病,目前确诊主要以实验室检测为依据。试验的19份山羊血清(17份成年羊,2份羔羊)采自于昌平区某养羊户新购入的羊,分别进行竞争性酶联免疫吸附试验(cELISA)和实时荧光定量PCR试验(real-time PCR),检测布氏杆菌抗体和抗原的水平。试验结果显示,c-ELIASA阳性率为57.89%,real-time PCR检测有2份阳性,17份阴性。此羊群血清学和病原学均为阳性,感染了布鲁氏杆菌,需立刻扑杀;real-time PCR检测快速灵敏,可结合血清学检测做进一步确诊。     

探讨血清学检测对羊布鲁氏菌病诊断的有效性

研究以奶山羊为研究对象,采用血清学检测对所采集的438份羊血清进行分析,探究血清学检测对羊布鲁氏菌病诊断的有效性。结果表明:基于虎红平板试验(RBT)初筛出147份阳性,试管凝集(SAT)和竞争性ELISA抗体检测对初筛的147份阳性进行复核,得136份阳性,其中92.5%的样本诊断为阳性。研究表明,血清学检测对于羊布病诊断具有有效性。     

应用荧光偏振检测方法对出口牛进行布氏杆菌病检测

荧光偏振试验(FPA)是一种新的试验方法。本文应用荧光偏振检测方法对出口哈萨克斯坦的321份牛进行布氏杆菌病检测,结果有25份牛血清样品检测为抗体阳性。同时对321份血清进行虎红平板凝集试验(RBPT)、试管凝集试验(SAT)、补体结合试验(CFT)、间接ELISA试验(I-ELISA)、竞争ELISA试验(C-ELISA)等比对试验,结果是FPA从敏感性和特异性上都优于或等于目前主要采用的RBPT、SAT、CFT、ELISA检测方法。FPA方法简便、快速、通量大,不需洗板,可在15分钟内完成92份样品的布氏杆菌病检测,极适合于大批量牛布氏杆菌病的检疫、筛查和疫病监控。     

口蹄疫病毒A型竞争ELISA抗体检测试剂盒在流行病学调查中的应用

为评价口蹄疫病毒A型竞争ELISA(cELISA)抗体检测试剂盒在流行病学调查中的应用前景,对2017年从福建省三明市采集的336份黄牛、奶牛、羊和猪血清样品,用A型cELISA抗体检测试剂盒进行抗体检测。结果显示,92份黄牛血清、92份羊血清、92份猪血清、60份奶牛血清的A型抗体阳性率分别为13.04%、11.96%、20.65%、86.67%。从上述4种血清中,各挑选10份血清(阴性、阳性各5份)共40份,采用口蹄疫病毒液相阻断ELISA(LPB-ELISA)抗体检测试剂盒进行验证。结果显示:cELISA检测为阳性的20份血清中,用LPB-ELISA检出阳性19份;cELISA检测为阴性的20份血清中,用LPB-ELISA检出阴性17份;两种方法的κ值为0.8,总符合率为90.00%。结果表明,A型cELISA试剂盒与LPB-ELISA试剂盒的符合率和一致性均较高,可用于口蹄疫流行病学调查和血清学监测。     

Ⅰ群禽腺病毒套式PCR检测方法的建立及应用

为了建立一种特异、敏感的Ⅰ群禽腺病毒(FAdV-Ⅰ)检测方法,根据GenBank中FAdV-Ⅰ不同血清型Hexon基因序列,选择保守区域设计内、外2对特异性检测引物,通过反应条件的优化,建立了FAdV-Ⅰ套式PCR检测方法。结果:该方法能够特异性扩增FADV-Ⅰ,而检测减蛋综合征病毒(EDSV)、鸡痘病毒(APV)、马立克病病毒(MDV)、鸡传染性喉气管炎病毒(ILTV)、鸭瘟病毒(DEV)、H9亚型禽流感病毒(AIV-H9)、新城疫病毒(NDV)、鸡传染性支气管炎病毒(IBV)均为阴性;检测下限为10~(2.0) TCID_(50),灵敏度是普通PCR方法的100倍;对3个不同浓度FAdV-Ⅰ的批内重复性检测和批间重复性检测的结果完全一致;相对于病毒分离鉴定方法的符合率为90.91%;用该方法检测91份临床病料样品,共检测出阳性样品58份,其中血清4型、8a型、8b型、11型阳性样品分别为38份、8份、7份、5份。结果说明本试验建立的FAdV-Ⅰ套式PCR检测方法具有良好的特异性、敏感性、重复性、准确性,能够用于FAdV-Ⅰ的临床检测。     

内蒙古部分地区放牧牛羊弓形虫血清学调查

[目的]了解内蒙古部分地区放牧牛羊弓形虫病感染情况。[方法]采用间接血凝试验(IHA)对阿拉善盟及呼伦贝尔市随机采集的286份放牧牛羊血清样本进行弓形虫抗体检测。[结果]绵羊血清弓形虫抗体总阳性率为1.68%,其中,阿拉善盟阳性率为4.84%,呼伦贝尔市阳性率为0.56%;经统计学分析,两个地区的绵羊血清弓形虫抗体阳性率差异不显著(P>0.05);牛血清弓形虫抗体总阳性率为0。[结论]内蒙古主要牧区放牧绵羊存在弓形虫感染,应引起足够的重视,并制定相应的弓形虫病防治措施。     

JEV、PPV、PRRSV、PRV多联PCR的应用研究

用 JEV、PRRSV二联 PCR,PPV、PRV二联 PCR以及这 4种病毒 4联 PCR对来自内蒙、广州、广西、天津、北京、吉林病料进行检测 ,共检了 1 4 6份病料。其中 PRRSV阳性 7份 ,PPV阳性 1 2份 ,PRV阳性 2 1份 ,PRV和 PPV混合感染 4份。并对 5份人工接种 JEV小鼠病料检测 ,其中 4份为阳性。随后对部分阳性 PCR扩增产物进行点杂交和核苷酸测序鉴定 ,证实了 PCR扩增准确性。对内蒙 PRRSV阳性扩增带测序结果显示 ,我国流行 PRRSV为美洲型 ,在扩增片段的核苷酸序列上有 3个碱基差异     

A survey on Dictyocaulus viviparus antibodies in bulk milk of dairy herds in Northern Germany

Parasitic bronchitis caused by the bovine lungworm, Dictyocaulus viviparus, occurs worldwide in temperate areas. The parasite is found predominantly in calves and heifers, but dairy cattle can suffer from lungworms when they become infected for the first time or if they have lost immunity due to lack of exposure to lungworm larvae during the grazing season. The present study was performed to determine the D. viviparus bulk milk antibody prevalence in dairy herds in the East Frisian region of northwestern Germany, Lower Saxony, by analysing bulk milk samples collected in January (860 samples), September (866 samples) and November (860 samples) 2008, thereby representing 906 dairy farms. These samples were tested for antibodies against D. viviparus by a milk ELISA. This test detects patent infections only since it is based on recombinant major sperm protein as antigen. While in January 12.8% of dairy farms were positive for D. viviparus antibodies, the bulk milk samples collected in September and November revealed 6.9% and 6.6% positive dairy herds. From the 906 dairy farms included in the study, 191 (21.1%) tested positive at least once for antibodies against lungworm. From 810 dairy farms from which bulk milk samples were obtained during all three samplings, 146 (18.0%) farms were positive at one sampling date, 27 (3.3%) at two, and 4 (0.5%) on all three sampling dates. The majority of the farms represented in the study belonged to four districts of East Frisia, which showed no significant difference in the proportion of positive dairy farms.     

Efficacy of injectable ivermectin against natural infections of Stephanurus dentatus in swine

Ivermectin (300 micrograms/kg of body weight) was given to swine subcutaneously in the neck to test its efficacy against the kidney worm, Stephanurus dentatus. Two separate field trials were conducted using 146 swine (40 males and 106 females). Urine was obtained before and after treatment and was examined for presence of S dentatus eggs. Stephanurus dentatus eggs were quantitated in positive samples. All treated swine positive for S dentatus eggs in the pretreatment urine samples (n = 54) were negative by 14 to 21 days after treatment with ivermectin. Adverse reactions caused by ivermectin injection were not noticed.     

Immunomagnetic isolation of canine circulating endothelial and endothelial progenitor cells

Background: Increased concentrations of circulating endothelial cells (CECs) are thought to be a biomarker of vascular injury in human patients with cardiovascular disease, neoplasia, vasculitis, sickle cell anemia, shock, and sepsis. Immunomagnetic isolation is a technique currently used to enumerate human CECs and can detect low numbers of cells. Objectives: The purpose of this study was to determine whether a standard protocol for immunomagnetic isolation could be used to obtain and enumerate CECs and a subpopulation of endothelial progenitor cells (EPCs) from canine whole blood. Methods: Cultured canine aortic endothelial cells were stained immunohistochemically with von Willebrand factor to verify morphology and number. Using magnetic beads conjugated with anti‐CD146, CECs/EPCs were isolated in culture and in canine whole blood. CD146‐positive cells were stained with fluorescein‐conjugated Ulex europaeus agglutinin 1 (UEA‐1) to confirm endothelial origin and cells were counted manually using a fluorescent microscope. The method was then applied to EDTA‐anticoagulated whole blood samples from 10 healthy client‐owned dogs. Results: The anti‐CD146–coated magnetic beads (>5/cell) bound the cultured canine aortic endothelial cells. Only rare UEA‐1–positive cells were obtained from whole blood, while >85–90% of cultured canine aortic endothelial cells were UEA‐1 positive. The percentage recovery of cultured canine aortic endothelial cells was >86%. CECs in canine whole blood had >8 beads attached to the surface and were 10–40 μm in size. Using immunomagnetic isolation, 43.4 ± 15.6 CECs/mL (range 24–70/mL) were isolated from canine whole blood samples. Conclusions: Immunomagnetic isolation is an acceptable method for enumerating canine CECs/EPCs in whole blood. Further studies are warranted to evaluate the clinical significance of CEC/EPC concentration in different canine diseases.     

Hemoplasmas in wild canids and felids in Brazil

Hemotropic mycoplasmas, epicellular erythrocytic bacterial parasites lacking a cell wall, are the causative agents of infectious anemia in numerous mammalian species. The presence of hemotropic mycoplasmas in blood samples of neotropical and exotic wild canids and felids from Brazilian zoos were recorded using molecular techniques. Blood samples were collected from 146 Brazilian wild felids, 19 exotic felids, 3 European wolves (Canis lupus), and from 97 Brazilian wild canids from zoos in the Brazilian states of S?o Paulo and Mato Grosso and the Federal District. Using conventional polymerase chain reaction (PCR), this work found 22 (13%) wild felids positive to Candidatus Mycoplasma haemominutum [4 jaguars (Panthera onca); 3 pumas (Puma concolor); 10 ocelots (Leopardus pardalis); 2 jaguarondis (Puma yagouaroundi); and 3 little spotted cats (Leopardus tigrinus)]. Only one little spotted cat (Leopardus tigrinus) was positive to Mycoplasma haemofelis, and none was positive to Candidatus Mycoplasma turicensis. Two bush dogs (Speothos venaticus) were positive for a Mycoplasma sp. closely related to Candidatus Mycoplasma haematoparvum, and two European wolves were positive for a Mycoplasma sp. closely related to Candidatus Mycoplasma haemominutum. This is the first study regarding the molecular detection of hemotropic mycoplasmas in wild canids.     

The relationship between the degree of thrombocytopenia and infection with Ehrlichia canis in an endemic area

Ehrlichia canis is the causative agent of canine monocytic ehrlichiosis. In order to evaluate platelet counts as a screening test for E. canis in an endemic area, 217 whole blood samples from dogs were divided into three groups: 71 non-thrombocytopenic samples (group A, platelet counts greater than 200 000/mL) and 146 thrombocytopenic samples (less than 200 000/mL). The thrombocytopenic group was further divided into 62 with platelet counts between 100 000-200 000/mL (Group B) and 84 samples with less than 100 000 platelets/mL (Group C). All samples were examined for the presence of a segment of the Ehrlichia canis 16S rRNA gene using a nested polymerase chain reaction. Sixty-seven of the 217 samples (30.9%) were positive for the presence of the E. canis 16S rRNA gene; 53 (63.1%) of the group C samples and 13 (21%) of group B. Only one (1.4%) of the non-thrombocytopenic samples (Group A) was positive. These data support the concept that platelet counts may be a good screening test for canine monocytic ehrlichiosis, and that the magnitude of thrombocytopenia may increase the reliability of diagnosis.     

Detection and typing of foot-and-mouth disease virus by enzyme-linked immunosorbent assay: a sensitive, rapid and reliable technique for primary diagnosis

A highly sensitive indirect sandwich enzyme-linked immunosorbent assay suitable for adoption as the routine diagnostic and typing test for foot-and-mouth disease virus of all seven serotypes is described. The assay uses rabbit and guinea pig antisera raised against inactivated 146S virus antigens. Strong homotypic and minimal heterotypic reactions with both whole virion 146S and derived virion subunit 12S antigens achieved a detection sensitivity approximately 125 times that of the complement fixation test. When applied to diagnostic material with positive-negative threshold criteria generated from testing a large number of negative samples, a positive result was obtained on 83.3 per cent of original virus-positive epithelia, three times the rate for the complement fixation test, and all were typed after one passage in culture.     

2017-2019年山东省部分地区猪瘟流行病学调查

为掌握近年来山东地区猪瘟病毒（CSFV）的流行及其遗传变异情况，本研究对2017年1月-2019年12月从山东省济南市、泰安市、聊城市、青岛市等10个地区采集的1 687份疑似猪瘟感染猪病料运用反转录PCR方法进行了病毒核酸检测，将CSFV检测为阳性的样品进行E2基因和CSFV全基因扩增和测序，利用生物信息学软件DNAStar和Mega 7.0对CSFV的E2蛋白以及全基因序列进行遗传进化分析。另外，将这些地区采集的4 866份血清样本进行了抗体ELISA检测。结果显示，1 687份病料中病原检测有188份阳性，总阳性率为11.1%，其中2017-2019年病原检测阳性率分别为8.5%、13.1%、15.1%，抗原阳性率逐年上升且呈不稳定性，提示规模化猪场猪瘟兔化弱毒疫苗的免疫存在一定的风险和压力；采集的4 866份血清样本中4 146份呈现抗体阳性（85.2%），统计得到2017-2019年抗体阳性率分别为84.3%、83.0%、92.3%，抗体阳性率呈现上升趋势，说明山东省规模化养殖场猪群猪瘟疫苗免疫保护的整体水平逐年提高；从阳性病料中扩增获得了5株CSFV全基因组序列和5株病毒E2基因序列，经过序列比对发现，5株临床分离毒株与2.1d亚型的CSFV全基因组序列同源性在97.5%~99.0%之间，表明5株分离毒株与2.1d亚型的CSFV亲缘性较近，分离毒株氨基酸位点的突变集中在102位（L→M）以及159位（K→R）氨基酸处。本研究分析了2017年1月-2019年12月山东部分地区猪群中CSFV毒株的流行与变异情况，为指导山东省CSFV预防与控制提供了有力的参考。     

An enzyme-linked immunosorbent assay (ELISA) for the primary diagnosis of foot-and-mouth disease. Characterization and comparison with complement fixation

A sandwich type ELISA for foot-and-mouth disease (FMD) virus types O, A and C was established, using a combination of rabbit anti-146 S and guinea pig hyperimmune antibodies. This method was found to be highly efficient for the detection of both 146 S particles and 12 S subunits. The ELISA was approximately 500 times more sensitive than complement fixation (CF) when examining epithelial samples of FMD vesicles. An early primary diagnosis of FMD was obtained by both CF and ELISA in 19 out of 21 confirmed cases. The remaining 2 cases were initially negative in CF but positive in ELISA.