马驽巴贝斯虫新疆伊犁株Bc48基因克隆、表达及其多克隆抗体的制备

为表达马驽巴贝斯虫新疆伊犁株Bc48基因,制备多克隆抗体,试验根据GenBank中马驽巴贝斯虫Bc48基因序列,设计合成1对特异性引物,以提取的新疆株Bc48基因组DNA为模板进行PCR扩增,将扩增产物克隆至原核表达载体pET-28a(+)中,阳性质粒转化至大肠杆菌BL21(DE3)感受态细胞中,经IPTG诱导后表达产物进行SDS-PAGE分析,切胶纯化蛋白后免疫6周龄雌性BALB/c小鼠,制备多克隆抗体;同时把该基因克隆至真核表达载体pCMV-N-flag中,将阳性质粒转化至大肠杆菌DH5α感受态细胞中,提取pCMV-N-flag-Bc48质粒转染到293T细胞中进行真核表达。结果显示,获得了大小约为15ku的融合蛋白,与预期目的蛋白大小相一致;免疫BALB/c小鼠制备的多克隆抗体经ELISA和Western blotting检测、验证,能特异地识别相应抗原,其抗体效价可达1∶128 000,说明该多克隆抗体有较高的抗体效价和特异性;真核表达质粒在293T细胞中表达Bc48蛋白。本试验可为进一步研究马驽巴贝斯虫功能基因及建立快速的检测方法奠定基础,在虫株的分类学研究及候选疫苗的研制中有重要意义。     

地方株马驽巴贝斯虫Bc48截短体单克隆抗体的制备及应用

本研究旨在建立马驽巴贝斯虫抗体的快速、准确检测方法。以纯化的马驽巴贝斯虫Bc48基因片段的原核表达产物免疫6周龄雌性BALB/c小鼠制备单克隆抗体,并利用重组抗原与单克隆抗体建立间接竞争ELISA(CI-ELISA)方法检测马驽巴贝斯虫抗体。结果显示:制备出3株能稳定分泌单克隆抗体的杂交瘤细胞株,命名为1H2、7F4、11F4,通过对CI-ELISA条件筛选得出,抗原最佳包被浓度为0.19μg·mL~(-1),单克隆抗体11F4的最佳工作浓度为1∶3.2×10~5,通过检测30份马驽巴贝斯虫阴性血清及20份阳性血清,确定该检测方法临界值为45%;特异性试验发现,该CI-ELISA方法不与感染马泰勒虫病的阳性血清发生反应,具有特异性;用所建立的CI-ELISA检测临床血清90份,与标准c-ELISA试剂盒总符合率为92.2%、阳性符合率92.1%、阴性符合率94.1%。试验结果表明,该CI-ELISA方法特异性强,敏感性高,稳定性和重复性好,操作简便。基于单克隆抗体(11F4)与重组蛋白(HIS-Bc48)所建立的CI-ELISA特异性、重复性好,可为新疆马驽巴贝斯虫的检测、监控提供有效手段。     

马驽巴贝斯虫新疆伊犁株Bc48基因克隆、表达及其多克隆抗体的制备

为表达马驽巴贝斯虫新疆伊犁株Bc48基因,制备多克隆抗体,试验根据GenBank中马驽巴贝斯虫Bc48基因序列,设计合成1对特异性引物,以提取的新疆株Bc48基因组DNA为模板进行PCR扩增,将扩增产物克隆至原核表达载体pET-28a（+）中,阳性质粒转化至大肠杆菌BL21（DE3）感受态细胞中,经IPTG诱导后表达产物进行SDS-PAGE分析,切胶纯化蛋白后免疫6周龄雌性BALB/c小鼠,制备多克隆抗体;同时把该基因克隆至真核表达载体pCMV-N-flag中,将阳性质粒转化至大肠杆菌DH5α感受态细胞中,提取pCMV-N-flag-Bc48质粒转染到293T细胞中进行真核表达。结果显示,获得了大小约为15 ku的融合蛋白,与预期目的蛋白大小相一致;免疫BALB/c小鼠制备的多克隆抗体经ELISA和Western blotting检测、验证,能特异地识别相应抗原,其抗体效价可达1：128 000,说明该多克隆抗体有较高的抗体效价和特异性;真核表达质粒在293T细胞中表达Bc48蛋白。本试验可为进一步研究马驽巴贝斯虫功能基因及建立快速的检测方法奠定基础,在虫株的分类学研究及候选疫苗的研制中有重要意义。     

新疆部分地区马驽巴贝斯虫病感染PCR检测调查

为了解新疆部分地区马匹感染马驽巴贝斯虫病的流行情况,通过PCR方法对不同地区所采集的样品进行检测,并对不同地区和不同年龄段马驽巴贝斯虫感染情况进行单因素方差分析。结果显示,所检测的504份马血样中,54份血样为阳性,阳性率为10.71%。其中北疆昭苏种马场、阿勒泰、吐鲁番托克逊马驽巴贝斯虫阳性率分别为20.61%、5.17%、0;巴音布鲁克牧场、和静地区马驽巴贝斯虫阳性率分别为0和8.59%。从阳性样品中都检测到了驽巴贝斯虫BC48基因片段,与已知序列的同源性为99%。X≤3,3X≤6,6X≤9,X9四个年龄段马驽巴贝斯虫感染率分别为9.76%、10.53%、11.21%和11.65%。单因素方差分析结果表明,昭苏种马场与阿勒泰、巴音布克牧场分别存在极显著性差异(P0.01);巴音布鲁克牧场与和静也存在极显著性差异(P0.01);昭苏种马场与吐鲁番托克逊存在显著性差异(P0.05),其余各地方差异不显著。各个年龄段马驽巴贝斯虫感染无显著性差异。本试验的研究数据可为研究新疆马驽巴贝斯虫分布特点提供数据支撑。     

南北疆部分地区马驽巴贝斯虫病血清学调查

为了解南北疆养殖马区域马匹感染驽巴贝斯虫情况,笔者采用竞争性酶联免疫吸附试验(eELISA)与血涂片镜检法相结合的试验法,对伊犁和巴州两地区马匹随机抽样(N--454),进行驽巴贝斯虫病血清学调查。经c-Elisa调查发现,驽巴贝斯虫状况(呈抗体阳性)总感染率为33.2%;其中北疆4县马驽巴贝斯虫感染率分别15.8%、7.9%、5.7%和2.6%;南疆和静县感染率为O.4%。经血液涂片检查发现极少部分抗体阳性全血发现虫体,其总阳性率6.6%。无临床症状的马匹血清抗体阳性率高说明该地区存在‘传染源'、传播蜱虫,故加以防范。     

云南部分地区马、驴、骡梨形虫感染情况调查

为了解云南地区马驴骡感染马泰勒虫和驽巴贝斯虫情况,采用双抗原夹心ELISA方法对云南8个地区马、驴、骡三种动物（n=969）进行了马泰勒虫和驽巴贝斯虫抗体阳性率的检测。结果显示：梨形虫总抗体阳性率为5.47%,其中马泰勒虫抗体阳性率为4.75%,驽巴贝斯虫抗体阳性率为4.85%,两者混合感染的抗体阳性率为4.13%;通过调查分析发现云南马、驴、骡存在梨形虫感染,且不同地区的不同动物的马梨形虫感染程度不同。本研究结果为马驴骡梨形虫病的流行防控提供了数据参考。     

内蒙古和新疆两地草原革蜱源性马驽巴贝斯虫病病原DNA检测

为探究来源于内蒙古西部某地的草原革蜱及新疆境内广泛分布的草原革蜱携带同一种病原-驽巴贝斯虫的情况。本试验借助光学、电子显微镜进行鉴定了采自于不同植被(包括内蒙古、新疆两地)的草原革蜱;并参考驽巴贝斯虫Bc48基因序列,合成引物,提取该种蜱携带的马驽巴贝斯虫病原DNA,进行PCR扩增、检测。结果显示,来自内蒙古(N=147)、新疆(N=200)两地的草原革蜱在假头基部、多孔区、齿式、气门板等超微结构形态方面基本相似。经PCR成功扩增出了草原革蜱所携带的驽巴贝斯虫病原DNA阳性条带452 bp;来源于内蒙古西部某地的草原革蜱携带驽巴贝斯虫的阳性率为8.16%(12/147),来源于新疆和静县平原的草原革蜱驽巴贝斯虫的阳性率为13.50%(27/200)。这些参数可为草原牧区优势分布蜱虫-草原革蜱所传播家畜血液原虫病的防治提供科学依据。     

新疆2种优势革蜱携带马梨形虫和饶氏立克次体病原的检测

本试验旨在探究新疆优势分布的2种硬蜱所携带马泰勒虫、马驽巴贝斯虫和饶氏立克次体病原的情况。对采集于昭苏、和静2个采样点的硬蜱样品(n=188)经过分子生物学方法进行了种属鉴定的同时,以马泰勒虫18S rRNA、马驽巴贝斯虫Bc48和饶氏立克次体16S rRNA为靶标,应用PCR进行检测,并将阳性产物测序比对。在188只蜱虫中,93只(昭苏县)为边缘革蜱,95只(和静县)为森林革蜱;在2种硬蜱中,马驽巴贝斯虫的PCR阳性率分别为昭苏县30.1%(28/93)、和静县6.3%(6/95);饶氏立克次体的检出率分别为昭苏县6.5%(6/93)、和静县36.8%(35/95)。试验鉴定归类了昭苏县与和静县采样点的优势种硬蜱,并发现两地的2种硬蜱均可携带马驽巴贝斯虫和饶氏立克次体,本试验为革蜱携带病原相关研究以及蜱传疾病的科学防控提供基础数据。     

新疆昭苏地区马梨形虫病病原及抗体检测

马梨形虫病是由马驽巴贝斯虫和马泰勒虫寄生于马属动物的红细胞内所引起的一类血液原虫病,呈全球性分布,尤其在新疆发病率更高,处于逐年上升趋势,对区域性马产业的发展影响极大。为了解2018年新疆昭苏养马区域马梨形虫的感染情况,随机采集昭苏县18个乡镇的马全血及血清各858份,采用PCR和间接ELISA分别进行检测,对两种方法检测的18个地区、不同年龄阶段的马驽巴贝斯虫、马泰勒虫及混合感染情况进行统计学分析。结果显示,PCR检测马驽巴贝斯虫、马泰勒虫及混合感染的阳性率分别为12.12%、13.87%和2.80%;间接ELISA检测马驽巴贝斯虫、马泰勒虫及混合感染的抗体阳性率分别为15.50%、10.14%和2.56%;不同年龄阶段筛查结果显示,在6岁以下的马匹感染马驽巴贝斯虫、马泰勒虫及混合感染的阳性率较高,并且不同地区的不同年龄阶段马匹的马梨形虫感染率存在不同程度的差异。此次获得的昭苏县马梨形虫感染情况的一线数据,可为当地养马区域马梨形虫病的综合防控提供技术支撑。     

血红扇头蜱和草原革蜱源性驽巴贝斯虫病原DNA检测

为了进一步鉴定、识别血红扇头蜱及草原革蜱的形态结构,探究来源于内地(广州某地区)的血红扇头蜱及新疆境内广泛分布的草原革蜱携带同一种病原驽巴贝斯虫的情况,试验对血红扇头蜱及草原革蜱进行扫描电镜观察、鉴定,并参考驽巴贝斯虫Bc48基因序列合成引物,提取该种蜱携带的驽巴贝斯虫病原DNA,进行PCR检测。结果表明:血红扇头蜱和草原革蜱分别在其孔区、齿式、气门板及基节外距等部位具有超微结构差异,可分别作为其同种蜱的鉴别要点。经PCR成功扩增出了血红扇头蜱和草原革蜱所携带的驽巴贝斯虫病原DNA阳性条带大小为452 bp,来源于广州某地区的血红扇头蜱携带驽巴贝斯虫的阳性率为8.16%(12/147),来源于本地的草原革蜱驽巴贝斯虫的阳性率为3.84%(6/156)。     

Diagnosis of equine piroplasmosis in Xinjiang province of China by the enzyme-linked immunosorbent assays using recombinant antigens

The prevalence of equine piroplasmosis in Xinjiang province, China, was examined by enzyme-linked immunosorbent assays (ELISAs). A total of 70 serum samples were taken from horses pastured on three farms in western Xinjiang, and examined for diagnosis of equine Babesia equi (B. equi) infection and B. caballi infection by ELISAs using recombinant equi merozoite antigen 1 (EMA-1) and recombinant P48 antigen, respectively. Of the 70 samples, 28 (40.0%) and 17 (24.3%) samples were positive for B. equi infection and B. caballi infection, respectively. In addition, 11 (15.7%) samples were positive for both B. equi and B. caballi infections. These results indicate that equine piroplasmosis is widespread and therefore a cause for serious concern in western Xinjiang. To our knowledge, this is the first report describing a survey on equine piroplasmosis in Xinjiang province, China.     

Seroepidemiological evidence for the possible presence of Babesia (Theileria) equi and Babesia caballi infections in donkeys in western Xinjiang, China

The prevalence of Babesia (Theileria) equi and B. caballi infections in donkeys in western Xinjiang China was investigated. In total, 93 serum samples were randomly taken from donkeys in the Kashi and Ili areas, and examined for B. equi and B. caballi infections by enzyme-linked immunosorbent assays using recombinant antigens. Of the 93 samples, 9 (9.6%) and 36 (38.7%) samples were positive for B. equi infection and B. caballi infection, respectively. In addition, 2 (2.2%) samples were positive for both B. equi and B. caballi infections. These results indicate that equine babesiosis might be extensively prevalent in donkeys in western Xinjiang.     

Seroepidemiologic studies on Babesia caballi and Babesia equi infections in Japan

Antibodies to Babesia caballi and Babesia equi were examined on a total of 2,019 horse serum samples that had been collected in 1971-1973 by the National Institute of Animal Health by enzyme-linked immunosorbent assay (ELISA) using recombinant proteins and by Western-blot analysis. Based on the criterion for positivity by ELISA, 5.4% (109/2,019) and 2.2% (44/2,019) had antibodies against B. caballi and B. equi, respectively. The ELISA-positive sera were further examined by Western blot; 30/109 for B. caballi and 2/ 44 for B. equi were positive for native B. caballi or B. equi, but none of them was seropositive for both infections. Based on the results of this study, further investigations should be required to survey horses that have arrived in Japan relatively recently and tick vectors of equine Babesia using ELISA with some recombinant protein, a parasite detection method in an in vitro culture of equine Babesia, and PCR testing.     

Evaluation of enzyme-linked immunosorbent assays with recombinant antigens for the serodiagnosis of equine Babesia infections

Two enzyme-linked immunosorbent assays (ELISA) with recombinant protein as antigens were evaluated by comparison with the indirect fluorescent antibody tests (IFAT) for the detection of specific antibodies to Babesia caballi and Babesia equi, respectively in 380 sera from experimentally infected, uninfected, and field horses. The high concordances of 92.4% (351/380) and 98.2% (373/380) between ELISA and IFAT for B. caballi and B. equi, respectively suggest that ELISA, especially for B. equi infection, could be alternative to the corresponding IFAT for serodiagnoses of equine piroplasmosis, although some improvements are required in ELISA for B. caballi.     

2020年北疆部分地区猪伪狂犬病毒gE蛋白抗体ELISA检测与分析

为了解2020年1—12月北疆部分地区猪伪狂犬病野毒株(PRV-gE)感染情况,采用间接酶联免疫吸附试验(ELISA)法对从北疆4个地区采集的657份猪血样进行PRV-gE蛋白抗体水平检测.结果显示,4个地区PRV-gE抗体阳性率为29.83％(196/657),乌苏地区与其他地区的抗体水平存在一定差异(P<0.05)...     

2016年-2017年新疆规模化猪场伪狂犬病抗体检测

为了解新疆地区不同规模化猪场伪狂犬病病毒(PRV)野毒感染情况,采用ELISA对2016年4月-2017年12月来自67个不同规模化猪场的2 516份血清进行了PRV-gE抗体血清学调查。结果显示,血清总阳性率为34.02%,2016年和2017年阳性率分别为17.77%和39.52%;哺乳仔猪、保育猪、育肥猪、后备母猪、妊娠母猪、哺乳母猪、公猪总阳性率分别为26.07%、22.35%、35.38%、32.70%、33.13%、31.06%和31.71%;猪场总阳性率为82.09%,北疆、南疆和东疆猪场阳性率分别为78.57%、100.00%和100.00%。结果表明,2016年-2017年新疆地区猪群中PRV感染率明显上升,南疆与东疆地区阳性率显著高于北疆地区,各个阶段猪群阳性率均显著升高,母猪与公猪感染加重是伪狂犬病持续存在的重要原因,对其进行防控与净化刻不容缓。     

Diagnosis of equine piroplasmosis in Brazil by serodiagnostic methods with recombinant antigens.

Serum samples from horses in the States of Sao Paulo and Mato Grosso do Sul, Brazil were examined for diagnosis of equine piroplasmosis by both the latex agglutination test (LAT) and enzyme-linked immunosorbent assay (ELISA) with recombinant antigens. Of the 47 samples analyzed, 38 (81%) and 42 (90%) samples were positive for B. equi infection and B. caballi infection, respectively. In addition, 35 (75%) samples were positive for both B. equi and B. caballi infections. These results indicate that equine piroplasmosis is widespread and therefore a cause for serious concern in the States of Sao Paulo and Mato Grosso do Sul, Brazil.     

Serological prevalence of Babesia caballi and Theileria equi in horses of Lara State, Venezuela

The main objective of this study was to demonstrate the occurrence of equine piroplasmosis (EP) in horses of Lara State, Venezuela, and to correlate it with the factors host's sex and age in order to know the epidemiology of this disease at the Venezuelan Centroccidental Region. Antibody levels to Babesia caballi and Theileria equi were assessed in 360 equine serum samples, collected from 9 municipalities of Lara State, using an ELISA technique with recombinant antigens and monoclonal antibodies (Mabs). Antibodies to B. caballi were found in 254 horses (70.6%), whereas 181 animals (50.3%) were detected as seropositives to T. equi. In addition, 128 samples (35.56%) were seropositives to both hemoparasites. There were no significant differences between the seropositivity to B. caballi and T. equi with the factors sex and age of the horses. These results show that Lara State is an enzootic area for equine piroplasmosis, and are a contribution to a partial knowledge of the dynamic of this disease in Venezuela.