光生物素标记DNA探针检测牛巴贝斯虫研究初报

光生物素标记重组质粒DNA(c51A)探针检测牛巴贝斯虫研究的初步试验结果表明,能特异地检出感染牛血中的巴贝斯虫,灵敏度为10pg质粒DNA,相当于约150个感染红细胞的虫体DNA。     

地高辛标记核酸探针检测牛粘膜病病毒

本课题建立了地高辛标记核酸探针检测牛病毒性腹泻－粘膜病病毒（ＢＶＤＶ）的诊断方法。根据ＢＶＤＶ基因序列的结构特点,在编码非结构蛋白ｐ１２５高度保守基因区内,设计合成一对特异性引物。以ＰＣＲ技术从ＢＶＤＶ基因重组质粒中扩增出了ＢＶＤＶ特异的、保守的４００ｂｐ核苷酸片段,经纯化后,地高辛标记,制备牛病毒性腹泻病毒的特异性核酸探针。通过检测ＢＶＤＶ细胞培养物、相关病毒及核酸载体和ＢＶＤＶ细胞培养物的总Ｒ     

地高辛标记核酸探针检测鸭瘟病毒

鸭瘟是鸭、鹅和其他雁形日禽类的一种急性、热性、啦血性传染病．特，征足流行广泛，传播迅速．发病率高和死亡率大。由于鸭瘟引起死亡、淘汰和产蛋下降，给发病地区的商品鸭和产蛋鸭造成很大的经济损失，目前．国内检症、诊断该病常用的方法有病毒分离、琼脂扩散试骑、酶联免疫吸附试验等。国内尚未见地高辛标记核酸探针检测鸭瘟病毒的报道。     

PCR制备地高辛标记的探针检测禽流感病毒核酸

用聚合酶链反应（PCR）技术,制备了广东禽流感无致病力分离株A/goose/China/24/96(H7N3）核蛋白基因片段（NPc）的地高辛标记的cDNA探针。建立并优化了检测禽流感病毒核酸的探针杂交法,探针杂交法能鉴别出非免疫鸡胚和SPF鸡胚尿囊液中的病毒,攻毒后第3天的SPF和非免疫鸡泄殖腔拭子中AIV的最大检出率为1/10,对临床样品中的AIV的最大检出率为1/7,而直接HA和HI法及AGP试验检不出临床样品的AIV。该探针具有较好的特异性和敏感性,为从分子水平探讨AIV的发病机理、临床早期快速诊断提供了新的研究手段。     

识别牛巴贝斯梨形虫的特异性DNA探针

用质粒ＰｕＮ１２１从墨西哥虫株Ｍ建立牛巴贝斯梨形虫ＤＮＡ的基因族，并克隆于大肠杆上，选择几个与标记的牛巴贝斯梨形虫基因ＤＮＡ杂交的重组质粒，进一步分析，发现ＰＭｕ－Ｂ１敏感性较高，能检测出２５ｐｇ纯净的牛巴贝斯梨形虫ＤＮＡ。１０μｌ感染全血中３００个梨形虫或０．０００２５％虫血症，ＰＭｕ－Ｂ１含有６．０ｋｂ牛巴贝斯梨形虫ＤＮＡ插人物，该插人物不与双芽巴贝斯梨形虫，伊氏锥虫，恶性疟原虫，边缘边虫，微     

地高辛标记的核酸探针检测EDS病毒的研究

地高辛标记的核酸探针检测ＥＤＳ病毒的研究王雪敏（邯郸农业高等专科学校牧医系河北永年０５７１５０）郑明球蔡宝祥陈溥言（南京农业大学动物医学院１材料与方法１．１地高辛标记的ＥＤＳ病毒核酸探针制备将ＥＤＳ病毒ＮＥ４株（南京农大传染病教研室提供）鸭胚尿囊液（...     

快速检测牛巴贝斯焦虫LAMP方法的建立

为提高牛巴贝斯焦虫(Babesia bovis)病检出率,本研究采用环介导等温扩增技术(LAMP)建立一种快速、灵敏、特异的检测B.bovis的方法。根据GenBank中登录的B.bovis细胞色素b基因(cyt b)序列,设计4条LAMP引物,优化反应体系条件,在Bst DNA聚合酶的作用下,62℃反应60min,加入EvaGreen后,肉眼观测。结果表明,该LAMP检测体系特异性强,与双芽巴贝斯焦虫(B.bigemina)DNA等不发生交叉反应;敏感性高,最小检测值为0.014fg(相当于1.58×10-3虫体的拷贝数),为一般PCR方法的1000倍。该方法具有简单、快速、低成本的特点,可用于B.bovis病的现场快速检测。     

南德文种牛发生巴贝斯虫病

本文报道南德文种牛发生双芽巴贝斯虫和牛巴贝斯虫病的诊断和治疗。对病牛主要采用血虫净按４ｍｇ／ｋｇ体重剂量,用无菌蒸馏水配成５％溶液肌注,每天１次,连用３天,大部分病牛停止便血尿,开始采食。     

光敏生物素标记核酸探针检测A群轮状病毒的研究

采用光敏生物素标记Ａ群轮状病毒（ＧＡＲＶ）全基因组ＲＮＡ,制备了ＧＡＲＶ群特异核酸探针,该探针与样品进行打点杂交,经亲和素－碱性磷酸酶（ＡＶ－ＡＰ）系统显色,可检出１００ｐｇ病毒ＲＮＡ序列。试验表明轮状病毒光敏生物素核酸探针具有特异、灵敏、稳定的特点,可用于ＧＡＲＶ的检测和进行基因相关性研究。     

Detection of IgM antibodies against Babesia bovis in cattle

The diagnosis of acute babesiosis by direct examination of blood smears has some limitations and the indirect serological methods currently in use are designed for detection of IgG, which may not be detectable at an early stage of infection. There is a need, therefore, for rapid and reliable procedures to diagnose acute infections. An ELISA system using a crude antigenic preparation of Babesia bovis was standardized for the detection of IgM antibodies. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkerboard titrations. Serum samples of cattle imported from tick-free areas collected before and during an immunization process were used to validate the tests. The specificity was 94% and sensitivity 100%. Specific IgM antibodies against B. bovis first appeared on the 11th day post-inoculation (p.i.) in animals infested with Boophilus microplus ticks and on the 19th day p.i. in animals which had been inoculated with infected blood. Antibody titers decreased after Day 33; however, all animals remained positive until the end of the experiment (124 days).     

牛巴贝斯虫PCR检测及体外培养试验

本试验通过PCR方法检测出牛巴贝斯虫单一感染的牛全血样本,并探讨了牛巴贝斯虫病患牛全血体外培养方法及培养条件。试验结果表明,PCR检测结果显示检测方法效果较好,单一感染样本的新鲜血液中虫体在含40%牛血清的完全培养液和37 ℃、5% CO₂的条件下能建立起牛巴贝斯虫的体外培养,虫体可以连续培养14 d,传代13次,最高染虫率可以达到12.5%,平均染虫率为4%。     

Attenuation of Babesia bovis by in vitro cultivation

A virulent strain of Babesia bovis was adapted to grow in erythrocyte culture in the presence of equine serum and in lieu of bovine serum. Four splenectomized calves inoculated with the adapted strain, 429, developed hematologic signs of infection and a low grade fever, but remained free of central nervous system (CNS) signs and recovered. All of six control animals inoculated with a virulent strain reacted severely and five showed CNS signs and died. The calves injected with the attenuated strain were solidly immune when challenged with the virulent strain at 44 or 78 days after vaccination.     

Immune control of Babesia bovis infection

Babesia bovis causes an acute and often fatal infection in adult cattle, which if resolved, leads to a state of persistent infection in otherwise clinically healthy cattle. Persistently infected cattle are generally resistant to reinfection with related parasite strains, and this resistance in the face of infection is termed concomitant immunity. Young animals are generally more resistant than adults to B. bovis infection, which is dependent on the spleen. Despite the discovery of B. bovis over a century ago, there are still no safe and effective vaccines that protect cattle against this most virulent of babesial pathogens. Immunodominant antigens identified by serological reactivity and dominant T-cell antigens have failed to protect cattle against challenge. This review describes the innate and acquired immune mechanisms that define resistance in young calves and correlate with the development of concomitant immunity in older cattle following recovery from clinical disease. The first sections will discuss the innate immune responses by peripheral blood- and spleen-derived macrophages in cattle induced by B. bovis merozoites and their products that limit parasite replication, and comparison of natural killer cell responses in the spleens of young (resistant) and adult (susceptible) cattle. Later sections will describe a proteomic approach to discover novel antigens, especially those recognized by immune CD4+ T lymphocytes. Because immunodominant antigens have failed to stimulate protective immunity, identification of subdominant antigens may prove to be important for effective vaccines. Identification of CD4+ T-cell immunogenic proteins and their epitopes, together with the MHC class II restricting elements, now makes possible the development of MHC class II tetramers and application of this technology to both quantify antigen-specific lymphocytes during infection and discover novel antigenic epitopes. Finally, with the imminent completion of the B. bovis genome-sequencing project, strategies using combined genomic and proteomic approaches to identify novel vaccine candidates will be reviewed. The availability of an annotated B. bovis genome will, for the first time, enable identification of non-immunodominant proteins that may stimulate protective immunity.     

牛巴贝斯虫TaqMan荧光定量PCR检测方法的建立

为建立一种灵敏、特异、快速的牛巴贝斯虫检测方法,针对牛巴贝斯虫Rap-1a基因设计引物进行PCR扩增,然后构建重组质粒制作标准品,经过优化反应体系、绘制标准曲线,建立了牛巴贝斯虫TaqMan荧光定量PCR方法,并进行灵敏度、特异性及稳定性检测,同时利用该方法对37份田间样品进行检测。结果显示：建立的牛巴贝斯虫TaqMan荧光定量PCR检测方法的标准曲线方程式为y=-3.362×Log（X）+43.32,相关系数R2=0.999,扩增效率为98.4%。该方法的灵敏度为1.0×102 copies/μL,是普通PCR（1.0×104 copies/μL）的100倍。该方法对牛双芽巴贝斯虫、大巴贝斯虫、卵形巴贝斯虫等7种常见的牛梨形虫病检测结果均为阴性,组内和组间重复试验的变异系数均小于2.5%,37份田间样品的阳性检出率为67.5%。结果表明,本试验建立的牛巴贝斯虫TaqMan荧光定量PCR灵敏度高,且特异、稳定,适用于牛巴贝斯虫的诊断,从而为其流行病学调查提供了快速有效的检测方法。     

Serological survey of Babesia bovis and Babesia bigemina in cattle in South Africa

A total of 719 serum samples collected from clinically healthy cattle from eight provinces located in different districts of South Africa were examined by the indirect enzyme-linked immunosorbent assay (ELISA) and the standard indirect fluorescent antibody test (IFAT) to determine the serological prevalence of Babesia bovis and Babesia bigemina. The results showed that 35.3% and 39.7% of cattle were positive for B. bovis and 30% and 36.5% were positive for B. bigemina antibodies on ELISA and IFAT, respectively. Mixed infections were detected in 18.2% and 26.3% of the samples using ELISA and IFAT, respectively. Consequently, the ELISAs with recombinant B. bovis spherical body protein-4 (BbSBP-4) and B. bigemina C-terminal rhoptry-associated protein-1 (BbigRAP-1/CT) were proven to be highly reliable in the serological diagnoses of bovine babesiosis in South African cattle, as evidenced by the significant concordance rates when the results were compared to those of IFAT. Moreover, the serological prevalence was significantly different among the tested provinces, in which the ranges exhibited between 15% and 73% for B. bovis infection and between 13% and 54% for B. bigemina infection. High sero-positive rates were present in Mpumalanga and KwaZulu-Natal provinces, while the lowest rate was in the North West province. Our data provide important information regarding the current seroprevalence of bovine babesiosis in South Africa, which might be beneficial in developing rational strategies for disease control and management.