Screening of Chinese Medicine Resisting Neospora caninum with ELISA

[Objective] The study was to screen the Chinese medicines with good resistance to Neospora caninum. [Method] Healthy Kunming mice pretreated with methylprednisolone for the purpose of immunity decrease were randomly divided into 15 groups, and each mouse was intraperitoneally inoculated with 1.0 ×104 N. caninum. Four hours later, the mice were gavaged with various Chinese medicines. Seven days post-administration, eyeball blood sampling was conducted and the serum was used for antibody level detection with ELISA method. [Result] Four among the 15 Chinese medicines showed lower antibody positive rates. [Conclusion] Scutellaria baicalensis, Stemona sessilifolia, Gastrodia elata and Coptis chinensis could enhance the immunity level of mice infected by N. caninum.

用重组蛋白NcSAG1t作为ELISA诊断抗原进行改良绒山羊Neospora caninum的血清学诊断

Ncaninum的表面抗原1(NcSAG1)在建立新孢子虫病的诊断上是一个重要的诊断抗原。为了建立有效的诊断方法,基因编码的截头(NcSAG1t)缺失一个信使缩氨酸和C-末端的疏水性区域的基因,象谷胱甘肽转移酶(GST)的融合蛋白一样被克隆和表达在escherichiacoli。纯化的GST-NcSAG1t作为ELISA诊断抗原被用在改良绒山羊N caninum抗体的检测中。经过对207份改良绒山羊血清样品进行检测,检出Ncaninum抗体阳性16份,阳性率为7.73%。结果初步说明青海省的改良绒山羊羊群中存在Ncaninum。

Histopathological and molecular study of Neospora caninum infection in bovine aborted fetuses

Objective:To estimate the extent to which abortion in dairy cows was associated with of Neospom caninum(N.caninum) and to determine the risk factors of neosporosis in dairy farms from 9 provinces in Iran.Methods:Polymerase chain reaction(PCR) test was used to detect Neospora infection in the brain of 395 bovine aborted fetuses from 9 provinces of Iran.In addition,the brains of aborted fetuses were taken for histopathological examination.To identify the risk factors associated with neosporosis,data analysis was performed by SAS.Results:N.caninum was detected in 179(45%) out of 395 fetal brain samples of bovine aborted fetuses using PCR.Among the PCR-positive brain samples,only 56 samples were suited for histopathological examination.The characteristic lesions of Neospora infection including non-suppurative encephalitis were found in 16(28%) of PCR-positive samples.The risk factors including season,parity of dam,history of bovine virus diarrhea and infectious bovine rhinotracheitis infection in herd,cow's milk production,herd size and fetal appearance did not show association with the infection.This study showed that Neospora caused abortion was significantly more in the second trimester of pregnancy than other periods.In addition,a significant association was observed between Neospora infection and stillbirth.Conclusions:The results showed N.caninum infection was detected in high percentage of aborted fetuses.In addition,at least one fourth of abortions caused by Neospora infection.These results indicate increasing number of abortions associated with the protozoa more than reported before in Iran.

Prevalence of Antibodies to <i>Toxoplasma gondii</i>and <i>Neospora caninum</i>in Pigs in Grenada, West Indies

The aim of the study was to estimate the prevalence of antibodies to Toxoplasma gondii (T. gondii) and Neospora caninum (N. caninum) in pigs in Grenada, West Indies. T. gondii is a serious zoonosis affecting the unborn fetus and immunocompromized individuals. N. caninum is a similar coccidian parasite, which is not zoonotic, but is the cause of abortion and neonatal mortality in livestock similar to T. gondii. An earlier study conducted in Grenada and using a modified agglutination test (MAT) revealed seropositivity to T. gondii in pigs. No information is available on N. caninum infection of pigs in the Caribbean islands including Grenada. Serum samples from 185 pigs in Grenada, West Indies were tested for antibodies to T. gondii and N. caninum using an indirect enzyme linked immunosorbent assay (ELISA). Antibodies to T. gondii were found in 24.3% of pigs (95% confidence interval (CI): 18.12% to 30.48%) as all the tested pigs were negative for antibodies to N. caninum. Although, seroprevalence for T. gondii was higher in females (25.75%) than in males (20.70%), this result was statistically insignificant (p = 0.57). The results were similar to a previous study in Grenada confirming the continuity of infection in pigs. Human Toxoplasmosis is transmitted mainly through ingestion of tissue cysts in contaminated raw or undercooked meat or sporulated oocysts in soil, water or vegetables. Education of farmers and the Grenadian community on epidemiology of these parasites is warranted to prevent infection in pigs and in humans. This is the first report on the seroprevalence of N. caninum in pigs in the Caribbean region.

Neospora caninum immune mapped protein 1(NcIMP1) is a novel vaccine candidate against neosporosis

The Neospora caninum immune mapped protein 1(Nc IMP1) was identified as a membrane protein,and a previous study indicated that Nc IMP1 could be a promising vaccine candidate against neosporosis. In this study, the immune response and protection efficacy of Nc IMP1 were evaluated. The coding sequence of Nc IMP1 was inserted into the eukaryotic expression vector pc DNA3.1(+), resulting in the recombination plasmid pc DNAIMP1, which was used for the intramuscular immunization of BALB/c mice. After immunization, the immune response was evaluated using a lymphoproliferative assay and cytokine and antibody measurements. Quantification of the cerebral parasite burden of mice challenged with 2106 N. caninum was performed 14 days after the last immunization. The results showed that the mice immunized with pc DNA-IMP1 developed a high level of specific antibody responses against recombinant Nc IMP1,with a mixed Ig G1/Ig G2 a response and a predominance of Ig G2 a production. The cellular immune response was associated with the production of IFN-γ, IL-2, IL-4 and IL-10 cytokines. The experiment was terminated 30 days p.i.,and the cerebral parasite burden in each mouse was assessed by quantitative PCR. The parasite burden was significantly reduced in the pc DNA-IMP1-vaccinated mice. These data suggest that IMP1 is a promising vaccine candidate against neosporosis.

Protective efficacy of vaccination with NcMIC3 and NcMIC8 against Neospora caninum infection in mice

Microneme proteins (MICs) are important for Apicomplexan parasite invasion due to their adhesion to host cells. Several studies have indicated that Neospora caninum MIC3 and MIC8 are important adhesion factors and potential vaccine candidates against neosporosis. In this study, we evaluated the protective efficacy of recombinant proteins and DNA vaccines of NcMIC3 and NcMIC8. BALB/c mice were immunized with rNcMIC3, rNcMIC8, pcDNA3.1-NcMIC3 and pcDNA3.1-NcMIC8 respectively, and challenged with N. caninum tachyzoites. The immune responses were evaluated through cytokine, antibody measurements and the parasite burden in the mice brain tissues. Serological analysis showed that recombinant protein vaccines induced higher levels of immunoglobulin G (IgG) than other groups. The percentage of IgGl and IgG2a in the recombinant protein groups was higher than the other groups, and with a predominance of IgGl over IgG2a, suggesting that recombinant protein vaccines elicited a Th2-type immune response, while DNA vaccines mainly produce a Th 1-type immune response. In addition, mice immunized with rNcMIC3 and rNcMIC8 a had lower parasite burden in brain tissue compared with the other groups. These results demonstrate that rNcMIC3 and rNcMIC8 could induce humoral and Th2-type immune response, leading to a considerable level of resistance against neosporosis.

新孢子虫硫氧还蛋白的生物信息学分析及其原核表达的鉴定

为研究新孢子虫硫氧还蛋白(Trx)的生物学特性,本研究利用PCR扩增犬新孢子虫Trx(NcTrx)基因的编码区序列,构建pET-32a(+)-NcTrx原核表达载体,经双酶切与测序鉴定正确后,利用系列生物信息学软件分析NcTrx基因及其编码氨基酸序列的生物信息学特征,并对其蛋白互作网络中排名前100的蛋白进行GO功能和KEGG信号通路的富集分析。重组质粒中NcTrx基因的测序结果显示,NcTrx基因片段为1 287 bp,编码428个氨基酸;该蛋白含1个信号肽,1个跨膜区,主要位于内质网上;NcTrx蛋白的二、三级结构主要由α-螺旋、无规则卷曲组成,有12个B细胞抗原表位;该蛋白属于硫氧还蛋白家族,存在典型的氧化还原基序“-CXXC-”,有38个磷酸化位点、10个O-糖基化位点;与NcTrx蛋白互作最强的为酪氨酸激酶样蛋白,二者通过氢键和静电作用实现相互对接;GO功能及KEGG信号通路的显著性富集分析结果显示,NcTrx与其互作的蛋白主要参与蛋白质转运、蛋白质二硫键异构酶活性等生物学过程,与内质网蛋白质加工、硫代谢等信号通路密切相关。将pET-32a(+)-NcTrx转化BL21(DE3)感受态细胞,并经IPTG诱导表达后采用镍亲和层析法纯化重组NcTrx蛋白(rNcTrx),经SDS-PAGE检测该蛋白的表达及纯化效果,采用Bradford法测定纯化蛋白的浓度。采用western blot鉴定rNcTrx的反应原性。SDS-PAGE结果显示,rNcTrx分子量约66 ku,主要以包涵体的形式表达,且纯化效果较好,浓度为0.8 mg/mL。Western blot检测结果显示,纯化的rNcTrx能够与鼠His MAb、新孢子虫感染的小鼠血清发生特异性反应,在66 ku处出现特异性条带。本研究首次经PCR扩增NcTrx基因,对NcTrx基因及其蛋白进行了生物信息学分析,并经原核系统表达及鉴定了NcTrx蛋白,为后续深入探究该蛋白的生物学功能提供参考依据。

犬新孢子虫miRNAs的鉴定与分析

为鉴定犬新孢子虫(Neospora caninum)特异表达的miRNAs,并研究其在宿主细胞中的表达情况,本研究收集N.caninum速殖子感染的山羊子宫内膜上皮细胞(caprine endometrial epithelial cells,EECs)24和48 h的样本,用Illumina高通量测序平台和生物信息学分析犬新孢子虫特异表达的miRNAs及其体外表达情况,并研究了一个上调的miRNA novel3_mature对犬新孢子虫在山羊EECs增殖的影响。结果发现,共鉴定出3404个犬新孢子虫特异表达的miRNAs(155个新预测miRNAs),其中77个(47个上调和30个下调)miRNAs显著差异表达;随机选取7个显著差异表达的miRNAs进行实时荧光定量PCR分析,结果与高通量测序结果一致;预测分析发现61个miRNAs的3978个靶基因,功能富集分析发现这些靶基因可能主要参与MAPK信号通路、突触导向、黏着斑、cMAP信号通路、钙信号通路等;转染novel3_mature的模拟物显著促进犬新孢子虫在山羊EECs中的增殖。犬新孢子虫存在多个特异表达的miRNAs,其在虫体增殖中发挥重要作用,研究成果为深入理解犬新孢子虫胞内生存提供了新的思路。

新孢子虫入侵对宿主细胞炎症反应与应答的调节

犬新孢子虫是一种寄生在动物细胞内的原虫,可引起新孢子虫病。该病临床表现为母牛的繁殖障碍,导致其流产或死胎,严重影响养殖业的正常发展。新孢子虫病广泛分布世界各地,但由于新孢子虫感染与宿主之间相互作用的机制尚未阐明,尚未研制出特效的药物与商品化疫苗。有研究表明,在虫体入侵宿主的过程中,宿主的天然免疫在抵抗新孢子虫感染中的作用不可忽视。论文主要阐述了犬新孢子虫入侵对宿主细胞免疫调节的影响以及虫体分泌的外泌体在新孢子虫入侵过程中的作用,为深入了解犬新孢子虫的致病机制以及新孢子虫病防治提供新的思路。

河南孟津地区6种引起奶牛流产病原的PCR检测及分析

为调查孟津地区引起奶牛流产的主要病原,本实验采集该地区8个规模化奶牛养殖场共52头新鲜流产胎牛,及其母体的血液、阴道分泌物和鼻腔分泌物。对所采集样本进行新孢子虫(%N.caninum)、弓形虫(T.gondii)、胎儿三毛滴虫(T.foetus)、布氏杆菌(B.abortus%)、牛传染性鼻气管炎病毒(IBRV)和牛病毒性腹泻病毒(BVDV)6种常见流产病原的PCR和RT-PCR检测。结果显示,被检样本总感染率为44.2%(23/52),其中新孢子虫感染率最高,为32.7%(17/52);IBRV次之,为13.5%(7/52);胎儿三毛滴虫、布氏杆菌、弓形虫和BVDV感染率分别为1.9%、1.9%、1.9%和0。11.5%(6/52)的被检样本存在混合感染,其中以新孢子虫+IBRV混合感染为主(50%,3/6)。结合被检流产奶牛的临床特征及饲养管理等因素,推断新孢子虫是导致该地区奶牛流产的主要病原。

新疆地区流产奶牛新孢子虫病和布氏杆菌病的流行病学调查

为了调查我国新疆地区流产奶牛新孢子虫病和布氏杆菌病的感染情况，应用酶联免疫吸附试验（ELISA），对新疆15个地区419份奶牛血清样本（有流产史奶牛）进行了牛新孢子虫病和布氏杆菌病的检测。结果显示新疆地区流产奶牛新孢子虫感染率为10．3％，布氏杆菌病抗体阳性率为2．7％，同时检测到既有新孢子虫抗体又有布氏杆菌抗体的奶牛血清，占总数的0．95％，各牛场所检流产奶牛血清新孢子虫抗体阳性率在0—50％，各个年龄段奶牛血清的抗体阳性率差异不显著（P〉0．05）。不同妊娠胎次的奶牛血清抗体阳性率差异显著（P〈0．05）。调查结果表明，新疆部分地区奶牛存在新孢子虫的感染且是导致流产的重要原因之一。此次调查研究为今后新疆地区有效预防和治疗新孢子虫病提供了一定的科学依据。

奶牛新孢子虫重组蛋白NcSRS2t间接ELISA方法的建立及初步应用

以纯化的犬新孢子虫(Neospora caninum)重组蛋白NcSRS2t为抗原包被酶标板,通过对间接ELISA各反应条件的优化,确定了最佳反应条件。采用已建立的间接ELISA反应条件,对218份阴性血清的检测结果进行统计学分析,确定了间接ELISA的判定标准,即OD450nm值大于等于0.32为阳性,小于0.32为阴性。应用建立的间接ELISA方法对128份阴性血清和50份阳性血清(经IDEXX公司新孢子虫抗体检测试剂盒证实)进行检测,结果表明,间接ELISA方法的特异性为93.0%,敏感性为90.0%。采用本试验建立的新孢子虫间接ELISA诊断方法对黑龙江省克山、海林、双城、甘南、克东、富裕、牡丹江、大庆等8个县(市)奶牛场的540份奶牛血清进行了检测。结果表明,被检的540份奶牛血清样本中奶牛新孢子虫的抗体阳性率为13.33%。该间接ELISA诊断方法的建立及初步应用为新孢子虫病诊断试剂盒的研制和新孢子虫病的预防研究奠定了基础。

牛新孢子虫病和弓形虫病的流行病学调查

为了调查我国牛新孢子虫和弓形虫的感染情况，应用新孢子虫酶联免疫吸附试验（ELISA）和弓形虫间接血凝试验（IAT）分别检测了来自国内10个省（市、自治区）的262份乳牛、10份肉牛和40份水牛血清。结果显示，乳牛新孢子虫的抗体阳性率为17．2％，弓形虫的抗体阳性率为2．3％，没有检测到既有新抱子虫抗体又有弓形虫抗体的乳牛血清。各牛场所检乳牛血清的新孢子虫抗体阳性率在0～34．4％之间。在水牛和肉牛血清中未检测到新孢子虫抗体和弓形虫抗体。流产乳牛血清的新孢子虫抗体阳性率为20．2％，未流产乳牛为16．1％，其中血清抗体阳性乳牛主要在妊娠中晚期流产。各个年龄段乳牛血清的抗体阳性率差异不显著（P〉0．05）。不同妊娠胎次的乳牛血清抗体阳性率差异不显著（P〉0．05）。确认新孢子虫病在国内大部分牛场存在。

牛新孢子虫病PCR检测方法的建立和应用

犬新孢子虫感染是导致妊娠母牛流产的主要原因之一,准确快速地诊断是有目的治疗该病的前提。本研究根据已知的犬新孢子虫种属特异性基因片段Nc-5基因序列,设计了一对特异性引物,建立了检测犬新孢子虫的PCR技术。利用本方法可以从感染牛胎儿的脑脊液及实质脏器中扩增出1条大小为350 bp的特异性核酸片段。在吉林省延边龙井地区的应用检测结果表明,流产牛胎儿中新孢子虫感染率为17%(15/90)。本方法在实际应用中快速、灵敏、特异性高,可以用于牛和其它动物新孢子虫病的快速诊断和流行病学调查。

新孢子虫dNcSRS2重组蛋白间接ELISA的建立及其应用

利用新孢子虫体外重组表面蛋白dNcSRS2蛋白作为包被抗原,对各项条件进行优化,确定判定标准,建立了检测新孢子虫血清抗体的间接ELISA方法。经对多例血清检测表明,所建立的诊断试剂盒重复性好、特异性强、灵敏度高,与进口的IFAT及两种商品化ELISA试剂盒的检测结果相比较,符合率均达到92%以上。应用建立的ELISA方法对236份奶牛血清的新孢子虫抗体进行检测,阳性率为22%。这是国内首次利用重组蛋白建立的诊断试剂盒,该方法的建立将为牛新孢子虫病的诊断与流行病学调查提供有效的技术手段。

乳牛流产胎儿中新孢子虫的鉴定

在血清学调查的基础上,进行了流产胎牛体内新孢子虫的鉴定。取新孢子虫血清抗体阳性乳牛的流产胎牛的脑、心、肺、脾、肝等组织进行病理学检查,经HE染色观察到脑组织中存在类似于新孢子虫包囊样结构,经免疫组织化学染色排除刚地弓形虫包囊的存在,确认该包囊为新孢子虫包囊。同时提取流产胎牛脑组织DNA,应用新孢子虫特异性引物Np6/Np21进行PCR扩增,扩增出的特异性目的条带的序列与新孢子虫标准株Nc-1的gene 5序列的一致性为98%。首次证实我国大陆流产胎牛脑组织中存在新孢子虫。

牛新孢子虫病LAMP检测方法的建立

为建立一种快捷、灵敏的牛新孢子虫病检测方法,根据GenBank上登录的新孢子虫NcSAG1基因(AF132217)序列设计合成了2对环介导等温扩增(LAMP)特异引物,建立了检测牛新孢子虫病的LAMP技术,并优化了LAMP的各反应条件,进行了敏感性和特异性试验。结果显示,LAMP方法扩增牛新孢子虫的电泳产物呈特征性梯状条带,显色反应呈现绿色荧光,酶切鉴定出现目的条带,LAMP方法检测的敏感性为5×105copies/μL;特异性试验显示,新孢子虫检测管显色后呈阳性,而瑟氏泰勒虫、弓形虫和附红细胞体等对照组均呈阴性。表明,本研究建立的LAMP方法具有灵敏、特异、快速等优点,适合于牛新孢子虫病的检测。

吉林株犬新孢子虫的分离与鉴定

将疑似感染犬新孢子虫而流产的牛胎儿脑组织接种于Vero细胞进行体外培养,同时感染长爪沙鼠进行动物试验。提取牛流产胎儿脑组织DNA、沙鼠脑组织DNA、细胞培养虫体DNA,用犬新孢子虫特异性引物进行PCR扩增。结果显示,PCR扩增出了1条约为681 bp的目的条带;测序结果表明,扩增出的特异性目的基因序列与GenBank中发表的犬新孢子虫NcSAG1基因(AF113004)核苷酸序列的同源性为99.4%。结果表明,在吉林省牛流产胎儿脑组织中分离出犬新孢子虫。

宁夏部分地区规模化奶牛场4种疫病的血清学调查分析

为了解宁夏主要奶牛养殖区规模化奶牛场牛传染性鼻气管炎(IBR)、牛病毒性腹泻(BVD)、新孢子虫抗体和布鲁菌病原的流行状况,采用酶联免疫吸附试验(ELISA)对未接种过IBR、BVD、新孢子虫疫苗、接种过布鲁菌疫苗的奶牛群体进行血清抗体检测,并对银川、吴忠和青铜峡地区的阳性状况进行分析。结果发现,76份流产牛血清中,IBR抗体阳性率为50.00%(38/76),BVD抗体阳性率为19.74%(15/76),新孢子虫抗体阳性率为27.63%(21/76),布鲁菌阳性率为19.74%(15/76),4种病原均感染的阳性率为34.21%(26/76),IBR和新孢子虫混合阳性率占总阳性率的34.62%;408份非流产牛血清中,BVD抗体阳性率为12.75%(52/408),IBR抗体阳性率为21.08%(86/408);IBR和BVD在宁夏的3个主要养殖区均有不同程度的流行,其中以银川地区阳性率最高。说明宁夏部分地区IBR和BVD在不同的地区存在着不同程度的感染,流产牛中普遍存在上述4种疫病的感染并且IBR和BVD阳性率明显高于非流产牛。

PCR诊断牛新孢子虫病的初步应用

目的以犬新孢子虫Nc-5基因为目的基因的PCR诊断方法,检测奶牛流产的胎牛脑组织的新孢子虫。方法根据GenBank发布的新孢子虫特异性基因片段Nc-5基因序列,设计1对特异性引物,以犬新孢子虫(Neospora caninum)标准株DNA为模板,PCR扩增Nc-5基因,与pMD18-T载体连接,转化大肠埃希菌JM109,获得阳性重组质粒pMD18-T-Nc-5,并测序。以环形泰勒虫、牛巴贝斯虫、刚地弓形虫、杜氏利什曼原虫以及犬新孢子虫标准株DNA为模板进行扩增以验证PCR的特异性,采用紫外分光光度计测定犬新孢子虫标准株DNA浓度和纯度,取高纯度的DNA样品用灭菌水稀释,分别取不同量的DNA进行PCR扩增,确定PCR方法的敏感性;利用该方法对32份奶牛流产的胎牛脑组织样品进行检测,同时,对其中23份流产的母牛血样进行ELISA血清学检测(作为对照),以评价犬新孢子虫PCR方法的检测效果。结果克隆的犬新孢子虫标准株目的基因大小为350bp,与GenBank(AY459289)中Nc-5基因序列一致性为98%,建立的犬新孢子虫PCR方法与环形泰勒虫、牛巴贝斯虫、刚地弓形虫和杜氏利什曼原虫均无交叉反应,最低能检测犬新孢子虫DNA3.125pg,检测流产胎牛脑组织阳性率为18.8%(6/32)。ELISA检验流产母牛血样抗体阳性率为17.4%(4/23),这4份阳性样品与其流产胎牛PCR检测结果均为阳性。结论建立的犬新孢子虫PCR诊断方法用于检测奶牛流产的胎牛脑组织新孢子虫的感染效果较好。