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争带的序列与新孢子虫标准株Nc-1的gene5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。     

Associations of Neospora caninum seropositivity with gestation number and pregnancy outcome in Danish dairy herds.

The prevalence and distribution of seropositivity towards the protozoan parasite Neospora caninum were studied in single blood samples from 1561 cows from 31 Danish dairy herds. Blood samples were analysed by an indirect enzyme-linked immunoassay and an indirect fluorescent-antibody test. Seroprevalence in 15 herds with previous abortions assigned to neosporosis ranged from 1% to 58%, with a mean frequency of 22%. In eight out of 16 herds without a history of N. caninum related abortions, no seroreactors were found. In the remaining eight herds, the seroprevalence ranged from 6% to 59%. The prevalence and distribution of seropositivity, gestation number prior to sampling, and breed were related to abortions and perinatal deaths using a random-effects logistic-regression model. Abortion risk was significantly increased in seropositive animals (OR=3) and in 2nd-gestation cows (OR=3). Perinatal death was significantly influenced by gestation number and breed, but not by serostatus. Reproductive performance and culling risk of cows were not affected by serostatus. Seropositivity increased with “age” (i.e. gestation number) (P=0.02). In open cows, seropositivity tended to decrease with distance from calving (P=0.05). The proportion of seropositive pregnant cows increased with trimester (P=0.02).     

奶牛新孢子虫病血清流行病学调查

本研究用酶联免疫吸附试验（ELISA）对采集自北京地区的94份奶牛血清（随机采集）和河北地区的55份奶牛血清（有流产史奶牛），进行Neospora caninum血清抗体检测。结果发现，北京地区随机采集的奶牛血清N．caninum抗体阳性率为18．1％（17／94），河北地区有流产史的奶牛N．caninum血清抗体阳性率为23．6％（13／55）。采用牛奶记录体系（DHI）对北京地区17头N．caninum血清抗体阳性牛进行了日产奶量、乳中蛋白率和乳脂率的测定，并与同群牛中134头阴性牛比较。结果表明，N．caninum血清抗体阳性牛日产奶量比阴性牛降低9．7％，乳中蛋白率和乳脂率分别降低20％和15．4％。初步证明N．caninum血清抗体阳性奶牛产奶量降低及奶品质的下降。对不同N．caninum抗体滴度阳性牛的泌乳期主要生产性能比较发现，其生产性能的变化与抗体滴度无明显相关性。     

福建省部分地区奶牛新孢子虫病血清学抗体检测

新孢子虫是造成怀孕母牛流产等繁殖障碍和新生犊牛运动障碍的重要寄生虫之一,目前尚未有有效的药物和疫苗来治疗和预防牛新孢子虫病。本研究使用新孢子虫抗体检测试剂盒对采自福建省漳州、南平、三明和龙岩4 个地区规模化奶牛场的1 472 份奶牛血清样品进行抗体检测,并通过SPSS 22.0软件进行生物统计学分析。结果表明,血清样品新孢子虫抗体总阳性率为7.47%,4 个地区样品阳性率分别为5.57%、5.12%、7.41%和12.97%;不同年龄阶段奶牛阳性率范围为2.93%~9.56%,不同胎次阳性率范围为4.83%~9.50%。经统计学分析,不同年龄阶段奶牛阳性率差异显著（P<0.05）。这表明福建省奶牛普遍存在新孢子虫感染,奶牛场应采取综合防控策略和制定有效的管理措施控制奶牛新孢子虫病。     

新疆伊犁部分地区奶牛新孢子虫病血清学调查

用新孢子虫地方虫株SRS2重组抗原作为ELISA诊断抗原,对新疆伊犁部分地区奶牛新孢子虫病进行了血清学调查。结果表明,从101份奶牛血清样品中检出牛新孢子虫抗体阳性血清12份,阳性率为11.9%,各种年龄段的奶牛均可感染,经统计学分析,差异不显著(P0.05)。本次血清学调查为新疆伊犁地区有效地控制该病的发生和蔓延提供了重要的理论依据。     

牛源犬新孢子虫NcGRA7基因的克隆及原核表达分析

为了解牛源犬新孢子虫NcGRA7基因的生物学特性,本实验采用PCR技术扩增牛源犬新孢子虫NcGRA7基因,并连接至pMD-18T载体中,构建重组质粒pMD 18-NcGRA7,经PCR、酶切鉴定及测序分析,将NcGRA7基因连接到原核表达载体pGEX-4T-1中,构建重组表达质粒pGEX-NcGRA7,转化大肠杆菌BL21,筛选阳性克隆,将PCR和酶切鉴定正确的重组质粒进行IPTG诱导表达,SDS-PAGE和westem blot分析表达产物.结果表明,扩增的NcGRA7基因片段大小为701 bp,编码233个氨基酸,与GenBank中登录的NcGRA7(AF176649)核苷酸序列同源性为98.7％;western blot分析表达蛋白的分子量为52 ku,具有较好的反应原性.本实验为犬新孢子虫NcGRA7基因疫苗研究奠定了基础.     

牛源犬新孢子虫孕鼠感染模型的建立

为建立牛源犬新孢子虫孕鼠感染模型,深入研究牛源犬新孢子虫对孕鼠的致病作用,本试验以雌性BALB/c小鼠为试验动物,分离Vero细胞中培养的牛源犬新孢子虫速殖子,分不同剂量组腹腔接种雌性BALB/c小鼠后,与雄性BALB/c小鼠合笼,每天观察小鼠临床症状和发病情况,观察主要脏器组织的病理变化,应用PCR方法检测孕鼠脑、肝脏、脾脏等脏器组织及胎盘中犬新孢子虫Nc5基因,并测定孕鼠胎盘湿重和胎盘系数。结果显示,感染模型小鼠的最佳攻虫剂量为10⁵个虫体;感染犬新孢子虫孕鼠先后出现精神不振、共济失调等临床症状,并有不同程度死亡;病理学观察模型小鼠脑、肝脏、脾脏等脏器组织出现充血、出血、肿大等病理变化;在模型小鼠脑、肝脏、脾脏等脏器组织及胎盘中检测到犬新孢子虫Nc5基因;随攻虫天数的增加,模型小鼠胎盘重量和胎鼠重量均不断增加,胎盘系数逐步降低,在第12、14、16天时,模型组与对照组相比,胎盘重量和胎盘系数均差异显著（P < 0.05）。本试验成功建立了牛源犬新孢子虫孕鼠感染模型,为犬新孢子虫致病机制研究奠定了基础。     

牛新孢子虫NcSAG1基因工程亚单位疫苗的免疫试验

为了解新孢子虫NcSAG1表面蛋白基因工程亚单位疫苗对实验动物的免疫效果,本试验提取延边黄牛新孢子虫野毒株基因组DNA,用PCR技术扩增新孢子虫NcSAG1表面蛋白基因,并在大肠杆菌中进行原核表达,将Western-blot鉴定具有免疫活性的重组蛋白与弗氏佐剂混合制备NcSAG1基因工程亚单位疫苗,免疫BALB/c小鼠,应用间接ELISA方法测定体液免疫水平,应用流式细胞技术测定细胞免疫水平,以此评价疫苗对实验动物的免疫应答反应。结果显示,制备的NcSAG1基因工程亚单位疫苗免疫BALB/c小鼠后,在三免后第3天时,检测抗体的OD450nm值达0.688;CD4+/CD8+值达3.650,均显著高于重组蛋白免疫组和PBS对照组,说明制备的基因工程亚单位疫苗能够提高实验动物的体液免疫和细胞免疫水平。本试验为牛新孢子虫NcSAG1表面蛋白基因工程亚单位疫苗的深入研究奠定了基础。     

Experimental Infection of Calves with Bovine Viral Diarrhoea Virus Type-2 (BVDV-2) Isolated from a Contaminated Vaccine

A non-cytopathic strain of BVDV-2 was isolated from a batch of live infectious bovine rhinotracheitis (IBR) vaccine, and inoculated intranasally into four 3-month-old calves. Severe signs of disease developed by days 4 and 6 in three of the calves, free of BVDV and antibodies to BVDV, that had been exposed to the virus. These calves survived the acute phase of the infection and progressively recovered. BVDV was consistently isolated, or the respective viral RNA was detected, in the buffy coats from blood samples collected starting from days 2 or 4 up to days 11 or 14 after the experimental infection. Viral RNA was also detected in sera from these infected calves until the presence in the serum of virus neutralizing antibodies was demonstrated. By contrast, the only calf having pre-existing neutralizing antibodies to BVDV at the start of the study was protected from the disease. No virus was detected at any time after experimental inoculation of this calf. Genomic characterization of the BVDV-2 isolated in cell cultures, or detected in sera from the experimentally infected animals, revealed 100% homology in the nucleotide sequence with the BVDV-2 detected as a contaminant of the live IBR virus vaccine. These findings provided evidence of the infective nature of the contaminant BVDV-2 and of its potential to generate disease outbreaks when inoculated into susceptible animals.