吉林株犬新孢子虫SAG1基因片段的克隆及蛋白抗原性预测

[目的]对犬新孢子虫SAG1基因进行克隆与序列分析。[方法]从细胞培养的吉林株虫体中提取犬新孢子虫DNA,根据已发表的犬新孢子虫鲥G1基因的核苷酸序列,设计并合成1对特异性引物,采用PCR技术,扩增大新孢子虫SAG1基因片段,并成功克隆到pMD18-T simple载体上,对经PCR、酶切鉴定为阳性的重组质粒进行序列分析。[结果]对犬新孢子虫SAG1基因进行克隆后,获得阳性重组质粒。克隆的SAG1基因片段长780bp,编码260个氨基酸,与已发表的美国株犬新孢子虫SAG1基因核苷酸序列（AF132217）的同源性为99．0％,氨基酸序列同源性为98．8％。通过分子生物学软件对翻译水平进行预测,发现吉林株核苷酸序列并不影响氨基酸翻译与蛋白质的表达。[结论]该试验成功克隆重组质粒,为建立犬新孢子虫的高效表达系统奠定了基础。     

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MAG1基因的克隆及在Vero细胞中表达

为了解牛源犬新孢子虫MAG1基因的生物学特性,本实验应用PCR技术扩增牛源犬新孢子虫MAG1基因,构建真核表达重组质粒pVAX-MAG1,将鉴定正确的pVAX-MAG1重组质粒转染Vero细胞,应用间接荧光检测方法(1FA)和western blot技术检测MAG1基因在Vero细胞中的表达.结果显示,扩增的牛源犬新孢子虫MAG1基因长度为1047bp,与GenBank中登录的MAG1 (EF580924.1)核苷酸序列同源性为99％,IFA检测MAG1基因在Vero细胞中获得瞬时表达,western blot分析表达蛋白的分子量为39 ku,具有较好的反应原性.本实验为新孢子虫病核酸疫苗与诊断试剂盒的研究奠定了基础.     

Isolation and characterization of a bovine isolate of Neospora caninum with low virulence

Neospora caninum tachyzoites were isolated from the brain of an asymptomatic naturally infected calf with precolostral-specific antibodies. The new isolate, named Nc-Spain 1H, was identified as a member of the N. caninum species based on its internal transcribed spacer 1 (ITS-1) sequence and was genetically characterized using microsatellite markers. Multilocus analysis showed that Nc-Spain 1H was genetically different from other N. caninum isolates. We compared the in vitro tachyzoite yield and viability rate of the Nc-Spain 1H and Nc-1 isolates in a plaque assay. The lower tachyzoite yields displayed by Nc-Spain 1H were complemented with a significantly lower viability rate. Moreover, in an in vitro tachyzoite–bradyzoite stage conversion assay, the percentage of Nc-Spain 1H bradyzoite conversion was similar to that of the cystogenic isolate Nc-Liv, with the exception that Nc-Spain 1H produced only intermediate bradyzoites. The pathogenicity of Nc-Spain 1H was examined in BALB/c mice, and the results demonstrated that Nc-Spain 1H failed to induce clinical signs or mortality and that no parasite DNA was detected in the brain during the chronic stage of infection. In a pregnant mouse model, Nc-1 infection resulted in high transplacental transmission, leading to a high neonatal mortality rate over time. In contrast, the offspring survival rate from Nc-Spain 1H-infected dams was almost 100%, and N. caninum DNA was detected in only one pup. These data show that Nc-Spain 1H appears to be a low virulence isolate and may be a suitable candidate for live vaccine development.     

Seroprevalence of <Emphasis Type="Italic">Neospora caninum</Emphasis> antibodies in beef cattle in three southern states of Mexico

The aims of the present study were to examine the seroprevalence of neosporosis in beef herds from three southern states of Mexico and determine the association with several risk factors. A cross-sectional serological survey for Neospora caninum was carried out by sampling a total of 596 animals from 31 herds in Chiapas, Veracruz, and Yucatan States and tested using an ELISA assay (IDEXX) to detect anti-N. caninum antibodies. The overall prevalence was 11.6%, (95% CI: 0.93–0.14), however, the prevalence for Chiapas was 15% (30/200) (95% CI: 0.11–0.21), in Yucatan 11.3% (21/186) (95% CI: 0.07–0.17) and in Veracruz 8.6% (18/210) (95% CI: 0.05–0.13). Of the 596 serum samples taken, 578 were from females and 18 were bulls., Only one bull sample was found seropositive from one herd in Veracruz State. The overall herd-prevalence was 70.9% (22/31) (95% CI: 0.53–0.84), and by State was: Chiapas 90% (9/10) (95% CI: 0.60–0.98); Yucatan 72.7% (8/12) (95% CI: 0.39–0.86); and Veracruz 50% (5/10) (95% CI: 0.24–0.76), respectively. The highest age-prevalences were found in animals 1 yr old (19.4% [95% CI: 0.01–0.35]) and 4 yrs old (19.6% [95% CI: 0.12–0.29]), and the lowest in animals 3 –yrs of age (6.2%,[95% CI: 0.02–0.20]). No association was found among all the variables tested in this study since most of the ranches have similar management conditions. In conclusion, N. caninum infection is common among beef herds in the Mexican tropics.     

牛新孢子虫内标双重荧光PCR检测方法的建立

为建立牛新孢子虫的快速准确检测方法,根据犬新孢子虫种属特异性基因Nc-5序列,设计高度保守的引物和荧光探针,通过引物设计和搭桥PCR法扩增,获得Nc-5荧光PCR内标模板。对内标模板的添加量和反应条件进行优化,建立了牛新孢子虫内标双重荧光PCR检测体系。该方法具有较好的特异性;可以检测到10个拷贝/PCR反应的核酸分子,与不加内标的荧光PCR检测灵敏度相当;通过对系列稀释的核酸样品的重复性检测,变异系数为0.50%～1.30%。通过对58份临床样品分别用该方法、不含内标的荧光PCR方法和普通PCR方法检测,结果显示,该方法与不含内标的荧光PCR方法的阳性检出率均为10.3%,比普通PCR方法阳性检出率（7.0%）高;表明该方法可用于临床样品中牛新孢子虫的快速检测,并能对实验室进行质量控制。     

ELISA法筛选抗新孢子虫中药的研究

[目的]筛选抗新孢子虫效果较好的中药。[方法]取健康昆明种小鼠,投服甲基氢化泼尼松,使其免疫力下降,然后随机分组,每只小鼠腹腔接种1.0×104个新孢子虫虫体,接种4 h后,各组小鼠灌服不同中药。给药7 d后,小白鼠眼球采血,收集血清,应用ELISA法检测其抗体水平。[结果]15个中药组中有4个中药组抗体阳性率较低。[结论]黄芩、百部、天麻、黄连可以提高感染新孢子虫小白鼠的免疫水平。