Animal Parasitic Diseases Laboratory, United States Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705, USA.
Vet Parasitol. 2012 Jun 8;187(1-2):28-35. doi: 10.1016/j.vetpar.2012.01.003. Epub 2012 Jan 8.
Improvements in the serological diagnosis of neosporosis are needed to differentiate acute versus chronic Neospora caninum infections. In the present study, N. caninum microneme protein 10 (NcMIC10), similar to other microneme proteins, was shown to be released in a calcium-dependent manner. NcMIC10 may be discharged during active invasion of host cells by the parasite, and thus represent an excellent marker for the diagnosis of neosporosis. In order to test this hypothesis, recombinant NcMIC10 (rNcMIC10) was expressed in Escherichia coli, and polyclonal antibodies were generated against non-overlapping fragments of the protein. A capture ELISA was developed using these antibodies, and was found to be highly accurate and reproducible with a detection range of 10-10,000 pg/ml. The anti-rNcMIC10 antibodies used in this study did not cross-react with the Toxoplasma gondii antigens. NcMIC10 was detected by the ELISA in sera of 9 out of 10 goats (90%) experimentally infected with N. caninum tachyzoites. In general, goats infected with a lower dose (10(4)) of the parasite displayed a peak in NcMIC10 levels between weeks 4 and 5 post infection. Goats infected with a higher parasite dose (10(6)) displayed a more rapid increase in NcMIC10 levels. In most animals, NcMIC10 decreased to undetectable levels by week 6 post infection. This is the first circulating Neospora antigen-based assay which may complement the existing antibody-based assays for a rapid and cost-effective definitive diagnosis of neosporosis in livestock.
需要改进弓形虫病的血清学诊断方法，以区分急性和慢性刚地弓形虫感染。本研究表明，刚地弓形虫微线蛋白 10（NcMIC10）与其他微线蛋白相似，以钙依赖的方式释放。NcMIC10 可能在寄生虫主动入侵宿主细胞时被排出，因此是弓形虫病诊断的一个极好的标志物。为了验证这一假设，在大肠杆菌中表达了重组 NcMIC10（rNcMIC10），并针对该蛋白的非重叠片段产生了多克隆抗体。使用这些抗体开发了捕获 ELISA，发现其具有高度的准确性和可重复性，检测范围为 10-10,000 pg/ml。本研究中使用的抗 rNcMIC10 抗体与刚地弓形虫抗原没有交叉反应。ELISA 检测到 10 只（90%）用刚地弓形虫速殖子实验感染的山羊血清中有 9 只（90%）检测到 NcMIC10。一般来说，感染较低剂量（10(4)）寄生虫的山羊在感染后 4 至 5 周时 NcMIC10 水平达到峰值。感染较高剂量（10(6)）寄生虫的山羊 NcMIC10 水平升高更快。在大多数动物中，NcMIC10 在感染后第 6 周下降到无法检测到的水平。这是第一个基于循环刚地弓形虫抗原的检测方法，可能会补充现有的基于抗体的检测方法，用于快速和具有成本效益的家畜弓形虫病的明确诊断。
