Ybañez Rochelle Haidee D, Terkawi Mohamad Alaa, Kameyama Kyohko, Xuan Xuenan, Nishikawa Yoshifumi
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan.
Clin Vaccine Immunol. 2013 Oct;20(10):1617-22. doi: 10.1128/CVI.00352-13. Epub 2013 Aug 21.
Neospora caninum is an apicomplexan parasite that causes abortion in cattle; hence, accurate diagnosis of this pathogen is important to the cattle farming industry. Our previous proteomics and immunoscreening analyses revealed that the N. caninum subtilisin-like serine protease 1 (NcSUB1) has potential as a serodiagnostic tool for Neospora. Consequently, we expressed two fragments containing five NcSUB1 tandem repeat copies covering amino acids (aa) 524 to 843 (NcSUB1t) and 555 to 679 (NcSUB1tr) to identify the antigenic regions. The serodiagnostic performances of NcSUB1t and NcSUB1tr were compared with that of N54, which contains a single copy of the repeats (aa 649 to 784), and with the truncated NcSAG1 (NcSAG1t), which lacks a signal peptide and C-terminal hydrophobic regions, as a positive reference. Serum samples from N. caninum experimentally infected cattle and mice and cattle from a farm with confirmed cases of Neospora abortion were tested by enzyme-linked immunosorbent assay (ELISA) with the four antigens. In the N. caninum experimentally infected cattle, the highest IgG1 antibody titers were detected against NcSUB1t, while specific IgG1 antibodies were detectable from 16 days postinfection (dpi), with levels peaking at 36 dpi for all of the antigens. On the other hand, the levels of anti-NcSUB1 IgG2 antibodies were lower than those of anti-SAG1t IgG2 antibodies. The ELISA with NcSUB1t and NcSUB1tr had good sensitivity (94.59 to 95.95%) and specificity (80 to 100%) with bovine serum field samples compared to NcSAG1t and showed no cross-reactions with sera from Toxoplasma gondii experimentally infected mice. Moreover, IgG antibodies against NcSUB1t were detected during parturition in the NcSAG1t antibody-positive cattle, and NcSUB1t-specific antibody transfer was observed from a mother to her calf. Our results show that the NcSUB1 tandem repeat is potentially useful for serodiagnosis of N. caninum.
犬新孢子虫是一种顶复门寄生虫，可导致牛流产；因此，准确诊断这种病原体对养牛业至关重要。我们之前的蛋白质组学和免疫筛选分析表明，犬新孢子虫枯草杆菌蛋白酶样丝氨酸蛋白酶1（NcSUB1）有潜力作为犬新孢子虫的血清学诊断工具。因此，我们表达了两个片段，分别包含五个NcSUB1串联重复拷贝，覆盖氨基酸（aa）524至843（NcSUB1t）和555至679（NcSUB1tr），以确定抗原区域。将NcSUB1t和NcSUB1tr的血清学诊断性能与包含单个重复拷贝（aa 649至784）的N54以及作为阳性对照的截短型NcSAG1（NcSAG1t，缺少信号肽和C端疏水区域）进行比较。用这四种抗原通过酶联免疫吸附测定（ELISA）检测来自犬新孢子虫实验感染牛和小鼠以及来自有确诊犬新孢子虫流产病例农场的牛的血清样本。在犬新孢子虫实验感染的牛中，针对NcSUB1t检测到最高的IgG1抗体滴度，而在感染后16天（dpi）可检测到特异性IgG1抗体，所有抗原的抗体水平在36 dpi时达到峰值。另一方面，抗NcSUB1 IgG2抗体水平低于抗SAG1t IgG2抗体水平。与NcSAG1t相比，用NcSUB1t和NcSUB1tr进行的ELISA对牛血清现场样本具有良好的敏感性（94.59%至95.95%）和特异性（80%至100%），并且与刚地弓形虫实验感染小鼠的血清无交叉反应。此外，在NcSAG1t抗体阳性的牛分娩期间检测到抗NcSUB1t的IgG抗体，并且观察到NcSUB1t特异性抗体从母体转移到其犊牛。我们的结果表明，NcSUB1串联重复序列在犬新孢子虫的血清学诊断中可能有用。
