Neospora caninum is a protozoan parasite (Phylum Apicomplexa) that has been recently suggested as a relevant cause of reproductive disorders in small ruminants. The aim of the present study is to develop and validate a new serological test based on time resolved fluorescency using N. caninum GRA7 recombinant antigen (GRA7-TRFIA) for the detection of N. caninum antibodies in sheep. A total of 346 serum samples (208 from experimentally infected sheep, 117 from a dairy farm with a previous history of Neospora-associated abortion, and 21 negative sera) were used. The validation of the new assay was performed by the evaluation of assay precision, analytical sensitivity (Se), accuracy and cross reactivity. In the experimentally infected sheep, antibody kinetics was compared between GRA7-TRFIA and an in house N. caninum tachyzoite soluble extract-based ELISA (NcSALUVET ELISA) by Wilcoxon matched-pairs signed rank test. The cut-off and diagnostic Se and specificity (Sp) of GRA7-TRFIA was estimated by ROC analysis with field samples. In addition, concordance and correlation between GRA7-TRFIA and a commercial ELISA and NcSALUVET ELISA were assessed by kappa value and Spearman correlation coefficient, respectively. Overall, GRA7-TRFIA showed an adequate precision, analytical Se and accuracy to detect anti-N. caninum antibodies in ovine serum, and no cross reactivity with the closely related protozoan Toxoplasma gondii. In naturally infected sheep, 100% Se and 95.35% Sp were obtained for a cut-off point of 62.68 Units of Fluorometry for N. caninum (UFN). Moreover, GRA7-TRFIA allowed earlier detection of N. caninum infection than NcSALUVET ELISA in experimentally infected sheep.

中文翻译：

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时间分辨荧光免疫测定法，用于检测绵羊抗新孢子虫抗体。

犬新孢子虫是一种原生动物寄生虫（Phylum Apicomplexa），最近被认为是小反刍动物繁殖障碍的相关原因。本研究的目的是开发和验证一种基于时间分辨荧光的新血清学测试，该测试使用犬新孢子虫GRA7重组抗原（GRA7-TRFIA）检测绵羊中的犬新孢子虫抗体。总共使用了346个血清样本（208个来自实验感染绵羊的血清，117个具有新孢子虫相关流产史的奶牛场和21个阴性血清）。通过评估分析精度，分析灵敏度（Se），准确性和交叉反应性来进行新分析的验证。在实验感染的绵羊中，比较了GRA7-TRFIA和内部N的抗体动力学。犬速殖子可溶性提取物为基础的酶联免疫吸附试验（NcSALUVET ELISA），采用Wilcoxon配对配对有符号秩检验。GRA7-TRFIA的临界值和诊断性Se和特异性（Sp）通过现场样品的ROC分析进行估算。另外，分别通过κ值和Spearman相关系数评估了GRA7-TRFIA与商业ELISA和NcSALUVET ELISA之间的一致性和相关性。总体而言，GRA7-TRFIA显示出足够的精密度，分析硒和检测抗N的准确度。羊血清中的犬抗抗体，与紧密相关的原生动物弓形虫无交叉反应。在天然感染的绵羊中，获得了100％Se和95.35％Sp的截止点，用于犬新孢子虫（UFN）的荧光法的临界值为62.68单位。此外，GRA7-TRFIA可以更早地检测到N。