Shrestha Aruna, Freudenschuss Barbara, Schwarz Lukas, Joachim Anja
Institute of Parasitology, Department of Pathobiology, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210, Vienna, Austria.
University Clinic for Swine, Department of Farm Animals and Veterinary Public Health, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210, Vienna, Austria.
Vet Parasitol. 2018 Jul 15;258:57-63. doi: 10.1016/j.vetpar.2018.06.008. Epub 2018 Jun 8.
The apicomplexan parasite Cystoisospora suis which causes neonatal porcine coccidiosis is one of the predominant parasite in suckling piglets. Currently, the immunofluorescence antibody test (IFAT) is the only available serological tool for detecting serum antibodies against C. suis which has several limitations, including bias from interpretation and low throughput. In the present study, an indirect enzyme-linked immunosorbent assay (ELISA) was developed using a previously characterized recombinant merozoite protein for the detection of specific immunoglobulin G (IgG) and IgA against C. suis. The recombinant protein was expressed in Escherichia coli as a N-terminal histidine fusion protein, and its specificity was confirmed in an immunoblot probed with highly positive anti-C. suis sera from experimentally infected piglets. Optimal dilutions of recombinant protein, sera and conjugate were determined by checkerboard titrations, and the serum dilution that gave the greatest ratio between the positive and the negative sera was selected for subsequent analyses. Agreement between the IFAT and the newly developed ELISA was assessed with kappa statistics. The receiver operating characteristic (ROC) curve analysis based on 185 serum samples with known C. suis exposure previously tested in the reference IFAT was used to determine the cut-off value, sensitivity and specificity of the ELISA. For IgG, the ELISA had an estimated cut-off value of 0.82 and sensitivity and specificity values of 94.7% and 98%, respectively, whereas for IgA the estimated cut-off value was 0.41 and sensitivity and specificity values were both100%. According to kappa coefficient, an excellent correlation (κ > 0.8) was found between IFAT and ELISA for both isotypes. The diagnostic accuracy of the test measured as the area under the ROC curve index scaled between 0.98 and 1.0, indicating high discriminatory capacity and its possible application as a serological tool for detecting antibody response in the host following C. suis exposure/immunization and large-scale surveillance studies.
引起新生仔猪球虫病的顶复门寄生虫猪等孢球虫是哺乳仔猪的主要寄生虫之一。目前，免疫荧光抗体试验（IFAT）是检测针对猪等孢球虫血清抗体的唯一可用血清学工具，但它有几个局限性，包括判读偏差和通量低。在本研究中，开发了一种间接酶联免疫吸附测定（ELISA），使用先前鉴定的重组裂殖子蛋白来检测针对猪等孢球虫的特异性免疫球蛋白G（IgG）和IgA。重组蛋白在大肠杆菌中作为N端组氨酸融合蛋白表达，其特异性在使用来自实验感染仔猪的高度阳性抗猪等孢球虫血清进行的免疫印迹中得到证实。通过棋盘滴定法确定重组蛋白、血清和结合物的最佳稀释度，并选择在阳性和阴性血清之间给出最大比例的血清稀释度用于后续分析。用kappa统计评估IFAT和新开发的ELISA之间的一致性。基于185份先前在参考IFAT中检测过的已知猪等孢球虫暴露情况的血清样本进行的受试者工作特征（ROC）曲线分析，用于确定ELISA的临界值、敏感性和特异性。对于IgG，ELISA的估计临界值为0.82，敏感性和特异性值分别为94.7%和98%，而对于IgA，估计临界值为0.41，敏感性和特异性值均为100%。根据kappa系数，发现IFAT和ELISA在两种同种型之间都有极好的相关性（κ>0.8）。以ROC曲线指数下面积衡量的试验诊断准确性在0.98至1.0之间，表明具有高鉴别能力，并且其可能作为一种血清学工具用于检测宿主在猪等孢球虫暴露/免疫后抗体反应以及大规模监测研究。
