Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Epidemiology, Südufer 10, 17493 Greifswald, Insel Riems, Germany.
Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Epidemiology, Südufer 10, 17493 Greifswald, Insel Riems, Germany.
Int J Parasitol. 2020 May;50(5):389-401. doi: 10.1016/j.ijpara.2019.12.010. Epub 2020 Mar 10.
Serological cross-reactions represent a serious problem in some currently available tests to diagnose Besnoitia infections in many species including cattle, caribou and donkeys. False-positive results are due to the low positive-predictive value of these serological tests for besnoitiosis. These tests therefore have clear limitations if large herds are screened in areas with low prevalence, since increased numbers of false-positive reactions require confirmatory testing by alternative serological methods, e.g. immunoblotting, which are time-consuming and create extra costs. To overcome this problem, we aimed to develop a highly sensitive and specific competitive ELISA (cELISA) using a panel of 12 monoclonal antibodies raised against the tachyzoite stage of Besnoitia besnoiti. A cELISA set up with one of these antibodies (Bb-cELISA1) was screened with a large panel of B. besnoiti-positive bovine sera to estimate the diagnostic sensitivity of the test. Sera from herds with Neospora caninum- or Sarcocystis spp.-infected cattle were used to estimate its diagnostic specificity. Relative to a reference standard, which combined the results obtained in a previously established highly sensitive and specific ELISA, in the immunofluorescence antibody test and in B. besnoiti tachyzoite and bradyzoite immunoblots, the new Bb-cELISA1 revealed a diagnostic sensitivity of 99.2% (95% confidence interval: 97.1-99.9%) and a diagnostic specificity of 99.9% (95% confidence interval: 97.7-100%). This novel assay was tested on a variety of proven Besnoitia-positive sera from other species, including B. besnoiti-infected cats, rabbits or Besnoitia bennetti-infected donkeys or Besnoitia tarandi-infected caribou. The results obtained with the new Besnoitia-cELISA for these animal species also corresponded almost perfectly with those of the reference tests, which included immunoblot and immunofluorescence antibody tests. In conclusion, the novel Besnoitia-cELISA represents a valuable tool for the diagnosis and control of bovine besnoitiosis and for studies on the epidemiology of Besnoitia infections in a variety of host species, including naturally exposed wildlife and experimental hosts.
血清学交叉反应是目前用于诊断包括牛、驯鹿和驴在内的许多物种贝氏巴贝斯虫病的一些检测方法中存在的严重问题。这些血清学检测方法对贝氏巴贝斯虫病的阳性预测值较低，因此导致假阳性结果。如果在流行率较低的地区对大量畜群进行筛查，这些检测方法就存在明显的局限性，因为假阳性反应的数量增加需要通过替代的血清学方法，如免疫印迹进行确认性检测，这既耗时又增加成本。为了解决这个问题，我们旨在使用针对贝氏巴贝斯虫速殖子阶段的 12 种单克隆抗体开发一种高度敏感和特异的竞争 ELISA（cELISA）。使用其中一种抗体（Bb-cELISA1）建立的 cELISA 用大量的贝氏巴贝斯虫阳性牛血清进行筛选，以评估该检测方法的诊断敏感性。使用感染有新孢子虫或肉孢子虫属的牛的畜群血清来估计其诊断特异性。与先前建立的高灵敏度和特异性 ELISA、免疫荧光抗体试验以及贝氏巴贝斯虫速殖子和缓殖子免疫印迹相结合的参考标准相比，新的 Bb-cELISA1 显示出 99.2%（95%置信区间：97.1-99.9%）的诊断敏感性和 99.9%（95%置信区间：97.7-100%）的诊断特异性。该新的检测方法已在其他物种的各种已证实的贝氏巴贝斯虫阳性血清中进行了测试，包括感染贝氏巴贝斯虫的猫、兔子或感染贝氏贝内特虫的驴或感染贝氏田鼠的驯鹿。用新的贝氏巴贝斯虫 cELISA 对这些动物物种进行的检测结果与包括免疫印迹和免疫荧光抗体检测在内的参考检测方法几乎完全一致。总之，新型贝氏巴贝斯虫 cELISA 为牛贝氏巴贝斯虫病的诊断和控制以及在包括自然暴露的野生动物和实验宿主在内的多种宿主物种中的贝氏巴贝斯虫感染的流行病学研究提供了一种有价值的工具。
