Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, China.
Department of Veterinary Parasitology, Faculty of Veterinary Sciences, Shaheed Benazir Bhutto University of Veterinary and Animal Sciences, Sakrand 67210, Sindh, Pakistan.
Genes (Basel). 2020 Jun 29;11(7):725. doi: 10.3390/genes11070725.
is an apicomplexan protozoan parasite that invades the liver and bile duct epithelial cells in rabbits and causes severe hepatic coccidiosis, resulting in significant economic losses in the domestic rabbit industry. Hepatic coccidiosis lacks the typical clinical symptoms and there is a lack of effective premortem tools to timely diagnose this disease. Therefore, in the present study we cloned and expressed the two microneme proteins i.e., microneme protein 1 () and microneme protein 3 () from and used them as recombinant antigens to develop a serodiagnostic method for an effective diagnosis of hepatic coccidiosis. The cDNAs encoding and were cloned and the mRNA expression levels of these two genes at different developmental stages of were determined by quantitative real-time PCR analysis (qRT-PCR). The immunoreactivity of recombinant EsMIC1 (rEsMIC1) and EsMIC3 (rEsMIC3) proteins were detected by Western blotting, and indirect enzyme-linked immunosorbent assays (ELISAs) based on these two recombinant antigens were established to evaluate their serodiagnostic potential. Our results showed that the proteins encoded by the ORFs of (711 bp) and (891 bp) were approximately 25.89 and 32.39 kDa in predicted molecular weight, respectively. Both and showed the highest mRNA expression levels in the merozoites stage of . Western blotting analysis revealed that both recombinant proteins were recognized by positive sera, and the indirect ELISAs using rEsMIC1 and rEsMIC3 were developed based on their good immunoreactivity, with 100% (48/48) sensitivity and 97.9% (47/48) specificity for rEsMIC1 with 100% (48/48) sensitivity and 100% (48/48) specificity for rEsMIC3, respectively. Moreover, rEsMIC1- and rEsMIC3-based indirect ELISA were able to detect corresponding antibodies in sera at days 6, 8, and 10 post infection, with the highest positive diagnostic rate (62.5% (30/48) for rEsMIC1 and 66.7% (32/48) for rEsMIC3) observed at day 10 post infection. Therefore, both and can be used as potential serodiagnostic candidate antigens for hepatic coccidiosis caused by .
是一种顶复门的原生动物寄生虫，它侵袭兔子的肝脏和胆管上皮细胞，导致严重的肝球虫病，给家兔产业造成重大经济损失。肝球虫病缺乏典型的临床症状，也缺乏有效的生前诊断工具来及时诊断这种疾病。因此，在本研究中，我们从克隆并表达了微线体蛋白 1（）和微线体蛋白 3（），并将它们作为重组抗原用于开发一种有效的肝球虫病血清学诊断方法。克隆了编码和的 cDNA，并通过定量实时 PCR 分析（qRT-PCR）确定了这两个基因在不同发育阶段的 mRNA 表达水平。通过 Western blot 检测了重组 EsMIC1（rEsMIC1）和 EsMIC3（rEsMIC3）蛋白的免疫反应性，并建立了基于这两种重组抗原的间接酶联免疫吸附试验（ELISA）来评估它们的血清学诊断潜力。我们的结果表明，ORF 编码的蛋白（711 bp）和（891 bp）的预测分子量分别约为 25.89 和 32.39 kDa。和在裂殖子阶段的 mRNA 表达水平最高。Western blot 分析显示，两种重组蛋白均被阳性血清识别，基于其良好的免疫反应性，建立了间接 ELISA，rEsMIC1 的敏感性为 100%（48/48），特异性为 97.9%（47/48），rEsMIC3 的敏感性为 100%（48/48），特异性为 100%（48/48）。此外，rEsMIC1 和 rEsMIC3 基于间接 ELISA 能够在感染后第 6、8 和 10 天检测到血清中的相应抗体，感染后第 10 天的阳性诊断率最高（rEsMIC1 为 62.5%（30/48），rEsMIC3 为 66.7%（32/48））。因此，和都可以作为由引起的肝球虫病的潜在血清学诊断候选抗原。
