Expression Analysis and Serodiagnostic Potential of Microneme Proteins 1 and 3 in Eimeria stiedai
Wenrui Wei, Nengxing Shen, Jie Xiao, Yuanyuan Tao, Yuejun Luo, Christiana Angel,Xiaobin Gu, Yue Xie, Ran He, Bo Jing, Xuerong Peng and Guangyou Yang
Abstract
Eimeria stiedai 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 (EsMIC1) and microneme protein 3 (EsMIC3) fromE. stiedai and used them as recombinant antigens to develop a serodiagnostic method for an effective diagnosis of hepatic coccidiosis. The cDNAs encoding EsMIC1 andEsMIC3 were cloned and the mRNA expression levels of these two genes at different developmental stages ofE. stiedaiwere determined by quantitative real-time PCR analysis (qRTPCR). The immunoreactivity of recombinantEsMIC1(rEsMIC1) andEsMIC3(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 ofEsMIC1(711 bp) andEsMIC3 (891 bp) were approximately 25.89 and 32.39 kDa in predicted molecular weight, respectively. Both EsMIC1 andEsMIC3 showed the highest mRNA expression levels in the merozoites stage ofE.stiedai. Western blotting analysis revealed that both recombinant proteins were recognized byE.stiedaipositive sera, and the indirect ELISAs usingrEsMIC1andrEsMIC3were developed based on their good immunoreactivity, with 100% (48/48) sensitivity and 97.9% (47/48) specificity forrEsMIC1with 100% (48/48) sensitivity and 100% (48/48) specificity forrEsMIC3, respectively. Moreover,rEsMIC1- andrEsMIC3-based indirect ELISA were able to detect corresponding antibodies in sera at days 6, 8, and 10 postE. stiedaiinfection, with the highest positive diagnostic rate (62.5% (30/48) forrEsMIC1and 66.7% (32/48) forrEsMIC3) observed at day 10 post infection. Therefore, both EsMIC1andEsMIC3can be used as potential serodiagnostic candidate antigens for hepatic coccidiosis caused byE. stiedai.
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Genes2020,11, 725; doi:10.3390/genes11070725
Read Full Text: https://www.mdpi.com/2073-4425/11/7/725
