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      Expression Analysis and Serodiagnostic Potential of Microneme Proteins 1 and 3 in Eimeria stiedai

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          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) from E. stiedai and used them as recombinant antigens to develop a serodiagnostic method for an effective diagnosis of hepatic coccidiosis. The cDNAs encoding EsMIC1 and EsMIC3 were cloned and the mRNA expression levels of these two genes at different developmental stages of E. stiedai 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 EsMIC1 (711 bp) and EsMIC3 (891 bp) were approximately 25.89 and 32.39 kDa in predicted molecular weight, respectively. Both EsMIC1 and EsMIC3 showed the highest mRNA expression levels in the merozoites stage of E. stiedai. Western blotting analysis revealed that both recombinant proteins were recognized by E. stiedai 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 E. stiedai 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 EsMIC1 and EsMIC3 can be used as potential serodiagnostic candidate antigens for hepatic coccidiosis caused by E. stiedai.

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          Most cited references 54

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          Microneme proteins in apicomplexans.

          Microneme secretion supports several key cellular processes including gliding motility, active cell invasion and migration through cells, biological barriers, and tissues. The modular design of microneme proteins enables these molecules to assist each other in folding and passage through the quality control system, accurately target to the micronemes, oligimerizing with other parasite proteins, and engaging a variety of host receptors for migration and cell invasion. Structural and biochemical analyses of MIC domains is providing new perspectives on how adhesion is regulated and the potentially distinct roles MICs might play in long or short range interactions during parasite attachment and entry. New access to complete genome sequences and ongoing advances in genetic manipulation should provide fertile ground for refining current models and defining exciting new roles for MICs in apicomplexan biology.
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            Independent translocation of two micronemal proteins in developing Plasmodium falciparum merozoites.

            Apical membrane antigen 1 of Plasmodium falciparum (PfAMA1) contains an N-terminal propeptide that is removed prior to the translocation of the mature protein onto the merozoite surface. We localized unprocessed PfAMA1 to the microneme organelles of the intraerythrocytic schizont. The results have suggested that the processed form of PfAMA1 translocates from the microneme compartment independently of another microneme protein, EBA175, which is also involved in the invasion of human erythrocytes.
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              Mix and match modules: structure and function of microneme proteins in apicomplexan parasites.

              Microneme organelles are found in the apical complex of all apicomplexan parasites and play an important role in the invasion process. The recent identification of microneme proteins from different apicomplexan genera has revealed a striking conservation of structural domains, some of which show functional complementation across species. This supports the idea that the mechanism of host cell invasion across the phylum is conserved not only morphologically, but also functionally at the molecular level. Here, we review and summarize these recent findings.
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                Author and article information

                Journal
                Genes (Basel)
                Genes (Basel)
                genes
                Genes
                MDPI
                2073-4425
                29 June 2020
                July 2020
                : 11
                : 7
                Affiliations
                [1 ]Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, China; wenruiwei1995@ 123456stu.sicau.edu.cn (W.W.); shennengxing@ 123456stu.sicau.edu.cn (N.S.); xiaojie@ 123456stu.sicau.edu.cn (J.X.); taoyuanyuan@ 123456stu.sicau.edu.cn (Y.T.); zhengyl2020@ 123456stu.sicau.edu.cn (Y.L.); zhengry2019@ 123456stu.sicau.edu.cn (C.A.); 13720@ 123456sicau.edu.cn (X.G.); 14265@ 123456sicau.edu.cn (Y.X.); ranhe1991@ 123456sicau.edu.cn (R.H.); zhangcy2018@ 123456stu.sicau.edu.cn (B.J.)
                [2 ]Department of Veterinary Parasitology, Faculty of Veterinary Sciences, Shaheed Benazir Bhutto University of Veterinary and Animal Sciences, Sakrand 67210, Sindh, Pakistan
                [3 ]Department of Chemistry, College of Life and Basic Science, Sichuan Agricultural University, Wenjiang 611130, China; 10533@ 123456sicau.edu.cn
                Author notes
                [* ]Correspondence: 10345@ 123456sicau.edu.cn
                [†]

                These authors contributed equally to this work.

                Article
                genes-11-00725
                10.3390/genes11070725
                7397282
                32610686
                © 2020 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

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