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Microneme proteins (MICs) play essential roles in host cell recognition and invasion by apicomplexan parasites and are also attractive candidates for vaccine development. However, comprehensive studies on\n                      <jats:italic>E. necatrix<\/jats:italic>\n                      MICs remain limited.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      <jats:italic>Eimeria necatrix MIC3<\/jats:italic>\n                      gene (En\n                      <jats:italic>MIC3<\/jats:italic>\n                      ) was amplified and expressed in\n                      <jats:italic>Escherichia coli<\/jats:italic>\n                      . The recombinant protein (rEnMIC3) was characterized via SDS-PAGE and Western blot. The antigenicity of rEnMIC3 and its localization in sporozoites (SZ) and second-generation merozoites (MZ-2) of\n                      <jats:italic>E. necatrix<\/jats:italic>\n                      were determined by Western blot and indirect immunofluorescence analyses (IFAs). The dynamic expression of EnMIC3 across different developmental stages and its impact on sporozoite invasion of host cells were analyzed. The immune protection provided by rEnMIC3 was evaluated in chickens using weight gain, lesion scores, oocyst production, anticoccidial index (ACI), and antibody levels.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      The open reading frame of En\n                      <jats:italic>MIC3<\/jats:italic>\n                      was 798\u00a0bp, encoding a 265-amino acid protein with a predicted molecular weight of 28.50\u00a0kDa. EnMIC3 contained a signal peptide and a single epidermal growth factor (EGF)-like domain. The rEnMIC3 with an approximate molecular weight of 36\u00a0kDa could be specifically recognized by convalescent sera from chickens infected with\n                      <jats:italic>E. necatrix<\/jats:italic>\n                      . The molecular mass of the native protein was approximately 35\u00a0kDa, and it localizes to the apical region in SZ but exhibits a cytoplasmic distribution in MZ-2. EnMIC3 mRNA was expressed at significantly higher levels in SZ than in MZ-2, whereas protein expression displayed an inverse pattern. Anti-rEnMIC3 polyclonal antibodies inhibited sporozoite invasion of DF-1 cells in a dose-dependent manner. Vaccination with rEnMIC3 conferred effective protection against\n                      <jats:italic>E. necatrix<\/jats:italic>\n                      challenge, with the high-dose group (200\u00a0\u00b5g) achieving the highest ACI value (171.32) and markedly elevated serum antibody levels.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>\n                      These findings not only offer a foundation for understanding the role of EnMIC3 protein in the host invasion of\n                      <jats:italic>E. necatrix<\/jats:italic>\n                      but also present a potential protective antigen of\n                      <jats:italic>E. necatrix<\/jats:italic>\n                      for the development of a subunit vaccine against avian coccidiosis.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s13071-026-07418-w","type":"journal-article","created":{"date-parts":[[2026,5,2]],"date-time":"2026-05-02T10:54:22Z","timestamp":1777719262000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Molecular characterization and immunoprotective potential of microneme protein 3 from Eimeria necatrix"],"prefix":"10.1186","volume":"19","author":[{"given":"Qianqian","family":"Feng","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Danli","family":"Yan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nianyu","family":"Xue","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dandan","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weimin","family":"Cai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuxin","family":"Zhou","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhaofeng","family":"Hou","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jinjun","family":"Xu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jianping","family":"Tao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,5,2]]},"reference":[{"key":"7418_CR1","doi-asserted-by":"publisher","DOI":"10.1016\/j.psj.2024.104663","volume":"104","author":"GF Mathis","year":"2025","unstructured":"Mathis GF, Lumpkins B, Cervantes HM, Fitz-Coy SH, Jenkins MC, Jones MK, et al. 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All animal procedures were performed in accordance with the guidelines and regulations of the Animal Experiment Ethics Committee of Yangzhou University, under License No. SYXK (SU) 2022-0044 for mice and SYXK (SU) 2021\u20130027 for chickens. Mice were anaesthetized by carbon dioxide (CO\u2082) inhalation prior to retro-orbital blood collection. CO\u2082 was introduced into the chamber at a gradual displacement rate of 20%\u201330% of the chamber volume per minute, allowing the animals to gradually lose consciousness and ensuring that sampling was conducted in a pain-free state. Chickens were killed via CO\u2082 inhalation using a gradual-fill method, in which the CO\u2082 concentration was steadily increased to 60\u201370% of the chamber volume, ensuring a humane and distress-free induction of unconsciousness.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"Not Applicable.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"The authors declare no competing interests.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"253"}}