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Currently, although a subunit vaccine (CoxAbic) targeting Eimeria maxima via maternal immunization is commercially available, no genetically engineered multivalent subunit vaccine exists against mixed infections caused by these four Eimeria species simultaneously. Therefore, we developed a tetravalent recombinant subunit vaccine (designated TEIN) by fusing key antigen genes (TA4, 3-1E, IMP1, NA4) from these four Eimeria species and expressing the construct in Pichia pastoris. A total of 500 chickens were randomly allocated into 25 experimental subgroups (n = 20 each), consisting of five groups (control, challenged, adjuvant, pPIC9K, and TEIN) and five challenge conditions (infection with Eimeria tenella, Eimeria acervulina, Eimeria maxima, Eimeria necatrix, or a mixture of four species). Immunization was performed via leg intramuscular injection at 14 and 21 days of age. At 28 days of age, all chickens except the controls were orally challenged with 1 \u00d7 104 sporulated oocysts. Statistical analysis was performed using one-way or two-way ANOVA as appropriate. Results showed that chickens vaccinated with the TEIN subunit vaccine exhibited significantly elevated serum levels of IgY, IL-2, IL-10, and IFN-\u03b3, as well as an increased splenic lymphocyte CD4+\/CD8+ ratio. The anticoccidial indices (ACI) against Eimeria tenella, Eimeria acervulina, Eimeria maxima, and Eimeria necatrix, and their mixed infection reached 174.82, 174.58, 174.41, 180.61, and 175.95, respectively. Moreover, no significant differences were observed in hematological parameters, serum biochemical markers, or histopathological findings between the vaccinated and control groups. These results demonstrate the vaccine\u2019s potential as a promising candidate for controlling mixed coccidial infections.<\/jats:p>","DOI":"10.3390\/ani16071087","type":"journal-article","created":{"date-parts":[[2026,4,1]],"date-time":"2026-04-01T14:59:01Z","timestamp":1775055541000},"page":"1087","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["A Tetravalent Recombinant Subunit Vaccine Provides Protection Against Mixed Challenges with Four Eimeria Species in Chickens"],"prefix":"10.3390","volume":"16","author":[{"given":"Xiao","family":"Ma","sequence":"first","affiliation":[{"name":"State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, College of Veterinary Medicine, Jilin University, Changchun 130062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0219-2424","authenticated-orcid":false,"given":"Xichen","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, College of Veterinary Medicine, Jilin University, Changchun 130062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jianhua","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, College of Veterinary Medicine, Jilin University, Changchun 130062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pengtao","family":"Gong","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, College of Veterinary Medicine, Jilin University, Changchun 130062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaocen","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, College of Veterinary Medicine, Jilin University, Changchun 130062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-1600-6007","authenticated-orcid":false,"given":"Xin","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, College of Veterinary Medicine, Jilin University, Changchun 130062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xu","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, College of Veterinary Medicine, Jilin University, Changchun 130062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tao","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, College of Veterinary Medicine, Jilin University, Changchun 130062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shuqin","family":"Cheng","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, College of Veterinary Medicine, Jilin University, Changchun 130062, China"},{"name":"Department of Agriculture and Animal Husbandry, Handan Vocational College of Science and Technology, Handan 056046, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nan","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, College of Veterinary Medicine, Jilin University, Changchun 130062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,1]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Mesa-Pineda, C., Navarro-Ru\u00edz, J.L., L\u00f3pez-Osorio, S., Chaparro-Guti\u00e9rrez, J.J., and G\u00f3mez-Osorio, L.M. 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