Abstract

Simple SummaryChicken coccidiosis is a widespread parasitic disease that causes major economic losses in poultry farming. In the field, chickens are often infected by several types ofEimeriaparasites at the same time, but currently available commercial vaccines are mainly live oocyst-based products, which carry potential safety risks including reversion to virulence and vaccine strain transmission. To tackle this problem, we developed a subunit vaccine called TEIN, which combines key proteins from four commonEimeriaspecies. Using molecular tools, recombinant proteins were produced, and the vaccine efficacy was tested under controlled experimental conditions. Vaccination significantly elevated serum levels of cytokines and increased the splenic lymphocyte CD4+/CD8+ratio. In homologous challenge experiments using each of the fourEimeriaspecies individually or in combination, vaccinated chickens showed better weight gain, lower parasite counts, and fewer intestinal lesions, demonstrating that the vaccine conferred strong protection. These results demonstrate that the TEIN vaccine provides effective protection against both single and mixedEimeriainfections under laboratory conditions, but its efficacy under field conditions requires further investigation.Chicken coccidiosis is a parasitic disease caused mainly byEimeria tenella,Eimeria acervulina,Eimeria maxima, andEimeria necatrix, with most cases presenting as mixed infections. Currently, although a subunit vaccine (CoxAbic) targetingEimeria maximavia maternal immunization is commercially available, no genetically engineered multivalent subunit vaccine exists against mixed infections caused by these fourEimeriaspecies simultaneously. Therefore, we developed a tetravalent recombinant subunit vaccine (designated TEIN) by fusing key antigen genes (TA4, 3-1E, IMP1, NA4) from these fourEimeriaspecies and expressing the construct inPichia pastoris. A total of 500 chickens were randomly allocated into 25 experimental subgro