Background: Eimeria necatrix , a member of the Apicomplexa phylum, is one of the most pathogenic parasites, causing high mortality in chickens. 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 E. necatrix MICs remain limited. Methods: Eimeria necatrix MIC3 gene (En MIC3 ) was amplified and expressed in Escherichia coli . 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 E. necatrix 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. Results: The open reading frame of En MIC3 was 798 bp, encoding a 265-amino acid protein with a predicted molecular weight of 28.50 kDa. EnMIC3 contained a signal peptide and a single epidermal growth factor (EGF)-like domain. The rEnMIC3 with an approximate molecular weight of 36 kDa could be specifically recognized by convalescent sera from chickens infected with E. necatrix . The molecular mass of the native protein was approximately 35 kDa, 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 E. necatrix challenge, with the high-dose group (200 µg) achieving the highest ACI value (171.32) and markedly elevated serum antibody levels. Conclusions: These findings not only offer a foundation for understanding the role of EnMIC3 protein in the host invasion of E. necatrix but also present a potential protective antigen of E. necatrix for the development of a subunit vaccine against avian coccidiosis.

Limosilactobacillus reuteri is one of the common lactic acid bacteria in the chicken gastrointestinal tract. Our previous study revealed that L. reuteri abundance declined in White Leghorn chickens following infection with Eimeria tenella, E. maxima, and E. necatrix, suggesting a potential role of this species in gut homeostasis during coccidiosis. In this study, eight L. reuteri strains were isolated from the intestinal tract of White Leghorn chickens. Strain S3 was selected as the most promising candidate after screening for acid and bile tolerance, adhesion ability, antagonism against pathogens. Oral administration of S3 strain at dose of 4 × 10⁷ CFU/bird, 1.2 × 10⁸ CFU/bird or 2 × 10⁸ CFU/bird for 18 days improved average daily gain (ADG) in both White Leghorn chickens and Suqin laying hens. The ADG value in groups treated with 4 × 10⁷ CFU/bird was significantly higher compared to that in the control group (p < 0.05). This treatment did not cause adverse effects on hepatic or renal function, but significantly increased the thymus index in both breeds and the levels of total protein and globulin in Suqin laying hens (p < 0.05). S3 strain also modulated gut microbiota composition in White Leghorn chickens, promoting beneficial lactobacilli colonization in the jejunum and achieving persistent colonization in the cecum. S3 strain selectively increased the proportion of CD3⁺CD4⁺ T lymphocytes in peripheral blood of Suqin laying hens. The anticoccidial efficacy of S3 strain was evaluated in Suqin laying hens challenged with E. tenella, E. maxima, and E. necatrix. The results showed that S3 strain alleviated clinical signs, reduced lesion scores, decreased oocyst output, and attenuated body weight loss. Anticoccidial index (ACI) values were 130.67, 148.06, and 163.40 for E. tenella, E. maxima, and E. necatrix infection, respectively. These results suggest that L. reuteri S3 is a safe chicken-derived probiotic with immunomodulatory and anticoccidial properties, and may serve as a feed additive for coccidiosis control in poultry production.