Eimeria magna is a common coccidia in the intestines of rabbits, causing anorexia, weight loss, diarrhea, and bloody stools. This study cloned and determined the expression levels of four Eimeria surface antigens (EmSAGs) at different developmental stages and showed that EmSAG10 and EmSAG11 are highly expressed at the merozoite stage. Rabbits were immunized with rEmSAG10 and rEmSAG11, and then challenged with E. magna after 2 weeks. Serum-specific antibodies and cytokine levels were detected using ELISA. Immune protection was evaluated based on the rate of the oocysts decrease, the output of oocysts (p < 0.05), the average weight gain, and the feed: meat ratio. Our results showed that rabbits immunized with rEmSAG10 and rEmSAG11 had a higher average weight gain (62.7%, 61.1%), feed; meat ratio (3.8:1, 4.5:1), and the oocysts decrease rate (70.8%, 81.2%) than those in the control group, and also significantly reduced intestinal lesions. The specific IgG level increased one week after the first rEmSAG10 and rEmSAG11 immunization and was maintained until two weeks after the challenge (p < 0.05). The TGF-β, IL-4, and IL-10 levels in the serum increased significantly after the secondary immunization with rEmSAG10 and rEmSAG11, while the IL-2 levels increased significantly after the secondary immunization with rEmSAG11 (both p < 0.05), suggesting that rEmSAG10 can induce a humoral and cellular immunity, while rEmSAG11 can only induce a humoral immunity. Therefore, rEmSAG10 is a candidate antigen for E. magna recombinant subunit vaccines.

Background Eimeria stiedae parasitizes the bile duct, causing hepatic coccidiosis in rabbits. Coccidiosis control using anticoccidials led to drug resistance and residues; therefore, vaccines are required as an alternative control strategy. Apical membrane antigen 1 (AMA1) and immune mapped protein 1 (IMP1) are surface-located proteins that might contribute to host cell invasion, having potential as candidate vaccine antigens. Methods Herein, we cloned and expressed the E. stiedae EsAMA1 and EsIMP1 genes. The reactogenicity of recombinant AMA1 (rEsAMA1) and IMP1 (rEsIMP1) proteins were investigated using immunoblotting. For the vaccination-infection trial, rabbits were vaccinated with rEsAMA1 and rEsIMP1 (both 100 μg/rabbit) twice at 2-week intervals. After vaccination, various serum cytokines were measured. The protective effects of rEsAMA1 and rEsIMP1 against E. stiedae infection were assessed using several indicators. Sera were collected weekly to detect the specific antibody levels. Results Both rEsAMA1 and rEsIMP1 showed strong reactogenicity. Rabbits vaccinated with rEsAMA1 and rEsIMP1 displayed significantly increased serum IL-2 (F(4, 25) = 9.53, P = 0.000), IL-4 (F(4, 25) = 7.81, P = 0.000), IL-17 (F(4, 25) = 8.55, P = 0.000), and IFN-γ (F(4, 25) = 6.89, P = 0.001) levels; in the rEsIMP1 group, serum TGF-β1 level was also elevated (F(4, 25) = 3.01, P = 0.037). After vaccination, the specific antibody levels increased and were maintained at a high level. The vaccination-infection trial showed that compared with the positive control groups, rabbits vaccinated with the recombinant proteins showed significantly reduced oocyst output (F(5, 54) = 187.87, P = 0.000), liver index (F(5, 54) = 37.52, P = 0.000), and feed conversion ratio; body weight gain was significantly improved (F(5, 54) = 28.82, P = 0.000). Conclusions rEsAMA1 and rEsIMP1 could induce cellular and humoral immunity, protecting against E. stiedae infection. Thus, rEsAMA1 and rEsIMP1 are potential vaccine candidates against E. stiedae. Graphic abstract

Eimeria magna is a common pathogen in rabbits, which results in lethargy, weight loss, diarrhea, and even death in severe cases after infection. The current method for preventing rabbit coccidiosis is to add anticoccidial drugs to the diet. However, there are many concerns about drug resistance and drug residues. In our study, the rEmMIC2 and rEmMIC3 proteins were cloned and expressed to evaluate potential as recombinant subunit vaccine candidate antigens. The protective effects of rEmMIC2 and rEmMIC3 were evaluated by the relative weight gain ratio, oocyst decrease rate, anticoccidial index, feed conversion ratio, pathological alterations, clinical symptoms, specific IgG antibody, and cytokine levels in rabbits. The molecular weights of rEmMIC2 and rEmMIC3 were 18.69 kDa and 17.47 kDa, respectively. After the coccidia challenge, the control groups showed anorexia and soft poop, whereas the experimental group showed few anorexia symptoms. Significantly different from the control group, the relative weight gain ratios of the immunized rEmMIC2 and rEmMIC3 groups were 78.37% and 75.29%, respectively, and the oocyst reduction was 77.95% and 76.09%, respectively, and the anticoccidial index was 171.12 and 169.29, respectively. IgG antibody, IFN-γ, IL-4, IL-10, and IL-17 levels were significantly increased in the experimental group. The results showed that rEmMIC2 and rEmMIC3 have potential as vaccine candidate antigens.