Chicken coccidiosis is a parasitic disease caused mainly by Eimeria tenella, Eimeria acervulina, Eimeria maxima, and Eimeria necatrix, with most cases presenting as mixed infections. 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 × 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-γ, 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’s potential as a promising candidate for controlling mixed coccidial infections.

The impact of neosporosis on the livestock industry has worsening each year. Globally, annual losses were estimated to exceed $2.4 billion, with abortion rates reaching up to 47 % in affected herds. But no effective vaccines or drugs are currently available for prevention and treatment. This study aimed to investigate the inhibitory effects of emodin on Neospora caninum (N. caninum) infection and its potential mechanisms. The appropriates safe concentration of emodin was determined by the Cell Counting Kit-8 (CCK-8) assay. The anti-N. caninum effects of emodin in Vero cells, MDBK cells, macrophages, as well as the combined effects of emodin and niclosamide in Vero cells, were further verified using qPCR or Giemsa staining. Subsequently, the effects of direct incubation of tachyzoites with emodin on N. caninum invasion and proliferation were also examined. The activation of necroptosis by emodin was evaluated by measuring Lactate Dehydrogenase (LDH) levels in the supernatant and assessing cellular pMLKL levels. The CCK-8 results indicated that emodin exhibited low toxicity to Vero cells, MDBK cells, and macrophages. The qPCR results showed that emodin reduced the number of N. caninum in Vero cells, MDBK cells, and macrophages. Giemsa results showed that the number of tachyzoites within the parasitophorous vacuole was decreased accordingly. Notably, emodin directly affected tachyzoites, reducing its invasion capability by 55 %-63 % and its proliferation capacity by 62 %-88 %. Additionally, the combination of emodin with niclosamide significantly enhanced its antiparasitic effects compared with emodin alone. Western blot analysis showed that emodin significantly increased pMLKL levels and enhanced LDH release, assisting N. caninum in activating necroptosis. However, the inhibitory effects of emodin on N. caninum invasion and proliferation were not significantly altered in Mlkl^-/- macrophages. In conclusion, emodin inhibited N. caninum infection may through a necroptosis-independent mechanism by directly targeting the parasite, highlighting its potential as a therapeutic candidate for neosporosis.