A recombinant adeno-associated virus expressing Eimeria stiedai HSP70 proteins exhibited a promising efficacy against rabbit coccidiosis.

Eimeria stiedai is a critical protozoan that colonizes in rabbit hepatocytes and cholangiocytes, causing severe hepatic coccidiosis and substantial economic loss in the rabbit industry. However, there are no effective vaccines against E. stiedai infection. In this study, the recombinant E. stiedai heat shock protein 70 (rEsHSP70) was produced via a prokaryotic expression system, and its polyclonal antibody was prepared. Immunofluorescence analysis demonstrated that EsHSP70 localized predominantly in the cytoplasm and enriched around the nucleus of sporozoites, while in merozoites it was distributed in the cytoplasm, displaying a peripheral and apical distribution pattern. Functional blocking assays demonstrated that anti-EsHSP70 polyclonal antibody significantly inhibited sporozoites invasion in primary rabbit hepatocytes, suggesting a potential role for EsHSP70 in this process. Furthermore, a recombinant adeno-associated virus serotype 9-ZsGreen1 expressing EsHSP70 (rAAV-ZsGreen1-EsHSP70) was successfully packaged and purified. The recombinant virus particles was observed by transmission electron microscopy (TEM) as 20 nm hexagonal structures, with a titer exceeding 1.0 × 1013 vg/mL. The recombinant viruses could infect 293 T cells and express EsHSP70 proteins. Immunization with recombinant rAAV-ZsGreen1-EsHSP70 significantly elevated the levels of specific IgG antibody, IL-2, IL-10, and IFN-γ, with reduced oocyst shedding by 69.8 %, alleviated liver lesion, resulting in an anticoccidial index (ACI) of 169.48. In conclusion, these results indicated that rAAV-ZsGreen1-EsHSP70 conferred a promising protective efficacy against E. stiedai infection, thereby providing valuable insights for the development of rabbit coccidiosis vaccines.