Cloning and expression of a cDNA coding for Eimeria acervulina 25 kDa protein associated with oocyst and sporocyst walls

The purpose of this study was to characterize a gene named EAH 00033530 identified by RNAseq analysis of sporulating Eimeria acervulina oocysts and its encoded protein. Quantitative RT-PCR analysis revealed peak expression of EAH 00033530 mRNA early (3-6 h) in sporulation followed by downregulation at 12-24 h. The gene for EAH 00033530 was expressed in Escherichia coli as a 70 kDa polyHis fusion protein (rEAH 00033530). Antisera prepared against rEAH 00033530 protein identified in immunoblotting a native 25 kDa E. acervulina protein (Ea25) that was present in oocyst-sporocyst extracts after treatment with the reducing agent DTT. Immunofluorescence staining using anti-rEa25 localized the protein to both E. acervulina oocyst and sporocyst walls, but not to sporozoites. The protein may be produced during in vivo oocyst development because immunostaining of duodenal tissue from E. acervulina-infected chickens revealed oocyst wall expression. As observed by ELISA, rEa25 protein appears to elicit a humoral immune response in chickens infected with non-irradiated or radiation-attenuated E. acervulina oocysts.