Available online 19 March 2022

Extracellular and intra-erythrocytic Theileria equi merozoites express RAP-1 proteins.

RAP-1 proteins elicit antibody responses in horses during Theileria equi infection.

RAP-1 antibodies correlate with a decline in parasite load during acute and persistent T. equi infection.

The RAP-1a protein is involved in invasion of equine erythrocytes.

N- and C- termini of the RAP-1b protein do not play a role in erythrocyte invasion.

Theileria equi is an obligate intracellular protozoan parasite that causes severe hemolytic anemia in most equid species. Similar to other apicomplexan parasites, T. equi contains rhoptries whose contents have been implicated in host cell invasion and formation of the parasitophorous vacuole that is crucial for survival of the species within cells. Despite their importance, the composition of T. equi rhoptries and their role(s) in host cell invasion remain unexplored. To gain insight into these issues, we evaluated the expression, immunogenicity, and functional roles of two T. equi rhoptry-associated proteins abbreviated as RAP-1a and RAP-1b. The full-length RAP-1a protein was expressed to perform the analysis but our efforts to express the full-length RAP-1b protein failed due to an unknown reason. We therefore generated synthetic immunogenic peptides that map onto the N- and C-termini of the RAP-1b protein as an alternative approach. Our findings show that both proteins are expressed in the extracellular and intra-erythrocytic merozoite stages of T. equi. Serological analyses show that T. equi-infected horses mount antibody responses that recognize both proteins and correlate with a decrease in T. equi load in both acutely and persistently infected horses. In vitro neutralization studies show that the T. equi RAP-1a protein contains neutralization-sensitive epitopes as antibodies developed against the protein significantly inhibited the parasites from invading equine erythrocytes. Conversely, antibodies developed against the RAP-1b synthetic peptides did not neutralize parasite invasion, showing that the protein regions on which the peptides were based are not required for T. equi invasion. Overall, the data shows that T. equi rhoptries and their contents are involved in invasion of host cells and supports T. equi RAP-1 proteins as candidates for developing novel serodiagnosis tools and vaccines.

Theileria equi

Merozoites

Rhoptries

RAP-1a

RAP-1b

Erythrocytes

Invasion

© 2022 Published by Elsevier Ltd on behalf of Australian Society for Parasitology.