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Research Project: Development of Detection and Control Strategies for Bovine Babesiosis and Equine Piroplasmosis

Location: Animal Disease Research

Title: Theileria equi RAP-1a and RAP-1b proteins contain immunoreactive epitopes and are suitable candidates for vaccine and diagnostics development

item ONZERE, CYNTHIA - Washington State University
item Fry, Lindsay
item BISHOP, RICHARD - Washington State University
item DA SILVA, MARTA - Washington State University
item MADSEN-BOUTERSE, SALLY - Washington State University
item BASTOS, REGINALDO - Washington State University
item KNOWLES, DONALD - Washington State University
item Suarez, Carlos

Submitted to: International Journal for Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/19/2022
Publication Date: 4/21/2022
Citation: Onzere, C.K., Fry, L.M., Bishop, R.P., Da Silva, M., Madsen-Bouterse, S.A., Bastos, R.G., Knowles, D.P., Suarez, C.E. 2022. Theileria equi RAP-1a and RAP-1b proteins contain immunoreactive epitopes and are suitable candidates for vaccine and diagnostics development. International Journal for Parasitology. 52(6):385-397.

Interpretive Summary: Theileria equi is a globally distributed, tick-transmitted apicomplexan parasite of horses. The parasite causes significant economic losses due to restriction of international movement of infected horses, reduced ability of infected horses to work (e.g. traction, riding, sporting, etc.), medical costs, and death of valuable animals. Apart from stringent testing efforts, prevention of equine piroplasmosis is largely based on tick control measures and anti-protozoal drug treatment, as there are not yet vaccines available to prevent infection or clinical disease. In this study, we examined two proteins (RAP-1a and RAP-1b) associated with specialized organelles found in apicomplexan parasites, called rhoptries, to determine if they are involved in parasite infection of host cells, and, if so, whether antibody neutralization of these proteins could preclude host cell infection. We found that RAP-1a, but not RAP-1b, is involved in the infection of host cells by the parasite, that pre-treatment of parasites with anti-RAP-1A antibodies inhibits parasite entry into cells, and that horses infected with T. equi develop a robust antibody response to RAP-1A. These findings indicate that RAP-1a may be a promising target for future vaccine and/or diagnostic assay development, and provide insight into blood cell invasion by T. equi.

Technical Abstract: Theileria equi is an obligate intracellular protozoan parasite that causes severe hemolytic anaemia 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 recognise both proteins and correlate with a decrease in T. equi load in both acutely and persistently infected horses. In vitro neutralisation studies show that the T. equi RAP-1a protein contains neutralisation-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 neutralise 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.