title: A novel neutralization sensitive and subdominant RAP-1-related antigen (RRA) is expressed by Babesia bovis merozoites.
abstract: OBJECTIVE
  The Babesia bovis genome encodes a rap-1 related gene denominated RAP-1 related antigen (RRA). In this study, we analysed the pattern of expression, immunogenicity and functional relevance of RRA.
  
  
  METHODS
  Phylogenetic analysis was performed using the program Phylip. Expression of rra was analysed by Northern blots, RT-PCR, immunoprecipitation, Western blots and immunofluorescence. RRA antigenicity was tested by T-cell proliferation and Western blot analysis, and functional relevance was determined in an in vitro neutralization assay.
  
  
  RESULTS
  RRA is more closely related to RAP-1b of Babesia bigemina than to B. bovis RAP-1, and it is highly conserved among distinct strains. Transcriptional analysis suggests lower numbers of rra transcripts compared to rap-1. Immunoprecipitation of metabolically labelled B. bovis proteins with antibodies against synthetic peptides representing predicted antigenic regions of RRA confirmed the expression of a ∼43 kDa RRA in cultured merozoites. Antibodies present in B. bovis hyperimmune sera, but not in field-infected cattle sera, reacted weakly with recombinant RRA, and no significant stimulation was obtained using recombinant RRA as antigen in T-cell proliferation assays, indicating that RRA is a subdominant antigen. Antibodies against RRA synthetic peptides reacted with merozoites using immunofluorescence, and were able to significantly inhibit erythrocyte invasion in in vitro neutralization tests, suggesting functional relevance for parasite survival.
  
  
  CONCLUSIONS
  B. bovis express a novel subdominant RAP-1-like molecule that may contribute to erythrocyte invasion and/or egression by the parasite.
authors.affiliation: Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, USA 99164-7040.
authors.forename: Carlos E
authors.initials: CE
authors.lastname: Suarez
authors.affiliation: Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, USA 99164-7040.
authors.forename: Jacob M
authors.initials: JM
authors.lastname: Laughery
authors.affiliation: Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, USA 99164-7040.
authors.forename: Reginaldo G
authors.initials: RG
authors.lastname: Bastos
authors.affiliation: Animal Disease Research Unit, Agricultural Research Service, United States Department of Agriculture, Pullman, WA, USA 99164-6630.
authors.forename: Wendell C
authors.initials: WC
authors.lastname: Johnson
authors.affiliation: Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, USA 99164-7040.
authors.forename: Junzo
authors.initials: J
authors.lastname: Norimine
authors.affiliation: Instituto Nacional de Tecnología Agropecuaria, Centro de Investigación en Ciencias Veterinarias y Agronómicas, Castelar, Argentina.
authors.forename: Gustavo
authors.initials: G
authors.lastname: Asenzo
authors.affiliation: Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, USA 99164-7040.
authors.forename: Wendy C
authors.initials: WC
authors.lastname: Brown
authors.affiliation: Instituto Nacional de Tecnología Agropecuaria, Centro de Investigación en Ciencias Veterinarias y Agronómicas, Castelar, Argentina.
authors.forename: Monica
authors.initials: M
authors.lastname: Florin-Christensen
authors.affiliation: Animal Disease Research Unit, Agricultural Research Service, United States Department of Agriculture, Pullman, WA, USA 99164-6630.
authors.forename: Will L
authors.initials: WL
authors.lastname: Goff
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics* / Q000378:metabolism; D016793:Babesia bovis / Q000235:genetics* / Q000378:metabolism*; D002417:Cattle; D049109:Cell Proliferation; D002478:Cells, Cultured; D000939:Epitopes; D005786:Gene Expression Regulation; D052940:Merozoites / Q000378:metabolism*; D008969:Molecular Sequence Data; D009500:Neutralization Tests / Q000662:veterinary; D010802:Phylogeny; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology; D016415:Sequence Alignment; D013601:T-Lymphocytes / Q000166:cytology
chemical_list: D000953:Antigens, Protozoan; D000939:Epitopes; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C097551:rhoptry-associated protein-1, Babesia