title: A novel 20-kilodalton protein conserved in Babesia bovis and B. bigemina stimulates memory CD4(+) T lymphocyte responses in B. bovis-immune cattle.
abstract: Acquired immunity against the hemoprotozoan parasite Babesia bovis is believed to depend on activation of antigen-specific CD4(+) T lymphocytes and IFN-gamma production. A strategy was employed to identify potentially protective antigens from B. bovis based on memory CD4(+) T lymphocyte recognition of fractionated merozoite proteins. Fractions of merozoites separated by continuous flow electrophoresis (CFE) that contained proteins of approximately 20 kDa were shown previously to stimulate memory CD4(+) lymphocyte responses in B. bovis-immune cattle with different MHC class II haplotypes. Expression library screening with rabbit antiserum raised against an immunostimulatory 20-kDa CFE fraction identified a 20-kDa protein (Bbo20) that contains a B lymphocyte epitope conserved in geographically distant B. bovis strains. An homologous 20-kDa protein that has 86.4% identity with Bbo20 and contains the conserved B cell epitope was identified in B. bigemina (Bbg20). Southern blot analysis indicated that both Babesia proteins are encoded by a single gene. Antibody against recombinant Bbo20 protein identified the antigen in CFE fractions shown previously to stimulate memory T lymphocyte responses in immune cattle. To verify Bbo20 as an immunostimulatory T lymphocyte antigen, CD4(+) T cell lines were propagated from B. bovis-immune cattle with merozoite antigen and shown to proliferate significantly against recombinant Bbo20 protein. Furthermore, Bbo20-specific CD4(+) T cell clones proliferated in response to several B. bovis strains and produced IFN-gamma. BLAST analysis revealed significant similarity of the Bbo20 and Bbg20 amino acid sequences with the hsp20/alpha-crystallin family.
authors.affiliation: Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, Washington State University, PO Box 647040, Pullman, WA 99164-7040, USA. wbrown@vetmed.wsu.edu
authors.forename: W C
authors.initials: WC
authors.lastname: Brown
authors.forename: B J
authors.initials: BJ
authors.lastname: Ruef
authors.forename: J
authors.initials: J
authors.lastname: Norimine
authors.forename: K A
authors.initials: KA
authors.lastname: Kegerreis
authors.forename: C E
authors.initials: CE
authors.lastname: Suarez
authors.forename: P G
authors.initials: PG
authors.lastname: Conley
authors.forename: R W
authors.initials: RW
authors.lastname: Stich
authors.forename: K H
authors.initials: KH
authors.lastname: Carson
authors.forename: A C
authors.initials: AC
authors.lastname: Rice-Ficht
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D001403:Babesia / Q000235:genetics / Q000276:immunology*; D016793:Babesia bovis / Q000235:genetics / Q000276:immunology; D001404:Babesiosis / Q000276:immunology / Q000469:parasitology / Q000662:veterinary*; D015139:Blotting, Southern; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D002417:Cattle; D002418:Cattle Diseases / Q000276:immunology* / Q000469:parasitology; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003459:Crystallins / Q000235:genetics; D006360:Heat-Shock Proteins / Q000235:genetics; D007106:Immune Sera / Q000276:immunology; D007156:Immunologic Memory; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D000953:Antigens, Protozoan; C446036:Bbo20 protein, Babesia bovis; D003459:Crystallins; D006360:Heat-Shock Proteins; D007106:Immune Sera; D015800:Protozoan Proteins