Identification of Babesia bovis merozoite antigens separated by continuous-flow electrophoresis that stimulate proliferation of helper T-cell clones derived from B. bovis-immune cattle

• Published in: Infection and Immunity Vol. 63; no. 8; pp. 3106 - 3116
• Main Authors: Brown, W.C. (Texas AandM University, College Station, TX.), Logan, K.S, Zhao, S, Bergman, D.K, Rice-Ficht, A.C
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01-08-1995
• Subjects: Animal bacterial diseases; Animals; ANTIGENE; ANTIGENOS; Antigens, Protozoan - chemistry; Antigens, Protozoan - isolation & purification; BABESIA BOVIS; Babesia bovis - immunology; Bacterial diseases; Biological and medical sciences; Cattle; Clone Cells; ELECTROFORESIS; ELECTROPHORESE; ETAPAS DE DESARROLLO; IDENTIFICACION; IDENTIFICATION; IMMUNITE CELLULAIRE; Infectious diseases; INMUNIDAD CELULAR; Lymphocyte Activation; Medical sciences; Molecular Weight; PESO MOLECULAR; POIDS MOLECULAIRE; REPONSE IMMUNITAIRE; RESPUESTA INMUNOLOGICA; STADE DE DEVELOPPEMENT; T-Lymphocytes - immunology; T-Lymphocytes, Helper-Inducer - immunology; Cell proliferation; Protozoa; Immunostimulation; Purification; Bovine; Gel electrophoresis; Helper cell; Antigen; Continuous process; Vertebrata; Mammalia; T-Lymphocyte; Merozoite; Artiodactyla; Babesia bovis; Sporozoa; Ungulata
• ISSN: 0019-9567; 1098-5522
• DOI: 10.1128/iai.63.8.3106-3116.1995

To characterize Babesia bovis merozoite antigens that stimulate anamnestic T helper (Th)-cell responses from B. bovis-immune cattle, B. bovis-specific Th-cell lines and clones, previously assigned to different antigenic groups (W. C. Brown, S. Zhao, A. C. Rice-Ficht, K. S. Logan, and V. M. Woods, Infect. Immun. 60:4364-4372, 1992), were tested in proliferation assays against fractionated merozoite antigens. The antigenic groups were determined by the patterns of response of Th clones to different parasite isolates and soluble or membrane forms of merozoite antigen. Soluble antigen fractionated by anion-exchange chromatography or gel filtration by using fast-performance liquid chromatography resolved two or three antigenic peaks, respectively. To enable fractionation of membrane-associated proteins and to resolve more precisely the proteins present in homogenized merozoites, a novel technique of continuous-flow electrophoresis was employed. Merozoite membranes or whole merozoites were homogenized and solubilized in sodium dodecyl sulfate-sample buffer, electrophoresed under reducing conditions on 15% or 10% acrylamide gels, eluted, and collected as fractions. Individual fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tested for the ability to stimulate Babesia-specific CD4+ T-cell lines and clones. CD4+ Th-cell lines from two cattle displayed differential patterns of reactivity and detected numerous peaks of antigenic activity, ranging from 14 to 76 kDa. Th-cell clones previously categorized into different antigenic groups detected antigenic peaks unique for clones representative of a given group. Antigens of 29, 51 to 52, and 85 to 95 kDa (group I), 40 kDa (group III), 20 kDa (group IV), 58 to 60 kDa (group VI), and 38, 45, and 83 kDa (group VII) were identified in the stimulatory fractions. Immunization of rabbits with selected fractions produced a panel of antisera that reacted specifically on Western blots (immunoblots)