Presence of common antigens, including major surface protein epitopes, between the cattle (intraerythrocytic) and tick stages of Anaplasma marginale

Abstract

Epitopes of major surface proteins of the intraerythrocytic cattle stage of Anaplasma marginale were demonstrated in the midgut stage of the organism within the infective tick host Dermacentor andersoni. These proteins were common to all A. marginale isolates tested and at all stages of parasitemia. Sera from cattle immunized with the tick midgut stage of A. marginale immunoprecipitated multiple-erythrocyte-stage proteins, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The major proteins recognized (primarily >14 and <200 kilodaltons [kDa]) included two major-erythrocyte-stage surface proteins of 36 and 105 kDa molecular size. To confirm the presence of common tick and erythrocyte A. marginale antigens with the immunized cattle sera, we purified the 36-kDa erythrocyte-stage protein by monoclonal immunoaffinity * Corresponding author. t Present address:

Immunization of cattle with a 36-kilodalton surface protein induces protection against homologous and heterologous Anaplasma marginale challenge

“…Immunization of cattle with purified AmF36. AmF36 was isolated from Florida isolate A. marginale-infected erythrocytes by using monoclonal immunoaffinity chromatography as previously described (26). Briefly, 1012 organisms were purified from erythrocytes and detergent-soluble proteins were extracted in a 50 mM Tris buffer (pH 8.0) containing 1.0% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 5 mM EDTA, 5 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, and 0.1 M N-ot-p-tosyl-L-lysyl-chloromethylketone.…”

“…At least two of these proteins, AmF36 and AmF105 (designated by abbreviations for genus, species, isolate, and apparent molecular mass in kilodaltons), bear epitopes highly conserved among A. marginale isolates from Israel, Kenya, and the United States (24, 25a, 28). In addition, epitopes from both surface proteins are shared with tick stages of A. marginale within Dermacentor andersoni ticks (26). Our strategy in vaccine development is to identify one or more antigens that induce protective immunity and to produce these antigens by recombinant DNA expression.…”

Expression and immune recognition of the conserved MSP4 outer membrane protein of Anaplasma marginale

“…Isolated proteins were dialyzed into 50 mM (NH4)2CO3 (pH 8.0) and 125I labeled with Iodobeads (Pierce) for 5 min following manufacturer's instructions. Immunoprecipitations were completed as described previously (30). In brief, lysed cells or isolated proteins were incubated with a monoclonal antibody, and the complexes were precipitated with protein G-Sepharose beads (Sigma).…”

“…A. marginale initial bodies were isolated from cryogenically preserves organisms (31) and biotin labeled with sulfosuccinimidobiotin (sulfo-NHS-biotin; Pierce) (13). Following immunoprecipitation (30) with AnaR76A1 or TryplEl, the proteins were isolated by SDS-polyacrylamide gel electrophoresis (PAGE) and electroblotted onto nitrocellulose membranes. The membranes were blocked with 3% bovine serum albumin (BSA) in TBS (0.5 M NaCl, 0.02 M Tris [pH 7.5]), incubated with horseradish peroxidase-labeled avidin (1:3,000 in TBS-1% BSA), and detected with 3,3'-diaminobenzidine and hydrogen peroxide after three TBS-0.02% Tween 20 washes (41).…”

Characterization of hemagglutinating components on the Anaplasma marginale initial body surface and identification of possible adhesins

“…The preparation of polyclonal and monoclonal antibodies has been described elsewhere (26,35,37). Antibodies reactive with A. marginale proteins are designated as follows: 22B1, a monoclonal antibody that is reactive with MSPla polypeptides and that neutralizes initial body infectivity in vitro (36); 76A1, a monoclonal antibody reactive with a 31-kDa surface polypeptide (MSP4) (31); 58A2, a monoclonal antibody reactive with MSP2 (26,36); R-783, polyclonal antiserum raised against purified initial bodies (37); R-883, polyclonal antiserum raised against immunoaffinitypurified MSP2 (35); 31B, polyclonal antiserum raised against partially purified MSP4 (31); anti-61, a polyclonal antibody raised against a partially purified 61-kDa A. marginale major surface protein; R-874, a polyclonal antibody raised against immunoaffinity-purified MSP1 complex (4); and Al-aF and A2-bF, rabbit polyclonal antibodies raised against recombinant MSP1a:GST and MSP1b:GST fusion proteins, respectively (23). Antibodies raised against non-A.…”