Determination of bovine lymphocyte responses to extracted proteins of Brucella abortus by using protein immunoblotting

Abstract

Isolation and identification of Brucella antigenic determinants important to cellular responses have been difficult. In this study, bovine peripheral blood mononuclear (PBM) cells from cattle vaccinated with Brucella abortus 19 proliferated to extracted bacterial proteins blotted onto nitrocellulose. Proteins were extracted from gamma-irradiated B. abortus 19 with a sodium dodecyl sulfate extraction buffer. The extracted proteins were * Corresponding author.

Lymphocyte proliferation in response to immunodominant antigens of Brucella abortus 2308 and RB51 in strain 2308-infected cattle

“…1). These results are similar to those reported for other studies that have shown that 94to 20-kDa proteins from S2308 (10) and most of the proteins from other smooth strains of B. aborus are contaminated with LPS 0 antigens, which is revealed when the proteins are separated by gel electrophoresis (1,12,14). No LPS 0 antigens were detected in the 22 protein fractions of SRB51 ( Fig.…”

“…An analysis of the antigens that cattle respond to during clinical infections but not after vaccination with S19 would more likely identify antigens that are critical for immunity to brucellosis. Furthermore, proteins isolated from B. abortus S19 are contaminated with lipopolysaccharide (LPS) 0 antigens (1,10,14), and cattle produce antibodies to these antigens when vaccinated with S19 (6,13). Therefore, previous reports of lymphocyte proliferation in S19-vaccinated cattle in response to isolated S19 proteins may have instead resulted from proliferation in response to LPS 0 antigens that contaminated the proteins.…”

Sodium dodecyl sulfate- and salt-extracted antigens from various Brucella species induce proliferation of bovine lymphocytes

“…Brucellergen, a salt extraction of B. melitensis 115 (2) used throughout the world (except in the United States) to identify Brucella-infected goats, sheep, and cattle, was also included. Stimulation of lymphocyte proliferation by proteins electroblotted onto nitrocellulose has been used as an innovative method for identifying immunogenic peptides from complex mixtures (5,10,12,14), including B. abortus S19 proteins (3,4). In the present study, we observed that both saltand SDS-extractions of different Brucella species produced similar bands stimulatory for bovine lymphocytes (Table 1) from cattle previously vaccinated with B. abortus 19 and not exposed to other Brucella species.…”

“…Proteins isolated from sodium dodecyl sulfate (SDS)extracted (3,4,26) gamma-irradiated ('37Cs) B. abortus S19, B. abortus 45/20, B. melitensis Rev 1, and B. melitensis Rev la and proteins from salt-extracted acetone-killed (2) B. abortus 45/20, B. abortus S19, B. abortus 2308, and B. melitensis 115 (Brucellergen [2]) were subjected to SDS-15% polyacrylamide gel electrophoresis (PAGE) analysis (11). Rev la was provided by M. Banai.…”

“…Results of the present studies illustrate that peripheral blood mononuclear cells from cattle sensitized to one strain of Brucella species (B. abortus 19) recognize and proliferate to antigens from other Brucella species. The lack of mitogenic activity of the Brucella components (Table 2) for bovine lymphocytes (3,4) and specificity of responding bovine lymphocytes to Bnrcella components (1,3,4) indicate the potential relevance of the different Brucella components to disease resistance or pathogenicity. The specific role of the identified Brucella components in disease resistance or pathogenicity, however, has not yet been determined.…”