Determinação da reatividade diferencial de anticorpos de bovinos vacinados com a cepa S19 de Brucella abortus e naturalmente infectados através de análises imunoproteômicas

Abstract

The aim of this study was to characterize a new soluble antigen of Brucella abortus obtained by Triton X-114 (TX-114) extraction by immunoproteomic analysis and immunoblot-avidity, using the detection conjugates anti-bovine IgG and Protein A labeled with peroxidase. Serum samples of three groups of bovine were studied: (GI) 30 nonvaccinated seropositive cows; (GII) 30 S19-vaccinated seropositive heifers; (GIII) 30 nonvaccinated seronegative cows. The one dimensional (1-D) and two dimensional (2-D) electrophoretic profiles of the TX-114 antigen revealed a broad spectrum of polypeptides (10 to 79 kDa), and 1-D immunoblot showed a widespread profile of reactivity by sera of GI compared with a more restricted profile by sera of GII, regardless of the detection conjugate. High avidity IgG antibodies were detected in sera of GI by both conjugates analyzed, but Protein A/peroxidase showed higher ability to detect low avidity IgG2 antibodies in sera of GII. Five major antigenic components (24, 28, 35, 47 and 50 kDa) were predominantly recognized by high avidity IgG antibodies in sera of GI, in addition to three minor antigenic components (10, 12 and 17 kDa) that were recognized exclusively by sera of this group, and so considered potential markers of infection and exclusion of vaccinal response. The proteomic characterization revealed 42 spots in 2-D gel, being possible to identify 22 hypothetical cytoplasmic proteins, while the 2-D immunoblot showed reactivity of polypeptides below 20 kDa exclusively with sera of GI. The analysis by mass spectrometry of these polypeptides was able to identify five antigenic proteins of B. abortus (Bfr, Dps/Ndpk-I, Sod, and Protein B of invasion), from which Ndpk-I and Protein B of invasion were identified in vaccinal S19 strain on the first time in the present study, and were related with the antigenicity in seropositive samples of non-vaccinated bovine. In conclusion, the immunoproteomic analysis of this new soluble antigen of B. abortus permitted the characterization of several antigenic proteins of the vaccinal S19-strain that could be candidates for the serodiagnosis of bovine brucellosis and the differentiation between vaccinated and infected animals. The evaluation of avidity of IgG2 antibodies to antigenic markers of the TX-114 antigen can also be considered an additional tool for a better serological distinction in the profile of functional affinity of IgG antibodies in response to infection and vaccination in bovine brucellosis.