Immunoproteomic identification of immunodominant antigens independent of the time of infection in Brucella abortus 2308-challenged cattle

Abstract

Brucellosis is a vital zoonotic disease caused by Brucella, which infects a wide range of animals and humans. Accurate diagnosis and reliable vaccination can control brucellosis in domestic animals. This study examined novel immunogenic proteins that can be used to detect Brucella abortus infection or as an effective subcellular vaccine. In an immunoproteomic assay, 55 immunodominant proteins from B. abortus 544 were observed using two dimensional electrophoresis (2DE) and immunoblot profiles with antisera from B. abortus-infected cattle at the early (week 3), middle (week 7), and late (week 10) periods, after excluding protein spots reacting with antisera from Yersinia enterocolitica O:9-infected and non-infected cattle. Twenty-three selected immunodominant proteins whose spots were observed at all three infection periods were identified using MALDI-MS/MS. Most of these proteins identified by immunoblot and mass spectrometry were determined by their subcellular localization and predicted function. We suggest that the detection of prominent immunogenic proteins during the infection period can support the development of advanced diagnostic methods with high specificity and accuracy; subsidiarily, these proteins can provide supporting data to aid in developing novel vaccine candidates.

Figure 1

2DE profile of B. abortus proteins and immunoblotting with antisera from B. abortus -infected cattle. (A) 2DE profile of proteins from B. abortus detected on silver-stained 2DE gels within the pI range 4–7. Immunoblotting analyses were performed with antisera from cattle after 3 (B), 7 (C), and 10 weeks (D) of challenge with B. abortus. Three replicates of 2DE analysis were performed in the independent experiments.

Figure 2

2DE analysis and the immunoblotting profile detected using sera from non-infected and Y. enterocolitica -infected cattle. A total of 25 immunoreactive dots were observed using the non-infected (A) and Y. enterocolitica-challenged (C) bovine sera, and the corresponding proteins are labeled on the 2DE gel [NC (B) and YP (D)]. The numbers represent the serial numbers of the immunoreactive proteins in immunoblotting analyses.

Figure 3

Comparative 2DE analysis of B. abortus proteins and immunoblotting profile of non-specific reactions. A total of 13 immunoreactive spots of common antigens that responded to the negative sera from non-infected cattle (A) and positive sera of Y. enterocolitica (B), and three types of sera from cattle after 3, 7 and 10 weeks of challenge with B. abortus were selected. The numbers represent the serial numbers of the immunoreactive proteins in immunoblot analyses.

Figure 4

Immunoblotting profile of B. abortus proteins responded with B. abortus -infected bovine antisera excluding non-specific proteins. Immunoblotting analyses were performed with antisera from cattle after 3, 7, and 10 weeks of challenge with B. abortus. After excluding non-specific reactions, a total of 101 (A), 84 (B), and 78 (C) immunoreactive dots, as well as 55 protein spots that reacted with antisera at all 3 stages (D , E, and F), were selected and labeled. The numbers represent the serial numbers of the immunoreactive proteins in immunoblot analyses.

Figure 5

Comparative 2DE analysis of B. abortus proteins and immunoblotting profiles of specific reactions. (A) A total of 55 immunoreactive spots of antigen that responded to antisera from cattle after 3, 7 and 10 weeks of challenge with B. abortus were selected. A total of 19, 10, and 4 immunoreactive spots of antigen responded to antisera at 2 time-points: (B) after 3 and 7 weeks of challenge, (C) after 3 and 10 weeks, and (D) after 7 and 10 weeks; these spots were selected and labeled on the 2DE gel. The numbers represent the serial numbers of the immunoreactive proteins in immunoblot analyses.