Serologic Assessment of Antigenic Type-V and other Outer Membrane Proteins from Brucella Species as Differential Diagnostic Targets for Brucellosis
thesisposted on 13.05.2017, 00:00 by Samantha D. Lambert
The Type V auto-secreting proteins of Gram-negative bacterial pathogens have been shown to be important surface-expressed molecules that facilitate colonization and in vivo survival. In particular, Brucella species have been shown to carry genes with the potential to express several Type V and Type V-like secreted proteins that are antigenic, possess putative virulence function, and may very well contribute to persistence of the microorganism in susceptible hosts (cattle, pigs, bison, sheep, and cervids [elk and deer]). Additionally, some of these proteins may be differentially surface-expressed and thus potentially represent species-specific markers. Experiments were therefore conducted to assess the feasibility of selected recombinant outer membrane proteins to be used to distinguish between B. abortus, B. melitensis, B. suis, and B. ovis infection in various host species. A total of four Brucella genes were previously cloned in E. coli and their recombinant products expressed and purified by established molecular procedures. Methods for determining sero-reactivity of these potential diagnostic targets consisted of SDS-PAGE, electroblotting, and immunolabelling with anti-sera from goats, sheep, cattle, and elk infected with either of the three pathogenic Brucella spp. Quantitative analysis of protein gels and Western blots was conducted by digital imaging. Results of this study reinforce the practicality of one or a number of these bacterial envelope proteins for use in a rapid serologic-based field assay for Brucellosis in wild and domestic animal hosts. Furthermore, these antigens may have utility in a test that can differentiate infection by pathogenic Brucella species specific to a target host.