Comprehensive Identification of Immunodominant Proteins of Brucella abortus and Brucella melitensis Using Antibodies in the Sera from Naturally Infected Hosts

Abstract

Brucellosis is a debilitating zoonotic disease that affects humans and animals. The diagnosis of brucellosis is challenging, as accurate species level identification is not possible with any of the currently available serology-based diagnostic methods. The present study aimed at identifying Brucella (B.) species-specific proteins from the closely related species B. abortus and B. melitensis using sera collected from naturally infected host species. Unlike earlier reported investigations with either laboratory-grown species or vaccine strains, in the present study, field strains were utilized for analysis. The label-free quantitative proteomic analysis of the naturally isolated strains of these two closely related species revealed 402 differentially expressed proteins, among which 63 and 103 proteins were found exclusively in the whole cell extracts of B. abortus and B. melitensis field strains, respectively. The sera from four different naturally infected host species, i.e., cattle, buffalo, sheep, and goat were applied to identify the immune-binding protein spots present in the whole protein extracts from the isolated B. abortus and B. melitensis field strains and resolved on two-dimensional gel electrophoresis. Comprehensive analysis revealed that 25 proteins of B. abortus and 20 proteins of B. melitensis were distinctly immunoreactive. Dihydrodipicolinate synthase, glyceraldehyde-3-phosphate dehydrogenase and lactate/malate dehydrogenase from B. abortus, amino acid ABC transporter substrate-binding protein from B. melitensis and fumarylacetoacetate hydrolase from both species were reactive with the sera of all the tested naturally infected host species. The identified proteins could be used for the design of serological assays capable of detecting pan-Brucella, B. abortus- and B. melitensis-specific antibodies.

Laboratory Diagnostic Procedures for Human Brucellosis: An Overview of Existing Approaches

“…The immunoblot-based assay showed several immunodominant proteins of B. abortus and B. melitensis in a previous study; this technique can be used to identify new candidate antigens for the serologic detection of brucellosis (68). Immunoproteomics of B. abortus RB51 (a mutant strain lacking the LPS portion) revealed several candidate antigens.…”

Isolate Specific Cold Response of Yersinia enterocolitica in Transcriptional, Proteomic, and Membrane Physiological Changes

“…The in-solution trypsin digestion of proteins was performed as described previously (Wareth et al, 2016). Briefly, 10 µg protein was used for acetone precipitation.…”

“…Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) measurements were carried out as described elsewhere (Wareth et al, 2016). Resultant peptides of trypsin digestion were desalted by solid phase extraction and the peptides were separated using Dionex Ultimate 3000 nanoLC (Dionex/Thermo Fisher Scientific, Idstein, Germany) on fritless silica micro-columns with an inner diameter of 100 µm.…”

Development of an interferon-γ release assay (IGRA) for detection of Brucella abortus and clinical diagnosis of brucellosis

“…The existing methods for the diagnosis of brucellosis, however, have been developed based mainly on serology, such as the Rose Bengal test (RBT), standard tube agglutination test (SAT), complement fixation test (CFT), fluorescence polarization test (FPA) and enzyme-linked immunosorbent assay (ELISA) [4]. Non-specific reactions are common in these approaches since specific epitopes of Brucella and parasitic bacterium are localized in the cell [5].…”