O-Polysaccharide Epitopic Heterogeneity at the Surface ofBrucellaspp. Studied by Enzyme-Linked Immunosorbent Assay and Flow Cytometry

Abstract

Smooth Brucella strains are classified into three serotypes, i.e., A+M−, A−M+, and A+M+, according to slide agglutination with A and M monospecific polyclonal sera. The epitopes involved have been located on the O-polysaccharide (O-PS) moiety of the smooth lipopolysaccharide (S-LPS), which represents the most exposed antigenic structure on the surface ofBrucella spp. By use of monoclonal antibodies (MAbs) a number of epitope specificities on the O-PS have been reported: A, M, and epitopes shared by both A and M dominant strains, which have been named common (C) epitopes. The latter have been further subdivided, according to relative MAb binding in enzyme-linked immunosorbent assays (ELISA) to A- and M-dominant Brucella strains and to cross-reacting Yersinia enterocolitica O:9, into five epitopic specificities: C (M>A), C (M=A), C/Y (M>A), C/Y (M=A), and C/Y (A>M). In the present study, we studied the occurrence of these epitopes at the surface of representatives of all Brucellaspecies and biovars including the live vaccine strains by analyzing the levels of MAb binding to whole Brucella cells in ELISA and flow cytometry assays. In ELISA, the level of MAb binding correlated well with the previously defined epitope specificity and the serotype defined by polyclonal sera for each Brucella species, biovar, or strain. However, MAbs to the C (M=A) and C (M>A) epitopes showed insignificant binding to B. suis biovar 2 strains and bound at lower titers to B. suis biovar 3 and B. neotomae than to the other Brucella strains. Some of the flow cytometry results were contradictory to those obtained by ELISA. In fact, it appeared by flow cytometry that all O-PS epitopes, including the A and M epitopes, are shared to different degrees byBrucella spp. which nevertheless show a high degree of O-PS heterogeneity according to MAb binding intensities. The subdivision of MAb specificities and Brucella serotypes was therefore less evident by flow cytometry than by ELISA. Whereas in ELISA the MAb specific for the A epitope showed insignificant binding to Y. enterocolitica O:9, this MAb bound strongly to Y. enterocolitica O:9 in flow cytometry. One of the two MAbs specific to the C (M=A) epitope also bound at a low but significant level to B. suis biovar 2 strains. However, as in ELISA the MAb specific for the C (M>A) epitope did not bind at all to B. suis biovar 2 strains in flow cytometry. Flow cytometry provided new information regarding specificity of the MAbs and may further explain some aspects of the capacity of passive protection of some MAbs against smooth Brucella infection in mice. As shown in the present study the occurrence of Brucella strains apparently completely devoid of one specific C O-PS epitope (e.g., B. suis biovar 2 devoid of the C [M>A] epitope) offers the possibility of obtaining vaccine strains devoid of a diagnostic O-PS epitope, which could further help to resolve the problem of discriminating infected from vaccinated animals that remains a major goal in brucellosis research.

WadD, a New Brucella Lipopolysaccharide Core Glycosyltransferase Identified by Genomic Search and Phenotypic Characterization

“…Enzyme-Linked Immunosorbent Assay using whole bacteria (sonicated cells) as the antigen were performed as described previously ( Cloeckaert et al, 1993a ). MoAbs used were directed against O -PS, R-LPS, and the OM lipoproteins Omp10, Omp16, and Omp19 ( Cloeckaert et al, 1990 , 1993a , b , 1998 ). The anti-R-LPS MoAbs used were A68/03F03/D05 (IgG2b), A68/10A06/B11 (IgM), A68/24D08/G09 (IgG1), and A68/24G12/A08 (IgG3).…”

Immunoproteomics of Brucella abortus reveals differential antibody profiles between S19‐vaccinated and naturally infected cattle

“…Brucellosis control programs are based on different protocols of vaccination with a live attenuated B. abortus strain 19 (S19) vaccine to decrease occurrence, followed by serodiagnosis and elimination of seropositive animals . S19 vaccine is antigenically similar to virulent strains, and hence serodiagnosis by LPS‐based conventional tests is not able to differentiate vaccinated from infected animals . Thus, diagnostic procedures for brucellosis are required to differentiate antibody response after vaccination from natural infection, and they should be inexpensive, simple, rapid, specific, and sensitive, being able to detect all stages of infection.…”

Development of an improved competitive ELISA based on a monoclonal antibody against lipopolysaccharide for the detection of bovine brucellosis

“…C epitopes have been subdivided and seven epitopes on the Brucella OPS have been defined, including: A, M, C (M > A), C (A = M), C/Y (M > A), C/Y (A = M), and C/Y (A > M) [ 20 , 21 ]. Further analysis of the MAb epitope specificity was performed with native rough phenotype Brucella and with smooth Brucella strains of three serotypes, i.e., A + M − , A − M + , and A + M + , corresponding to strains expressing mainly the A (A-dominant) or M (M-dominant) antigen or both antigens in nearly equivalent amounts [ 22 ] (Fig.…”

“…However, other bacterial infections often disturb the serological examination of brucellosis [ 12 , 13 ], due to the antigens of cross-reaction among the bacteria. It is known that smooth Brucella LPS contains O-polysaccharide (OPS) moiety, which has been divided into seven epitopic specificities: A, M, C (M > A), C (A = M), C/Y (M > A), C/Y (A = M), and C/Y (A > M) [ 20 , 21 ]. Research has shown that the cross-reactions in the brucellosis serological detection mainly occur as a result of the similar structures that A and C/Y epitopes share with the OPS of Y. enterocolitica O:9 and other bacteria [ 14 , 15 ].…”

“…Brucella suis, which possess five biovars of three serotypes were not used due to the lack of the strains in the laboratory. As Brucella suis biovar 2 displayed unique reactivity with MAbs of C (A = M) and C (M > A) [ 21 ], identification of MAbs with Brucella suis biovar 2 may provide some interesting results. Almost all of the Mabs were against the C/Y or M epitopes, with the exception of MAb 3 F9.…”