Enzyme-linked immunosorbent assay with Brucella native hapten polysaccharide and smooth lipopolysaccharide.

Brucella melitensis native haptens (NH) are polysaccharides identical to the O-side chain of the smooth lipopolysaccharide (S-LPS) (E. Moreno, H. Mayer, and I. Moriyón, Infect. Immun. 55:2850-2853, 1987) which precipitate with sera from infected cattle but not from strain 19-vaccinated cattle. In the present work, NH was extracted by the hot-water method (R. Díaz, J. Toyos, M.D. Salvo, and M.L. Pardo, Ann. Rech. Vet. 12:35-39, 1981) and purified free of S-LPS and protein. Purified NH lacked the ability to coat polystyrene and sheep erythrocytes. In contrast, NH acylated with stearoyl chloride bound to both polystyrene and erythrocytes. By hemagglutination and enzyme-linked immunosorbent assay (ELISA), S-LPS and acylated NH gave similar results with blood sera from brucellosis-free, strain 19-vaccinated, and infected cattle. Moreover, a significant correlation between the results of NH ELISA and S-LPS ELISA was demonstrated with milk sera. However, in a competitive ELISA with milk sera, S-LPS in the liquid phase abrogated the binding of antibodies to acylated NH adsorbed to polystyrene, while NH in the liquid phase did not influence the binding of antibodies to polystyrene-adsorbed S-LPS. It is hypothesized that the different precipitations of NH and S-LPS with sera from infected or strain 19-vaccinated cattle are due to differences in the affinity of the antibodies produced upon vaccination or infection and in the physical state of aggregation of NH and S-LPS in aqueous solutions

Enzyme-linked immunosorbent assay with Brucella native hapten polysaccharide and smooth lipopolysaccharide

Brucella melitensis native haptens (NH) are polysaccharides identical to the O-side chain of the smooth lipopolysaccharide (S-LPS) (E. Moreno, H. Mayer, and I. Moriyón, Infect. Immun. 55:2850-2853, 1987) which precipitate with sera from infected cattle but not from strain 19-vaccinated cattle. In the present work, NH was extracted by the hot-water method (R. Díaz, J. Toyos, M.D. Salvo, and M.L. Pardo, Ann. Rech. Vet. 12:35-39, 1981) and purified free of S-LPS and protein. Purified NH lacked the ability to coat polystyrene and sheep erythrocytes. In contrast, NH acylated with stearoyl chloride bound to both polystyrene and erythrocytes. By hemagglutination and enzyme-linked immunosorbent assay (ELISA), S-LPS and acylated NH gave similar results with blood sera from brucellosis-free, strain 19-vaccinated, and infected cattle. Moreover, a significant correlation between the results of NH ELISA and S-LPS ELISA was demonstrated with milk sera. However, in a competitive ELISA with milk sera, S-LPS in the liquid phase abrogated the binding of antibodies to acylated NH adsorbed to polystyrene, while NH in the liquid phase did not influence the binding of antibodies to polystyrene-adsorbed S-LPS. It is hypothesized that the different precipitations of NH and S-LPS with sera from infected or strain 19-vaccinated cattle are due to differences in the affinity of the antibodies produced upon vaccination or infection and in the physical state of aggregation of NH and S-LPS in aqueous solutions.</jats:p

Evaluation of Lipopolysaccharides and Polysaccharides of Different Epitopic Structures in the Indirect Enzyme-Linked Immunosorbent Assay for Diagnosis of Brucellosis in Small Ruminants and Cattle

Brucella abortus and Brucella melitensis have surface lipopolysaccharides and polysaccharides carrying B. melitensis-type (M) and B. abortus-type (A) epitopes as well as common (C) epitopes present in all smooth Brucella biotypes. Crude lipopolysaccharides, hydrolytic O polysaccharides, and native hapten polysaccharides of MC or AC specificity were evaluated in indirect enzyme-linked immunosorbent assays with polyclonal, monoclonal, or protein G conjugates by using sera from cattle, sheep, and goats infected with AC, MC, or AMC Brucella biotypes. Regardless of the antigen, the levels of antibodies were lower in goats than in sheep and highest in cattle. The diagnostic performance of the assay was not affected by the absence of lipid A-core epitopes, the presence of contaminating outer membrane proteins, the AC or MC epitopic structure of the absorbed antigen, or the conjugate used. Moreover, with sera from cattle vaccinated with B. abortus S19 (AC) or from sheep and goats vaccinated with B. melitensis Rev 1 (MC), AC and MC antigens showed similar levels of reactivity. The results show that antibodies to the C epitopes largely dominate in infection, and this is consistent with the existence of multiple overlapping C epitopes (V. Weynants, D. Gilson, A. Cloeckaert, A. Tibor, P. A. Denoel, F. Godfroid, J. N. Limet, and J.-J. Letesson, Infect. Immun. 65:1939–1943, 1997) rather than with one or two C epitopes. It is concluded that, by adaptation to the corresponding antibody levels, brucellosis in cattle, sheep, and goats can be diagnosed by immunosorbent assay with a single combination of conjugate and antigen

Serological response to the outer membrane lipoprotein in animal brucellosis.

The presence of antibodies to Brucella outer membrane lipoprotein was investigated in cattle and rams. Low but significant amounts of antibody were detected in sera from B. abortus-infected cattle and from B. ovis-infected rams which had developed epididymitis. Strain-19-vaccinated cattle also showed a weak albeit transient antibody response

Evaluation of Lipopolysaccharides and Polysaccharides of Different Epitopic Structures in the Indirect Enzyme-Linked Immunosorbent Assay for Diagnosis of Brucellosis in Small Ruminants and Cattle

ABSTRACT Brucella abortus and Brucella melitensis have surface lipopolysaccharides and polysaccharides carrying B. melitensis -type (M) and B. abortus -type (A) epitopes as well as common (C) epitopes present in all smooth Brucella biotypes. Crude lipopolysaccharides, hydrolytic O polysaccharides, and native hapten polysaccharides of MC or AC specificity were evaluated in indirect enzyme-linked immunosorbent assays with polyclonal, monoclonal, or protein G conjugates by using sera from cattle, sheep, and goats infected with AC, MC, or AMC Brucella biotypes. Regardless of the antigen, the levels of antibodies were lower in goats than in sheep and highest in cattle. The diagnostic performance of the assay was not affected by the absence of lipid A-core epitopes, the presence of contaminating outer membrane proteins, the AC or MC epitopic structure of the absorbed antigen, or the conjugate used. Moreover, with sera from cattle vaccinated with B. abortus S19 (AC) or from sheep and goats vaccinated with B. melitensis Rev 1 (MC), AC and MC antigens showed similar levels of reactivity. The results show that antibodies to the C epitopes largely dominate in infection, and this is consistent with the existence of multiple overlapping C epitopes (V. Weynants, D. Gilson, A. Cloeckaert, A. Tibor, P. A. Denoel, F. Godfroid, J. N. Limet, and J.-J. Letesson, Infect. Immun. 65:1939–1943, 1997) rather than with one or two C epitopes. It is concluded that, by adaptation to the corresponding antibody levels, brucellosis in cattle, sheep, and goats can be diagnosed by immunosorbent assay with a single combination of conjugate and antigen. </jats:p

Serological response to the outer membrane lipoprotein in animal brucellosis

The presence of antibodies to Brucella outer membrane lipoprotein was investigated in cattle and rams. Low but significant amounts of antibody were detected in sera from B. abortus-infected cattle and from B. ovis-infected rams which had developed epididymitis. Strain-19-vaccinated cattle also showed a weak albeit transient antibody response.</jats:p

Serological response to the outer membrane lipoprotein in animal brucellosis

The presence of antibodies to Brucella outer membrane lipoprotein was investigated in cattle and rams. Low but significant amounts of antibody were detected in sera from B. abortus-infected cattle and from B. ovis-infected rams which had developed epididymitis. Strain-19-vaccinated cattle also showed a weak albeit transient antibody response

Evaluation of serological tests for diagnosis of Brucella melitensis infection of goats

Five serological assays were evaluated for the diagnosis of brucellosis in goats: the rose bengal test (RBT), complement fixation test (CFT), radial immunodiffusion (RID) with Brucella and Yersinia enterocolitica O:9 polysaccharides, counterimmunoelectrophoresis (CIEP) with cytosol, and enzyme-linked immunosorbent assay (ELISA) with polyclonal and protein G conjugates and smooth lipopolysaccharide (S-LPS), native hapten polysaccharide (NH), or cytosol antigens. For optimal sensitivity, RBT had to be used with sera-antigen at a 3:1 dilution. In the RID test, Brucella melitensis biotype 1 NH could not be replaced by Brucella abortus biotype 1 or Y. enterocolitica 0:9 polysaccharides. In the ELISA, S-LPS and NH gave similar results and the protein G conjugate increased the specificity. With the sera from 55 B. melitensis culture-positive goats, the sensitivity was 100% for RBT, CFT (titer &gt; or = 4), and ELISA with S-LPS or NH; 94% for RID; and 93% for CIEP. All tests were negative (100% specific) when testing the sera from 127 brucella-free goats. Larger discrepancies among the results of the serological tests were obtained with sera from goats of areas where brucellosis is endemic. When the sera of 20 young goats vaccinated subcutaneously (10(9) CFU of B. melitensis Rev 1) and bled 6 months later were examined, the specificities were as follows: NH ELISA, 60%; CFT and S-LPS ELISA, 75%; RBT, 80%; CIEP, 90%; and RID, 94%. With the sera from 10 young goats vaccinated conjunctivally (10(9) CFU of B. melitensis Rev 1) all tests were 100% specific 4 months after vaccination. The proportion of goats giving a positive reaction after vaccination decreased faster in RID than in other tests.</jats:p