Identification of protective outer membrane antigens of Brucella ovis by passive immunization of mice with monoclonal antibodies.

Outer membrane proteins (OMPs) and rough lipopolysaccharide (R-LPS), the main surface antigens of Brucella ovis, display surface-exposed epitopes. Mixtures of monoclonal antibodies (mAbs) to both antigens were previously shown to protect mice against a B. ovis challenge. To further identify the antigens involved, seven mAbs against Brucella OMPs (Omp10, Omp16, Omp19, Omp25, Omp31, Omp2b and Omp1) and three to R-LPS were tested for protection either individually or in combinations. Significant reduction in spleen infection in challenged mice, relative to controls, was used as the protection criteri. Controls included nonimmunized mice and mice given an irrelevant, anti-O-polysaccharide (OPS), mAb. For comparison, a group received a mouse serum containing antibodies to both OMPs and R-LPS; this serum was prepared by immunization with a B. ovis hot-saline extract which, as described previously, induces protective immunity in mice and rams. Significant protection was observed with both mAbs to OMPs and R-LPS. mAbs to Omp16, Omp19 and Omp31 afforded the highest protection and prevented the development of splenomegaly. The protective effect of mAb to Omp31 was not interfered with by nonprotective mAbs in different mixtures. The data presented confirm the protective role of antibodies to OMPs and R-LPS against B. ovis, and identify several OMPs, especially Omp31, which are promising candidates for a subunit vaccine against ram epididymitis.

Effect of early antibiotic treatment on the antibody response to cytoplasmic proteins of Brucella melitensis in mice.

To test whether antibiotic therapy hampers the antibody response to Brucella antigens, 30 BALB/c mice were infected with Brucella melitensis H38 and randomized for treatment with doxycycline administered intraperitoneally for 42 days starting at 7 or 28 days postinfection (p.i.) (groups DOX7 and DOX28, respectively) or for no treatment (control group). Antibodies to smooth lipopolysaccharide (LPS) reached peak levels (mean optical density [OD] = 2.618) between days 56 and 70 p.i. in the control group, and similar peak levels (mean OD = 2.486) were observed in the DOX28 group, but significantly lower peak levels (mean OD = 0.821) were observed at 28 days p.i. in the DOX7 group. The antibody response against cytoplasmic proteins depleted of LPS (CPs) reached maximal levels (mean OD = 2.402) between days 56 and 70 p.i. in the control group, but no response was detected in the DOX7 group. Anti-CP antibodies were detected in only three animals from the DOX28 group, at levels significantly lower than those in the control group (mean maximal OD = 0.791). The pattern of antibody response to an 18-kDa cytoplasmic protein of Brucella spp. was similar to that against the CP antigen. This study shows that early antibiotic treatment affects the antibody response of mice to cytoplasmic proteins of Brucella and, to a lesser extent, to LPS.

Effect of bovine non-agglutinating antibodies on the blood clearance of 131I-labelled Brucella abortus strain 45/20.

Non-agglutinating anti-Brucella abortus S45/20 antibodies were isolated and purified from sera of immunized cattle by means of immunoadsorption and ion-exchange chromatography (DEAE-Sephadex A-50). They corresponded to the IgG1 isotype as shown by immunoelectrophoresis using monospecific anti-IgG1 and anti-bovine gamma globulin sera. These antibodies failed to agglutinate the antigen. They were detected by the anti-bovine gamma globulin test, showing higher titres than those of agglutinating antibodies during the whole period of the experiment. Blood clearance of 131I-S45/20 in mice, was slower in those groups which had received non-agglutinating antibodies than in the control group.

Kinetic analysis of cattle anti-Brucella abortus S45/20 non-agglutinating antibodies in antibody-dependent cell-mediated cytotoxicity.

Calves inoculated with Brucella abortus S45/20 produced, against surface antigens, non-agglutinating antibodies (NAAb) which were isolated and purified. A kinetic analysis was carried out of NAAb in antibody-dependent cell-mediated cytotoxicity (ADCC) using sheep red blood cells labelled with surface antigen from B. abortus S45/20 as target cells. Three parameters were examined: time of incubation, effector cell:target cell (E:T) ratio and NAAb dose. It was found that the NAAb were not able to mediate ADCC with bovine spleen cells.

[Detection in bovine serum of non-agglutinating antibodies directed against occult antigens of Brucella abortus strain 19]. / Detección en suero bovino de anticuerpos no aglutinantes dirigidos hacia antígenos ocultos de Brucella abortus cepa 19.

Injection of different animal species with particulate T-dependent antigens induces the appearance of two antibody populations belonging to IgG class, one of them with agglutinating activity and the other lacking this capacity, that is, non-agglutinating. In this paper, 3 adult female Hereford were inoculated subcutaneously, fortnightly, four times, with 5 x 10(10) heat killed smooth S19 Brucella abortus cells. Blood samples were taken ten days after the last inoculation and were individually processed. Serum agglutinating titres against the homologous antigen and the Coombs test score were similar, indicating that nonagglutinating antibody titres directed against surface antigens of the smooth phase were not significant (Table 1). When sera were incubated with a suspension of Brucella abortus S45/20 (a rough strain) they showed poor agglutinating ability, but they contained non-agglutinating activity at high level, as determined by the anti-gamma-globulin test (Table 1). Similar results were obtained when the same sera were adsorbed with Brucella abortus S19 up to elimination of the antibodies directed against surface antigens, and then incubated with a suspension of strain 45/20 (Table 1). These antibodies were isolated from sera by immunoadsorption, using a thick suspension of S45/20 and then dissociated with glycine-CHl buffer 0.1M pH 3. These antibodies showed little agglutinating capacity for S45/20; however non-agglutinating antibodies were bound to the bacteria up to 1.5 micrograms g as demonstrated by Coombs test. Apart from the incomplete antibodies against external antigens of smooth Brucella abortus strains detected in long-term stimulated cattle, non-agglutinating antibodies with specificity for deeper antigens of the smooth bacteria were evidenced.(ABSTRACT TRUNCATED AT 250 WORDS)

Detección suero bovino de anticuerpos no aglutinantes dirigidos hacia antígenos acultos de Brucella abortus cepa 19 / Detection in bovine serun of non-agglutinating antibodies to occult antigenos of Brucella abortus strain 19

Bovinos hembras de raza Hereford, inoculados con Brucella obortus cepa 19 en fase lisa, elaboraron anticuerpos no aglutinantes que se unieron específicamente a la cepa rugosa 45/20. Los sueros de estos animales, adsorbidos con B. abortus cepa 19 hasta eliminación de los anticuerpos dirigidos contra antígenos de superfície de la cepa lisa, mostraron contener una población no aglutinante de anticuerpos que se unieron a la cepa rugosa 45/20, siendo detectados mediante reacción de Cooms hasta un límite de 1,5 ug. Al margens de los anticuerpos incompletos producidos hacia antígenos externos de cepas lisas de B. abortus, se pone en evidencia este tipo de anticuerpos, con especificidad por antígenos más profundos que también estarían presentes en la superficie de la cepa rugosa. Estos anticuerpos, cuya participación en la depuración sanguínea y degradación del agente etiológico de la brucelosis bovina sería poco significativa, están siendo estudiados minuciosamente con el objeto de establecer la importancia que pudieran tener en el diagnóstico y pronóstico de brucelosis

Detección en suero bovino de anticuerpos no aglutinantes dirigidos hacia antígenos ocultos de Brucella abortus cepa 19. / [Detection in bovine serum of non-agglutinating antibodies directed against occult antigens of Brucella abortus strain 19]

Injection of different animal species with particulate T-dependent antigens induces the appearance of two antibody populations belonging to IgG class, one of them with agglutinating activity and the other lacking this capacity, that is, non-agglutinating. In this paper, 3 adult female Hereford were inoculated subcutaneously, fortnightly, four times, with 5 x 10(10) heat killed smooth S19 Brucella abortus cells. Blood samples were taken ten days after the last inoculation and were individually processed. Serum agglutinating titres against the homologous antigen and the Coombs test score were similar, indicating that nonagglutinating antibody titres directed against surface antigens of the smooth phase were not significant (Table 1). When sera were incubated with a suspension of Brucella abortus S45/20 (a rough strain) they showed poor agglutinating ability, but they contained non-agglutinating activity at high level, as determined by the anti-gamma-globulin test (Table 1). Similar results were obtained when the same sera were adsorbed with Brucella abortus S19 up to elimination of the antibodies directed against surface antigens, and then incubated with a suspension of strain 45/20 (Table 1). These antibodies were isolated from sera by immunoadsorption, using a thick suspension of S45/20 and then dissociated with glycine-CHl buffer 0.1M pH 3. These antibodies showed little agglutinating capacity for S45/20; however non-agglutinating antibodies were bound to the bacteria up to 1.5 micrograms g as demonstrated by Coombs test. Apart from the incomplete antibodies against external antigens of smooth Brucella abortus strains detected in long-term stimulated cattle, non-agglutinating antibodies with specificity for deeper antigens of the smooth bacteria were evidenced.(ABSTRACT TRUNCATED AT 250 WORDS)

Detección suero bovino de anticuerpos no aglutinantes dirigidos hacia antígenos acultos de Brucella abortus cepa 19 / Detection in bovine serun of non-agglutinating antibodies to occult antigenos of Brucella abortus strain 19

Bovinos hembras de raza Hereford, inoculados con Brucella obortus cepa 19 en fase lisa, elaboraron anticuerpos no aglutinantes que se unieron específicamente a la cepa rugosa 45/20. Los sueros de estos animales, adsorbidos con B. abortus cepa 19 hasta eliminación de los anticuerpos dirigidos contra antígenos de superfície de la cepa lisa, mostraron contener una población no aglutinante de anticuerpos que se unieron a la cepa rugosa 45/20, siendo detectados mediante reacción de Cooms hasta un límite de 1,5 ug. Al margens de los anticuerpos incompletos producidos hacia antígenos externos de cepas lisas de B. abortus, se pone en evidencia este tipo de anticuerpos, con especificidad por antígenos más profundos que también estarían presentes en la superficie de la cepa rugosa. Estos anticuerpos, cuya participación en la depuración sanguínea y degradación del agente etiológico de la brucelosis bovina sería poco significativa, están siendo estudiados minuciosamente con el objeto de establecer la importancia que pudieran tener en el diagnóstico y pronóstico de brucelosis (AU)