Protection of mice against Brucella abortus infection by inoculation with monoclonal antibodies recognizing Brucella O-antigen

Monoclonal antibodies recognizing the O-polysaccharide portion of Brucella abortus strain 2308 provided BALB/c mice with passive protection against challenge exposure with the homologous strain. Numbers of colony-forming organisms in the spleen were reduced by IgM and IgG monoclonal antibodies. Active immunization of mice, using B abortus 2308S lipopolysaccharide, resulted in production of IgM antibody at 14 days. Clearance of organisms in the actively immunized mice after challenge exposure at 14 days was nearly identical to that in passively immunized mice. Mice either passively or actively immunized were effectively protected from 0 to 28 days. Bacterial colonization of the spleen was observed to increase in both groups of mice at 56 days and indicated that humoral responses were effective in eliminating the organism in the early stages of infection, but other immune mechanisms were necessary for protection of mice in the later stage of infection with virulent strains of B abortus.     

Regulated upon activation normal T-cell expressed and secreted (RANTES) contributes to abortion caused by Brucella abortus infection in pregnant mice

Brucella abortus (B. abortus) is a facultative intracellular pathogen that can survive inside macrophages and trophoblast giant cells, and the causative agent of brucellosis. In the present study, we found that production of regulated upon activation normal T-cell expressed and secreted (RANTES) due to B. abortus infection contributes to abortion in pregnant mice. B. abortus infected pregnant interferon-gamma (IFN-gamma) knockout mice died within 15 days of infection, but non-pregnant IFN-gamma knockout mice were still alive. With infection by wild type B. abortus, a large amount of RANTES production was observed in pregnant IFN-gamma knockout mice, and induction of RANTES was also observed in normal pregnant mice infected with the wild type, but not in those infected with the intracellular replication-defective mutant. Production of RANTES and IFN-gamma were inhibited in mice inoculated with the respective RANTES or IFN-gamma antibody. Neutralization of RANTES, induced by B. abortus infection, served to prevent abortion. These results indicate that the production and function of RANTES are correlated with IFN-gamma in pregnant mice infected with B. abortus.     

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.     

Protective activity to murine experimental brucellosis conferred by monoclonal antibody ISS/32 anti-B. abortus.

The protective properties of the monoclonal antibody ISS/32 anti-B. abortus were estimated by splenic infection with B. abortus 544. Five groups of Balb/c mice were used: two groups, previously vaccinated with a 45/20 antigen and a-LPS antigen, were challenged after 30 days intravenously by inoculation of 2.10(5) cells of B. abortus 544, one group was challenged with the same dose of B. abortus 544 preincubated with MAb-ISS/32 and another one with B. abortus 544 incubated with negative serum; the fifth group infected with B. abortus 544 only served as control. The results, expressed as an index of splenic infection, show significant protective properties of monoclonal antibody ISS/32. The infection index in the MAb-ISS/32 group of mice was a bit lower than in B. abortus 45/20 vaccine group.     

Bovine monoclonal antibody specific for Brucella abortus lipopolysaccharide

The development of a bovine monoclonal antibody against Brucella abortus smooth lipopolysaccharide (BM-8) by interspecies fusion of bovine peripheral lymphocytes from an immunized cow and a murine plasmacytoma cell line is described. The twice cloned cell line secreted bovine IgG1 subclass antibody. Ascites fluid was prepared in pristane treated nu/nu mice by intraperitoneal injection. The pooled ascites fluid was purified by affinity chromatography and the functions of the antibody assessed in various serological tests. The BM-8 antibody did not agglutinate well at a neutral pH, however, under acid conditions it was efficient at agglutinating B. abortus cells. The antibody did not precipitate B. abortus LPS in double agar gel immunodiffusion but was very active in the direct complement fixation test and the indirect enzyme immunoassay, although it was unable to compete with a murine monoclonal antibody in a competitive enzyme immunoassay.     

Variations in immunoglobulin isotype produced during the antibody response to Brucella abortus and Staphylococcus aureus vaccines in sheep

Experiments in sheep were carried out to examine factors modifying the immunoglobulin (Ig) isotype of the antibody response to Brucella abortus. Live B abortus (S19) stimulated higher titres of agglutinating antibody and IgG1 and IgG2 antibody than did killed B abortus. Live B abortus stimulated a more protracted synthesis of IgG2 antibody during the primary and secondary responses than did the killed S19 vaccine. In a second experiment, the capacity of live and killed Staphylococcus aureus to modify the antibody response to killed B abortus was examined. Both live and killed S aureus enhanced production of anti-brucella antibodies; this response was attributed to the adjuvant properties of S aureus. Killed S aureus enhanced production of anti-brucella antibody to a greater extent than live S aureus. Live S aureus did not preferentially enhance production of IgG2 anti-brucella antibody. The results suggested that the enhanced production of IgG2 antibody induced by live vaccines does not depend solely on a pyogenic lesion at the vaccination site.     

Monoclonal antibodies directed against Brucella abortus cell surface antigens

A panel of monoclonal antibodies (MAb) has been raised against Brucella abortus cell surface antigens from mice immunized with either heat/phenol treated or UV killed bacterial suspensions of B. abortus. The hybridomas were screened by either a microagglutination procedure or by an indirect enzyme immunoassay (EIA) on sonicated bacterial preparations. From a large number of MAb generated by various procedures, two distinct types of MAb emerged. The most numerous type was capable of agglutinating B. abortus and reacting with a soluble preparation of lipopolysaccharide (LPS). A second type was not capable of agglutinating the bacterial suspensions or of binding to the soluble LPS preparation but reacted with an antigen present in bacterial sonicates. Two MAb of this type react differentially with sonicates prepared from virulent and avirulent strains of B. abortus. There appeared to be sufficient evidence from our analysis of the relative degree of cross reaction with antigens present on a range of B. abortus strains and Brucella and xenogenic bacterial species to conclude that each of the seven MAb was recognising a separate antigenic site on the B. abortus cell surface.     

Temporal cell-mediated immune responses of cattle following experimental and natural exposure to living Brucella abortus.

A study on cell-mediated immune responses in cattle with different exposure experiences to Brucella abortus was conducted by an in vitro lymphocyte stimulation assay. The purpose of this study was to determine how soon the cell-mediated immune responses would be detected following experimental exposure to B. abortus and to study the cell-mediated immune trend following experimental and natural exposure of cattle to B. abortus. The first positive cell-mediated immune responses occurred one to two weeks after experimental inoculation with living B. abortus strain 2308. The cell-mediated immune responses in these animals appeared at least one week before the appearance of of B. abortus serum agglutinating antibodies. Animals which were naturally infected with B. abortus biotypes 1 and 2 demonstrated positive cell-mediated immune responses throughout the study.     

The bovine immune response to Brucella abortus I. A water soluble antigen precipitated by sera of some naturally infected cattle.

Selected sera from cattle naturally infected with Brucella abortus precipitate water soluble antigens extracted by sonication from B. abortus. One of these antigens resembles antigen E (Baughn and Freeman) as it is excluded from Sephadex G-200 gels, migrates anodally when electrophoresed at pH 8.6, resists heating at 100 degrees C for ten minutes and appears to be susceptible to papain digestion. Precipitins specific for this antigen remained in sera from which all detectable Brucella agglutinating antibody had been removed by adsorption with live or heat killed B. abortus. The antigen has been extracted from smooth and rough strains of B abortus. Precipitins specific for this antigen have been detected in antisera produced against Brucella canis.     

Enzyme-linked immunosorbent assay for differentiation of the antibody response of cattle naturally infected with Brucella abortus or vaccinated with strain 19

Purified O chain of Brucella abortus was passively attached to polystyrene to differentiate antibody responses of cattle vaccinated with B abortus strain 19 from those of naturally infected cattle. In the indirect assay, using O polysaccharide as antigen, a single serum dilution was used and mouse monoclonal antibody to bovine L chain conjugated with horseradish peroxidase was the detection reagent. Measurable antibody was not found in sera of vaccinated cattle, except for 3 sera from cattle that were persistently infected with strain 19. Sera from 25 cattle infected with pathogenic strains contained antibody on the basis of results of indirect enzyme immunoassay, using smooth lipopolysaccharide or O chain as antigens, or results of competitive enzyme immunoassay, using the O-chain antigen. Results in sera from calves with experimentally induced Yersinia enterocolitica serotype 0:9 infection or inoculated with a low dose of B abortus strain 2308 were comparable with those in sera of cattle that were vaccinated with strain 19. The data correlated with those from competitive enzyme immunoassay, using one serum dilution and horseradish peroxidase-conjugated mouse monoclonal antibody to smooth lipopolysaccharide. On the basis of results of the indirect enzyme immunoassay, all sera (except those samples obtained before inoculation) contained antibody to smooth lipopolysaccharide.     

Immunological responses of fluke-infected and fluke-free cattle to Salmonella dublin and other antigens.

Immune responses to heat-killed Brucella abortus strain 19 and to ovalbumin were compared in 15 fluke-infected and 15 fluke-free Friesian heifers. B abortus was injected 16 weeks and ovalbumin 19 weeks after the oral administration of 1000 metacercariae of Fasciola hepatica. Agglutinating antibody responses to B abortus were similar in both groups. Immediate type hypersensitivity to ovalbumin was apparently suppressed in fluke-infected animals when assessed by active and passive cutaneous anaphylaxis two weeks after sensitisation. However, when assessed by Schultz-Dale responses of intestine, in vitro, 36 weeks after sensitisation there was no difference between the groups. The heifers were subsequently given live Salmonella dublin intravenously. The fluke-infected animals which became carriers of S dublin had the most persistently elevated titres of agglutinating antibodies in their sera and the highest incidence of immediate-type hypersensitivity, as assessed by Schultz-Dale responses of intestine, but the weakest cutaneous delayed hypersensitivity reactions to S dublin. The latter might have been related to lymphopenia which developed after fluke infection. The increased susceptibility of fluke-infected cattle to S dublin cannot be attributed to impaired agglutinin responses but may result from effects on cell-mediated mechanisms.     

Duration of strain 2308 infection and immunogenicity of Brucella abortus lipopolysaccharide in five strains of mice

A study was conducted to compare immunogenicity of a Brucella abortus lipopolysaccharide (LPS) and the duration of infection in 5 strains of mice. Mice of strains CBA/NJ, BALB/c, CD-1, C3H/HeN, and C3H/HeJ were allotted into 2 large groups (vaccinated with proteinase K-treated LPS or nonvaccinated) and 6 subgroups based on the intervals between challenge exposure to B abortus strain 2308 and the week the response data were obtained. Criteria used in comparing responses between the various strains of mice as well as between vaccinated and nonvaccinated mice were splenomegaly, colony-forming units (CFU) from spleens, and antibody titers. Responses were evaluated at 1, 2, 3, 5, 8, and 12 weeks after challenge exposure. Results indicated that all strains of mice became infected and maintained infection throughout the 12-week period, the percentages of mice infected were significantly (P less than 0.05) less in vaccinated mice for the first 5 weeks after challenge exposure, and there were no direct correlations between increased immunoglobulins (IgM and IgG titers) and reduction in CFU. Vaccinated mice of strains BALB/c, CD-1, C3H/HeN, and C3H/HeJ had increased titers when challenge exposed and also had significantly (P less than 0.05) smaller spleens and lower CFU. Vaccinated CBA/NJ mice did not have marked antibody titers. The overall results indicated that vaccination with LPS offers some initial protection against B abortus strain 2308 infection, but this protection disappears gradually and in various degrees in the 5 strains of mice studied.     

Response of the badger (Meles meles) to infection with Brucella abortus

Three badgers exposed to conjunctival instillation of Brucella abortus strain 544 developed a mild subclinical infection accompanied by the production of agglutinating and complement fixing antibodies. Six months after inoculation, the infection could not be detected in one badger and was confined to lymphoid tissue in the other two. No cell mediated immune response to B abortus antigens could be detected by in vitro lymphocyte transformation tests or intradermal tests for delayed hypersensitivity during the observation period.     

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.     

Cellular and humoral responses of Brucella abortus-infected bovine fetuses

Fifty-nine bovine fetuses naturally and experimentally infected with Brucella abortus were studied. Lymphoid hyperplasia in multiple lymph nodes, lymphoid depletion in the thymic cortex, adrenal cortical hyperplasia, and disseminated inflammatory foci composed mainly of large mononuclear leukocytes were present in infected fetuses. Histopathologic changes in naturally infected fetuses were indistinguishable from those infected fetuses inoculated in utero. Fetuses inoculated with 1.0 X 10(3) to 1.0 X 10(5) colony-forming units of strain 2308 B abortus were aborted on postinoculation day (PID) 7 to 19. Fetuses obtained by PID 9 and 10 had increased immunoglobulin concentrations and antibody. Increased cortisol values were present in fetuses obtained as early as PID 6. The initial fetal inflammatory response was composed of large mononuclear leukocytes. In fetuses obtained by PID 9 to 10, moderate numbers of neutrophils mixed with mononuclear leukocytes were present in the inflammatory foci. This shift in the initial inflammatory reaction coincided with the appearance of agglutinating antibody.     

Detection of antigenic fractions from Brucella abortus S45/20 which bind to non-agglutinating antibodies using electroblotting and enzyme-linked antibody probes

Outer membrane antigens which bind to non-agglutinating antibodies (NAAb) elicited by smooth (S19) and rough (S45/20) Brucella abortus strains, were extracted from S45/20 by stirring in cold 2.5% NaCl and then analyzed by SDS-PAGE, electroblotting and enzyme-linked antibody test. Eight bands were observed in the gel stained with Coomassie blue. Seven antigenic fractions were transferred to nitrocellulose by blotting. A 27-kd band was recognized by bovine anti-S45/20 non-agglutinating serum and not by purified NAAb against surface antigens. Bands 10 kd and 14.3 kd bound to bovine anti-S45/20 NAAb from calves immunized with either S19 or S45/20. A 12.0-kd band was recognized by the serum and NAAb from calves immunized with S45/20 but not by those injected with S19. There are thus antigenic fractions shared by S19 and S45/20 which bind in vitro to NAAb.     

The serological response of cattle immunized with Yersinia enterocolitica O:9 or O:16 to Yersinia and Brucella abortus antigens in enzyme immunoassays

Sera from calves immunized with Yersinia enterocolitica serotypes O:9 or O:16 were tested by indirect enzyme linked immunosorbent assay (ELISA) using lipopolysaccharide (LPS) preparations from Brucella abortus or Y. enterocolitica O:9 or O:16 for their antibody content of the IgG1 or IgG2 subclasses. High IgG1 responses were present with the three antigens in both groups although some individual variations between animals were noted. The IgG2 responses were modest and in some cases not above background 'noise'. Thus IgG2 antibody was not measurable in sera from serotype O:9 injected calves when using serotype O:16 LPS or in serotype O:16 injected calves when using B. abortus or serotype O:9 LPSs. A competitive ELISA using B. abortus O-polysaccharide and a monoclonal antibody to B. abortus LPS (initially designed to differentiate the antibody responses of cattle naturally infected with B. abortus from those vaccinated with strain 19) was used on sera from both groups of calves. Using this test, no antibody was detected in the group immunized with serotype O:16 and except for one animal in the serotype O:9 immunized group, only low levels of antibody were transiently in evidence. One animal in this group responded with quite high levels of competing antibody which, however, declined towards the end of the test period. The competitive ELISA may prove a useful serological tool for differentiating vaccinal and field infection titers to B. abortus and also to eliminate cross-reactions observed with Y. enterocolitica serotypes.     

Characterization of a murine model of intranasal infection suitable for testing vaccines against C. abortus

Mouse models have been widely used to test candidate vaccines against Chlamydophila abortus infection in mice. Although the induction of a systemic infection by endogenous or intraperitoneal inoculation is a useful tool for understanding the immune mechanism involved in the protection conferred by the vaccination, a different approach is necessary to understand other factors of the infection, such as mucosal immunity or the colonization of target organs. To test whether C. abortus intranasal model of infection in mice is a useful tool for testing vaccines in a first group of experiments mice, were infected intranasally with C. abortus to characterize the model of infection. When this model was used to test vaccines, two inactivated experimental vaccines, one of them adjuvated with QS-21 and another with aluminium hydroxide, and a live attenuated vaccine (strain 1B) were used. Non-vaccinated control mice died within the first 8 days, after displaying substantial loss of weight. Histologically, the mice showed lobar fibrinopurulent bronchointerstitial pneumonia. Prior immunization with QS-21 adjuvated vaccine or 1B vaccine presented mortality and the recipients showed a greater number of T cells in the lesions, especially CD8(+) T cells, than the control mice and mice immunized with vaccine adjuvated with aluminium hydroxide. The results confirm that the C. abortus intranasal model of infection in mice is a useful tool for testing vaccines.     

Cleavage of bovine immunoglobulin G1 in whey by an extracellular material from Brucella abortus.

Culture extracts of in vitro grown Brucella abortus were demonstrated to cleave a part of the Fc portion of bovine immunoglobulin G1 in whey but not in serum or as a purified protein from serum. Supernates from Strains 19 and 2308 of B. abortus were both capable of this hydrolysis whereas living cells were not. The cleavage process was independent of antibody activity to B. abortus, appeared to require factor(s) found only in some whey samples and was ineffective with the other bovine immunoglobulins.     

Persistent plaque formation in experimental murine brucellosis

Primary plaque forming cells (PFC) are present in spleens of mice 150 days or more following an infection with Brucella abortus. The development of primary plaques in mice long after antigenic challenge is an uncommon phenomenon, unlike the plaque formation (PF) induced by a non-living antigen. The mechanism of this persistent PF has been now investigated in light of a prolonged persistence of the corresponding antigen in tissues. Living E. coli, inoculated in massive dose into mice, survived in their organs for a brief time, while concomitantly PFC disappeared by day sixteen. Infection with B. abortus, in contrast, induced persistent presence of bacteria in the organs of inoculated mice and stimulated long lasting plaque formation. Only direct plaques were found during all stages of infection. Repeated inoculations of dead B. abortus also induced continuous production of primary plaques, whereas an interval in supply of the antigen resulted in disappearance of PFC. Rifampin (40 mg/kg) eliminated bacteria from the treated mice, which resulted in the disappearance of primary PFC. It seems likely that long lasting PF in B. abortus infected mice is connected with a constant antigenic stimulus operating in the carrier state.