Killing of Brucella abortus by bovine serum

Studies of the serum bactericidal system in bovine brucellosis were undertaken to investigate the role of the humoral immune response in protection of cattle against the facultative intracellular parasite Brucella abortus. Fresh sera from normal control cattle, infected cattle, and cattle immunized with B. abortus cell envelopes were collected before treatment and during the course of immunization or infection. Normal fresh bovine serum or fresh agammaglobulinemic serum from colostrum-deprived calves was effective in killing smooth virulent B. abortus 2308, but rough strains RB51 (a rough mutant of strain 2308) and 45/20 were much more sensitive to serum. The difference in susceptibility to serum was shown to be correlated with differences in lipopolysaccharide chemotype, with the more resistant strain 2308 having O polysaccharide and the more susceptible strains 45/20 and RB51 lacking O side chains. By treatment of fresh serum with MgCl2 and EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] killing was shown to occur via the classical pathway of complement activation. When antibody to B. abortus was present, killing of strain RB51 increased but killing of smooth strain 2308 decreased. The earliest antibody response in serum from infected animals did not interfere with killing. When affinity-purified bovine immunoglobulins specific for B. abortus smooth lipopolysaccharide were added to fresh normal bovine serum, immunoglobulin G1 (IgG1) and IgG2 isotypes blocked killing but IgM and IgA isotypes did not. Thus, it appears that serum from previously unexposed animals or animals early during infection can kill smooth B. abortus, an appropriate defense mechanism before the organism becomes intracellular. At later stages of infection, blocking antibodies predominate.     

Immune and pathologic responses in mice infected with Brucella abortus 19, RB51, or 2308.

Immune and pathologic responses were measured for 20 weeks after infection of mice with Brucella abortus 19, RB51, or 2308. Live bacteria and bacterial antigens of 19 and RB51 persisted in spleens for 10 and 4 weeks after infection, respectively, whereas 2308 bacteria and bacterial antigens persisted for at least 20 weeks. Small germinal centers and profound lymphoid depletion occurred in spleens of mice during the first 4 weeks of infection with strain 19 or 2308; however, mice infected with strain RB51 had much larger germinal centers but no lymphoid depletion. At 4 weeks, only spleen cells from RB51-infected mice proliferated when incubated with 2308 bacteria. Large germinal centers in the spleen and spleen cell proliferative responses to 2308 did not appear in strain 19-infected mice until 6 weeks or in strain 2308-infected mice until 10 weeks. Similar proliferative responses to 2308 occurred in mice infected with strain 19 or RB51 at 6 weeks and in mice infected with strain 19, RB51, or 2308 at 10 weeks. However, at 20 weeks, spleen cell proliferative responses to 2308 occurred in mice infected with strain 19 or 2308 but not in mice infected with strain RB51. Mice infected with strain RB51 had lower and less persistent antibody titers to 2308 than did mice infected with strain 19 or 2308. Collectively, these results indicate that RB51-infected mice have less persistent immune responses to 2308 than do mice infected with 19 or 2308. The shorter duration of the responses probably resulted because RB51 is considerably less pathogenic and is cleared more rapidly from mice than are 19 and 2308.     

Overexpression of protective antigen as a novel approach to enhance vaccine efficacy of Brucella abortus strain RB51

Brucella abortus strain RB51 is an attenuated rough strain that is currently being used as the official live vaccine for bovine brucellosis in the United States and several other countries. We reasoned that overexpression of a protective antigen(s) of B. abortus in strain RB51 should enhance its vaccine efficacy. To test this hypothesis, we overexpressed Cu/Zn superoxide dismutase (SOD) protein of B. abortus in strain RB51. This was accomplished by transforming strain RB51 with a broad-host-range plasmid, pBBR1MCS, containing the sodC gene along with its promoter. Strain RB51 overexpressing SOD (RB51SOD) was tested in BALB/c mice for its ability to protect against challenge infection with virulent strain 2308. Mice vaccinated with RB51SOD, but not RB51, developed antibodies and cell-mediated immune responses to Cu/Zn SOD. Strain RB51SOD vaccinated mice developed significantly (P < 0.05) more resistance to challenge than those vaccinated with strain RB51 alone. The presence of the plasmid alone in strain RB51 did not alter its vaccine efficacy. Also, overexpression of SOD did not alter the attenuation characteristic of strain RB51.     

Lymphocyte proliferation in response to Brucella abortus 2308 or RB51 antigens in mice infected with strain 2308, RB51, or 19.

Lymphocyte proliferation to 22 protein fractions (106 to 18 kDa) of Brucella abortus 2308 or the lipopolysaccharide O-antigen-deficient mutant of 2308, strain RB51, was measured for 20 weeks after infection of mice with strain 2308, RB51, or 19. Throughout the 20-week study, the 22 protein fractions of 2308 and RB51 induced a similar pattern of proliferation when they were incubated with lymphocytes from the infected mice. In addition, during the 20 weeks, lymphocytes from all groups of infected mice exhibited the highest proliferation when the lymphocytes were incubated with 18-kDa or smaller proteins from either 2308 or RB51. Lymphocytes obtained from mice at 6 weeks after infection with strain RB51 or 19 exhibited similar proliferation to the 18-kDa proteins of S2308 or SRB51. Lymphocytes from strain 2308-infected mice did not proliferate to these proteins until 10 weeks after infection, and the responses were similar to those in strain RB51-infected mice but lower than those in strain 19-infected mice. Lymphocytes obtained from mice at 20 weeks after infection with strain 19 or 2308 proliferated to most of the 22 fractions of 2308 or RB51, which contained 106- to 18-kDa proteins. However, lymphocytes obtained from strain RB51-infected mice at 20 weeks did not proliferate to any of these fractions. These results indicate that mice infected with RB51 have less-persistent lymphocyte proliferative responses to 2308 proteins than do mice infected with 2308 or 19. In addition, all 2308 proteins that stimulate lymphocyte proliferation appear to be present in RB51.     

Induction of antigen-specific Th1-type immune responses by gamma-irradiated recombinant Brucella abortus RB51

Brucella abortus strain RB51 is an attenuated rough mutant used as the live vaccine against bovine brucellosis in the United States and other countries. We previously reported the development of strain RB51 as a bacterial vaccine vector for inducing Th1-type immune responses against heterologous proteins. Because safety concerns may preclude the use of strain RB51-based recombinant live vaccines, we explored the ability of a gamma-irradiated recombinant RB51 strain to induce heterologous antigen-specific immune responses in BALB/c mice. Exposure of strain RB51G/LacZ expressing Escherichia coli beta-galactosidase to a minimum of 300 kilorads of gamma radiation resulted in complete loss of replicative ability. These bacteria, however, remained metabolically active and continued to synthesize beta-galactosidase. A single intraperitoneal inoculation of mice with 10(9) CFU equivalents of gamma-irradiated, but not heat-killed, RB51G/LacZ induced a beta-galactosidase-specific Th1-type immune response. Though no obvious differences were detected in immune responses to B. abortus-specific antigens, mice vaccinated with gamma-irradiated, but not heat-killed, RB51G/LacZ developed significant protection against challenge with virulent B. abortus. In vitro experiments indicated that gamma-irradiated and heat-killed RB51G/LacZ induced maturation of dendritic cells; however, stimulation with gamma-irradiated bacteria resulted in more interleukin-12 secretion. These results suggest that recombinant RB51 strains exposed to an appropriate minimum dose of gamma radiation are unable to replicate but retain their ability to stimulate Th1 immune responses against the heterologous antigens and confer protection against B. abortus challenge in mice.     

Vaccination with Brucella abortus rough mutant RB51 protects BALB/c mice against virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis.

Vaccination of BALB/c mice with live Brucella abortus RB51, a stable rough mutant, produced protection against challenge with virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis. Passive-transfer experiments indicated that vaccinated mice were protected against B. abortus 2308 through cell-mediated immunity, against B. ovis PA through humoral immunity, and against B. melitensis 16M through both forms of immunity. Live bacteria were required for the induction of protective cell-mediated immunity; vaccination with whole killed cells of strain RB51 failed to protect mice against B. abortus 2308 despite development of good delayed-type hypersensitivity reactions. Protective antibodies against the heterologous species were generated in vaccinated mice primarily through anamnestic responses following challenge infections. Growth of the antigenically unrelated bacterium Listeria monocytogenes in the spleens of vaccinated mice indicated that nonspecific killing by residual activated macrophages contributed minimally to protection. These results encourage the continued investigation of strain RB51 as an alternative vaccine against heterologous Brucella species. However, its usefulness against B. ovis would be limited if, as suggested here, epitopes critical for protective cell-mediated immunity are not shared between B. abortus and B. ovis.     

Complement fixation test to assess humoral immunity in cattle and sheep vaccinated with Brucella abortus RB51.

The live attenuated Brucella abortus strain RB51 is a rifampin-resistant, lipopolysaccharide (LPS) O-chain-deficient mutant of virulent B. abortus 2308. The reduced O-chain content in RB51 prevents this bacterium from inducing antibodies detectable by the conventional serologic tests for bovine brucellosis diagnosis that mainly identify antibodies to LPS. The absence of available serologic tests for RB51 also complicates the diagnosis of possible RB51 infections in humans exposed to this strain. The purpose of this study was to evaluate the suitability of a complement fixation (CF) test performed with the rough strain B. abortus RB51, previously deprived of anticomplementary activity, in detecting anti-B. abortus RB51 antibodies in cattle and sheep experimentally vaccinated with this strain. The results of this study showed that a CF test with RB51 as the antigen is able to specifically detect antibodies following RB51 vaccination in cattle and sheep. In addition, this method could be a useful tool for detecting B. abortus RB51 infection in humans.     

Immune responses and resistance to brucellosis in mice vaccinated orally with Brucella abortus RB51.

Immune responses and resistance to infection with Brucella abortus 2308 (S2308) were measured in mice following oral or intraperitoneal (i.p.) vaccination with strain RB51 (SRB51). Bacteria persisted in the parotid lymph node for 4 weeks following oral vaccination of mice with 5 x 10(8) or 5 x 10(6) CFU of SRB51. Bacteria did not appear in the spleen during 12 weeks after oral vaccination, whereas they did appear in the spleen for 8 weeks following i.p. vaccination of mice with SRB51 (5 x 10(8) or 5 x 10(6) CFU). Increased resistance to S2308 infection occurred at 12 to 20 weeks in mice vaccinated i.p. with SRB51 (5 x 10(8) or 5 x 10(6) CFU) but occurred at 12 weeks only in mice vaccinated orally with SRB51 (5 x 10(8) CFU). Oral SRB51 vaccination induced lower levels of antibodies to the surface antigens of intact SRB51 bacteria than did i.p. vaccination. However, neither route of vaccination induced anamnestic antibody responses to the surface antigens of intact S2308 bacteria after challenge infection of the vaccinated mice with S2308. Mice vaccinated orally with SRB51 and challenged with S2308 at 12 to 20 weeks had lower and less persistent spleen cell proliferation and production of gamma interferon in response to S2308 and certain immunodominant S2308 proteins (32 to < or = 18 kDa) than did mice vaccinated i.p. with SRB51. However, mice vaccinated orally or i.p. with SRB51 and challenged with S2308 had similar spleen cell tumor necrosis factor alpha production. These results indicate that oral vaccination of mice with SRB51 was effective in inducing protective immunity to S2308 infection, although the immunity was lower and less persistent than that induced by i.p. vaccination. The lower protective immunity induced by oral vaccination may have resulted from lower and less persistent cell-mediated immunity and gamma interferon production in response to S2308 and S2308 proteins.     

Complementation of Brucella abortus RB51 with a functional wboA gene results in O-antigen synthesis and enhanced vaccine efficacy but no change in rough phenotype and attenuation

Brucella abortus RB51 is a stable rough, attenuated mutant vaccine strain derived from the virulent strain 2308. Recently, we demonstrated that the wboA gene in RB51 is disrupted by an IS711 element (R. Vemulapalli, J. R. McQuiston, G. G. Schurig, N. Srirauganathan, S. M. Halling, and S. M. Boyle, Clin. Diagn. Lab. Immunol. 6:760-764, 1999). Disruption of the wboA gene in smooth, virulent B. abortus, Brucella melitensis, and Brucella suis results in rough, attenuated mutants which fail to produce the O polysaccharide (O antigen). In this study, we explored whether the wboA gene disruption is responsible for the rough phenotype of RB51. We complemented RB51 with a functional wboA gene, and the resulting strain was designated RB51WboA. Colony and Western blot analyses indicated that RB51WboA expressed the O antigen; immunoelectron microscopy revealed that the O antigen was present in the cytoplasm. Crystal violet staining, acryflavin agglutination, and polymyxin B sensitivity studies indicated that RB51WboA had rough phenotypic characteristics similar to those of RB51. Bacterial clearance studies of BALB/c mice indicated no increase in the survival ability of RB51WboA in vivo compared to that of RB51. Vaccination of mice with live RB51WboA induced antibodies to the O antigen which were predominantly of the immunoglobulin G2a (IgG2a) and IgG3 isotypes. After in vitro stimulation of splenocytes with killed bacterial cells, quantitation of gamma interferon in the culture supernatants indicated that RB51WboA immunization induced higher levels of gamma interferon than immunization with RB51. Mice vaccinated with RB51WboA were better protected against a challenge infection with the virulent strain 2308 than those vaccinated with RB51. These studies indicate that in addition to the disruption of the wboA gene there is at least one other mutation in RB51 responsible for its rough phenotype. These studies also suggest that the expressed O antigen in RB51WboA is responsible either directly or indirectly for the observed enhancement in the T-cell response.     

Role in virulence of a Brucella abortus protein exhibiting lectin-like activity

Brucella abortus is a facultative, intracellular zoonotic pathogen which can cause undulant fever in humans and abortions in cattle. A 14-kDa protein of B. abortus was previously identified to be immunogenic in animals infected with Brucella spp. In this study, we discovered that the 14-kDa protein possessed immunoglobulin binding and hemagglutination properties that appeared to be based on the protein's lectin-like properties. Hemagglutination inhibition experiments suggested that the 14-kDa protein has affinity towards mannose. Disruption of the gene encoding the 14-kDa protein in virulent B. abortus strain 2308 induced a rough-like phenotype with an altered smooth lipopolysaccharide (LPS) immunoblot profile and a significant reduction in the bacterium's ability to replicate in mouse spleens. However, the mutant strain was stably maintained in mouse spleens at 2.0 to 2.6 log(10) CFU/spleen from day 1 to week 6 after intraperitoneal inoculation with 4.65 log(10) CFU. In contrast to the case for the smooth virulent strain 2308, in the rough attenuated strain RB51 disruption of the 14-kDa protein's gene had no effect on the mouse clearance pattern. These findings indicate that the 14-kDa protein of B. abortus possesses lectin-like properties and is essential for the virulence of the species, probably because of its direct or indirect role in the synthesis of smooth LPS.     

Comparative protection of mice against virulent and attenuated strains of Brucella abortus by passive transfer of immune T cells or serum.

Passively transferred immune serum provided significantly greater protection to BALB/c mice against attenuated Brucella abortus 19 than against virulent strain 2308, whether serum donors had been infected with strain 19 or 2308. In contrast, immune T cells conferred better protection upon recipients challenged with the homologous strain of B. abortus. It is hypothesized that strain 2308, but not strain 19, can survive in macrophages after opsonization and that epitopes which induce protective cell-mediated immunity may differ between strains 19 and 2308.     

Validation of the abbreviated Brucella AMOS PCR as a rapid screening method for differentiation of Brucella abortus field strain isolates and the vaccine strains, 19 and RB51

The Brucella AMOS PCR assay was previously developed to identify and differentiate specific Brucella species. In this study, an abbreviated Brucella AMOS PCR test was evaluated to determine its accuracy in differentiating Brucella abortus into three categories: field strains, vaccine strain 19 (S19), and vaccine strain RB51/parent strain 2308 (S2308). Two hundred thirty-one isolates were identified and tested by the conventional biochemical tests and Brucella AMOS PCR. This included 120 isolates identified as B. abortus S19, 9 identified as B. abortus strain RB51, 57 identified as B. abortus biovar 1, 15 identified as B. abortus bv. 2, 1 identified as B. abortus bv. 2 (M antigen dominant), 7 identified as B. abortus bv. 4, and 22 identified as B. abortus S2308 and isolated from experimentally infected cattle. The Brucella AMOS PCR correctly identified each isolate as RB51/S2308, S19, or a field strain of Brucella.     

Comparison of spleen cell proliferation in response to Brucella abortus 2308 lipopolysaccharide or proteins in mice vaccinated with strain 19 or RB51.

Mice vaccinated with Brucella abortus 19 (S19) or RB51 (SRB51) had spleen cells which proliferated in response to proteins of 32, 27, 18, and < 18 kDa but not in response to proteins of 106, 80, and 49 kDa from B. abortus 2308 (S2308) following vaccination and challenge infection with S2308. Spleen cells from mice vaccinated with S19 but not with SRB51 had increased proliferation in response to S2308 lipopolysaccharide (LPS) following challenge infection with S2308. We previously reported that mice vaccinated with S19 or SRB51, which were analyzed in the current study, have increased resistance to infection with S2308 and that only mice vaccinated with S19 produce antibody to S2308 LPS (M. Stevens, S. Olsen, G. Pugh, Jr., and D. Brees, Infect. Immun. 63:264-270, 1995). The results from our current and previous studies support the contention that vaccination of mice with S19 or SRB51 induces protection from infection with S2308 by cell-mediated immune responses to the same immunodominant (32, 27, 18, and < 18 kDa) protein antigens of S2308. In addition, the absence of S2308 LPS-responsive spleen cells and antibody to S2308 LPS in mice vaccinated with SRB51 suggests that immune responses to LPS have no role in SRB51-induced protective immunity.     

A DNA vaccine encoding Cu,Zn superoxide dismutase of Brucella abortus induces protective immunity in BALB/c mice

This study was conducted to evaluate the immunogenicity and protective efficacy of a DNA vaccine encoding Brucella abortus Cu,Zn superoxide dismutase (SOD). Intramuscular injection of plasmid DNA carrying the SOD gene (pcDNA-SOD) into BALB/c mice elicited both humoral and cellular immune responses. Animals injected with pcDNA-SOD developed SOD-specific antibodies which exhibited a dominance of immunoglobulin G2a (IgG2a) over IgG1. In addition, the DNA vaccine elicited a T-cell-proliferative response and also induced the production of gamma interferon, but not interleukin-10 (IL-10) or IL-4, upon restimulation with either recombinant SOD or crude Brucella protein, suggesting the induction of a typical T-helper-1-dominated immune response in mice. The pcDNA-SOD (but not the control vector) induced a strong, significant level of protection in BALB/c mice against challenge with B. abortus virulent strain 2308; the level of protection was similar to the one induced by B. abortus vaccine strain RB51. Altogether, these data suggest that pcDNA-SOD is a good candidate for use in future studies of vaccination against brucellosis.     

The role of integrase/recombinase xerD and monofunctional biosynthesis peptidoglycan transglycosylase genes in the pathogenicity of Brucella abortus infection in vitro and in vivo

Brucella abortus clones identified previously using a green fluorescence protein reporter system after 4h macrophage infection provided insight regarding possible genes involved in early host-pathogen interaction. Among identified genes were an integrase/recombinase (xerD) gene involved in cell division, and a monofunctional biosynthesis peptidoglycan transglycosylase (mtgA) gene that catalyzes the final stages of the peptidoglycan membrane synthesis. Here, we evaluate the in vitro and in vivo survival of B. abortus xerD and mtgA insertional mutants. B. abortus xerD::kan and B. abortus mtgA::kan demonstrated no significant growth defects in broth culture when compared to the parental strain, S2308. Also, neither gene was required for B. abortus S2308 replication in RAW 264.7 macrophages. However, experimental evidence using interferon regulatory factor 1 knockout mice, a mouse strain highly susceptible to virulent Brucella, revealed that mice infected with B. abortus xerD::kan or B. abortus mtgA::kan survived longer than mice infected with S2308. Additionally, in immunocompetent BALB/c mice, B. abortus xerD::kan had a significantly lower level of bacterial survival when compared to S2308. Together, these results suggest that B. abortus xerD and mtgA genes play a role during the initial phase of infection in mice.     

Characterization of specific immune responses of mice inoculated with recombinant vaccinia virus expressing an 18-kilodalton outer membrane protein of Brucella abortus

Using the shuttle vector pMCO2 and the vaccinia virus wild-type WR strain, we constructed a recombinant virus expressing an 18-kDa outer membrane protein of Brucella abortus. BALB/c mice inoculated with this virus produced 18-kDa protein-specific antibodies, mostly of immunoglobulin G2a isotype, and in vitro stimulation of splenocytes from these mice with purified maltose binding protein-18-kDa protein fusion resulted in lymphocyte proliferation and gamma interferon production. However, these mice were not protected against a challenge with the virulent strain B. abortus 2308. Disruption of the 18-kDa protein's gene in vaccine strain B. abortus RB51 did not affect either the strain's protective capabilities or its in vivo attenuation characteristics. These observations suggest that the 18-kDa protein plays no role in protective immunity.     

Interleukin-10 downregulates protective immunity to Brucella abortus.

In vivo neutralization of interleukin-10 (IL-10) with an anti-IL-10 monoclonal antibody resulted in up to 10-fold fewer bacteria in the spleens of BALB/c mice infected with the virulent Brucella abortus strain 2308. In vitro neutralization of endogenous IL-10 in brucella antigen-stimulated cultures of splenocytes from infected mice resulted in increased gamma interferon production in these cultures, whereas exogenous recombinant IL-10 inhibited the ability of peptone-elicited peritoneal macrophages to control intracellular brucellae. These data suggest that IL-10 may be downregulating the immune response to B. abortus by affecting both macrophage effector function and the production of the protective Th1 cytokine gamma interferon.     

Protective properties of rifampin-resistant rough mutants of Brucella melitensis

Vaccination against Brucella infections in animals is usually performed by administration of live attenuated smooth B. abortus strain S19 and B. melitensis strain Rev1. They are proven effective vaccines against B. abortus in cattle and against B. melitensis and B. ovis in sheep and goats, respectively. However, both vaccines have the main drawback of inducing O-polysaccharide-specific antibodies that interfere with serologic diagnosis of disease. In addition, they retain residual virulence, being a cause of abortion in pregnant animals and infection in humans. To overcome these problems, one approach is to develop defined rough mutant Brucella strains lacking O antigen of lipopolysaccharide. B. abortus rough strain RB51, a rifampin-resistant mutant of virulent strain B. abortus 2308, is used as a vaccine against B. abortus infection in cattle in some countries. However, RB51 is not effective in sheep, and there is only preliminary evidence that it is effective in goats. In this study, we tested the efficacies of six rifampin-resistant rough strains of B. melitensis in protecting BALB/c mice exposed to B. melitensis infection. The protective properties, as well as both humoral and cellular immune responses, were assessed in comparison with those provided by B. melitensis Rev1 and B. abortus RB51 vaccines. The results indicated that these rough mutants were able to induce a very good level of protection against B. melitensis infection, similar to that provided by Rev1 and superior to that of RB51, without inducing antibodies to O antigen. In addition, all B. melitensis mutants were able to stimulate good production of gamma interferon. The characteristics of these strains encourage further evaluation of them as alternative vaccines to Rev1 in primary host species.     

Comparison of immune responses and resistance to brucellosis in mice vaccinated with Brucella abortus 19 or RB51.

Immune responses and resistance to infection with Brucella abortus 2308 (S2308) were measured in mice following vaccination with B. abortus 19 (S19) or the lipopolysaccharide (LPS) O-antigen-deficient mutant, strain RB51 (SRB51). Live bacteria persisted for 8 weeks in spleens of mice vaccinated with 5 x 10(6) or 5 x 10(8) CFU of SRB51, whereas bacteria persisted for 12 weeks in mice vaccinated with 5 x 10(6) CFU of S19. Mice vaccinated with 5 x 10(6) or 5 x 10(8) CFU of SRB51 had increased resistance to infection with S2308 at 12, 16, and 20 weeks after vaccination, but the resistance was lower than that induced by vaccinating mice with 5 x 10(6) CFU of S19. Spleen cells obtained from mice vaccinated with S19 or SRB51 generally exhibited similar proliferative responses to S2308 bacteria or bacterial proteins (106 to 18 kDa) following challenge of mice with S2308 at 12, 16, or 20 weeks after vaccination. Mice vaccinated with S19 had antibody to S2308 bacteria and S2308 smooth LPS at 4, 8, and 12 weeks after vaccination. In contrast, mice vaccinated with either dose of SRB51 did not produce antibody to S2308 smooth LPS. In addition, only mice vaccinated with the highest dose of SRB51 (5 x 10(8) CFU) had antibody responses to S2308 bacteria, although the responses were lower and less persistent than those in mice vaccinated with S19. Collectively, these results indicate that SRB51-vaccinated mice have similar cell-mediated immune responses to S2308 but lower resistance to infection with S2308 compared with S19-vaccinated mice. The lower resistance in SRB51-vaccinated mice probably resulted from a combination of rapid clearance of SRB51 and an absence of antibodies to S2308 LPS.     

Lymphocyte proliferation in response to Brucella abortus RB51 and 2308 proteins in RB51-vaccinated or 2308-infected cattle.

Cattle vaccinated with Brucella abortus strain RB51 (SRB51) or infected with strain 2308 (S2308) had lymph node lymphocytes which proliferated most when incubated with 32-, 27-, 18-, or <18-kDa proteins of either SRB51 or S2308. Some S2308-infected cattle but no SRB51-vaccinated cattle had lymphocytes which proliferated in response to 80- and 49-kDa proteins of SRB51 and S2308. These results suggest that cattle vaccinated with SRB51 or infected with S2308 have lymphocytes which proliferate in response to most of the same S2308 proteins and that the immunodominant protein antigens of SRB51 and S2308 have similar molecular masses of 32, 27, 18, and <18 kDa.