Antibody-mediated protection against Brucella abortus in BALB/c mice at successive periods after infection: variation between virulent strain 2308 and attenuated vaccine strain 19.

In BALB/c mice antibodies specific for the O polysaccharide (OPS) as well as T lymphocytes mediate protective immunity to Brucella abortus. We performed quantitative analyses of isotypes of OPS antibodies generated during primary infections, and tested the protective qualities of antisera at successive stages of infection against B. abortus strain 2308, representative of the wild type, and attenuated vaccine strain 19. IgM antibodies predominated during the first 3-4 weeks of infection. IgG3 antibodies increased slowly for the first 3 weeks but then rose rapidly and persisted at high levels (> 300 micrograms/ml). IgG1, IgG2a and IgG2b antibodies had increased slightly by week 4 and then remained at low to moderate levels (< 70 micrograms/ml). Week 2 serum pools (IgM high, IgG3 low or undetectable) transferred substantial protection against 2308 (> or = 1 log unit) which increased relatively little (to 1.2-1.5 log units) with later sera that were high in IgG antibodies. In contrast, week 2 sera conferred low levels of protection against 19 (< 0.6 log units), but protection was dramatically increased (to > or = 2.3 log units) with sera obtained 1 week later that had slightly increased IgG antibodies. Monoclonal IgM antibodies also provided better protection against 2308 than 19, while monoclonal IgG3 antibodies protected much better against 19. Strain 19 opsonized with antibodies taken at any stage of infection was killed within normal macrophages, whereas comparably opsonized 2308 underwent intracellular replication. Phagocytosis of 2308 was better than of 19 when brucellae were opsonized with either polyclonal IgM or IgG3 antibodies, and the difference between strains was more extreme following IgM opsonization. The data suggest an explanation for differences in the growth curves of 2308 and 19 in spleens of BALB/c mice. Higher numbers achieved by 19 at week 2 could result from extracellular replication owing to ineffectual opsonization by IgM antibodies, while the precipitous decline of 19 beginning at week 3 could be caused by the increase in more effective IgG3 opsonins that facilitate its rapid intracellular destruction.     

Alteration of protective and serologic responses in BALB/c mice vaccinated with chemically modified versus nonmodified proteins of Brucella abortus 19.

A study was conducted to determine whether the covalent chemical modification of Brucella abortus 19 salt-extractable proteins (BCSP) and BCSP derivatives would modulate the immune responses in BALB/c mice. Salt-extractable proteins BCSP 0-70 and BCSP 70-100 were modified with acetoacetic anhydride, and recombinant proteins rBCSP20 (20 kDa), rBCSP31 (31 kDa), and rBCSP45 (45 kDa) were modified with succinic and dodecanoyl anhydrides. Four weeks after mice were vaccinated with the different preparations, principal and control mice were challenge exposed with a virulent culture of B. abortus 2308, and mice were necropsied 2 weeks later. Serum samples were obtained immediately before mice were challenge exposed and at necropsy. Sera were tested for specific immunoglobulin M (IgM) and G (IgG) antibodies by using an enzyme-linked immunosorbent assay. Acylation decreased the immune responses (increased IgG antibodies and reduced spleen CFU and splenomegaly) induced by both BCSP 0-70 and BCSP 70-100. Modification of the recombinant proteins by dodecanoyl and succinic anhydrides had no effect on the protection induced; however, the IgG serologic responses to the homologous and heterologous proteins were altered. Monophosphoryl lipid A markedly enhanced the immunogenicity of BCSP 0-70.     

Aerosol infection of BALB/c mice with Brucella melitensis and Brucella abortus and protective efficacy against aerosol challenge

Brucellosis is a zoonotic disease with a worldwide distribution that can be transmitted via intentional or accidental aerosol exposure. In order to engineer superior vaccine strains against Brucella species for use in animals as well as in humans, the possibility of challenge infection via aerosol needs to be considered to properly evaluate vaccine efficacy. In this study, we assessed the use of an aerosol chamber to infect deep lung tissue of mice to elicit systemic infections with either Brucella abortus or B. melitensis at various doses. The results reveal that B. abortus causes a chronic infection of lung tissue in BALB/c mice and peripheral organs at low doses. In contrast, B. melitensis infection diminishes more rapidly, and higher infectious doses are required to obtain infection rates in animals similar to those of B. abortus. Whether this difference translates to severity of human infection remains to be elucidated. Despite these differences, unmarked deletion mutants BAΔasp24 and BMΔasp24 consistently confer superior protection to mice against homologous and heterologous aerosol challenge infection and should be considered viable candidates as vaccine strains against brucellosis.     

Comparison of living and nonliving vaccines for Brucella abortus in BALB/c mice.

The BALB/c mouse was selected as a model for infection with Brucella abortus on the basis of protracted nonclinical infection produced by strain 2308, virulent for cattle, and relatively rapid clearance of strain 19, an attenuated strain used to vaccinate cattle. Protection in mice vaccinated with strain 19 was compared with that obtained with nonliving vaccines at early (1 week) and later (4 weeks) intervals after challenge with strain 2308 and assessed by enumeration of B. abortus organisms in the spleen. Mice challenged 4 weeks after vaccination with strain 19 exhibited significant protection at 1 and 4 weeks postinfection (p.i.), with an increased magnitude of protection at the later time. When challenged 6 weeks after vaccination with strain 19, the level of protection diminished between 1 and 4 weeks p.i. and at the later time was not always significantly different from controls. Mice immunized 4 weeks earlier with nonliving vaccines in mineral oil with t trehalose dimycolate (TDM) and muramyl dipeptide (MDP) demonstrated patterns of protection similar to those obtained following the 6 week vaccination-challenge interval with strain 19. Vaccination with cell envelopes derived from strain 2308 produced equivalent protection at 1 week p.i. whether administered in phosphate-buffered saline, incomplete Freund adjuvant, or the TDM and MDP adjuvant. Equivalent protection also followed vaccination with strain 2308 killed whole cells, cell envelopes, or outer membrane proteins in phosphate-buffered saline or in the TDM and MDP adjuvant. The TDM and MDP adjuvant alone induced nonspecific resistance, which peaked at 1 day p.i. and was still present at 1 week p.i., although by this time its magnitude was significantly less than the protection induced by antigen combined with the adjuvant. These data, together with the results of antibody assays and passive and adoptive transfer studies, suggested that protection at 1 week p.i. could be accounted for largely by an effect of O antibodies, with T cell-mediated immune responses having a subsidiary role.     

Brucella abortus requires the heme transporter BhuA for maintenance of chronic infection in BALB/c mice

The gene annotated BAB2_1150 in the Brucella abortus 2308 genome sequence is predicted to encode a homolog of the well-characterized heme transporter ShuA of Shigella dysenteriae and accordingly has been given the designation bhuA (Brucella heme utilization). Phenotypic analysis of an isogenic bhuA mutant derived from B. abortus 2308 verified that there is a link between BhuA and the ability of the parent strain to use heme as an iron source in in vitro assays. Maximum expression of bhuA in B. abortus 2308 is observed during stationary phase when this strain in cultivated in low-iron minimal medium, and a comparison of the growth characteristics of the B. abortus bhuA mutant and 2308 in this medium suggested that heme serves as an important iron source for the parent strain during stationary phase. The B. abortus bhuA mutant HR1703 exhibits significant attenuation in cultured murine macrophages compared to strain 2308, and unlike its parent strain, the B. abortus bhuA mutant is unable to maintain a chronic spleen infection in experimentally infected BALB/c mice. These experimental findings suggest that heme and/or heme-containing proteins represent important iron sources for B. abortus 2308 during its residence in the mammalian host and that BhuA is required for efficient utilization of these iron sources.     

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.     

Brucella abortus deficient in copper/zinc superoxide dismutase is virulent in BALB/c mice.

The gene encoding the Cu/Zn superoxide dismutase (SOD) of Brucella abortus strain 2308 was identified in a Brucella genomic library utilizing a combination of Western blotting and native gel electrophoresis. The Cu/Zn SOD gene was inactivated in vitro by ligation of a kanamycin resistance gene into the open reading frame encoding SOD. The plasmid born construct was introduced back into B. abortus by electroporation. Replacement of the wild-type Cu/Zn SOD by recombination was demonstrated by showing that both the KnR gene and the Cu/Zn SOD gene hybridized to the same band in a Southern analysis of genomic DNA. In addition, KnR strains were deficient in Cu/Zn SOD activity as assessed by lack of Cu/Zn SOD activity on a native gel and by lack of reactivity with specific serum in a Western analysis. Either strain 2308 or the Cu/Zn SOD deficient mutant injected intraperitoneally into BALB/c mice, exhibited no differences in their ability to colonize the spleen at 7 and 28 days post-inoculation. Thus, the inability to produce Cu/Zn SOD by B. abortus does not significantly impair its virulence in mice.     

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.     

Intact purine biosynthesis pathways are required for wild-type virulence of Brucella abortus 2308 in the BALB/c mouse model

Brucella abortus 2308 derivatives with mini-Tn5 insertions in purE, purL, and purD display significant attenuation in the BALB/c mouse model, while isogenic mutants with mini-Tn5 insertions in pheA, trpB, and dagA display little or no attenuation in cultured murine macrophages or mice. These experimental findings confirm the importance of the purine biosynthesis pathways for the survival and replication of the brucellae in host macrophages. In contrast to previous reports, however, these results indicate that exogenous tryptophan and phenylalanine are available for use by the brucellae in the phagosomal compartment.     

A DNA vaccine encoding lumazine synthase from Brucella abortus induces protective immunity in BALB/c mice

This study was conducted to evaluate the immunogenicity of the Brucella abortus lumazine synthase (BLS) gene cloned into the pcDNA3 plasmid, which is driven by the cytomegalovirus promoter. Injection of plasmid DNA carrying the BLS gene (pcDNA-BLS) into BALB/c mice elicited both humoral and cellular immune responses. Antibodies to the encoded BLS included immunoglobulin G1 (IgG1) IgG2a, IgG2b, IgG3, and IgM isotypes. Animals injected with pcDNA-BLS exhibited a dominance of IgG2a over IgG1. In addition, spleen cells from vaccinated animals produced interleukin-2 and gamma interferon but not IL-10 or IL-4 after in vitro stimulation with recombinant BLS (rBLS), suggesting the induction of a Th1 response. Protection was evaluated by comparing the levels of infection in the spleens of vaccinated mice challenged with B. abortus 544. Immunization with pcDNA-BLS- reduced the bacterial burden relative to those in the control groups. Mice immunized with rBLS produced a significant humoral response but did not show a specific cellular response or any protection from challenge. Altogether, these data suggest that pcDNA-BLS is a good immunogen for the production of humoral and cell-mediated responses in mice and is a candidate for use in future studies of vaccination against brucellosis.     

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.     

Brucella abortus RB51 induces protection in mice orally infected with the virulent strain B. abortus 2308

Brucellae are gram-negative, facultative intracellular bacteria which are one of the most common causes of abortion in animals. In addition, they are the source of a severe zoonosis. In this trial, we evaluated the effect of oral inoculation of Brucella abortus RB51 in mice against a challenge infection with B. abortus 2308. First, we showed that a gastric acid neutralization prior to the oral inoculation contributed to a more homogeneous and consistent infection with both vaccine strain B. abortus RB51 and virulent strain B. abortus 2308. Successively, we assessed the clearance and the immune response following an oral infection with B. abortus RB51. Oral inoculation gave a mild infection which was cleared 42 days after infection, and it induced a delayed humoral and cell-mediated immune response. Finally, we immunized mice by oral inoculation with B. abortus RB51, and we challenged them with the virulent strain B. abortus 2308 by an oral or intraperitoneal route 42 days after vaccination. Oral inoculation of B. abortus RB51 was able to give protection to mice infected with the virulent strain B. abortus 2308 by the oral route but not to mice infected intraperitoneally. Our results indicate that oral inoculation of mice with B. abortus RB51 is able to give a protective immunity against an oral infection with virulent strains, and this protection seems to rely on an immune response at the mucosal level.     

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.     

Histopathologic changes induced by vaccination in experimental cutaneous leishmaniasis of BALB/c mice.

Highly susceptible BALB/c mice became partially resistant to Leishmania mexicana amazonensis infection after intravenous immunization with solubilized homologous promastigote antigen. Immunized BALB/c mice exhibited mixed mononuclear cell reactions, with granulomatous inflammation, collagen deposition, and fibrinoid necrosis at the site of infection. In contrast, naive animals displayed a monomorphic picture composed of largely vacuolated and parasitized macrophages with areas of coagulative necrosis. Electron microscopy revealed an increased number of eosinophils, sometimes in close contact with parasitized macrophages, in immunized animals. These findings illustrate that histologic changes reflect host immune status in cutaneous leishmaniasis, and that susceptibility of BALB/c mice to L m amazonensis, although dependent on genetic background, can be artificially modified.     

Anti-thyroglobulin antibodies induced with recombinant reovirus infection in BALB/c mice.

BALB/c mice that are infected with reovirus Type 1 develop thyroiditis. Viral antigens were seen in the cytoplasm of epithelial cells but not in the surrounding colloidal space of the thyroid. Examination of sera from the infected mice revealed autoantibodies that, by immunofluorescence, reacted with second antigens in the colloid (ground-glass staining pattern) and thyroglobulin (puffy staining pattern). An enzyme-linked immunosorbent assay designed to identify the reactive antigens showed the autoantibodies to direct against thyroglobulin. Synthetic serum thymic factor (FTS) suppressed autoantibody production to the thyroid after reovirus Type 1 infection. Reovirus Type 3, in contrast to reovirus Type 1, did not induce autoantibodies to react against thyroglobulin. By the use of recombinants between reovirus Type 1 and Type 3, the segment of the reovirus genome responsible for the induction of autoantibodies to thyroglobulin was identified. Virus containing the S1 genome segment from reovirus Type 1, which codes the sigma 1 polypeptide (i.e. haemagglutinin), infected epithelial cells in the thyroid and induced autoantibodies against the thyroglobulin. However, virus containing the S1 gene segment from reovirus Type 3 failed to infect cells in the thyroid and did not induce autoantibodies against thyroglobulin. In this study, reovirus Type 1 induces thyroiditis and autoimmunity, and the S1 gene segment is required for the induction of autoantibodies against thyroglobulin.     

Antibody responses to Brucella abortus 2308 in cattle vaccinated with B. abortus RB51.

Cattle vaccinated with Brucella abortus rough strain RB51 (SRB51) produced small amounts of serum immunoglobulin G (IgG) but no IgM antibody to smooth strain 2308 (S2308) bacteria and produced no IgG or IgM antibody to S2308 lipopolysaccharide (LPS). Western immunoblot analysis revealed that antiserum from SRB51-vaccinated cattle contained IgG antibody that reacted with S2308 proteins of 84 to <20 kDa. However, antiserum from the vaccinated cattle did not contain agglutinating B. abortus antibody in the tube agglutination test for brucellosis. These results suggest that SRB51-vaccinated cattle produced no antibody to S2308 LPS, although they did produce nonagglutinating IgG antibody that reacted with S2308 bacteria and bacterial proteins of 84 to <20 kDa.     

Evaluation of protection afforded by Brucella abortus and Brucella melitensis unmarked deletion mutants exhibiting different rates of clearance in BALB/c mice

Research for novel Brucella vaccines has focused upon the development of live vaccine strains, which have proven more efficacious than killed or subunit vaccines. In an effort to develop improved vaccines, signature-tagged mutant banks were screened to identify mutants attenuated for survival. Mutants selected from these screens exhibited various degrees of attenuation characterized by the rate of clearance, ranging from a failure to grow in macrophages after 24 h of infection to a failure to persist in the mouse model beyond 8 weeks. Ideal vaccine candidates should be safe to the host, while evoking protective immunity. In the present work, we constructed unmarked deletion mutants of three gene candidates, manBA, virB2, and asp24, in both Brucella abortus and Brucella melitensis. The Deltaasp24 mutants, which persist for extended periods in vivo, are superior to current vaccine strains and to other deletion strains tested in the mouse model against homologous challenge infection after 12, 16, and 20 weeks postvaccination. The Deltaasp24 mutants also display superior protection compared to DeltamanBA and DeltavirB2 mutants against heterologous challenge in mice. From this study, a direct association between protection against infection and cytokine response was not apparent between all vaccine groups and, therefore, correlates of protective immunity will need to be considered further. A distinct correlation between persistence of the vaccine strain and protection against infection was corroborated.     

Interferon-gamma is crucial for surviving a Brucella abortus infection in both resistant C57BL/6 and susceptible BALB/c mice

Brucella abortus is an intracellular bacterial pathogen that causes chronic infections in humans and a number of agriculturally important species of animals. It has been shown that BALB/c mice are more susceptible to infections with virulent strains of Brucella abortus than C57BL/6 or C57BL/10 strains. In experiments described here, gene knock-out mice were utilized to elucidate some of the salient components of resistance. Resistant C57BL/6 mice with gene deletions or disruptions in the interferon-γ (IFN-γ), perforin or β₂-microglobulin genes had decreased abilities to control intracellular infections with B. abortus strain 2308 during the first week after infection. However, only the IFN-γ knock-out mice had a sustained inability to control infections and this resulted in death of the mice at approximately 6 weeks post-infection. These mice had a continual increase in the number of bacterial colony-forming units (CFU) in their spleens until death. When BALB/c mice with the disrupted IFN-γ gene were infected they had more splenic CFU at one week post-infection than control mice but the increase was not statistically significant and by 3 weeks they did not have more CFU than control mice. Moreover, the number of splenic bacteria did not increase in the BALB/c IFN-γ knock-out mice between 6 and 10·5 weeks, although they died at 10·5 weeks, the time by which normal BALB/c mice were clearing the infection. Death in both strains of IFN-γ gene disrupted mice coincided with symptoms of cachexia and macrophages comprised ≥75% of the splenic leucocytes.     

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.     

The siderophore 2,3-dihydroxybenzoic acid is not required for virulence of Brucella abortus in BALB/c mice

2,3-Dihydroxybenzoic acid (DHBA) is the only siderophore described for Brucella, and previous studies suggested that DHBA might contribute to the capacity of these organisms to persist in host macrophages. Employing an isogenic siderophore mutant (DeltaentC) constructed from virulent Brucella abortus 2308, however, we found that production of DHBA is not required for replication in cultured murine macrophages or for the establishment and maintenance of chronic infection in the BALB/c mouse model.