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Brucella spp. are intracellular bacteria that cause an infectious disease called brucellosis in humans and many domestic and wildlife animals. B. suis primarily infects pigs and is pathogenic to humans. The macrophage- Brucella interaction is critical for the establishment of a chronic Brucella infection. Our studies showed that smooth virulent B. suis strain 1330 (S1330) prevented programmed cell death of infected macrophages and rough attenuated B. suis strain VTRS1 (a vaccine candidate) induced strong macrophage cell death. To further investigate the mechanism of VTRS1-induced macrophage cell death, microarrays were used to analyze temporal transcriptional responses of murine macrophage-like J774.A1 cells infected with S1330 or VTRS1. In total 17,685 probe sets were significantly regulated based on the effects of strain, time and their interactions. A miniTUBA dynamic Bayesian network analysis predicted that VTRS1-induced macrophage cell death was mediated by a proinflammatory gene (the tumor necrosis factor alpha [TNF-α] gene), an NF-κB pathway gene (the IκB-α gene), the caspase-2 gene, and several other genes. VTRS1 induced significantly higher levels of transcription of 40 proinflammatory genes than S1330. A Mann-Whitney U test confirmed the proinflammatory response in VTRS1-infected macrophages. Increased production of TNF-α and interleukin 1β (IL-1β) were also detected in the supernatants in VTRS1-infected macrophage cell culture. Hyperphosphorylation of IκB-α was observed in macrophages infected with VTRS1 but not S1330. The important roles of TNF-α and IκB-α in VTRS1-induced macrophage cell death were further confirmed by individual inhibition studies. VTRS1-induced macrophage cell death was significantly inhibited by a caspase-2 inhibitor but not a caspase-1 inhibitor. The role of caspase-2 in regulating the programmed cell death of VTRS1-infected macrophages was confirmed in another study using caspase-2-knockout mice. In summary, VTRS1 induces a proinflammatory, caspase-2- and NF-κB-mediated macrophage cell death. This unique cell death differs from apoptosis, which is not proinflammatory. It is also different from classical pyroptosis, which is caspase-1 mediated.

The penicillin-binding proteins (PBPs) are enzymes that regulate the assembly of the peptidoglycan layer of the bacterial cell wall. The genome of Brucella melitensis strain 16 M possesses seven pbp genes: three in pbp-1 family (designated as 1A , 1B , and 1C ); one in pbp-2 family; and three in pbp-6 family (designated as 6A , 6B , and 6C ). We investigated the importance of pbp-1 and pbp-2 genes to viability, cell morphology and infectivity of B. melitensis . A recombinant B. melitensis strain (designated 16 MΔ pbp1C ) was generated by disrupting the pbp-1C of strain 16 M by allelic exchange. This strain produced nearly 20% smaller colonies on trypticase soy agar plates, and grew slower in trypticase soy broth compared to the strain 16 M. Electron microscopy revealed that strain 16 M exhibited native cocco-bacillus morphology, while 16 MΔ pbp1C possessed a spherical morphology. Strain 16 MΔ pbp1C did not differ from strain 16 M in terms of recovery from infected mouse macrophage cell line J774.1, or recovery from spleens of infected BALB/c mice, suggesting that pbp-1C is dispensable for intracellular persistence of B. melitensis . Expression of mRNA of fixR , the gene downstream of pbp-1C was similar between the strains 16 M and 16 MΔ pbp1C suggesting that disruption of pbp-1C did not induce any polar effects. Multiple attempts to mutate pbp-1A , pbp-1B , or pbp-2 genes failed, most probably because these genes are indispensable for viability of B. melitensis . Our findings suggest that pbp-1C regulates in vitro growth and cell morphology, whereas pbp-1A , pbp-1B , and pbp-2 are essential for viability of B. melitensis .

To avoid potentiating the spread of an antibiotic resistance marker, a plasmid expressing a leuB gene and a heterologous antigen, green fluorescent protein (GFP), was shown to complement a leucine auxotroph of cattle vaccine strain Brucella abortus RB51, which protected CD1 mice from virulent B. abortus 2308 and elicited GFP antibodies.

Burkholderia mallei is the etiologic agent of glanders in solipeds (horses, mules and donkeys), and incidentally in carnivores and humans. Little is known about the molecular mechanisms of B. mallei pathogenesis. The putative carboxy-terminal processing protease (CtpA) of B. mallei is a member of a novel family of endoproteases involved in the maturation of proteins destined for the cell envelope. All species and isolates of Burkholderia carry a highly conserved copy of ctpA. We studied the involvement of CtpA on growth, cell morphology, persistence, and pathogenicity of B. mallei. A sucrose-resistant strain of B. mallei was constructed by deleting a major portion of the sacB gene of the wild type strain ATCC 23344 by gene replacement, and designated as strain 23344DeltasacB. A portion of the ctpA gene (encoding CtpA) of strain 23344DeltasacB was deleted by gene replacement to generate strain 23344DeltasacBDeltactpA. In contrast to the wild type ATCC 23344 or the sacB mutant 23344DeltasacB, the ctpA mutant 23344DeltasacBDeltactpA displayed altered cell morphologies with partially or fully disintegrated cell envelopes. Furthermore, relative to the wild type, the ctpA mutant displayed slower growth in vitro and less ability to survive in J774.2 murine macrophages. The expression of mRNA of adtA, the gene downstream of ctpA was similar among the three strains suggesting that disruption of ctpA did not induce any polar effects. As with the wild type or the sacB mutant, the ctpA mutant exhibited a dose-dependent lethality when inoculated intraperitoneally into CD1 mice. The CD1 mice inoculated with a non-lethal dose of the ctpA mutant produced specific serum immunoglobulins IgG1 and IgG2a and were partially protected against challenge with wild type B. mallei ATCC 23344. These findings suggest that CtpA regulates in vitro growth, cell morphology and intracellular survival of B. mallei, and a ctpA mutant protects CD1 mice against glanders.

A 97-kDa purified aminopeptidase N (PepN) of Brucella melitensis was previously identified to be immunogenic in humans. The B. melitensis pepN gene was cloned, expressed in Escherichia coli and purified by affinity chromatography. The recombinant PepN (rPepN) exhibited the same biochemical properties, specificity and susceptibility to inhibitors as the native PepN. rPepN was evaluated as a diagnostic antigen in an indirect enzyme-linked immunosorbent assay (ELISA) using sera from patients with acute and chronic brucellosis. The specificity of the ELISA was determined with sera from healthy donors. The ELISA had a cutoff value of 0.156 with 100% specificity and 100% sensitivity. Higher sensitivity was obtained using rPepN compared with crude extract from B. melitensis. Anti-PepN sera did not exhibit serological cross-reaction to crude extracts from Rhizobium tropici, Ochrobactrum anthropi, Yersinia enterocolitica 09 or E. coli O157H7.

Brucellaspp. are facultative intracellular bacteria that cause brucellosis in humans and other animals.Brucellaspp. are taken up by macrophages, and the outcome of the macrophage-Brucellainteraction is a basis for establishment of a chronicBrucellainfection. Microarrays were used to analyze the transcriptional response of the murine macrophage-like J774.A1 cell line to infection with virulentBrucella melitensisstrain 16M. It was found that most significant changes in macrophage gene transcription happened early following infection, and global macrophage gene expression profiles returned to normal between 24 and 48 h postinfection. These findings support the observation that macrophages kill the majority ofBrucellacells at the early infection stage, but the survivingBrucellacells are able to avoid macrophage brucellacidal activity inside replicative phagosomes at the later infection stage. At 4 h postinfection, macrophage genes involved in cell growth, metabolism, and responses to endogenous stimuli were down-regulated, while the inflammatory response (e.g., tumor necrosis factor alpha and Toll-like receptor 2), the complement system, the responses to external stimuli, and other immune responses were up-regulated. It is likely that the most active brucellacidal activity happened between 0 and 4 h postinfection. Mitochondrion-associated gene expression, which is involved in protein synthesis and transport, electron transfer, and small-molecule transfer, and many other mitochondrial functions were significantly down-regulated at 4 h postinfection. Although there were both pro- and antiapoptosis effects,B. melitensis16M appears to inhibit apoptosis of macrophages by blocking release of cytochromecand production of reactive oxygen species in the mitochondria, thus preventing activation of caspase cascades.

Neospora caninum, an apicomplexan parasite, is a leading cause of bovine abortions worldwide. The efficacy of gamma-irradiated N. caninum strain NC-1 tachyzoites as a vaccine for neosporosis was assessed in C57BL6 mice. A dose of 528 Gy of gamma irradiation was sufficient to arrest replication but not host cell penetration by tachyzoites. Female C57BL6 mice were vaccinated with two intraperitoneal inoculations of 1 x 10(6) irradiated tachyzoites at 4-wk intervals. When stimulated with N. caninum tachyzoite lysates, splenocytes of vaccinated mice, cultured 5 and 10 wk after vaccination, secreted significant (P

Brucella abortusstrain 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 expressingEscherichia coliβ-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 β-galactosidase. A single intraperitoneal inoculation of mice with 109CFU equivalents of gamma-irradiated, but not heat-killed, RB51G/LacZ induced a β-galactosidase-specific Th1-type immune response. Though no obvious differences were detected in immune responses toB. abortus-specific antigens, mice vaccinated with gamma-irradiated, but not heat-killed, RB51G/LacZ developed significant protection against challenge with virulentB. 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 againstB. abortuschallenge in mice.

The putative carboxyl-terminal processing protease (CtpA) ofBrucella suis1330 is a member of a novel family of endoproteases involved in the maturation of proteins destined for the cell envelope. TheB. suisCtpA protein shared up to 77% homology with CtpA proteins of other bacteria. A CtpA-deficientBrucellastrain (1330ΔctpA), generated by allelic exchange, produced smaller colonies on enriched agar plates and exhibited a 50% decrease in growth rate in enriched liquid medium and no growth in salt-free enriched medium compared to the wild-type strain 1330 or thectpA-complemented strain 1330ΔctpA[pBBctpA]. Electron microscopy revealed that in contrast to the native coccobacillus shape of wild-type strain 1330, strain 1330ΔctpApossessed a spherical shape, an increased cell diameter, and cell membranes partially dissociated from the cell envelope. In the J774 mouse macrophage cell line, 24 h after infection, the CFU of the strain 1330ΔctpAdeclined by approximately 3 log10CFU relative to wild-type strain 1330. Nine weeks after intraperitoneal inoculation of BALB/c mice, strain 1330ΔctpAhad cleared from spleens but strain 1330 was still present. These observations suggest that the CtpA activity is necessary for the intracellular survival ofB. suis. Relative to the saline-injected mice, strain 1330ΔctpA-vaccinated mice exhibited 4 to 5 log10CFU of protection against challenge with virulentB. abortusstrain 2308 orB. suisstrain 1330 but no protection againstB. melitensisstrain 16 M. This is the first report correlating a CtpA deficiency with cell morphology and attenuation ofB. suis.

In Gram-negative bacteria, autotransporters are secreted proteins able to translocate themselves through the inner- and outer-membranes to the cell surface or to the extracellular environment. The influence of the putative outer membrane autotransporter (OmaA) protein to the persistence of Brucella suis was investigated. Sequence analyses revealed that the OmaA protein of B. suis strain 1330 consists of a signal peptide, a passenger alpha-domain, and a transporter beta-domain, which are the characteristic components of an autotransporter protein. The transporter beta-domain consists of 14 individual amphipathic beta-strands, and a 46-amino acid long alpha-helix lies upstream of the transporter domain, indicating that the B. suis OmaA is a type-I classical autotransporter. BLAST search and phylogenetic analyses revealed that the B. suis OmaA protein shares more similarities with adhesin autotransporter proteins than with protease autotransporter proteins of other bacteria. An OmaA-deficient strain (1330DeltaomaA) was generated by disrupting the DNA region encoding the passenger alpha-domain of the OmaA protein of B. suis wild type strain 1330. The omaA gene encoding the full-length OmaA protein was cloned and used to complement the OmaA-deficient strain. The OmaA-deficient strain did not differ from the wild type strain in terms of persistence in J774 macrophage cell line 24 and 48 h after inoculation, or clearance from the spleens of BALB/c mice at 1 week after intraperitoneal inoculation. These observations suggest that the function of the OmaA protein is dispensable during the acute phase of B. suis infection. However, the OmaA-deficient strain was cleared from the spleens of BALB/c mice faster than the wild type strain between the third and the ninth week after intraperitoneal inoculation, indicating that the OmaA may be important during the chronic phase of B. suis infection. Relative to the BALB/c mice injected with saline, those vaccinated with the OmaA-deficient strain exhibited 3.0-3.9log10 colony forming units protection against a challenge with B. suis strain 1330. This study is the first report correlating an autotransporter protein deficiency with persistence of B. suis in vitro and in vivo.

The Brucella abortus L7/L12 gene encoding ribosomal protein L7/L12 and the Listeria monocytogenes partial hly gene encoding the protective region of the hemolysin (partial listeriolysin, pLLO) were cloned into vaccinia virus by homologous recombination to produce recombinants WRL7/L12 and WRpLLO, respectively. The ability of these recombinants to induce humoral, cell mediated and protective immune response in mice was assessed. Although mice inoculated with WRL7/L12 recombinant produced antibodies specific to vaccinia virus and L7/L12 antigens, they were not protected against a virulent challenge with B. abortus 2308 strain. In contrast, mice inoculated with WRpLLO were protected against a challenge with virulent L. monocytogenes. Stimulation with purified fusion listeriolysin protein (MBP-LLO), but not with unrelated control protein (MBP), induced splenocytes from WRpLLO-inoculated mice to secrete significantly higher amounts of IFN-gamma than saline inoculated mice. Mice inoculated with either WRpLLO or WRL7/L12 recombinants produced predominantly IgG2a isotype antibody responses, indicative of a Th1 type of immune response. The protective potential of the WRpLLO recombinant correlated with the level of IFN-gamma produced in these mice.

Virulent strains of the bovine opportunistic pathogen Haemophilus somnus ( Histophilus somni ) cause multi-systemic diseases in cattle. One of the reported virulence factors that H. somnus may use to persist in the host is resistance to intracellular killing. We report here that H. somnus significantly ( P 2 − ) by bovine mammary and alveolar macrophages as well as by polymorphonuclear leukocytes. Inhibition of O 2 − was time- and dose-dependent and did not occur after incubation with Escherichia coli , H. influenzae , or Brucella abortus . Non-viable H. somnus , purified lipooligosaccharide, or cell-free supernatant from mid-log phase cultures did not inhibit O 2 − production, indicating that O 2 − inhibition required contact with live H. somnus . Furthermore, preincubation of phagocytic cells with cytochalasin B to prevent phagocytosis did not decrease the ability of H. somnus to inhibit O 2 − production. Some H. somnus isolates from the prepuce of healthy bulls were less capable or incapable of inhibiting macrophage O 2 − production compared to isolates tested from disease sites. Our results suggest that inhibition of O 2 − may be an important virulence factor exploited by pathogenic strains of H. somnus to resist killing by professional phagocytic cells.

An immunogenic aminopeptidase was purified from Brucella melitensis strain VTRM1. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps. This procedure resulted in a yield of 29% and a 144-fold increase in specific activity. The aminopeptidase appeared to be a monomeric enzyme with a molecular mass of 96 kDa and an isoelectric point of 4.8. Its activity was optimal at pH 7.0 at 40°C. The enzyme was strongly inhibited by EDTA, 1,10-phenathroline, and divalent cations (Zn 2+ and Hg 2+ ), suggesting that this protein was a metalloaminopeptidase. The enzyme showed preference for alanine at the N termini of aminoacyl derivatives. The K m values for l -alanine- p -nitroanilide (Ala- p NA) and Lys- p NA were 0.35 and 0.18 mM, respectively. The N-terminal sequence of aminopeptidase was used for a homologous search in the genomes of B. melitensis 16M and Brucella suis 1330. The analysis revealed an exact match of the probe sequence (36 bp) with an open reading frame of 2,652 bp encoding a protein predicted to be alanyl aminopeptidase (aminopeptidase N). Collectively, these data suggest designation of the B. melitensis enzyme as an aminopeptidase N. The aminopeptidase was recognized by sera from patients with acute and chronic brucellosis, suggesting that the enzyme may have important diagnostic implications.

The first effective Brucella vaccine was based on live Brucella abortus strain 19, a laboratory-derived strain attenuated by an unknown process during subculture. This induces reasonable protection against B. abortus, but at the expense of persistent serological responses. A similar problem occurs with the B. melitensis Rev.1 strain that is still the most effective vaccine against caprine and ovine brucellosis. Vaccines based on killed cells of virulent strains administered with adjuvant induced significant protection but also unacceptable levels of antibodies interfering with diagnostic tests. Attempts were made to circumvent this problem by using a live rough strain B. abortus 45/20, but this reverted to virulence in vivo. Use of killed cells of this strain in adjuvant met with moderate success but batch to batch variation in reactogenicity and agglutinogenicity limited application. This problem has been overcome by the development of the rifampicin-resistant mutant B. abortus RB51 strain. This strain has proved safe and effective in the field against bovine brucellosis and exhibits negligible interference with diagnostic serology. Attempts are being made to develop defined rough mutant vaccine strains that would be more effective against B. melitensis and B. suis. Various studies have examined cell-free native and recombinant proteins as candidate protective antigens, with or without adjuvants. Limited success has been obtained with these or with DNA vaccines encoding known protective antigens in experimental models and further work is indicated.

Brucella abortus resists the microbicidal mechanisms of macrophages, and the expression of its heat shock proteins (HSPs) such as GroEL, GroES and HtrA may play a role in this resistance. Bacterial HSPs can be very immunogenic, inducing protective immunity in various types of bacterial infections. However, the significance of immune responses directed against B. abortus HSPs in the protection against brucellosis is currently unresolved. To elucidate the role of these proteins in protection against Brucella challenge, individual, divalent or trivalent baculovirus (BV) recombinants of B. abortus GroEL, GroES and/or HtrA were injected into BALB/c mice either as protein-expressing whole cells or as purified proteins. The preparations were given to mice in combination with Freund's or Ribi adjuvant, respectively. In addition, some mice were primed with a vaccinia virus-GroEL recombinant, followed by inoculation with purified GroEL-Ribi adjuvant combination. Antibodies were observed against B. abortus GroEL and HtrA, but not against GroES. Cellular immune response was demonstrated by observing significant IFN-gamma release by lymphocytes of mice immunized with the purified HtrA-Ribi adjuvant combination. However, none of the mice inoculated with individual, divalent or trivalent HSP-expressing cells combined with complete Freund's adjuvant or inoculated with purified B. abortus HSPs combined with Ribi adjuvant, were protected against challenge with B. abortus virulent strain 2308. Priming with vaccinia virus-GroEL recombinant and boosting with GroEL-Ribi combination did not induce protective immunity. Based on the results obtained, we suggest that although humoral and cell-mediated immune responses are induced, but protective immune response is not induced by B. abortus HSPs.

The members of the genusBrucellaare gram-negative, facultatively intracellular bacterial pathogens that cause brucellosis in many animal species and humans. Although live, attenuated vaccines are available to protect several animal species from the disease, there is no safe and effective vaccine for human use. Here we report that a bacterium that is closely related toBrucellaspecies,Ochrobactrum anthropi, can be used as a vaccine vector for the delivery ofBrucellaantigens to mice, leading to the elicitation of protective immunity against brucellosis.Brucella abortusCu,Zn superoxide dismutase (SOD), a protectiveBrucellaantigen, was expressed in large amounts inO. anthropistrain 49237 by use of the broad-host-range plasmid pBBR1MCS. NeitherO. anthropistrain 49237 nor the recombinantO. anthropistrain 49237SOD, expressingB. abortusCu,Zn SOD, provided protection against virulentBrucellainfection in mice. Analysis of immune responses indicated that strains 49237 and 49237SOD stimulated a mix of Th1 and Th2 type responses in the mice. After the immune response was switched to a Th1-biased response by addition of oligonucleotides containing unmethylated CpG motifs, bothO. anthropistrain 49237 and the recombinantO. anthropistrain 49237SOD induced protection in mice. However, the protection conferred by strain 49237SOD was significantly better than that induced by the parental strain, 49237.

A safe, more sensitive, nonradioactive, neutral red uptake assay was adopted to replace the traditional51Cr release assay for detection ofBrucella-specific cytotoxic T lymphocyte (CTL) activity. Our studies indicated thatBrucella abortusstrain RB51 vaccination of mice induced specific CTLs against both strain RB51- and strain 2308-infected J774.A1 macrophages but not againstListeria monocytogenes-infected J774.A1 cells. The antigen-specific cytotoxic activity was exerted by T lymphocytes but not by NK cells. CD3+CD4+T cells secreted the highest level of gamma interferon (IFN-γ) and were able to exert a low but significant level of specific lysis ofBrucella-infected macrophages. They also exerted a low level of nonspecific lysis of noninfected macrophages. In contrast, CD3+CD8+T cells secreted low levels of IFN-γ but demonstrated high levels of specific lysis ofBrucella-infected macrophages with no nonspecific lysis. These findings indicate thatB. abortusstrain RB51 vaccination of mice induces specific CTLs and suggest that CD3+CD4+and CD3+CD8+T cells play a synergistic role in the anti-Brucellaactivity.

Brucellaspp. are gram-negative intracellular pathogens that survive and multiply within phagocytic cells of their hosts. Smooth organisms present O polysaccharides (OPS) on their surface. These OPS help the bacteria avoid the bactericidal action of serum. ThewboAgene, coding for the enzyme glycosyltransferase, is essential for the synthesis of O chain inBrucella. In this study, the sensitivity to serum of smooth, virulentBrucella melitensis16M andB. abortus2308, roughwboAmutants VTRM1, RA1, and WRR51 derived from these twoBrucellaspecies, and theB. abortusvaccine strain RB51 was assayed using normal nonimmune human serum (NHS). The deposition of complement components and mannose-binding lectin (MBL) on the bacterial surface was detected by flow cytometry. RoughB. abortusmutants were more sensitive to the bactericidal action of NHS than were roughB. melitensismutants. Complement components were deposited on smooth strains at a slower rate compared to rough strains. Deposition of iC3b and C5b-9 and bacterial killing occurred when bacteria were treated with C1q-depleted, but not with C2-depleted serum or NHS in the presence of Mg-EGTA. These results indicate that (i) OPS-deficient strains derived fromB. melitensis16M are more resistant to the bactericidal action of NHS than OPS-deficient strains derived fromB. abortus2308, (ii) both the classical and the MBL-mediated pathways are involved in complement deposition and complement-mediated killing ofBrucella, and (iii) the alternative pathway is not activated by smooth or rough brucellae.

The lipooligosaccharide (LOS) of Haemophilus somnus undergoes antigenic phase variation, which may facilitate evasion from the bovine host immune response and/or colonization and dissemination. However, LOS antigenic diversity in H. somnus has not been adequately investigated. In this study, monoclonal antibodies (MAbs) specific to various LOS epitopes were used to investigate antigenic variation and stability in LOS from H. somnus strains and phase variants. Clinical isolates of H. somnus exhibited intrastrain, as well as interstrain, antigenic heterogeneity in LOS when probed with MAbs to outer core oligosaccharide epitopes in an enzyme-linked immunosorbent assay (ELISA). However, epitopes reactive with MAbs directed predominately to the inner core heptose region were highly conserved. At least one epitope, which was expressed in few strains, was identified. One LOS component affected by phase variation was identified as phosphorylcholine (PCho), which is linked to the primary glucose residue. Inhibition ELISA, immunoblotting, and electrospray-mass spectrometry were used to confirm that MAb 5F5.9 recognized PCho. LOS reactivity with MAb 5F5.9 was associated with loss of most of the outer core oligosaccharide, indicating that reactivity with PCho was affected by phase variation of the glycose residues in this region. Our results indicate that outer core epitopes of H. somnus LOS exhibit a high degree of random, phase-variable antigenic heterogeneity and that such heterogeneity must be considered in the design of vaccines and diagnostic tests.

This work is a part of an ongoing effort to develop vaccinia virus recombinants expressing various Brucella abortus proteins. The B. abortus groEL gene encoding the antigenic heat shock protein GroEL was subcloned into vaccinia virus via homologous recombination and expression confirmed by Western blotting. Female BALB/c mice inoculated with recombinant vaccinia virus/GroEL produced GroEL and vaccinia virus specific antibodies. Mice were challenged 8 weeks post-inoculation with virulent B. abortus strain 2308 and protection measured by the rate of clearance of live Brucella from spleens. Although induction of specific immune response to GroEL and vaccinia virus was demonstrated by the appearance of antibodies in mice, no significant level of protection was demonstrable.

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 ofBrucella 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 strainB. abortus2308. Disruption of the 18-kDa protein's gene in vaccine strainB. abortusRB51 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.

Brucella abortus vaccine strain RB51 is a natural stable attenuated rough mutant derived from the virulent strain 2308. The genetic mutations that are responsible for the roughness and the attenuation of strain RB51 have not been identified until now. Also, except for an assay based on pulsed-field gel electrophoresis, no other simple method to differentiate strain RB51 from its parent strain 2308 is available. In the present study, we demonstrate that the wboA gene encoding a glycosyltransferase, an enzyme essential for the synthesis of O antigen, is disrupted by an IS 711 element in B. abortus vaccine strain RB51. Exploiting this feature, we developed a PCR assay that distinguishes strain RB51 from all other Brucella species and strains tested.

We constructed a rough mutant of Brucella abortus 2308 by transposon (Tn 5 ) mutagenesis. Neither whole cells nor extracted lipopolysaccharide (LPS) from this mutant, designated RA1, reacted with a Brucella O-side-chain-specific monoclonal antibody (MAb), Bru-38, indicating the absence of O-side-chain synthesis. Compositional analyses of LPS from strain RA1 showed reduced levels of quinovosamine and mannose relative to the levels in the parental, wild-type strain, 2308. We isolated DNA flanking the Tn 5 insertion in strain RA1 by cloning a 25-kb Xba I genomic fragment into pGEM-3Z to create plasmid pJM6. Allelic exchange of genomic DNA in B. abortus 2308 mediated by electroporation of pJM6 produced kanamycin-resistant clones that were not reactive with MAb Bru-38. Southern blot analysis of genomic DNA from these rough clones revealed Tn 5 in a 25-kb Xba I genomic fragment. A homology search with the deduced amino acid sequence of the open reading frame disrupted by Tn 5 revealed limited homology with various glycosyltransferases. This B. abortus gene has been named wboA . Transformation of strain RA1 with a broad-host-range plasmid bearing the wild-type B. abortus wboA gene resulted in the restoration of O-side-chain synthesis and the smooth phenotype. B. abortus RA1 was attenuated for survival in mice. However, strain RA1 persisted in mice spleens for a longer time than the B. abortus vaccine strain RB51, but as expected, neither strain induced antibodies specific for the O side chain.

To identify Brucella antigens that are potentially involved in stimulating a protective cell-mediated immune response, a gene library of Brucella abortus 2308 was screened for the expression of antigens reacting with immunoglobulin G2a antibodies from BALB/c mice vaccinated with B. abortus RB51. One selected positive clone (clone MCB68) contained an insert of 2.6 kb; nucleotide sequence analysis of this insert revealed two open reading frames (ORFs). The deduced amino acid sequences of the first and second ORFs had significant similarities with the YajC and SecD proteins, respectively, of several bacterial species. Both the YajC and SecD proteins were expressed in Escherichia coli as fusion proteins with maltose binding protein (MBP). In Western blots, sera from mice vaccinated with B. abortus RB51 recognized YajC but not SecD. Further Western blot analysis with purified recombinant YajC protein indicated that mice inoculated with B. abortus 19 or 2308 or B. melitensis RM1 also produced antibodies to YajC. In response to in vitro stimulation with recombinant MBP-YajC fusion protein, splenocytes from mice vaccinated with B. abortus RB51 were able to proliferate and produce gamma interferon but not interleukin-4. This study demonstrates, for the first time, the involvement of YajC protein in an immune response to an infectious agent.

Brucella abortus is a Gram-negative, facultative intracellular pathogen for several mammals, including humans. Live attenuated B. abortus strain RB51 is currently the official vaccine used against bovine brucellosis in the United States and several other countries. Overexpression of protective B. abortus antigen Cu/Zn superoxide dismutase (SOD) in a recombinant strain of RB51 (strain RB51SOD) significantly increases its vaccine efficacy against virulent B. abortus challenge in a mouse model. An attempt has been made to better understand the mechanism of the enhanced protective immunity of RB51SOD compared to its parent strain RB51. We previously reported that RB51SOD stimulated enhanced Th1 immune response. In this study, we further found that T effector cells derived from RB51SOD-immunized mice exhibited significantly higher cytotoxic T lymphocyte activity than T effector cells derived from RB51-immunized mice against virulent B. abortus-infected target cells. Meanwhile, the macrophage responses to these two strains were also studied. Compared to RB51, RB51SOD cells had a lower survival rate in macrophages and induced lower levels of macrophage apoptosis and necrosis. The decreased survival of RB51SOD cells correlates with the higher sensitivity of RB51SOD, compared to RB51, to the bactericidal action of either Polymyxin B or sodium dodecyl sulfate (SDS). Furthermore, a physical damage to the outer membrane of RB51SOD was observed by electron microscopy. Possibly due to the physical damage, overexpressed Cu/Zn SOD in RB51SOD was found to be released into the bacterial cell culture medium. Therefore, the stronger adaptive immunity induced by RB51SOD did not correlate with the low level of innate immunity induced by RB51SOD compared to RB51. This unique and apparently contradictory profile is likely associated with the differences in outer membrane integrity and Cu/Zn SOD release. NIAID-NIHR-21 [AI076812, AI057875]; University of Michigan Unit for Laboratory Animal Medicine; American Society of Laboratory Animal Practitioners Foundation; Undergraduate Research Opportunity Program (UROP) at the University of MichiganUniversity of Michigan System This project was supported by NIAID-NIHR-21 grants AI076812 and AI057875 and to Yongqun He. We would like to acknowledge the contributions of Megan Ann Ramaker, Kimberly Quandt, and Jered M. Wendte whose externship in our lab was funded by the University of Michigan Unit for Laboratory Animal Medicine and the American Society of Laboratory Animal Practitioners Foundation. Kimberly P. Ku was supported by the Undergraduate Research Opportunity Program (UROP) at the University of Michigan.

Infection by members of the Gram-negative bacterial genus Brucella causes brucellosis in a variety of mammals. Brucellosis in swine remains a challenge, as there is no vaccine in the USA approved for use in swine against brucellosis. Here, we developed an improved recombinant Brucella abortus vaccine strain RB51 that could afford protection against Brucella suis infection by over-expressing genes encoding homologous proteins: L7/L12 ribosomal protein, Cu/Zn superoxide dismutase [SOD] and glycosyl-transferase [WboA]. Using strain RB51leuB as a platform and an antibiotic-resistance marker free plasmid, strains RB51leuB/SOD, RB51leuB/SOD/L7/L12 and RB51leuB/SOD/WboA were constructed to over-express the antigens: SOD alone, SOD and ribosomal protein L7/L12 or SOD and glycosyl-transferase, respectively. The ability of these vaccine candidates to protect against a virulent B. suis challenge were evaluated in a mouse model. All vaccine groups protected mice significantly (P < 0.05) when compared to the control group. Within the vaccine groups, the mice vaccinated with strain RB51leuB/SOD/WboA were significantly better protected than those that were vaccinated with either strain RB51leuB/SOD or RB51leuB/SOD/L7/L12. These results suggest that Brucella antigens can be over-expressed in strain RB51leuB and elicit protective immune responses against brucellosis. Since the plasmid over-expressing homologous antigens does not carry an antibiotic resistance gene, it complies with federal regulations and therefore could be used to develop safer multi-species vaccines for prevention of brucellosis caused by other species of Brucella.

Pregnant balb/c mice received various doses of either Brucella abortus strain 19, a smooth vaccine strain, or B abortus strain RB51, a stable rough organism, intraperitoneally on day 9 of gestation to compare the relative pathogenicity of the two attenuated strains. Nine days after inoculation, spleens and placentas were collected for bacteriological and histopathological examination. A dose of 107·5 strain 19 organisms produced a severe necrosuppurative placentitis occasionally accompanied by fetal death. This dose resulted in a 10-fold higher level of splenic infection than did a dose of 10 9·5 strain RB51 organisms, which produced only mild to minimal placentitis not associated with fetal death. Strain 19 infected mice showed seroconversion in the standard tube agglutination test in contrast to the seronegative titre of strain RB51 infected mice. The results of this study corroborate previous investigations on the relative pathogenicity and the serological response of the non-pregnant mouse to strain RB51.

A recombinant 60 kDa Brucella abortus protein expressed in Escherichia coli was recognized in immunoblots by sera from mice experimentally infected with B. abortus and a dog experimentally infected with B. canis. Sera from humans and dogs with naturally acquired brucellosis also recognized this protein, which was designated BA60K. The gene encoding BA60K was localized within an 18 kb B. abortus genomic fragment and its direction of transcription determined by subcloning and maxicell analysis of selected restriction fragments. The nucleotide sequence of 1800 bases encompassing the predicted gene location was determined, revealing an open reading frame encoding a protein of 546 amino acids (predicted relative molecular mass of 57515). Solid phase micro-sequencing of BA60K eluted from two-dimensional polyacrylamide gels confirmed the predicted amino acid sequence. Comparison of the predicted amino acid sequence of BA60K with a protein sequence database revealed that BA60K shares 67.9% identity with the GroEL protein of E. coli, a member of the Hsp60 family of chaperonins. The immunodominant Hsp60 homologs from Legionella pneumophila, Chlamydia trachomatis and Mycobacterium tuberculosis were also found to share greater than 59% amino acid sequence identity with the BA60K protein. The identification of BA60K as a member of the Hsp60 family of chaperonins supports its role in stimulating a prominent host immune response during the course of Brucella infections. It also indicates that BA60K is an important candidate for studies aimed at identifying the antigens responsible for eliciting the protective immune response to brucellosis.

The possibility of expressing a homologous antigen and a heterologous antigen simultaneously in an attenuated Brucella melitensis strain was investigated. The Brucella wboA gene encoding a mannosyltransferase involved in biosynthesis of lipopolysaccharide O-antigen, and the Bacillus anthracis pag gene encoding the protective antigen (PA) were cloned into plasmid pBBR4MCS. The resulting plasmid was introduced into O-antigen deficient B. melitensis strain WRRP1 to produce strain WRSPA. Strain WRSPA produced O-antigen and a series of PA products, induced protection in BALB/c mice against challenge with B. melitensis strain 16M, but failed to protect A/J mice against challenge with B. anthracis Sterne strain.

Neospora caninum, an obligate intracellular protozoan parasite, is the causative agent of bovine neosporosis, an important disease affecting the reproductive performance of cattle worldwide. Currently there is no effective vaccine available to prevent N. caninum infection in cattle. In this study, we examined the feasibility of developing a live, recombinant N. caninum vaccine using Brucella abortus vaccine strain RB51 as the expression and delivery vector. We generated two recombinant RB51 strains each expressing SRS2 (RB51/SRS2) or GRA7 (RB51/GRA7) antigens of N. caninum. BALB/c mice immunized by single intraperitoneal inoculation of the recombinant RB51 strains developed IgG antibodies specific to the respective N. caninum antigen. In vitro stimulation of splenocytes from the vaccinated mice with specific antigen resulted in the production of interferon-gamma, but not IL-5 or IL-10, suggesting the development of a Th1 type immune response. Upon challenge with N. caninum tachyzoites, mice vaccinated with strain RB51/SRS2, but not RB51/GRA7, showed significant resistance to cerebral infection when compared to the RB51 vaccinated mice, as determined by the tissue parasite load using a real-time quantitative TaqMan assay. Interestingly, mice vaccinated with either strain RB51 or RB51/GRA7 also contained significantly lower parasite burden in their brains compared to those inoculated with saline. Mice vaccinated with strain RB51/SRS2 or RB51/GRA7 were protected to the same extent as the strain RB51 vaccinated mice against challenge with B. abortus virulent strain 2308. These results suggest that a recombinant RB51 strain expressing an appropriate protective antigen(s), such as SRS2 of N. caninum, can confer protection against both neosporosis and brucellosis.

Background In prokaryotes, the ureases are multi-subunit, nickel-containing enzymes that catalyze the hydrolysis of urea to carbon dioxide and ammonia. The Brucella genomes contain two urease operons designated as ure 1 and ure 2. We investigated the role of the two Brucella suis urease operons on the infection, intracellular persistence, growth, and resistance to low-pH killing. Results The deduced amino acid sequence of urease-α subunits of operons-1 and -2 exhibited substantial identity with the structural ureases of alpha- and beta-proteobacteria, Gram-positive and Gram-negative bacteria, fungi, and higher plants. Four ure deficient strains were generated by deleting one or more of the genes encoding urease subunits of B. suis strain 1330 by allelic exchange: strain 1330Δure 1K (generated by deleting ureD and ureA in ure 1 operon), strain 1330Δure 2K (ureB and ureC in ure 2 operon), strain 1330Δure 2C (ureA, ureB, and ureC in ure 2 operon), and strain 1330Δure 1KΔure 2C (ureD and ureA in ure 1 operon and ureA, ureB, and ureC in ure 2 operon). When grown in urease test broth, strains 1330, 1330Δure 2K and 1330Δure 2C displayed maximal urease enzyme activity within 24 hours, whereas, strains 1330Δure 1K and 1330Δure 1KΔure 2C exhibited zero urease activity even 96 h after inoculation. Strains 1330Δure 1K and 1330Δure 1KΔure 2C exhibited slower growth rates in tryptic soy broth relative to the wild type strain 1330. When the BALB/c mice were infected intraperitoneally with the strains, six weeks after inoculation, the splenic recovery of the ure deficient strains did not differ from the wild type. In contrast, when the mice were inoculated by gavage, one week after inoculation, strain 1330Δure 1KΔure 2C was cleared from livers and spleens while the wild type strain 1330 was still present. All B. suis strains were killed when they were incubated in-vitro at pH 2.0. When the strains were incubated at pH 2.0 supplemented with 10 mM urea, strain 1330Δure 1K was completely killed, strain 1330Δure 2C was partially killed, but strains 1330 and 1330Δure 2K were not killed. Conclusion These findings suggest that the ure 1 operon is necessary for optimal growth in culture, urease activity, resistance against low-pH killing, and in vivo persistence of B. suis when inoculated by gavage. The ure 2 operon apparently enhances the resistance to low-pH killing in-vitro.

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 log10 CFU/spleen from day 1 to week 6 after intraperitoneal inoculation with 4.65 log10 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. The members of the Brucella genus are small, nonmotile, gram-negative, facultatively intracellular bacteria that can cause brucellosis in a variety of mammals. Brucellosis is a chronic zoonotic disease resulting in undulant fever in humans and abortion and/or infertility in affected animals. There are six species of Brucella that are currently recognized based on host specificity. They include Brucella abortus (cattle), B. melitensis (goats), B. suis (hogs), B. canis (dogs), B. ovis (sheep), and B. neotomae (wood rats) (7). Recently, Brucella has been isolated from a variety of marine mammals, including cetaceans (e.g., dolphins), seals, and otters (11).

Brucella abortus strain RB51 is an attenuated rough strain, currently being used as the official live vaccine for bovine brucellosis in the USA and several other countries. In strain RB51, the wboA gene, encoding a glycosyltransferase required for the O-side chain synthesis, is disrupted by an IS711 element. Recently, we have demonstrated that strain RB51WboA, RB51 complemented with a functional wboA gene, remains rough but expresses low quantities of O-side chain in the cytoplasm. Mice vaccinated with strain RB51WboA develop greatly enhanced resistance against challenge with B. abortus virulent strain 2308. We have also demonstrated that overexpression of Cu/Zn superoxide dismutase (SOD) in strain RB51 (RB51SOD) significantly increases its vaccine efficacy against strain 2308 challenge. In this study, we constructed a new recombinant strain, RB51SOD/WboA, that over expresses SOD with simultaneous expression of O-side chain in the cytoplasm. We tested the vaccine potential of strains RB51SOD, RB51WboA, RB51SOD/WboA against challenge with virulent Brucella melitensis 16M and B. abortus 2308 in mice. In comparison with strain RB51, strain RB51SOD induced better protection against strain 2308, but not strain 16M, challenge. Similar to strain RB51WboA, vaccination with strain RB51SOD/WboA resulted in complete protection of the mice from infection with strain 2308. When challenged with strain 16M, mice vaccinated with either strain RB51WboA or strain RB51SOD/WboA were significantly better protected than those vaccinated with strain RB51 or RB51SOD. These results suggest that strains RB51WboA and RB51SOD/WboA are good vaccine candidates for inducing enhanced protection against B. melitensis infection.

A recombinant vaccine was constructed for piscine mycobacteriosis utilizing a Brucella abortus strain RB51 vector expressing a mammalian Mycobacterium sp. 85A antigen. Juvenile striped bass were inoculated with the resulting construct at doses equivalent to 106, 107, 108, 109, and 1010 colony-forming units/fish. Blood and tissue samples from these fish demonstrated significant specific humoral and cell-mediated immune responses towards the 85A antigen in a dose-dependent manner. However, survival studies determined that inoculated fish failed to demonstrate cross-protective responses after live Mycobacterium marinum challenge 70 days post-inoculation.

Brucella abortus vaccine strain RB51 is an attenuated, stable rough mutant that is being used in many countries to control bovine brucellosis. Our earlier study demonstrated that the protective efficacy of strain RB51 can be significantly enhanced by overexpressing Cu–Zn superoxide dismutase (SOD), a homologous protective antigen. We have also previously demonstrated that strain RB51 can be engineered to express heterologous proteins and mice vaccinated with such recombinant RB51 strains develop a strong Th1 type of immune response to the foreign proteins. The present study is aimed at combining these two characteristics to generate new recombinant RB51 vaccines with enhanced abilities to protect against brucellosis and simultaneously able to protect against infections by Mycobacterium spp. We constructed two recombinant RB51 strains, RB51SOD/85A which overexpresses SOD with simultaneous expression of the 85A, a protective protein of Mycobacterium spp., and RB51ESAT which expresses ESAT-6, another protective protein of M. bovis, as a fusion protein with the signal sequence and few additional amino terminal amino acids of SOD. Mice vaccinated with these recombinant strains developed specific immune responses to the mycobacterial proteins and significantly enhanced protection against Brucella challenge compared to the mice vaccinated with strain RB51 alone.

Objective—To develop a novel oral vaccine delivery system for swine, using the rough vaccine strain of Brucella abortus. Animals—56 crossbred pigs from a brucellosis-free facility. Procedure—In 3 separate experiments, pigs were orally vaccinated with doses of 1 × 109 to > 1 × 1011 CFU of strain RB51 vaccine. The vaccine was placed directly on the normal corn ration, placed inside a whole pecan, or mixed with cracked pecans and corn. Results—Oral vaccination of pigs with vaccine strain RB51 resulted in a humoral immune response to strain RB51 and short-term colonization of the regional lymph nodes. Conclusions and Clinical Relevance—A viscous liquid such as Karo corn syrup in association with pecans that scarify the oral mucosa are necessary when placing the live vaccine directly onto corn or other food rations. Doses of > 1 × 1011 CFU of RB51 organisms/pig in this mixture ensures 100% colonization of regional lymph nodes via the oral route. This method may allow an efficient and economical means to vaccinate feral swine for brucellosis. (Am J Vet Res 2001;62:1328–1331)

The current work has the overall objectives of genetically engineering mono and poly-valent, IL-12 producing vaccinia/Brucella recombinant vaccines using synthetic E/L promoters and to test such vaccinia recombinants for their ability to induce a protective immune response against challenge with virulent Brucella in a mouse model. For an effective and strongly protective immune response it may he necessary to combine vaccination with vaccinia/Brucella recombinants with the inoculation of DNA vaccines. At this stage the following has been achieved: 1. Demonstrated Brucella Cu/Zn SOD to he a protective antigen. 2. Produced first WRvaccinia/Brucella antigen recombinants simultaneously producing IL-12. 3. Produced first WRvaccinia/Brucella recombinants producing a fusion protein of two protective Brucella antigens (SOD and L7/L12). 4. Initiated protection studies with WRvaccinia/Brucella antigen recombinants producing IL-12. i Detected additional protective Brucella antigens (BMCP24, BaBSC31 and BaPAL16.5) ready for cloning into vaccinia. 6. Produced several Brucella DNA vaccines to he used in combination with WRvaccinia/Brucella antigen recombinants and 7. Initiated production of MVA vaccinia/Brucella antigen recombinants. Based on the identification of protective antigens and the role of IL- 12, we are confident that the use of vaccinia/Brucella recombinants producing IL-12 will induce protective immunity. We are now able to produce such recombinants.

Brucella abortusRB51 is a stable rough, attenuated mutant vaccine strain derived from the virulent strain 2308. Recently, we demonstrated that thewboAgene in RB51 is disrupted by an IS711element (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 thewboAgene in smooth, virulentB. abortus,Brucella melitensis, andBrucella suisresults in rough, attenuated mutants which fail to produce the O polysaccharide (O antigen). In this study, we explored whether thewboAgene disruption is responsible for the rough phenotype of RB51. We complemented RB51 with a functionalwboAgene, 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 thewboAgene 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.

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.

One hundred and seven pregnant cows, which had been calfhood vaccinated with Brucella abortus strain 19 (S-19) were revaccinated with either S-19 or strain RB51 (S-RB51). All S-19-revaccinated animals seroconverted, while none of the RB51-revaccinated animals seroconverted. Two out of 25 (8%) S-19-revaccinated animals aborted, while none of the 57 RB51-revaccinated group aborted. Four of the S-19-revaccinated animals shed S-19 in the milk for at least 7 days, while only 1 cow shed S-RB51 for at least 3 days (but

Brucella abortusstrain RB51 is a stable, rough, attenuated mutant widely used as a live vaccine for bovine brucellosis. Our ultimate goal is to develop strain RB51 as a preferential vector for the delivery of protective antigens of other intracellular pathogens to which the induction of a strong Th1 type of immune response is needed for effective protection. As a first step in that direction, we studied the expression of a foreign reporter protein, β-galactosidase ofEscherichia coli, and the 65-kDa heat shock protein (HSP65) ofMycobacterium bovisin strain RB51. We cloned the promoter sequences ofBrucella sodCandgroEgenes in pBBR1MCS to generate plasmids pBBSODpro and pBBgroE, respectively. The genes for β-galactosidase (lacZ) and HSP65 were cloned in these plasmids and used to transform strain RB51. An enzyme assay in the recombinant RB51 strains indicated that the level of β-galactosidase expression is higher under thegroEpromoter than under thesodCpromoter. In strain RB51 containing pBBgroE/lacZ, but not pBBSODpro/lacZ, increased levels of β-galactosidase expression were observed after subjecting the bacteria to heat shock or following internalization into macrophage-like J774A.1 cells. Mice vaccinated with either of the β-galactosidase-expressing recombinant RB51 strains developed specific antibodies of predominantly the immunoglobulin G2a (IgG2a) isotype, and in vitro stimulation of their splenocytes with β-galactosidase induced the secretion of gamma interferon (IFN-γ), but not interleukin-4 (IL-4). A Th1 type of immune response to HSP65, as indicated by the presence of specific serum IgG2a, but not IgG1, antibodies, and IFN-γ, but not IL-4, secretion by the specific-antigen-stimulated splenocytes, was also detected in mice vaccinated with strain RB51 containing pBBgroE/hsp65. Studies with mice indicated that expression of β-galactosidase or HSP65 did not alter either the attenuation characteristics of strain RB51 or its vaccine efficacy againstB. abortus2308 challenge.

Vaccination of mice with Escherichia coli expressing Brucella Cu/Zn superoxide dismutase (SOD) [ E. coli (pBSSOD)] induced a significant level of protection against virulent Brucella abortus challenge, although this level was not as high as the one reached with B. abortus vaccine strain RB51. In addition, vaccination with E. coli (pBSSOD) induced antibodies to Cu/Zn SOD and a strong proliferative response of splenocytes when stimulated in vitro with a thioredoxin-Cu/Zn SOD fusion protein.

Genes encoding Brucella abortus Cu/Zn superoxide dismutase (SOD) and a 54 kDa Escherichia coli HtrA homologue were cloned into shuttle plasmids pUV-1 and pSC11, and transfected into vaccinia virus to develop recombinants vUBSOD and vSB54. Control vaccinia virus recombinants vUV-1 and vSC11, carrying only the beta-gal reporter gene but no B. abortus DNA were also developed. Recombinants were analyzed in Western blotting using a polyclonal B. abortus immune serum. vUBSOD expressed a protein of apparent molecular weight of 28 kDa, composed of the 20 kDa B. abortus Cu/Zn-SOD and a protein approximately 8 kDa encoded by a portion of the vaccinia virus TK gene. vSB54 expressed a 54 kDa protein corresponding to the 54 kDa HtrA homologue. Recombinants vUSV-1 and vSC11 did not express B. abortus proteins. Groups of mice were inoculated intraperitoneally with 10(7) TCID50 of 1 of the 4 different recombinant vaccinia viruses and 5 weeks later their sera were analyzed for antibodies against vaccinia virus and B. abortus proteins. Each group of mice responded with antibodies to vaccinia virus. Sera of vSB54-inoculated mice recognized the 54 kDa HtrA homologue. vUBSOD did not induce a humoral immune response. These results represent the first report on the expression of B. abortus proteins by vaccinia virus recombinants and the first demonstrated immune response against a B. abortus protein expressed by such a recombinant.

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.

A rifampin-resistant mutant of Brucella abortus, designated RB51, was derived by repeated passage of strain 2308 on Trypticase soy supplemented with 1.5% agar and varying concentrations rifampin or penicillin. The RB51 colonies absorbed crystal violet and RB51 cell suspensions autoagglutinated, indicating a rough type colonial morphology for this strain. No O-chain component was detected in lipopolysaccharide (LPS) extracted from RB51 on SDS-PAGE gels stained with silver. Western blot analysis with the monoclonal antibody BRU 38, which is specific for the perosamine homopolymer O-chain of smooth Brucella LPS, indicated that the LPS of RB51 is highly deficient in O-chain when compared with the parenteral smooth strain 2308 or rough strain 45/20. Biochemically, RB51 resembles parental strain 2308 in its ability to utilize erythritol. Intraperitoneal inoculation of RB51 into mice results in a splenic colonization which is cleared within four weeks post infection. RB51 does not revert to smooth colony morphology upon passage in vivo (mice) or in vitro. Mice infected with RB51 produce antibodies against B. abortus antigens including class 2 and 3 outer membrane proteins but not against the O-chain. Furthermore, rabbits, goats and cattle hyperimmunized with sonicates of RB51 develop antibodies to B. abortus cellular antigens but do not develop antibodies specific for the O-chain. Immunization of mice with 1 x 10(8) viable RB51 organisms confers significant protection against challenge with virulent B. abortus strain 2308.

Extracts of Brucella abortus strains 2308,RB51,45/20 and ST 19 had no significant differences in superoxide dismutase (SOD) activity as measured by the epinephrine assay. These B. abortus strains represent smooth, intermediate and rough colony forms. SOD activity was inhibited 60 to 75% by 2 mM KCN and suggests the presence of Cu/Zn SOD. The SOD activities were similar when the strains were grown in trypticase soy broth containing either 0.5% glucose or erythritol. There were two distinct SOD activity bands in native polyacrylamide gel electrophoresis with identical mobilities for each of the strains. When the native gel was stained for SOD activities in the presence of 2 mM KCN, the SOD band that co-migrated with the bovine erythrocyte Cu/Zn SOD activity disappeared. The band of SOD activity that migrated similar to E. coli iron SOD activity was unaffected by KCN. There were no significant differences in either the total SOD or Cu/Zn SOD activities among the strains. As the Brucella strains represent ranges of virulence, it is difficult to associate any primary role for SOD as a virulence factor.