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The pathological consequences of exposure to the vaccine strain Brucella abortus S19 were evaluated in 30 employees from vaccine-manufacturing plants. Active brucellosis was diagnosed in 21 subjects, of whom only five recalled an accidental exposure. Clinical manifestations were mild, and only one patient presented a complication. After antimicrobial therapy, initially symptomatic patients either experienced clinical remission or had mild persistent symptoms. This is the first study reporting infection by B. abortus S19 among workers from vaccine-manufacturing plants, which in many cases was acquired from unnoticed exposures. Measures to improve the safety of B. abortus S19 handling should be implemented.

Antibodies to cytoplasmic proteins (CP) of Brucella have been shown to be useful for the diagnosis of human brucellosis; however, some early-diagnosed patients lack such an antibody response while having high titers of antibodies to lipopolysaccharide (LPS). To address which factors determine this serological discrepancy in the early stages of brucellosis we examined the antibody response to CP and LPS of 21 patients involved in an outbreak of B. melitensis infection who had a short duration of clinical illness at diagnosis (3-40 d). At diagnosis, antibodies to LPS (IgM and/or IgG) were found in all patients, while anti-CP antibodies were detected in 16 subjects (76%). At 6 weeks post-diagnosis IgG to CP (with or without IgM) had been detected in 13 patients and IgM alone had been found in 4; however, 4 other patients (19%) had no response to CP. No significant differences were found between these 3 groups in terms of age, gender, antimicrobial agents or factors that could hamper the immune response. Notably, however, the 4 non-responders and 3 of the 4 patients having only IgM to CP had started antibiotic therapy within 14 d post-symptoms, while treatment was started later in 9 of 13 patients who developed anti-CP IgG. In addition, maximum titers of IgG to CP tended to be lower in early-treated patients. These results suggest that very early antibiotic therapy hampers the antibody response to Brucella CP but has little impact on the anti-LPS response. Given the higher specificity of the former and the higher sensitivity of the latter, both reactivities should be measured in order to diagnose human brucellosis.

The characterization of proteins from Brucella spp, the causative agent of brucellosis, has been the subject of intensive research. We have described an 18-kDa cytoplasmic protein of Brucella abortus and shown the potential usefulness of this protein as an antigen for the serologic diagnosis of brucellosis. The amino acid sequence of the protein showed a low but significant homology with that of lumazine synthases. Lumazine is an intermediate product in bacterial riboflavin biosynthesis. The recombinant form of the 18-kDa protein (expressed in E. coli) folds like the native Brucella protein and has lumazine-synthase enzymatic activity. Three-dimensional analysis by X-ray crystallography of the homolog Bacillus subtilis lumazine synthase has revealed that the enzyme forms an icosahedral capsid. Recombinant lumazine synthase from B. abortus was crystallized, diffracted X rays to 2.7-A resolution at room temperature, and the structure successfully solved by molecular replacement procedures. The macromolecular assembly of the enzyme differs from that of the enzyme from B. subtilis. The Brucella enzyme remains pentameric (90 kDa) in its crystallographic form. Nonetheless, the active sites of the two enzymes are virtually identical at the structural level, indicating that inhibitors of these enzymes could be viable pharmaceuticals across a broad species range. We describe the structural reasons for the differences in their quaternary arrangement and also discuss the potential use of this protein as a target for the development of acellular vaccines.

The diagnosis of human neurobrucellosis usually relies on the detection of antibodies to Brucella lipopolysaccharide (LPS) in cerebrospinal fluid (CSF) by agglutination tests or enzyme-linked immunosorbent assay (ELISA). Here we describe the detection of immunoglobulin G (IgG) to cytoplasmic proteins (CP) of Brucella spp. by ELISA and Western blotting in seven CSF samples from five patients with neurobrucellosis. While IgG to CP (titers of 200 to 12, 800) and IgG to LPS (800 to 6,400) were found in the CSF of these patients, these antibodies were not detected in CSF samples from two patients who had systemic brucellosis without neurological involvement. The latter, however, had serum IgG and IgM to both LPS and CP. No reactivity to these antigens was found in CSF samples from 14 and 20 patients suffering from nonbrucellar meningitis and noninfectious diseases, respectively. These findings suggest that, in addition to its usefulness in the serological diagnosis of human systemic brucellosis, the ELISA with CP antigen can be used for the specific diagnosis of human neurobrucellosis.

An antigenic capture ELISA was developed to measure serum antibodies against an 18 kDa cytoplasmic protein of Brucella. This assay was used to detect anti-18 kDa reactivity in sera from 30 dogs having confirmed or suspected brucellosis. Antibodies against the 18 kDa protein were found in 26 of them, which were also positive by the slide agglutination test (2ME-RSAT). The overall correlation (positive and negative results) between the ELISA and 2ME-RSAT tests was 93.3%. Additionally, these sera were assayed by an indirect ELISA using a whole extract of cytoplasmic proteins of B. abortus (LPS-free CYT). The results of both ELISAs were coincident in 28 of 30 (93.3%) dogs having confirmed or suspected brucellosis. When a serological follow-up was performed on some dogs having confirmed brucellosis, antibody titers measured by both ELISAs showed a parallel progression. On the other hand, the capture ELISA showed good specificity, since a positive result was obtained only in 2 of 103 sera from healthy dogs. These preliminary results show that the ELISA for detecting serum antibodies against the 18 kDa cytoplasmic protein of Brucella could be useful for the diagnosis of canine brucellosis. This study also shows that the results obtained with this single protein of Brucella are equivalent to those obtained with the whole extract of cytoplasmic proteins.

A serological 2-year follow-up of 35 patients who had brucellosis with different clinical outcomes was performed. Patients were followed up by standard tube agglutination (STA) and 2-mercaptoethanol STA (2ME-STA) tests and by two enzyme-linked immunosorbent assays (ELISAs) to independently measure values of serum IgG to either lipopolysaccharide (LPS) or cytoplasmic proteins of Brucella. Whereas STA and 2ME-STA tests revealed characteristic antibody profiles for patients who recovered (group I), this was not the case for patients with persistent illness (group II) or patients with relapses (group III). On the other hand, titers measured by the 2ME-STA test were low or negative in 10 patients who had positive blood cultures. Levels of IgG antibodies to LPS and proteins in each group of patients exhibited characteristic changes. For group I, however, antibodies to proteins were better predictors of recovery, since titers reached negative values for five patients. Levels of IgG antibodies to LPS and proteins were persistently high in group II patients, and peaks in these antibody levels corresponded with relapses in group III patients. The determination of values of IgG antibodies to proteins by ELISA appears useful for the serological follow-up of patients with brucellosis.

A preparation of Brucella abortus cytoplasmic proteins was depleted of lipopolysaccharide (LPS) by immunoadsorption with a monoclonal antibody (BC68) specific for the O antigen of B. abortus smooth LPS. This extract was used as antigen in an indirect ELISA for measuring antiprotein humoral immune response in dogs suffering from brucellosis and in healthy controls. All of the affected dogs studied showed IgG antiprotein response, while 2% (2 of 103) of the healthy dogs used as controls gave a positive result. All sera found to be positive by ELISA were also positive by the rapid slide agglutination test. These preliminary results show that the ELISA using B. abortus cytoplasmic proteins could be useful for the specific diagnosis of canine brucellosis.

An outbreak of Brucella melitensis in a family was studied. From the fourteen family members who ate unpasteurized goat cheese nine became ill. Patients included four females and five males of 8 to 75 years old. In seven of the patients the diagnosis was confirmed by positive blood culture for B. melitensis biovar 1. All the patients were analyzed by standard tube agglutination (STA) and standard tube agglutination with 2-mercaptoethanol (STA-2ME) tests at the time of diagnosis. In six of the patients, ELISA assays were used to assess the humoral immune anti-protein and anti-lipopolysaccharide (LPS) responses. Anti-LPS IgG antibodies were detected in all of the patients. Anti-proteins IgG antibodies were present at significant levels in all the studied patients including the STA-2ME negative ones.

Some anticytoplasmic protein monoclonal antibodies (MAbs) from mice immunized by infection with Brucella ovis cells have been obtained. One of these MAbs, BI24, was used to purify by immunoaffinity a protein with a pI of 5.6 and a molecular mass of 18 kDa. This protein was present in all of the rough and smooth Brucella species studied, but it could not be detected in Yersinia enterocolitica 09. Three internal peptides of this protein were partially sequenced; no homology with other bacterial proteins was found. The immunogenicity of the 18-kDa protein was studied with both human and bovine sera by a capture enzyme-linked immunosorbent assay system with MAb BI24.

A preparation of Brucella abortus cytoplasmic proteins was depleted of lipopolysaccharide (LPS) by immunoadsorption with a monoclonal antibody (MAb), BC68, specific for the O antigen of B. abortus smooth LPS. Two enzyme-linked immunosorbent assay (ELISA) systems were developed and used in this study. The first system includes an LPS-free cytoplasmic protein preparation; the second one was based on antigen capture on MAb BC68. By using these systems, we have demonstrated that 94% (33 of 35) of the brucellosis patients studied showed immunoglobulin G antiprotein response and also that all of the patients showed a strong anti-LPS reactivity. Thirty-six serologically positive individuals with no active infection at the time of examination (SPI) were also included. No immunoglobulin G antibodies against proteins were detected in 34 of them (92%), whereas 31 SPI (86%) showed various degrees of anti-LPS reactivity. The use of the LPS-free protein extract in ELISAs made it possible to establish differential reactivity patterns between active and inactive brucellosis.

Results indicating that analysis of the immune humoral response of brucellosis patients by immunoblotting provides useful information for the characterization of antigenic fractions of possible diagnostic importance in human brucellosis are presented. Sera of 90 patients were obtained: 23 suffering from chronic brucellosis, 20 from acute brucellosis and 47 belonging to the group of serologically positive individuals (SPI) without clinical evidence of active infection at the time of examination, and 35 healthy volunteers. They were tested against three antigenic fractions: cytoplasmic (CYT), outer membrane (OM) and inner membrane (IM). These fractions, which include virtually all the bacterial protein components, were prepared from Brucella abortus 1119/3 strain by detergent solubilization, enzymatic digestion and ultracentrifugation. Results obtained with these fractions showed the existence of antigens that permit the detection of brucellosis patients and their differentiation from SPI patients, with very high sensitivity.