Isabelle Jacques

Evaluation of a Multiplex PCR Assay (Bruce-ladder) for Molecular Typing of All Brucella Species, Including the Vaccine Strains

ABSTRACT An evaluation of a multiplex PCR assay (Bruce-ladder) was performed in seven laboratories using 625 Brucella strains from different animal and geographical origins. This robust test can differentiate in a single step all of the classical Brucella species, including those found in marine mammals and the S19, RB51, and Rev.1 vaccine strains.

MLVA-16 typing of 295 marine mammal Brucella isolates from different animal and geographic origins identifies 7 major groups within Brucella ceti and Brucella pinnipedialis

BackgroundSince 1994, Brucella strains have been isolated from a wide range of marine mammals. They are currently recognized as two new Brucella species, B. pinnipedialis for the pinniped isolates and B. ceti for the cetacean isolates in agreement with host preference and specific phenotypic and molecular markers. In order to investigate the genetic relationships within the marine mammal Brucella isolates and with reference to terrestrial mammal Brucella isolates, we applied in this study the Multiple Loci VNTR (Variable Number of Tandem Repeats) Analysis (MLVA) approach. A previously published assay comprising 16 loci (MLVA-16) that has been shown to be highly relevant and efficient for typing and clustering Brucella strains from animal and human origin was used.Results294 marine mammal Brucella strains collected in European waters from 173 animals and a human isolate from New Zealand presumably from marine origin were investigated by MLVA-16. Marine mammal Brucella isolates were shown to be different from the recognized terrestrial mammal Brucella species and biovars and corresponded to 3 major related groups, one specific of the B. ceti strains, one of the B. pinnipedialis strains and the last composed of the human isolate. In the B. ceti group, 3 subclusters were identified, distinguishing a cluster of dolphin, minke whale and porpoise isolates and two clusters mostly composed of dolphin isolates. These results were in accordance with published analyses using other phenotypic or molecular approaches, or different panels of VNTR loci. The B. pinnipedialis group could be similarly subdivided in 3 subclusters, one composed exclusively of isolates from hooded seals (Cystophora cristata) and the two others comprising other seal species isolates.ConclusionThe clustering analysis of a large collection of marine mammal Brucella isolates from European waters significantly strengthens the current view of the population structure of these two species, and their relative position with respect to the rest of the Brucella genus. MLVA-16 is confirmed as being a rapid, highly discriminatory and reproducible method to classify Brucella strains including the marine mammal isolates. The Brucella2009 MLVA-16 genotyping database available at http://mlva.u-psud.fr/ is providing a detailed coverage of all 9 currently recognized Brucella species.

Protection conferred on mice by monoclonal antibodies directed against outer-membrane-protein antigens of Brucella.

Twenty-six monoclonal antibodies (MAbs) directed against seven brucella outer-membrane proteins (OMPs) of 10, 16.5, 19, 25-27, 31-34, 36-38 and 89 Kda were screened for passive protection of mice; three MAbs (directed against 16.5, 25-27 and 36-38 Kda) reduced significantly the initial colonisation of the spleen measured 7 days after challenge. Although significant, the reduction in numbers of Brucella organisms in the spleen was low compared with that conferred by MAbs against lipopolysaccharide of smooth specificity (S-LPS). The three most protective MAbs belonged to two isotypes (IgG1 and IgG2a) and were specific for three different OMPs. No relationship between protection and binding of MAbs to the challenge S strain or its rough mutant was observed in the mouse model. The humoral protection depended mainly on antibodies directed against S-LPS, although some MAbs against OMPs had weak protective activity.

Rough mutants defective in core and O-polysaccharide synthesis and export induce antibodies reacting in an indirect ELISA with smooth lipopolysaccharide and are less effective than Rev 1 vaccine against Brucella melitensis infection of sheep

Classical brucellosis vaccines induce antibodies to the O-polysaccharide section of the lipopolysaccharide that interfere in serodiagnosis. Brucella rough (R) mutants lack the O-polysaccharide but their usefulness as vaccines is controversial. Here, Brucella melitensis R mutants in all main lipopolysaccharide biosynthetic pathways were evaluated in sheep in comparison with the reference B. melitensis Rev 1 vaccine. In a first experiment, these mutants were tested for ability to induce anti-O-polysaccharide antibodies, persistence and spread through target organs, and innocuousness. Using the data obtained and those of genetic studies, three candidates were selected and tested for efficacy as vaccines against a challenge infecting 100% of unvaccinated ewes. Protection by R vaccines was 54% or less whereas Rev 1 afforded 100% protection. One-third of R mutant vaccinated ewes became positive in an enzyme-linked immunosorbent assay with smooth lipopolysaccharide due to the core epitopes remaining in the mutated lipopolysaccharide. We conclude that R vaccines interfere in lipopolysaccharide immunosorbent assays and are less effective than Rev 1 against B. melitensis infection of sheep.

Protection conferred on mice by combinations of monoclonal antibodies directed against outer-membrane proteins or smooth lipopolysaccharide of Brucella

The effect of monoclonal antibodies (MAbs) injected alone or in combination on brucella splenic infection in CD-1 mice was tested 7 and 21 days after a challenge with virulent Brucella abortus 544. Passive immunisation of mice with anti-25-27-kDa MAb alone, or mixed with protective anti-16.5 and anti-36-38-kDa MAbs, or with MAbs of the same specificity which were previously demonstrated to have no activity on CD-1 mice, produced a significant reduction of spleen counts of B. abortus (p less than 0.01). Other combinations of MAbs did not reduce splenic infection in comparison with the untreated control group. BALB/c mice were used to test the possible interference of the immune response of CD-1 mice against MAbs that were produced in BALB/c mice. No reduction of splenic infection was shown with anti-25-27- or -36-38-kDa MAbs, whereas anti-lipopolysaccharide (LPS) MAb which was produced in CBA mice was effective. Combination of anti-protein MAbs with the anti-LPS MAb produced only the effect of the anti-LPS MAb at 7 and 21 days after challenge.

Induction of antibody and protective responses in mice by Brucella O-polysaccharide-BSA conjugate

A Brucella conjugate vaccine was synthesized by covalently coupling the O-polysaccharide (PS) obtained from Brucella melitensis 16M, to bovine serum albumin (BSA). The conjugate (PS-BSA) was non-toxic for mice. When injected at two doses (10 or 1 micrograms) in mice, it induced the production of antibodies and the boost was shown to be efficient if administered after 10 weeks. Immunization of mice with 10 micrograms of conjugate conferred significant protection against virulent challenge with B. melitensis H38, similar to that obtained with the reference vaccine H38.

Immunogenicity of recombinant Escherichia coli expressing the omp31 gene of Brucella melitensis in BALB/c mice

BALB/c mice were immunized with recombinant Escherichia coli expressing the omp31 gene of Brucella melitensis, a gene coding for a major outer membrane protein. Immunization resulted in the production of specific antibodies to B. melitensis in the serum, the production of which was considerably increased after boosting with a dose ten times lower than the first. A significant specific proliferative response of immune spleen cells to B. melitensis was observed 5 weeks after the first immunization but this response did not persist. Despite the induction of systemic humoral and cellular immune responses by recombinant E. coli expressing the B. melitensis omp31 gene, no significant protection against a challenge with smooth B. melitensis H38S was observed in immunized mice. These results demonstrate that despite the strong antibody response induced in mice, immunization with the recombinant Omp31 of B. melitensis does not confer any protective effect against a virulent smooth B. melitensis. However, its potential protective effect for protection against rough Brucella would be worth testing.

Efficacy of ELISA compared to conventional tests (RBPT and CFT) for the diagnosis of Brucella melitensis infection in sheep

An indirect ELISA (iELISA) using a B. abortus smooth lipopolysaccharide (S-LPS) as coating antigen and a polyclonal anti-sheep IgG peroxydase-labeled serum as conjugate was developed. The iELISA was assessed in comparison to the Food and Agriculture Organisation/International Atomic Energy Agency (FAO/IAEA) standard bovine ELISA kit (IAEA kit) using an anti-bovine IgG conjugate that cross-reacts with sheep antibodies, and with complement fixation test (CFT) and Rose Bengal Plate Test (RBPT). The evaluation was performed on sera from ewes experimentally infected with Brucella melitensis 53H38 (H38), using negative and positive sheep reference sera. No significant difference was found as regards to the specificity, the lower limit of detection and the estimated sensitivity of the two iELISAs. This suggests that an anti-bovine conjugate could allow the development of only one ELISA protocol for all ruminants. The iELISA, if well standardized, proved to be a good screening test able to be used either alone or in addition to RBPT.

Pulmonary evolution of cystic fibrosis patients colonized by Pseudomonas aeruginosa and/or Burkholderia cepacia

We analysed the pulmonary evolution (radiological scores and pulmonary function) of 81 cystic fibrosis (CF) patients colonized by Pseudomonas aeruginosa (PA), by Burkholderia cepacia (BC) or by both these bacteria, compared to a control group. Pulmonary function was compared in the age bracket 6–13 years. Functional vital capacity (FVC) and forced expiratory volume (FEV1) values for PA colonized patients were significantly worse than for the control group but better than for children colonized by both organisms. In this last group, the evolution of radiological scores and pulmonary function showed a greater decline 2 years after the first colonization compared to the other groups. FVC and FEV1 values in patients colonized by BC were not worse than these of patients colonized by PA. Moreover, BC affected older patients with advanced lung disease and often previously colonized with PA. These results suggested that co-colonization by PA and BC could be a more deleterious factor on the pulmonary evolution than the isolated colonization by PA or BC, and that BC could be a severity marker rather than a cause. In addition, after starting the utilization of mouthpieces with filter at single use for spirometry in 1993 (without any other change in preventive measures already taken during hospitalization), incidence of BC decreased from 8.2% to zero, and no new case of BC colonization has been observed over the last 4 years.ConclusionCo-colonization of CF patients by PA and BC is more deleterious for pulmonary evolution than colonization by one of these bacteria alone. Re-inforcement of environmental measures during hospitalization (e.g. use of disposable mouthpieces for spirometry) was sufficient to reduce the transmission of BC.

Monoclonal antibodies to Brucella rough lipopolysaccharide: characterization and evaluation of their protective effect against B. abortus

We characterized 4 monoclonal antibodies (mAb) specific for rough lipopolysaccharide (R-LPS) of Brucella. mAb were selected by enzyme-linked immunosorbent assay (ELISA) on whole B. abortus 45/20 rough cells and R-LPS from B. melitensis B115 rough cells. Specificity was confirmed by immunoblot analysis using R-LPS and smooth LPS (S-LPS) preparations. Anti-R-LPS revealed the low molecular mass R-LPS molecules below 20.1 kDa in the R-LPS and S-LPS preparations as well as the typical A and M patterns in high molecular mass S-LPS molecules (between 21.5 and 66 kDa) in the S-LPS preparations. An O-polysaccharide-specific mAb revealed only high molecular mass S-LPS molecules in the S-LPS preparation. In ELISA the anti-R-LPS mAb bound better on rough than on smooth B. abortus 544 whole cells, and this was confirmed by immunoelectron microscopy. Protective activity of anti-R-LPS mAb of different isotypes was tested on mice and compared with an S-LPS-specific mAb. Only the IgG3 mAb reduced significantly the splenic infection but did not reach the level of protection conferred by the S-LPS-specific mAb.