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See detailIdentification of Brucella spp. genes involved in intracellular trafficking.
Delrue, R. M.; Martinez-Lorenzo, M.; Lestrate, P. et al

in Cellular Microbiology (2001), 3(7), 487-97

After uptake by host cells, the pathogen Brucella transits through early endosomes, evades phago-lysosome fusion and replicates in a compartment associated with the endoplasmic reticulum (ER). The ... [more ▼]

After uptake by host cells, the pathogen Brucella transits through early endosomes, evades phago-lysosome fusion and replicates in a compartment associated with the endoplasmic reticulum (ER). The molecular mechanisms underlying these processes are still poorly understood. To identify new bacterial factors involved in these processes, a library of 1800 Brucella melitensis 16M mini-Tn5catkm mutants was screened for intracellular survival and multiplication in HeLa cells and J774A.1 macrophages. Thirteen mutants were identified as defective for their intracellular survival in both cell types. In 12 of them, the transposon had inserted in the virB operon, which encodes a type IV-related secretion system. The preponderance of virB mutants demonstrates the importance of this secretion apparatus in the intracellular multiplication of B. melitensis. We also examined the intracellular fate of three virB mutants (virB2, virB4 and virB9) in HeLa cells by immunofluorescence. The three VirB proteins are not necessary for penetration and the inhibition of phago-lysosomal fusion within non-professional phagocytes. Rather, the virB mutants are unable to reach the replicative niche and reside in a membrane-bound vacuole expressing the late endosomal marker, LAMP1, and the sec61beta protein from the ER membrane, proteins that are present in autophagic vesicles originating from the ER. [less ▲]

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See detailIdentification and characterization of in vivo attenuated mutants of Brucella melitensis.
Lestrate, P.; Delrue, R. M.; DANESE, Isabelle ULg et al

in Molecular Microbiology (2000), 38(3), 543-51

Brucella melitensis 16M is a Gram-negative alpha2-proteobacterium responsible for abortion in goats and for Malta fever in humans. This facultative intracellular pathogen invades into and survives within ... [more ▼]

Brucella melitensis 16M is a Gram-negative alpha2-proteobacterium responsible for abortion in goats and for Malta fever in humans. This facultative intracellular pathogen invades into and survives within both professional and non-professional phagocytes. Signature-tagged mutagenesis (STM) was used to identify genes required for the in vivo pathogenesis of Brucella. A library of transposon mutants was screened in a murine infection model. Out of 672 mutants screened, 20 were not recovered after a 5 day passage in BALB/c mice. The attenuation of 18 mutants was confirmed using an in vivo competition assay against the wild-type strain. The 18 mutants were characterized further for their ability to replicate in murine macrophages and in HeLa cells. The sequences disrupted by the transposon in the mutants have homology to genes coding for proteins of different functional classes: transport, amino acid and DNA metabolism, transcriptional regulation, peptidoglycan synthesis, a chaperone-like protein and proteins of unknown function. The mutants selected in this study provide new insights into the molecular basis of Brucella virulence. [less ▲]

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See detailGenetic organisation of the lipopolysaccharide O-antigen biosynthesis region of brucella melitensis 16M (wbk).
Godfroid, F.; Cloeckaert, A.; Taminiau, Bernard ULg et al

in Research in Microbiology (2000), 151(8), 655-68

Brucella spp. are Gram-negative, facultative intracellular bacteria that cause a zoonotic world-wide disease. As in other Gram-negative bacteria, its S-LPS (smooth lipopolysaccharide) is a major ... [more ▼]

Brucella spp. are Gram-negative, facultative intracellular bacteria that cause a zoonotic world-wide disease. As in other Gram-negative bacteria, its S-LPS (smooth lipopolysaccharide) is a major determinant of virulence. The Brucella melitensis 16M LPS O-antigen is a homopolymer of 4-formamido-4,6, dideoxymannose. In this study, the previously cloned 14-kb wbk gene cluster was sequenced, and seven open reading frames (ORFs) as well as four insertion sequences were identified. Six of the seven ORFs are homologous to LPS biosynthesis genes from other organisms. The gmd, per and wbkC gene products are predicted to be involved in 4-formamido-4,6,dideoxymannose synthesis. By deletion experiments, we demonstrated that the putative formyltransferase WbkC is absolutely required for the O-side-chain production. The wbkA gene product is similar to several mannosyltransferases and is probably involved in the polymerisation of the B. melitensis O-side-chain. We also identified two genes (wzm and wzt) encoding proteins with high similarity to several two-component ABC (ATP-binding cassette) transporters. Their implication in O-antigen translocation across the inner membrane was confirmed by gene replacement. Finally, no function has been assigned to the wbkB gene either by homology search or functionally, because deletion of wbkB did not interfere with the O-antigen structure. The seven ORFs have a low G + C content, indicating that they might have been acquired by lateral transfer from a progenitor with more A + T rich DNA. [less ▲]

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See detailDiagnosis of bovine brucellosis by skin test: conditions for the test and evaluation of its performance.
Saegerman, Claude ULg; Vo, T. K.; De Waele, L. et al

in Veterinary Record : Journal of the British Veterinary Association (1999), 145(8), 214-8

Brucellergene OCB (Rhone-Merieux) was used as an allergen to define the intrinsic parameters of a skin test and to compare its properties with serology for the diagnosis of bovine brucellosis. The skin ... [more ▼]

Brucellergene OCB (Rhone-Merieux) was used as an allergen to define the intrinsic parameters of a skin test and to compare its properties with serology for the diagnosis of bovine brucellosis. The skin test was also evaluated for its capacity to solve problems associated with false positive reactions in serological tests. The optimal reading delay for the skin test was 72 hours. The brucellosis allergic reaction was two to three times less intense than the tuberculosis allergic reaction. An increase of 1.1 mm or more in the skin thickness was therefore considered to be an adequate cut-off. The specificity calculated for 1192 brucellosis-free animals (including animals from brucellosis-free herds in which false positive serological reactions had been reported) was 99-83 per cent (95 per cent confidence interval [CI] 99-40 to 99-98 per cent). The sensitivity determined from 27 experimentally infected heifers ranged from 93 per cent (95 per cent CI 76 to 100 per cent) to 78 per cent (95 per cent CI 58 to 91 per cent) when measured respectively one and six months after the infection. Allergic reactions could be detected in vaccinated animals up to four-and-a-half years after the vaccination. On the other hand, no sensitisation was recorded in naive animals after up to eight monthly injections of the allergen. The skin test gave valuable information, in combination with the serological tests, in both acute and chronic brucellosis. The skin test discriminated brucellosis clearly from false positive serological reactions due to infections with Yersinia enterocolitica O9. [less ▲]

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See detailIdentification of the perosamine synthetase gene of Brucella melitensis 16M and involvement of lipopolysaccharide O side chain in Brucella survival in mice and in macrophages.
Godfroid, F.; Taminiau, Bernard ULg; DANESE, Isabelle ULg et al

in Infection and Immunity (1998), 66(11), 5485-93

Brucella organisms are facultative intracellular bacteria that may infect many species of animals as well as humans. The smooth lipopolysaccharide (S-LPS) has been reported to be an important virulence ... [more ▼]

Brucella organisms are facultative intracellular bacteria that may infect many species of animals as well as humans. The smooth lipopolysaccharide (S-LPS) has been reported to be an important virulence factor of these organisms, but the genetic basis of expression of the S-LPS O antigen has not yet been described. Likewise, the role of the O side chain of S-LPS in the survival of Brucella has not been clearly defined. A mini-Tn5 transposon mutant library of Brucella melitensis 16M was screened by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies (MAbs) directed against the O side chain of Brucella. One mutant, designated B3B2, failed to express any O side chain as confirmed by ELISA, Western blot analysis, and colony coloration with crystal violet. Nucleotide sequence analysis demonstrated that the transposon disrupted an open reading frame with significant homology to the putative perosamine synthetase genes of Vibrio cholerae O1 and Escherichia coli O157:H7. The low G+C content of this DNA region suggests that this gene may have originated from a species other than a Brucella sp. The survival of B. melitensis mutant strain B3B2 in the mouse model and in bovine macrophages was examined. The results suggested that S-LPS or, more precisely, its O side chain is essential for survival in mice but not in macrophages. [less ▲]

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See detailExpression of Lamp-1 and Lamp-2 and their interactions with galectin-3 in human tumor cells.
Sarafian, V.; Jadot, Michel ULg; Foidart, Jean-Michel ULg et al

in International Journal of Cancer = Journal International du Cancer (1998), 75(1), 105-11

Lysosomal-membrane-associated glycoproteins (Lamps) 1 and 2 are rarely found on the plasma membranes of normal cells. There is evidence suggesting an increase in their cell-surface expression in tumor ... [more ▼]

Lysosomal-membrane-associated glycoproteins (Lamps) 1 and 2 are rarely found on the plasma membranes of normal cells. There is evidence suggesting an increase in their cell-surface expression in tumor cells, with some data indicating that the adhesion of some cancer cells to the extracellular matrix is partly mediated by interactions between Lamps and E-selectin and between Lamps and galectins (endogenous-galactoside-binding lectins). The present study examined the expression of Lamp-1 and Lamp-2 and their interactions with galectin-3 in different human tumor cell lines. Indirect immunofluorescence staining revealed accumulation of Lamp molecules at the edges of A2058 human metastasizing melanoma cells suggesting that these glycoproteins could participate in cell adhesion. Flow cytometry showed the presence of cell-surface Lamps in A2058, HT1080 (human fibrosarcoma) and CaCo-2 (human colon-adenocarcinoma) cells. Treatment with 2 mM sodium butyrate for 24 and 48 hr resulted in a significant increase in Lamps surface expression. A strong binding of A2058 to recombinant galectin-3 was detected by FACS. The application of 2 and 5 mM butyrate for the same incubation period enhanced galectin-3 binding to Lamps-expressing cells. Our results support the idea that Lamps may be considered a new family of adhesive glycoproteins participating in the complex process of tumor invasion and metastasis. [less ▲]

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See detailInfection of cattle with Yersinia enterocolitica O:9 a cause of the false positive serological reactions in bovine brucellosis diagnostic tests.
Weynants, V.; Tibor, A.; Denoel, P. A. et al

in Veterinary Microbiology (1996), 48(1-2), 101-12

During the last four years, an increasing number of cattle herds were classified positive by brucellosis screening tests in areas of Belgium and France free of the disease. No clinical symptom of ... [more ▼]

During the last four years, an increasing number of cattle herds were classified positive by brucellosis screening tests in areas of Belgium and France free of the disease. No clinical symptom of brucellosis was reported in these animals and no Brucella abortus strains were isolated. After two years, no brucellosis outbreak was registered in all of the herds concerned. On this basis, all the serological reactions observed were classified as false positive. An ELISA using Yersinia Outer membrane Proteins (YOPs) as antigens was developed in order to discriminate between a Yersinia enterocolitica O:9 infection and a Brucella abortus infection. Antibodies against YOPs were detected in sera from Y. enterocolitica O:9 experimentally infected cattle (n = 4) but not in sera from B. abortus experimentally infected cattle (n = 4). In a field study, 66.7% of the 174 serum samples from cattle presenting false positive serological reactions showed anti-YOPs antibodies whereas only 10% of 454 sera, classified negative by the brucellosis screening tests, showed anti-YOPs antibodies. Our results suggest that infections with Y. enterocolitica O:9 may cause false positive reactions in brucellosis testing. [less ▲]

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See detailAssessment of the cell-mediated immunity in cattle infection after bovine herpesvirus 4 infection, using an in vitro antigen-specific interferon-gamma assay.
Godfroid, J.; Czaplicki, G.; Kerkhofs, P. et al

in Veterinary Microbiology (1996), 53(1-2), 133-41

The cell-mediated immunity (CMI) following bovine herpesvirus 4 (BHV4) infection has been poorly investigated in cattle. The in vivo response measured by a delayed type of hypersensitivity (DTH) assay has ... [more ▼]

The cell-mediated immunity (CMI) following bovine herpesvirus 4 (BHV4) infection has been poorly investigated in cattle. The in vivo response measured by a delayed type of hypersensitivity (DTH) assay has been reported to be positive in only few animals showing serological evidences of BHV4 infection. We have investigated the CMI following BHV4 infection by an in vitro antigen-specific interferon gamma (IFN-gamma) release assay, as an indicator of an actively acquired immunity to BHV4. Our preliminary results using a partially purified antigen suggest that there was a measurable CMI in 75 out of 168 animals (44.4%) originating from a farm with a clinical history and serological evidences (76.3% seropositivity) of BHV4 infection. If the results of serological tests and BHV4 IFN-gamma test are interpreted in parallel, 81.5% of the animals are classified positive, demonstrating the complementarity of these tests. The specificity of the BHV4 IFN-gamma test was supported by the absence of a measurable CMI in 41 animals originating from a farm with no clinical history or serological evidence of BHV4 infection. In an allied study, we developed a bovine herpesvirus 1 (BHV1) IFN-gamma test. This allowed us to measure the antigen specific IFN-gamma release after stimulation with a mixture of BHV1 and BHV4 antigens. Animals that were classified negative by the BHV4 IFN-gamma test and by the BHV1 IFN-gamma test, were classified negative after stimulation with a mixture of both antigens. Animals that were classified positive by the BHV4 IFN-gamma test or the BHV1 IFN-gamma test, were classified positive after stimulation with a mixture of both antigens. Taken together these results suggest that the in vitro assessment of the CMI after BHV4 infection should be further investigated as a specific and valuable alternative to the DTH assay. [less ▲]

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See detailInfection of cattle with Yersinia enterocolitica O:9 a cause of the false positive serological reactions in bovine brucellosis diagnostic tests.
Weynants, V.; Tibor, A.; Denoel, P. A. et al

in Veterinary Microbiology (1996), 48(1-2), 101-12

During the last four years, an increasing number of cattle herds were classified positive by brucellosis screening tests in areas of Belgium and France free of the disease. No clinical symptom of ... [more ▼]

During the last four years, an increasing number of cattle herds were classified positive by brucellosis screening tests in areas of Belgium and France free of the disease. No clinical symptom of brucellosis was reported in these animals and no Brucella abortus strains were isolated. After two years, no brucellosis outbreak was registered in all of the herds concerned. On this basis, all the serological reactions observed were classified as false positive. An ELISA using Yersinia Outer membrane Proteins (YOPs) as antigens was developed in order to discriminate between a Yersinia enterocolitica O:9 infection and a Brucella abortus infection. Antibodies against YOPs were detected in sera from Y. enterocolitica O:9 experimentally infected cattle (n = 4) but not in sera from B. abortus experimentally infected cattle (n = 4). In a field study, 66.7% of the 174 serum samples from cattle presenting false positive serological reactions showed anti-YOPs antibodies whereas only 10% of 454 sera, classified negative by the brucellosis screening tests, showed anti-YOPs antibodies. Our results suggest that infections with Y. enterocolitica O:9 may cause false positive reactions in brucellosis testing. [less ▲]

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See detailSpecific bovine brucellosis diagnosis based on in vitro antigen-specific gamma interferon production.
Weynants, V.; Godfroid, J.; Limbourg, B. et al

in Journal of Clinical Microbiology (1995), 33(3), 706-12

In order to improve the specificity of the diagnosis of bovine brucellosis, we developed a test which can be regarded as an in vitro correlate of the delayed-type hypersensitivity test (DTH). A mixture of ... [more ▼]

In order to improve the specificity of the diagnosis of bovine brucellosis, we developed a test which can be regarded as an in vitro correlate of the delayed-type hypersensitivity test (DTH). A mixture of cytoplasmic proteins from Brucella melitensis B115 was used as a specific antigenic stimulus in bovine whole blood culture. Supernatants harvested at 18 to 24 h after the in vitro antigenic stimulus were assayed for their gamma interferon (IFN-gamma) content by using a commercial sandwich enzyme-linked immunosorbent assay kit. The IFN-gamma assay was evaluated with 10 heifers during the course (80 days) of an experimental infection and with 14 cows from an ongoing brucellosis outbreak. All of these animals were slaughtered, and pertinent organs were subjected to classical bacteriological analyses. In addition, we analyzed 23 field cases in which false-positive serological reactions occurred. The IFN-gamma results were compared with those of the standard DTH and a battery of serological assays, and they were correlated with bacteriological data. Both for the experimental infection and for the field brucellosis outbreak, the IFN-gamma assay detected infection in more animals than any combination of the serological tests, and it detected infection earlier than these tests. Finally, none of the samples from cows showing false-positive serological reactions was classified as positive by the IFN-gamma assay, attesting to its specificity and to its usefulness in interpreting ambiguous serological results. A rapid and convenient alternative to the DTH, the IFN-gamma assay appears to be an ideal method that is complementary to the serological diagnosis protocols. [less ▲]

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See detailSpecific bovine brucellosis diagnosis based on in vitro antigen-specific gamma interferon production.
Weynants, V.; Godfroid, J.; Limbourg, B. et al

in Journal of Clinical Microbiology (1995), 33(3), 706-12

In order to improve the specificity of the diagnosis of bovine brucellosis, we developed a test which can be regarded as an in vitro correlate of the delayed-type hypersensitivity test (DTH). A mixture of ... [more ▼]

In order to improve the specificity of the diagnosis of bovine brucellosis, we developed a test which can be regarded as an in vitro correlate of the delayed-type hypersensitivity test (DTH). A mixture of cytoplasmic proteins from Brucella melitensis B115 was used as a specific antigenic stimulus in bovine whole blood culture. Supernatants harvested at 18 to 24 h after the in vitro antigenic stimulus were assayed for their gamma interferon (IFN-gamma) content by using a commercial sandwich enzyme-linked immunosorbent assay kit. The IFN-gamma assay was evaluated with 10 heifers during the course (80 days) of an experimental infection and with 14 cows from an ongoing brucellosis outbreak. All of these animals were slaughtered, and pertinent organs were subjected to classical bacteriological analyses. In addition, we analyzed 23 field cases in which false-positive serological reactions occurred. The IFN-gamma results were compared with those of the standard DTH and a battery of serological assays, and they were correlated with bacteriological data. Both for the experimental infection and for the field brucellosis outbreak, the IFN-gamma assay detected infection in more animals than any combination of the serological tests, and it detected infection earlier than these tests. Finally, none of the samples from cows showing false-positive serological reactions was classified as positive by the IFN-gamma assay, attesting to its specificity and to its usefulness in interpreting ambiguous serological results. A rapid and convenient alternative to the DTH, the IFN-gamma assay appears to be an ideal method that is complementary to the serological diagnosis protocols. [less ▲]

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