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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 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 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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