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Brucella melitensis 16M is a Gram-negative α[sub 2]-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 viva 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 viva 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. [ABSTRACT FROM AUTHOR]

Bacterial differentiation is often associated with the asymmetric localization of regulatory proteins, such as histidine kinases. PdhS is an essential and polarly localized histidine kinase in the pathogenic alphaproteobacterium Brucella abortus. After cell division, PdhS is asymmetrically segregated between the two sibling cells, highlighting a differentiation event. However, the function(s) of PdhS in the B. abortus cell cycle remains unknown. We used an original approach, the pentapeptide scanning mutagenesis method, to generate a thermosensitive allele of pdhS. We report that a B. abortus strain carrying this pdhS allele displays growth arrest and an altered DivK-yellow fluorescent protein (YFP) polar localization at the restrictive temperature. Moreover, the production of a nonphosphorylatable PdhS protein or truncated PdhS proteins leads to dominant-negative effects by generating morphological defects consistent with the inhibition of cell division. In addition, we have used a domain mapping approach combined with yeast two-hybrid and fluorescence microscopy methods to better characterize the unusual PdhS sensory domain. We have identified a fragment of the PdhS sensory domain required for protein-protein interaction (amino acids [aa] 210 to 434), a fragment sufficient for polar localization (aa 1 to 434), and a fragment (aa 527 to 661) whose production in B. abortus correlates with the generation of cell shape alterations. The data support a model in which PdhS acts as an essential regulator of cell cycle progression in B. abortus and contribute to a better understanding of the differentiation program inherited by the two sibling cells.

Peripheral blood mononuclear cells (PBMC) isolated from cattle infected with Brucella secreted gamma-interferon (IFN-gamma) after antigen-specific stimulation with Brucellergene, which is a mixture of cytoplasmic proteins of rough Brucella melitensis B115. Following the depletion of the monocyte-macrophages from the PBMC, the enriched lymphocyte populations stimulated with Brucellergene did not produce IFN-gamma. Two-colour immunofluorescence staining of intracellular IFN-gamma and bovine cell surface molecules identified the cells producing IFN-gamma among the PBMC stimulated with Brucellergene. Moreover, this method could be used to estimate the number of T-cells specifically producing IFN-gamma. For a given animal, there is a significant correlation (p < 0.05) between the production of IFN measured by an ELISA of the supernatant of whole blood stimulated with Brucellergene and the number of T-cells producing IFN-gamma after in vitro stimulation with Brucellergene. The development of the immunofluorescence staining technique provides a new tool for analysing and for measuring the T-cell immune response in cattle.

Most of the B cells from bovine leukemia virus (BLV) infected cows in persistent lymphocytosis (PL) were known to express the CD5 T-cell marker but it was not known whether this peculiar membrane phenotype relates to an activation state. It was demonstrated that these B cells were also flagged by two other membrane markers normally borne by cells belonging to the myeloid lineage (namely CD11b and CD11c). Moreover, cell cycle analysis illustrated that a significant percentage of these B cells (greater than 15%) left their resting (G0/G1) status and progressed through the cell cycle. In addition, T-cell-depleted peripheral blood mononuclear cells from animals in PL were shown to proliferate in response to a IL-2-containing supernatant (MLA 144). These results indicate that the CD5+ B cells from BLV-infected cows in PL are activated cells.

This study reports on the functional characteristics of a bovine T-cell differentiation antigen recognized by the monoclonal antibody (mAb) 8C11. This mAb has previously been found to react with a 67-kD molecule shared by thymocytes and peripheral blood T cells and to be undetectable on the B cells of healthy animals. This antigen is also largely expressed on the B cells from bovine leukemia virus-infected animals. Molecules with a similar cell distribution have been described in other species (mouse, human, rat and sheep), and were termed CD5 molecules. In order to confirm the CD5-like nature of the target molecule recognized by 8C11, functional T-cell assays were carried out. We report here that this mAb, like its human and murine homologues, enhances the proliferative responses of T cells to mitogens or alloantigens but does not directly stimulate T-cell division. Moreover, we have shown an enhancing effect of this 8C11 mAb on bovine interleukin-2 production by concanavalin A-stimulated bovine peripheral blood mononuclear cells.

Trypanosoma theileri is an ubiquitous blood parasite of cattle. This parasite grows easily in RPMI medium and perturbs lymphocyte tests based on [3H]thymidine uptake. This paper reports that 0.5 microgram ml-1 amphotericin B in the medium is adequate to get Trypanosoma growth inhibition without alteration of either mitogenic or allogenic stimulation of bovine lymphoid cells in vitro.

Monoclonal antibodies have been produced that recognize a conformation of homopolygalacturonic acid (pectic acid) induced by an optimum concentration of calcium and sodium of about 1 and 150 millinormal, respectively. The epitope recognized is probably part of the dimers of pectin chains associated according to the ;egg box' model.