Brucella abortus strain RB51 infection of bovine trophoblast epithelial cells: characterization of receptor signaling and bacterial outer membrane proteins

In 1995, a bovine brucellosis vaccine containing Brucella abortus strain RB51 was accepted for calfhood vaccination. However there is no objective serologic tests, such as ELISA, which detects antibodies specific for RB51-vaccinated cattle, and intravenous inoculation of pregnant animals with strain RB51 is followed by placentitis;To create an objective diagnostic test, antigenic outer membrane proteins from strain RB51 were isolated by Concanavalin A lectin affinity chromatography. One eluted protein of 35 kDa reacted in western blot analysis to every individual serum from strain RB51 and 2308 infected cattle. This protein may be a useful candidate antigen in developing a specific test for detecting antibody to RB51 in RB51-vaccinated cattle;Invasion and replication of bacteria in trophoblastic epithelial cells are key events in placentitis. To identify the receptor involved in adhesion of strain RB51 to bovine trophoblastic epithelial cells and the signal pathway that mediates bacterial phagocytosis, a bioassay and double immunofluorescence labeling were used. In bioassays, adhesion of B. abortus to trophoblastic cells was inhibited by fibronectin, RGDS peptide, and antibodies to [alpha] 5 or [beta] 1 integrin subunits. Uptake of bacteria was inhibited by genistein, an inhibitor of tyrosine kinases. Ultrastructurally, intracellular bacteria were located in the rough endoplasmic reticulum or in phagolysosomes of trophoblasts. These results indicate that strain RB51 binds to the trophoblastic epithelial cell surface by an [alpha] 5[beta] 1 integrin, that uptake is mediated by tyrosine kinases, and that bacteria replicate intracellularly within cisternea of the rough endoplasmic reticulum;The consequences of Brucella induced-placentitis on the binucleate trophoblastic cell population has never been studied. Numbers of Binucleate cells stained with Dolichos Biflorus agglutinin lectin were quantified by morphometric analysis in placentomes from non-infected and strain RB51-infected pregnant cattle. There was diffuse decreased numbers of cells in sections of placentomes with placentitis. Immunohistochemistry for proliferating cell nuclear antigen and terminal deoxy dUTP nick end-labeling showed reduced proliferation of progenitor mononuclear cytotrophoblasts in arcade zone, that could explain binucleate cell depletion, but not increased programmed cell death. Because binucleate cells produce placental lactogen, decreased numbers of cells could participate to mechanisms of abortion