Actin assessment in addition to specific immuno-fluorescence staining to demonstrate rickettsial growth in cell culture.

Rickettsiae are able to spread within infected cell mono-layers by modifying intra-cellular actin formations. The study analyzes whether a visualization of actin modifications in addition to specific immuno-fluorescence staining of rickettsiae might facilitate the proof of rickettsial growth in cell culture. Cell mono-layers of Vero E6 und BGM cells were infected with Rickettsia honei. Intra-cellular actin was fluorescence stained with TRITC-(tetra-methyl-5,6-isothiocyanate)-labeled phalloidin in addition to specific immuno-fluorescence staining of rickettsiae with FITC-(fluorescein-isothiocyanate)-labeled antibodies. DNA of bacteria and cells was counter-stained with DAPI (4´,6-diamino-2-phenyl-indole). Cell cultures infected with Vaccinia virus were used as positive controls, cell cultures infected with Coxiella burnetii as negative controls. High concentrations of R. honei are necessary to demonstrate characteristic modifications of the intra-cellular actin. This effect is more pronounced in Vero E6 cells than in BGM cells. Actin staining with phalloidin is not suited for an early proof of rickettsial growth in cell culture but may confirm unclear findings in specific immuno-fluorescence staining in case of sufficient bacterial density.

Epithelial and mesenchymal cells in the bovine colonic mucosa differ in their responsiveness to Escherichia coli Shiga toxin 1.

Bovine colonic crypt cells express CD77 molecules that potentially act as receptors for Shiga toxins (Stx). The implication of this finding for the intestinal colonization of cattle by human pathogenic Stx-producing Escherichia coli (STEC) remains undefined. We used flow cytometric and real-time PCR analyses of primary cultures of colonic crypt cells to evaluate cell viability, CD77 expression, and gene transcription in the presence and absence of purified Stx1. A subset of cultured epithelial cells had Stx receptors which were located mainly intracellularly, with a perinuclear distribution, and were resistant to Stx1-induced apoptosis and Stx1 effects on chemokine expression patterns. In contrast, a population of vimentin-positive cells, i.e., mesenchymal/nonepithelial cells that had high numbers of Stx receptors on their surface, was depleted from the cultures by Stx1. In situ, CD77(+) cells were located in the lamina propria of the bovine colon by using immunofluorescence staining. A newly established vimentin-positive crypt cell line with high CD77 expression resisted the cytolethal effect of Stx1 but responded to Stx1 with a significant increase in interleukin-8 (IL-8), GRO-alpha, MCP-1, and RANTES mRNA. Combined stimulation with lipopolysaccharide and Stx1 increased IL-10 mRNA. Our results show that bovine colonic crypt cells of epithelial origin are resistant to both the cytotoxic and modulatory effects of Stx1. In contrast, some mucosal mesenchymal cells, preliminarily characterized as mucosal macrophages, are Stx1-responsive cells that may participate in the interaction of STEC with the bovine intestinal mucosa.

Cytoskeletal changes in Eimeria bovis-infected host endothelial cells during first merogony.

The first merogony of Eimeria bovis takes place in lymphatic endothelial cells of the ileum, resulting in the formation of macromeronts up to 250 microm. In this study, we investigated the host cell cytoskeleton (actin filaments, microtubules, spectrin, vimentin intermediate filaments) associated with parasitic development in vitro by confocal laser scanning microscopy (CLSM) using primary bovine umbilical vein endothelial cells (BUVEC) and bovine spleen lymphatic endothelial cells (BSLEC) as host cells. No prominent changes in the host cell cytoskeleton were detected 1-3 days after E. bovis sporozoite invasion. With ongoing meront maturation a significant increase in microtubules and actin filaments close to the parasitophorous vacuole (PV) was found. Mature macromeronts within the PV were completely enclosed by these cytoskeletal elements. Our findings suggest, that in order to guarantee the survival of the host cell on the enlargement of macromeronts, E. bovis needs not only to augment but also to rearrange its cytoskeletal system.

Comparison of four commercially available assays for the detection of IgM phase II antibodies to Coxiella burnetii in the diagnosis of acute Q fever.

Four commercially available serological assays for the detection of IgM phase II antibodies in patients with acute Q fever infection were compared using a panel of 23 serum samples from patients with acute Q fever and 88 control sera from blood donors.