cytochalasin-d and Mastitis--Bovine

Bovine mastitis caused by Escherichia coli has traditionally been viewed as a transient infection. However, E. coli can also cause clonal persistent intramammary infection (IMI) in dairy cows. In this study, we explored the possibility that E. coli strains associated with persistent IMI are better able to adhere to, invade, survive and replicate in cultured mammary epithelial cells (MAC-T) than transient strains, and examined their serotype, overall genotype, phylogenetic group, and the presence of known virulence genes. Both transient and persistent E. coli strains adhered to MAC-T cells, but persistent strains invaded MAC-T cells 2.6-63.5 times more than transient strains. Blocking the adhesin/invasin FimH with mannose diminished but did not eliminate adhesion and invasion of any strain. Cytoskeletal and protein kinase inhibitors cytochalasin D, colchicine, genistein and wortmannin dramatically reduced invasion of MAC-T cells by both strains. All of the persistent strains, but only one transient strain, were able to survive and replicate intracellularly in MAC-T cells over 48 h. Transient and persistent strains displayed heterogeneous serotypes and overall genotypes, but similar phylogeny (group A), and lacked virulence genes of invasive E. coli. We have found that E. coli strains associated with persistent IMI are better able to invade and replicate within cultured mammary epithelial cells than transient strains. The invasion process involves the host cytoskeleton and signaling cascades and is not FimH dependent. Our findings suggest that the invasion of mammary epithelial cells and intracellular survival play an important role in the pathogenesis of persistent E. coli mastitis.

Topics: Analysis of Variance; Animals; Bacterial Adhesion; Cattle; Cells, Cultured; Colchicine; Cytochalasin D; Enzyme Inhibitors; Epithelial Cells; Escherichia coli; Escherichia coli Infections; Female; Genistein; Genotype; In Situ Hybridization, Fluorescence; Mammary Glands, Animal; Mastitis, Bovine; Phylogeny; Serotyping; Virulence

The purpose of this study was to investigate the interaction between Escherichia coli and primary mammary epithelial cell cultures derived from cows with persistent intramammary infection (IMI). Two strains of E. coli, isolated from the milk of two different cows suffering from persistent E. coli IMI were tested for adhesion to and invasion of three primary mammary epithelial cell cultures derived from mammary biopsies of the two infected cows. Intracellular E. coli were detected during five days post infection in vitro. Both strains of E. coli adhered to and invaded monolayers of all three primary mammary epithelial cell cultures. One strain adhered less but invaded more than the other. Comparison with other mammary pathogens indicated that E. coli invaded the cells less efficiently than Staphylococcus aureus, about as efficiently as Streptococcus dysgalactiae and more efficiently than Streptococcus uberis. The mechanism of E. coli invasion was studied using the cytoskeleton disrupting agents colchicine and cytochalasin D. These compounds inhibited the invasion of E. coli. Invasion of E. coli could also be inhibited by the phosphokinase inhibitors genistein and staurosporin in a dose-dependent fashion. Phorbol-myristyl-acetate (PMA) had no effect on the invasion of E. coli. Histology of mammary tissue revealed chronic inflammatory changes in quarters that were persistently infected by E. coli. Intracellular bacteria were not detected in mammary tissue sections. Polymerase chain reaction (PCR) analysis suggested that the two strains of E. coli lacked genes encoding for bundle-forming pili (bfpA), intimin (eae) and translocated intimin receptor (tir), which are characteristic for enteropathogenic E. coli (EPEC).

Topics: Animals; Bacterial Adhesion; Cattle; Cells, Cultured; Colchicine; Cytochalasin D; Enzyme Inhibitors; Epithelial Cells; Escherichia coli; Escherichia coli Infections; Female; Genistein; Histocytochemistry; Mammary Glands, Animal; Mastitis, Bovine; Polymerase Chain Reaction; Staphylococcus aureus; Streptokinase

Staphylococcus aureus is one of the most important pathogens of the bovine mammary gland. The interaction of S. aureus with cells of the bovine mammary gland is considered to play an essential role in the pathogenesis. In this study, we identified a new target cell for S. aureus adhesion and invasion. For that purpose, cells which compose the alveoli of the mammary gland were cultured. In these cultures, two morphologically different cell types, elongated and cubic cells, were observed. Adhesion and invasion of S. aureus was studied using microscopical and microbiological methods. S. aureus adhered specifically and in large numbers (about 300 bacteria/cell) to the elongated cell type. No adhesion to the cubic cell type was observed. In addition, bacteria were also found intracellularly in the elongated cells, and enclosed in membrane vesicles. Adhesion and invasion were time dependent and reached maximum levels after 4 h. Invasion was strongly reduced by staurosporine and genistein. The newly identified target cell was further characterized.

Topics: Animals; Antibodies, Monoclonal; Bacterial Adhesion; Cattle; Cell Culture Techniques; Colchicine; Cytochalasin D; Enzyme Inhibitors; Female; Immunohistochemistry; Lactation; Mammary Glands, Animal; Mastitis, Bovine; Microscopy, Electron; Nucleic Acid Synthesis Inhibitors; Staphylococcal Infections; Staphylococcus aureus; Staurosporine; Tropism