Date of Award
Master of Science
Veterinary Microbiology and Preventive Medicine
Evelyn A. Dean-nystrom
James A. Roth
Shiga toxin (Stx) binding to polymorphonuclear neutrophils (PMN) is hypothesized to play a role in the pathogenesis of Shiga toxin-producing Escherichia coli (STEC) disease in humans. Pigs are an excellent model for studying the role of PMN in STEC disease because, like humans, they are susceptible to Stx-mediated disease and Stx binds to porcine PMN in vitro. The objectives of this study were to develop a flow cytometric assay to monitor Stx2 binding under conditions required for functional studies (37yC in cell culture medium) and to evaluate the functional effects of Stx2 on porcine granulocytes. Binding and internalization of Stx2, or the non-toxic binding subunit of Stx2 (Stx2B), by isolated granulocytes, and the effects on apoptosis and oxidative burst were monitored by flow cytometry. Granulocyte preparations contained PMN and eosinophils (EOS) which bound and internalized Stx2 and Stx2B. Similar percentages of Stx2+ granulocytes were detected when Stx2 incubation was performed at 4yC and 37yC. Incubation with Stx2, but not with Stx2B, increased the mean percentage of apoptotic cells and reduced the mean percentage of cells capable of an oxidative burst. Pre-incubation of Stx2 with monoclonal antibodies against the Stx2B prevented Stx2 binding and effects on apoptosis and oxidative burst. The demonstration that Stx interacts with both porcine PMN and EOS in vitro extends the evidence that granulocytes, may contribute to STEC pathogenesis. The availability of a porcine Stx-mediated disease model will facilitate further investigations of the role of granulocytes in STEC disease.
Scherer, Amanda, "Effects of Shiga toxin 2 on isolated porcine granulocytes" (2009). Graduate Theses and Dissertations. 10604.