[beta]-hemolysin and the pathogenesis of Serpulina hyodysenteriae infection

Abstract

A murine model of Serpulina hyodysenteriae infection was utilized to explore the pathogenesis of lesion formation induced by that organism. Detailed characterization of gross and microscopic lesions in that model demonstrated that mice fed Teklad diet 85420 experienced the onset of severe gross and histologic lesions much sooner (within 24 hours) following infection with S. hyodysenteriae than did mice fed conventional rodent chow. However, gross and microscopic features of the induced lesions were no different in mice fed either diet. The model was then used to document the progression of morphologic changes that occur in the cecal mucosa of mice infected with S. hyodysenteriae. Mice were necropsied hourly following infection and examined by transmission electron microscopy. Early ultrastructural changes consisted of microvillus effacement, intercellular fluid accumulation and increased internalization of luminal bacteria. These changes progressed to include epithelial cell degeneration and necrosis, translocation of massive numbers of bacteria to perivascular lamina proprial phagocytic and stromal cells, and changes in lamina propria capillaries indicative of increased permeability. Experiments were then performed in order to assess the role of the 6-hemolysin of S. hyodysenteriae in the development of the described ultrastructural changes. Purified hemolysin was introduced intraluminally into surgically created murine cecal loops and tissues were examined by transmission electron microscopy. It was shown that the hemolysin induced pronounced microvillus loss and vacuolation of superficial epithelial cells. Cells became detached, often contained bacteria and were occasionally necrotic. Many detached cells appeared apoptotic. Studies were undertaken to purify the hemolysin and identify its gene. Crude hemolysin was purified by electrophoresis and electroelution and submitted for N-terminal amino acid sequencing. A single sequence was obtained indicating the presence of a single peptide. A degenerate oligonucleotide probe was constructed based on the amino acid sequence and used to screen a genomic library of S. hyodysenteriae in