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In vitro studies of Brachyspira pilosicoli pathogenesis

Naresh, Ram (2010) In vitro studies of Brachyspira pilosicoli pathogenesis. PhD thesis, Murdoch University.

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Brachyspira pilosicoli is an intestinal spirochaete that colonizes the large intestine of a variety of species of birds and animals, including human beings. Colonization can lead to local inflammation and to diarrhoea in a condition known as “intestinal spirochaetosis”. This infection has been described in many countries throughout the world. In the colonization process the bacterium must cross the thick mucus blanket overlaying the colonic epithelium. Characteristically, B. pilosicoli then attaches by one cell end to the underlying epithelium, forming a dense “false brush border”. The mechanisms involved in moving through the mucus layer, attaching to enterocytes and inducing local cellular damage are poorly understood. The lack of in vitro models to study these events has been a major constraint to understanding the pathogenesis of B. pilosicoli infections.
The work described in this thesis deals with i) the development of an in vitro model of spirochaete attachment by using cells in suspension (erythrocytes) and cell monolayers (Caco-2), ii) the attraction of B. pilosicoli to mucin, and iii) the effects of norepinephrine exposure on expression of virulence traits by B. pilosicoli.
Attachment assays conducted with erythrocytes from different species at different ratios and time intervals identified one human isolate (WesB) that adhered to goose and chicken erythrocyte at a 1:1000 ratio. This same strain, and an isolate from a pig (95/1000) also attached to Caco-2 cells. Transmission and scanning electron microscopy confirmed that the attachment resembled the in vivo situation. Exposure of the Caco-2 cells to B. pilosicoli resulted in actin rearrangements, damaged cell junctions and apoptosis. Caco-2 cells that were colonized with B. pilosicoli also demonstrated a significant up-regulation of interleukin-1ß (IL-1ß) and IL-8 expression, helping to confirm that the spirochaetes were inducing pathological changes in the cultured cells. Treatment of the monolayers with B. pilosicoli sonicates caused significant up-regulation of IL-1ß, TNF-α, and IL-6, but culture supernatants and non-pathogenic Brachyspira innocens did not altered cytokine expression. Hence Il-8 expression was specifically associated with exposure to live B. pilosicoli cells.
For mucin attraction, 15 B. pilosicoli strains isolated from humans, pigs, chickens and dogs, and a control strain of Brachyspira hyodysenteriae, were analysed for their ability to enter solutions of hog gastric mucin in an in vitro capillary tube assay. Attraction started in a 2 % mucin solution, and then increased with increasing concentrations to peak at around 6 - 8 % mucin. Attraction varied from strain to strain. B. pilosicoli strain 95/1000 and B. hyodysenteriae strain B204 also were attracted to viscous solutions of polyvinylpyrillodone (PVP), in a manner mirroring the response to mucin. This suggested that as well as chemotaxis to mucin components, “viscotaxis” is involved in the attraction to mucin.
Finally, exposure of B. pilosicoli to norepinephrine enhanced the attachment to Caco-2 cells, chemotactic response to mucin, and spirochaete growth. Taken together, these in vitro studies have shed new light onto the pathogenic processes that are involved in intestinal spirochaetosis caused by B. pilosicoli.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Veterinary and Biomedical Sciences
Supervisor(s): Hampson, David
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