Ulrike Heise: Pathogen-host-interactions in the large intestinal mucosa of pigs with experimentally induced swine dysentery
Pigs with experimentally induced swine dysentery may develop severe diarrhea with mucus and blood and fibrinous to diphtheroid colitis a few days after inoculation with Brachyspira hyodysenteriae and recover spontaneously after a few days to several weeks. The objective of this investigation was to characterize the cellular reactions in the large intestinal mucosa during the acute disease to gain insights into pathogen-host interactions.
The multiple immune fluorescence method was adapted to differentiate inflammatory and immune cells in the colonic mucosa of the pig. Using this method, quantity and distribution of inflammatory and immune cells was first compared in the mucosa at four representative localizations along the large intestine (cecum, proximal colon, central flexure, descending colon) of clinically healthy pigs. Then the cellular reactions were characterized in the altered large intestinal mucosa during the acute phase of swine dysentery.
Multiple immune fluorescence protocols were established for the combination of MHCII, CD16 and Brachyspira hyodysenteriae as well as CD4, CD8 and Brachyspira hyodysenteriae.
This allowed to differentiate granulocytes, dendritic cells, CD4+, CD8+ and CD4+CD8+ T-lymphocytes. Macrophages and IgA+ plasma cells each were labeled in combination with Brachyspira hyodysenteriae. This method was used, because many cell types in pigs can be conclusively identified by the combined labeling of epitopes on the cells only and because it allowed to study the interaction of Brachyspira hyodysenteriae with the various cell types.
Data analysis was pixel based using the image analysis program ImageJ. Accidental and real co-localizations of epitopes on cells were distinguished by mathematical comparison.
In the control pigs, the quantity of the different cell types in the full thickness of the mucosa varied only minor between the large intestinal localizations examined. There were, however, significant differences between basal and apical regions of the mucosa in all four sites: the number of intraepithelial CD8+ T lymphocytes was significantly higher in the apical regions, whereas the quantity of IgA+ plasma cells was significantly higher in the basal regions. This is
SUMMARY 194
comparable to the distribution of these cells in villi versus crypts in the small intestine. In addition, the quantity of granulocytes and macrophages was higher in the basal region of the cecum than that of CD4+ and CD4+ CD8+ T lymphocytes in the basal region of the proximal colon. In contrast, more macrophages, dendritic cells, CD4+ and CD4+ CD8+ T lymphocytes were present in the apical region of the mucosa in the descending colon.
In pigs with experimentally induced swine dysentery, quantity and distribution of inflammatory and immune cells were specifically determined at sites with diphtheroid colitis in the proximal colon. Cellular reactions were characterized by an increased number of granulocytes in the apical region of the mucosa that was most severely damaged. The overall quantity of macrophages and dendritic cells was decreased and their distribution in the mucosa had changed. Large, partly disintegrated macrophages were present in necrotic mucosa. Dendritic cells were found in close proximity to neutrophils and Brachyspira hyodysenteriae. Macrophages and dendritic cells demarcated areas of necrotic mucosa. Many Brachyspira hyodysenteriae had invaded the areas of necrotic mucosa and there was co-localisation of Brachyspira hyodysenteriae with granulocytes and macrophages suggesting uptake of bacteria or bacterial antigen by these cells. There was a marked reduction of CD4+ T lymphocytes and intraepithelial CD8+ T lymphocytes, the effectors of cellular immunity, whereas quantity and distribution of IgA+ plasma cells and labeling of basal crypt epithelial cells for IgA was unchanged.
Methods and findings of this investigation will be the basis for further investigations of the quantity and distribution of inflammatory and immune cells in the large intestinal mucosa in pigs with experimentally induced swine dysentery. They may serve for comparison of reactions during recovery, after inoculation with Brachyspira hyodysenteriae strains of different virulence and after vaccination and challenge.
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