Resende, Talita Pilar2020-08-252020-08-252019-08https://hdl.handle.net/11299/215076University of Minnesota Ph.D. dissertation. August 2019. Major: Veterinary Medicine. Advisors: Connie Gebhart, Fabio Vannucci. 1 computer file (PDF); xiv, 152 pages.The intracellular intestinal bacterium Lawsonia intracellularis infects epithelial cells of small and large intestines of pigs and other animals, causing proliferative enteropathy. The hallmark lesion of proliferative enteropathy is crypt hyperplasia, which results in thickening of the affected intestinal mucosa. Proliferative enteropathy is described in pig herds worldwide and leads to poor growth performance. Although two commercial vaccines are available to prevent proliferative enteropathy, some producers still control the disease by using in feed antimicrobials. The current trend to reduce the use of feed antimicrobials stresses the need of alternative approaches to control livestock bacterial diseases, which are more effective when designed to block essential mechanisms of pathogenesis. The investigation of L. intracellularis pathogenesis has been hindered by the lack of an adequate in vitro model that accurately reproduces the infection and proliferation, and hence, the development of strategically designed methods of controlling proliferative enteropathy is limited. The overall goal of this thesis was to evaluate the effectiveness of in vitro models in reproducing cellular proliferation, the hallmark lesion of proliferative enteropathy in vivo and other changes observed in proliferative enteropathy, based on the characterization of the intestinal epithelial changes induced by L. intracellularis infection in the intestine. We performed an indirect quantification and analysis of the impact of L. intracellularis infection on the intestinal epithelial cells by immunofluorescence and special stains. Besides confirming the increased numbers of cells in the proliferative stage, increased numbers of transit amplifying cells and decreased numbers of mucin producing goblet cells, we also observed decreased numbers of enteroendocrine cells in pigs severely affected by proliferative enteropathy. As the proportion of enterocytes is not changed through the course of proliferative enteropathy, and the number of secretory cells is decreased, we hypothesize that L. intracellularis infection modifies the mechanisms involved in the epithelial differentiation towards secretory cells, most likely the Notch signaling. The next step was to investigate the feasibility of traditional cell cultures as in vitro models for L. intracellularis infection. Cell line cultures, composed of a single cell type, are widely used to investigate pathogenesis of bacterial infections, especially of intracellular bacteria such as L. intracellularis. We used various assays to investigate whether L. intracellularis infection would result in higher cellular proliferation in intestinal epithelial cells, cultured under standard and stressed conditions. Our results showed that none of the three intestinal epithelial cell lines proliferate when infected by L. intracellularis, regardless of the culture condition. Intestinal organoids, or enteroids, considered the ultimate in vitro model of intestinal epithelium, were then evaluated as in vitro models for L. intracellularis infection. First, tridimensional mouse enteroids were microinjected with L. intracellularis suspensions and assayed for the ability of L. intracellularis to infect the cells. The success of L. intracellularis propagation in the mouse enteroids was limited, but L. intracellularis antigen was observed in the basal side of the enteroid cells, which was an intriguing finding. The limited success of L. intracellularis infection and propagation in mouse enteroids drove the efforts towards the development of swine enteroids. In the two-dimensional swine enteroids, L. intracellularis infection and propagation was successful and, thus, two-dimensional swine enteroids are the most promising in vitro model to investigate L. intracellularis pathogenesis. Our last attempt in revealing important aspects of L. intracellularis infection was using a vaccinology approach to identify possible candidates for recombinant vaccines and/or antigen for serology assays. Based on in silico analysis, three protein candidates were produced, purified and tested against pig sera and intestinal washes for antigenicity. Although the in silico analysis indicated that the proteins were good vaccine/serological assay candidates, the results showed that although antigenic, the tested proteins were not immunogenic, since proliferative enteropathy naïve pigs also have antibodies against L. intracellularis. In summary, this thesis investigated the pathogenesis of the obligately intracellular bacterium, L. intracellularis, using various in vitro models, most notably swine enteroids. Additionally, it led to the hypothesis that L. intracellularis infection may modify the mechanisms involved in epithelial cell differentiation.enThesis or Dissertation