Salmonellosis remains a major cause of gastroenteritis worldwide and novel control strategies are urgently sought for the livestock industry. Probiotics are one such novel control strategy that has become evermore popular since the ban of antimicrobial growth promoters in the EU. This project was undertaken to develop the current understanding of the mechanisms employed by probiotic bacteria for the control of S. Typhimurium infection in pigs. Probiotics are thought to act as competitive exclusion (CE) agents and have been demonstrated to be efficacious for the control of foodbome pathogens such as S. Typhimurium. The aim of this project was to isolate and characterise a porcine Lactobacillus strain that could be used as a probiotic to control S. Typhimurium infection in pigs. These studies also extended to understanding the mechanism of action exerted by this putative probiotic strain using novel in vitro models of the porcine intestine. In this study, an L. plantarum strain, lCI, was isolated from the faeces of a clinically healthy pig; the taxonomic identity of the L. plantarum strain lC I was confirmed and found to meet the current EU requirements for use as a commercial feed additive. Furthermore, in vitro, the antimicrobial spectrum of L. plantarum lC I was evaluated against S. Typhimurium in broth culture and shown to have bacteriostatic activity, significantly inhibiting the growth of S. Typhimurium. The inhibition of S. Typhimurium growth was conferred in part by the presence of L-Iactic acid and was determined as pH dependent by viable counts of Salmonella following exposure to the cell free supematant (CFS) from L. plantarum lC 1 adjusted with inorganic acid to pH 4.5. Furthermore, Multi-Dimensional Protein Identification Technology (MuD PIT) was used to identify differentially expressed proteins in S. Typhimurium following exposure 2 to the CFS adjusted with inorganic acid to pH 4.5. In these studies the Salmonella type three secretion system proteins SipB, SipC, SopB and InvG along with the virulence determinants VirK, LepA, LepB and SomA (Ybjx) were significantly down regulated. L. plantarum lC 1 was evaluated for its ability to CE S. Typhimurium in mono layers, or 3D aggregates of the porcine jejunal epithelial cell line (IPEC-J2) and the cell crown in vitro organ culture (CCIVOC) assay. S. Typhimurium invasion into 3D cells was reduced following competition CE assays with L. plantarum lC 1, but did not reduce the association of S. Typhimurium to porcine intestinal tissue. Moreover, following a protection CE assay L. plantarum did not reduce changes in the porcine proteome induced by S. Typhimurium infection. However, ex vivo, L. plantarum lC 1 acted as a potent secretagogue of acidic mucins from porcine jejunal and colonic tissues maintained using CCIVOC. In addition, L. plantarum lC I induced changes in the relative abundance of tubulin-a which may indicate that probiotics can induce host cytoskeletal rearrangements. In this study both porcine 3D cell culture and CCIVOC were developed as novel surrogate models of the porcine gastrointestinal tract and proved efficacious for the characterization of probiotics, although further studies are required to elucidate how the results obtained in these models translate to the in vivo environment of the pig.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:553086 |
| Date | January 2010 |
| Creators | Collins, James William |
| Publisher | University of Reading |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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