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.

The recent association between S. Enteritidis PT4 and poultry products has caused a great deal of concern from adverse publicity and with resulting national and international requirements to control the major food-poisoning Salmonella serotypes at the breeder and layer levels in order to ensure that poultry products are Salmonella-free. The exact mechanism whereby these serotypes are able to colonise the intestine of chickens is still exactly unknown. Indeed, there is increasing evidence that colonisation is not solely a metabolic function but that some form of physical association with cells or an organ in the gut is involved. Thus, invasion and fimbrial genes required for colonisation have been identified (Clayton et al., 2008, Morgan et al., 2004) suggesting physical contact was required. An alternative approach would be to analyse the patterns of gene expression by microarray analysis at the site of colonisation (caeca). This has been done for a number of niches and is now being applied to intra-cellular infection but has not so far been applied to the intestine. The S. Enteritidis transcriptome during the colonisation of the caeca of one day chicks was characterised by Agilent microarray. The microarray results were evaluated by real-time PCR with 96% compatibility. The pattern of gene transcription was different in the intestine compared with broth culture. Thirty four percent of the genes showed a significant change in level of expression. Major changes occured from adaptation to the caecal environment with up-regulation of genes required for energy generation and carbohydrate metabolism/transport, while amino acids and nucleotide metabolism, translation, replication and cell wall biogenesis genes were among the down-regulated genes. Fumarate respiratory and osmotic response genes were selected from the up-regulated genes and were mutated and tested in the lab for their inhibitory effect and for competitive growth under anaerobic and osmotic environments showing variable responses. Association between chicken colonisation phenotype and gene mutation indicated that genes associated with osmolarity was more important than tri carboxylic acid (TCA)-associated genes in their contribution to the colonisation phenotype. There is considerable scope for improvement in inactivated vaccines through a more rational approach. An inactivated vaccine prepared by formalising S. Enteritidis harvested directly from the chicken caeca was thought to be more protective than bacteria grown in vitro. Unfortunately this was not the case. Expected reasons for this failure are explained, and alternative approach to producing a proper effective inactivated vaccine is suggested.

Salmonella is a major cause of enteric illness in both humans and animals. The ability of Salmonella isolates to cause disease in animals and humans encompasses a spectrum of host specificity and disease severity. In terms of evolution Salmonella isolates have remained relatively similar in terms of genetic content. It would therefore seem paradoxical that the diversification of many ecological niches and the invocation of very different disease symptoms by Salmonella have been possible. This infers great importance on differences in gene content between Salmonella isolates. Consultation of genomic sequence data from various Salmonella isolates has confirmed that a large proportion of strain-specific DNA is laterally transferred. More specifically a large proportion of this laterally transferred DNA was found to be bacteriophage-associated. The assertion could therefore be made that infections of isolates by temperate phages may play a significant role in Salmonella evolution and differentiation. Indeed, it has been suggested that a variable assortment of prophages provides a transferable repertoire of pathogenic determinants in Salmonella. The temperate phages present in Salmonella enterica serovar Typhimurium isolates have been most extensively investigated to date. However, how variable is the excisable prophage content between different Salmonella species, subspecies, serovars and individual isolates? In the current study the temperate phages present in 102 Salmonella isolates from a wide variety of species, subspecies and serovars were characterised by various methods. To assess the diversity of excisable temperate phages present in Salmonella isolates phages were induced with mitomycin C and viewed by electron microscopy. Temperate phages were identified in 81.4 % of 102 Salmonella isolates and the most commonly isolated phages belonged to the Myoviridae morphology family. The host ranges of the induced phages varied greatly. Sixty-five lysates contained phages that could infect one other Salmonella isolate from a panel of 24 indicator isolates. Six different methods of phage DNA extraction were tested. The amount of phage DNA extracted from lysates varied considerably depending on the Salmonella isolate from which the lysate was made. Fifty-three Hind III restriction digest profiles were obtained from eighty-six lysates and thirty-seven of these profiles were found to be unique. Phages with identical restriction profiles were identified and one of these phages was associated with clinical Salmonella isolates. Finally PCR profiling of the phage DNA and chromosomal DNA obtained from Salmonella isolates was used to assess the diversity of functional and defective phages present in the isolates. Genes associated with the ST64B phage were most commonly identified in the phage DNA extracted from lysates and in the chromosomal DNA from Salmonella isolates. Great diversity was observed in the temperate phage content of Salmonella isolates for all of the characterisation methods utilised. PCR profiling was determined to be the most sensitive method to identify temperate phages in Salmonella isolates. Assessment of the inducible and non-inducible prophage content of isolates by PCR profiling was also shown to have potential for the characterisation of Salmonella strains.

Bibliography: leaves 126-171 / xiv, 171 leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1988