One of the critical challenges in the transmissible spongiform encephalopathy (TSE) field is to understand the molecular basis of prion propagation and decipher the enigma of prion strains and their role in TSEs. The research approach adopted in this dissertation tackled different subjects of keen interest for prion characterisation and diagnostics. A high throughput enzyme-linked immunosorbent assay (ELISA) was developed for detection of disease-related prion protein (PrPSc) using the protease thermolysin. Thermolysin allowed isolation of protease resistant PrPSc in its full-length form, while cellular prion protein (PrPC) was digested. With further extraction and precipitation of PrPSc with sodium phosphotungstic acid (NaPTA) and in conjunction with using monoclonal antibodies that recognise distinct epitopes, PrPSc was detected and quantified successfully. Molecular strain typing of ruminant TSEs was investigated using Western blotting and depending on the resistance of PrPSc to digestion with proteinase-K (PK) and thermolysin. The methods discriminated clearly between classical ovine scrapie and experimental ovine BSE. In contrast, experimental CH1641-like isolates showed heterogenous molecular profiles. In addition, the findings from this study demonstrated the existence of thermolysin-sensitive PrP isoforms which are resistant to PK and their presence varied between individual sheep and brain regions. When studying prion propagation using the serial protein misfolding cyclic amplification (sPMCA) technique, different strains/isolates of ruminant prions were successfully amplified in vitro from as little as 0.01 ng of brain seed. Furthermore, ovine BSE was readily amplified in vitro in brain substrates from sheep with homozygous VRQ or AHQ Prnp genotype. In contrast, the CH1641 strain was refractory to such amplification. This method allowed for differentiation of experimental BSE from CH1641 prion strains within an ovine host, providing hope for the potential of sPMCA as a strain typing assay. The use of bacterially expressed recombinant PrPsen (rPrPsen) as substrate in PMCA reactions (rPrP-PMCA) was assessed. The use of the substrate improved the sensitivity, specificity, practicality and speed of sPMCA assays for detecting a range of ovine prion isolates. Expression and purification of recombinant Syrian hamster prion protein (Sha rPrP) and VRQ ovine PrP (VRQ rPrP) provided substrate for detecting PrPSc in scrapie affected brain samples. Although both substrates had the same level of sensitivity, rSha PrPsen had better specificity than VRQ rPrP. There were variations in amplification efficiency between different batches of the same rPrP. This study recommends further investigations looking at the use of a range of experimental CH1641 and BSE samples, as well as using panels of CH1641-like field isolates for sPMCA reaction to establish (such) strain typing methodology. Furthermore, applying the rPrP-PMCA assay to detect PrPSc in secreta and excreta of infected sheep in the pre-clinical phase of the disease may provide a non invasive ante-mortem test for scrapie.

Dysregulated IL-1 activity has been implicated in the pathogenesis of several acute and chronic inflammatory as well as autoimmune diseases. Genomic analysis has identified additional IL-1 family members and also expanded the IL-1 receptor family. To date, there are eleven IL-1 family members and nine IL-1 receptor family members. Information regarding the biological activity and immunological role of the newer IL-1 family members is still very limited. The novel IL-1 cytokines, IL-1F6, IL-1F8 and IL-1F9 (recently renamed IL-36α, IL-36β and IL-36γ respectively) have been shown to signal via IL-1 receptor related protein 2 (IL-1Rrp2, recently renamed IL-36R). The main aims of this study were: (i) to investigate the expression of IL-1Rrp2 by human myelomonocytic and non-myelomonocytic immune cells as well as other human cells, (ii) to determine the possible function of IL-1Rrp2 and (iii) to determine the effect of IL-1Rrp2 expression on T lymphocytes. Results reported in this thesis indicate that among human myelomonocytic cells, constitutive IL-1Rrp2 expression is unique to dendritic cells (DCs). IL-1Rrp2 was expressed by monocyte- derived DCs (MDDCs) and plasmacytoid DCs (pDCs) but not by peripheral blood type 1 or type 2 myeloid DCs (mDC1 or mDC2). IL-1Rrp2 expression was regulated in response to both classical (IL-1β) and novel IL-1 cytokines (IL-1F8 and IL-1F9). Similarly to IL-1β and bacterial LPS, novel IL-1 cytokines mediated DC maturation as shown by DC phenotypic changes (e.g. upregulation of HLA-DR expression and decreased CD1a expression following culture of DCs with IL-1F8) and cytokine production. Among non-myelomonocytic cells, constitutive IL-1Rrp2 expression was observed in lamina propria tissue, T lymphocytes, NCI/ADR-RES cells and HT 29 cells. IL-1F8-matured human DCs were fully functional as they induced proliferation of IFN-γ-producing TH1 subsets. Results suggest that novel IL-1 cytokines play a role in inflammatory responses involving human DCs, with possible implications for inflammatory disease.

Background: Pneumonia is a disease of the lungs that is caused by a number of pathogens including Gram-negative Pseudomonas aeruginosa and Gram-positive Streptococcus pneumoniae. These pathogens are prevalent causes of hospital-acquired pneumonia, and P. aeruginosa is particularly problematic with regards to chronic pulmonary infection in patients with Cystic Fibrosis (CF). Antibodies are known to provide a component of host-defence against this microbe, but recent evidence suggests that cells secreting the pro-inflammatory cytokine interleukin-17 (IL-17), namely T helper 17 (Th17) cells, are also significant in these responses. Aim: To investigate the sources of IL-17 and related cytokines during P. aeruginosa and S. pneumoniae infection, and to investigate if IL-1β has a role in Th17 formation during infection with these pathogens. Furthermore, to investigate the cytokine secretion of dendritic cells (DCs) derived from different sources following infection with these pathogens, and their roles at inducing Th17 secretion from naive CD4+ T cells. Methods & Results: Dendritic cells from mucosal sites were found to be better than GM-CSF derived bone marrow dendritic cells (BMDCs) at inducing Th17 cells from naive T cells. Th17 cells were derived in response to both P. aeruginosa and S. pneumoniae, with the role of IL-1β seeming to be negligible for P. aeruginosa. However, the role of IL-1β during Th17 cell induction during S. pneumoniae is unclear and needs further investigation. γδ T cells were found to be a source of IL-17 during P. aeruginosa infection in a IL-23 dependent manner. Furthermore, γδ T cells were also found to be a source of IL-22, yet the majority of cells were either IL-17 or IL-22 producing, not double producers as expected. In vivo infection with these pathogens identifed γδ T cells to be a main source of IL-17 during P. aeruginosa infection, with Th17 cells having more of a role during S. pneumoniae infection, yet the main sources of IL-17 still need to be identified. Preliminary infections with S. pneumoniae in IL-17RKO mice identified IL-17 as a key mediator in downstream inflammatory responses in the lung during infection. Conclusions: This study demonstrates that IL-17 responses are induced in response to infection with the respiratory pathogens P. aeruginosa and S. pneumoniae. Ex vivo, mucosal DCs were found to induce more robust Th17 cell responses compared to GM-CSF derived BMDCS. In vivo, Th17 cells appear to have a role in S. pneumoniae infection, with γδ T cells appearing to be the dominant source of IL-17 during P. aeruginosa infection. Furthermore, in vitro investigations of γδ T cells found them to be differentially IL-17 and IL-22 producing in response to P. aeruginosa infection, in a DC contact independent manner.

Furthermore, these studies provide a better understanding of the therapeutic potential of this parasite molecule in inflammatory diseases such as CIA and asthma and collectively, they have demonstrated the potent immunomodulatory effects of ES-62.

Infiltration of inflammatory cells into the tissue during the inflammatory response is beneficial to the host. Chemokines and their receptors are instrumental in this process by influencing the migration and behaviour of leukocytes in the tissue. However, prolonged inflammation is associated with many diseases. In recent years, a family of atypical receptors have emerged which do not seem to signal. One of these receptors, D6, is able to internalise and degrade 12 pro-inflammatory CC chemokines and has a role in the resolution of the inflammatory response. Here, using a murine transgenic approach, the potential therapeutic role of D6 in suppressing cutaneous inflammation in vivo has been investigated, using a well-characterised model of skin inflammation. In addition, expression of D6 in a range of inflammatory disorders has also been characterised. Transgenic mice were generated (K14D6), using an epidermis-specific transgene, in which expression of the D6 transgene was driven by the human keratin 14 promoter in epidermal keratinocytes. K14D6 mice were validated and we have shown that D6 is expressed in K14D6 but not in wild-type epidermal keratinocytes. The K14D6 transgene was shown to be functional as only K14D6 keratinocytes were able to bind CCL2 and progressively deplete extracellular CCL3. K14D6 mice can dampen down cutaneous inflammation in response to a topical application of TPA. In addition, K14D6 mice displayed reduced infiltration of epidermal T cells and mast cells compared to wild-type mice. Using a microarray approach, we examined the transcriptional consequences of non-ligated D6 and after ligand binding in primary murine keratinocytes from K14D6 and wild-type mice. Although limited conclusions could be made from the microarray data, our results suggest the possibility that non-ligated D6 in murine keratinocytes may have a negative impact on the transcription of some genes, such as chemokines. In a previous study, D6 null mice displayed a human psoriasis-like pathology after chemical induced skin inflammation, suggesting a possible involvement of D6-dysfunction as a contributing factor in the pathogenesis of psoriasis. We have investigated the possible correlation between D6 expression levels and cutaneous disease development. Analysis of skin biopsies revealed that D6 mRNA levels were 8-fold higher in uninvolved psoriatic skin compared to matching psoriatic lesional skin, atopic dermatitis and control skin. In PBMCs, there was no significant difference in D6 mRNA expression in psoriasis patients compared to control. A preliminary study examining surface D6 expression on leukocytes from control and rheumatoid arthritis patients revealed enhanced D6 expression on B cells and myeloid DCs. In this study, we have shown for the first time that increased expression of D6 in vivo can limit cutaneous inflammation, therefore providing a rationale for exploring the therapeutic potential of D6 in human inflammatory diseases. In addition, we provide evidence that D6 expression is dysregulated in inflammatory disorders further suggesting an involvement of this receptor in the pathogenesis of these diseases.

CCX-CKR is an atypical chemokine receptor for the homeostatic chemokines CCL19, CCL21 and CCL25. CCL19 and CCL21 are also ligands for CCR7 and are crucial for the induction of antigen specific immunity and tolerance, whereas CCL25 is the sole ligand for CCR9 and is involved in the recruitment of immune effector cells to the small intestine. CCX-CKR does not signal after binding its ligands, as determined by a failure to induce the rapid increase in intracellular calcium that is typical of G-protein mediated signalling. CCX-CKR also does not become desensitised to chemokine binding and therefore is proposed to act as a scavenger receptor that can regulate the activity of CCR7 and CCR9 by affecting the availability of their ligands in vivo. At the time of starting my project, there were no published reports describing the biological function of CCX-CKR in vivo and the principal aim of my thesis was to characterise the immune system of the recently generated CCX-CKR KO mouse with particular focus on the intestinal immune compartment where all three of the chemokine ligands are expressed. Firstly, as described in Chapter 3, I analysed the cellular composition of the secondary lymphoid organs of CCX-CKR KO mice. These studies revealed normal proportions and absolute numbers of lymphocytes, CD11c+ dendritic cells (DC), macrophages and natural killer (NK) cells in the absence of CCX-CKR. The proliferative responses of lymphocytes to mitogenic or TCR stimulation in vitro were also normal, although there was a decreased production of IFNγ by CD4+ T cells from CCX-CKR KO mice. Although most of the phenotypic subsets of conventional DC were present in comparable numbers in the mesenteric lymph nodes (MLN) of CCX-CKR KO and WT mice, there was a consistent and dramatic reduction in the numbers of CD11cloPDCA-1+ plasmacytoid DC (pDC) in CCX-CKR KO MLN. In parallel, fewer CD11cloB220+ cells from CCX-CKR KO MLN than WT expressed CCR9, despite this marker being expressed normally by lymphocytes in these mice. The proportions of pDC in CCX-CKR KO inguinal lymph nodes (ILN) were also significantly reduced compared to WT and pDC from CCX-CKR KO MLN, ILN and spleen all appeared to express higher levels of class II MHC than WT pDC. These data suggest that CCX-CKR may play an important role in the recruitment and/or survival of pDC in the LN and that in its absence, pDC in secondary lymphoid organs may have a more mature phenotype. In Chapter 4, I examined the cellularity of the intestinal immune compartment in resting CCX-CKR KO mice, as well as the effects of Flt3L administration in vivo. Although CCX-CKR KO mice displayed no histological abnormalities in their small intestinal architecture and had normal numbers of T cells in the lamina propria, they did have significantly reduced numbers of intra-epithelial lymphocytes (IEL) as well as decreased proportions of CD19+ B cells and increased proportions of CD11c+ cells in the lamina propria compared with WT mice. The proportions and absolute numbers of CD103+ DC were normal in the lamina propria and MLN of CCX-CKR KO mice, suggesting that CCX-CKR has little to no role in regulating DC migration from the lamina propria to the MLN, a process that is critically dependent on CCR7. Although the proportions and absolute numbers of B cells and CD11c+ cells were normal in CCX-CKR KO Peyer’s patches (PP), there were significantly decreased proportions of pDC compared with WT PP. In vivo treatment of CCX-CKR KO mice with the DC differentiation factor Flt3L, expanded CD11c+ DC numbers dramatically in both CCX-CKR KO and WT small intestinal lamina propria, ILN, spleen, MLN and PP. Although Flt3L abolished the apparent defects in pDC populations in the ILN and PP, this was not the case for CCX-CKR KO MLN, which remained significantly deficient in pDC compared to WT MLN. Work in Chapter 6 examined parallel effects in the blood and bone marrow. As the CCX-CKR ligands CCL19 and CCL21 are involved in the development of all adaptive immune responses, and together with the other CCX-CKR ligand, CCL25, orchestrate immune responses to antigen encountered in the gut, I next investigated the development of antigen specific immunity and tolerance in CCX-CKR KO mice. There were no significant differences in systemic immune responses to subcutaneous immunisation with ovalbumin (OVA) emulsified in complete Freunds adjuvant (CFA) between CCX-CKR KO and WT mice when assessed in vivo or in vitro. However, the development of oral tolerance in CCX-CKR KO mice was impaired, with no suppression of OVA specific delayed type hypersensitivity (DTH) responses or of serum OVA specific IgG2a as was seen in WT mice. In parallel with defective systemic tolerance after feeding OVA, CCX-CKR KO mice appeared to be more susceptible to priming of systemic and local antibody responses after feeding OVA with cholera toxin (CT) as a mucosal adjuvant. In addition, there was some evidence of priming of OVA specific antibody responses in CCX-CKR KO mice fed OVA alone, which was not seen in WT mice. Despite this evidence of abnormal mucosal immunity, CCX-CKR KO mice developed DSS colitis normally, with all indices of disease being identical in CCX-CKR KO and WT animals. Together, these data suggest that there are selective defects in the regulation of antigen specific mucosal immune responses in the small intestine of CCX-CKR KO mice that may predispose these animals towards exaggerated active immune responses. Finally, I performed some preliminary experiments to try and relate DC function to the immune dysregulation I observed in CCX-CKR KO mice and to explore the basis of the pDC defect in the MLN. Bone marrow derived and splenic DC from CCX-CKR KO mice showed a reduced ability to process and present intact protein antigens although endocytic activity was normal. MLN DC from normal and Flt3L treated CCX-CKR KO mice responded similarly to in vitro stimulation with the synthetic TLR7 agonist R848, showing an expansion in the numbers of CD11chiPDCA-1+ cells that nearly all expressed CD40 and CD86. In addition, in vivo administration of R848 triggered identical migration of CD11chiclassIIMHChiCD103+ DC into the MLN of CCX-CKR KO and WT mice, suggesting that lamina propria pDC can effectively mobilise local DC to the MLN in the absence of CCX-CKR. There were no differences in the proportions or absolute numbers of pDC in the liver of CCX-CKR KO and WT mice despite the fact that liver pDC have been implicated in other studies of oral tolerance. Although the proportions of pDC were normal in the bone marrow of resting and Flt3L treated CCX-CKR KO mice, there did appear to be a defective recruitment of Flt3L expanded pDC into the blood of these animals. I used an adoptive transfer approach to study the in vivo localisation of pDC into lymphoid organs and although these experiments were not entirely conclusive, they indicated that WT pDC could enter the MLN and other lymphoid tissues of CCX-CKR KO mice normally and that pDC from CCX-CKR KO mice may have a defect in their ability to enter WT MLN. Taken together, my data suggest that CCX-CKR is involved in the entry and/or survival of pDC in secondary lymphoid organs, a process that normally involves migration across high endothelial venules (HEV). This was associated with impaired oral tolerance induction and heightened immune responses to antigen delivered orally, indicating that CCX-CKR contributes to the regulation of mucosal immunity and tolerance by an as yet unclear mechanism. Further study of these animals will hopefully better define the relationship between pDC and the regulation of mucosal immune responses.

By advancing our knowledge of these key signals in regulating tolerance and priming at the single cell level in vitro and in vivo, we will therefore increase our understanding of an important physiological process at the molecular level, ultimately leading to identification of potential targets for enhancing or inhibiting immunity and tolerance.

The integrin αVβ5 is a receptor for sCD23 molecule and the αVβ5-CD23 interaction sustains proliferation of the pre-B cell line SMS-SB. This thesis describes further investigation into the role of αVβ5 integrin during human B cell development. B cell development in the bone marrow involves stepwise maturation of progenitor cells through different defined stages. A tight regulation of proliferation and differentiation mediates the progression of progenitor cells through this developmental pathway. A variety of signals from soluble molecules and adhesive interactions regulate this balance of proliferation and differentiation. The main aim of this work was to assess the importance of the integrin αVβ5 during B cell development by defining its expression and function during specific stages of B cell development in the bone marrow. The αVβ5 integrin was expressed by B cell precursors in the bone marrow, by different pre-B cell lines and the αVβ5-CD23 interaction sustained proliferation of some pre-B cell lines. The pre-B cell lines SMS-SB, RS4;11 and 697 showed a significant proliferative response to αVβ5 stimulation by sCD23, a sCD23-derived long peptide and anti-αVβ5 MAb 15F11. Transitional and more mature B cell lines down-regulated αVβ5 expression and did not show a proliferative response. Both αVβ5 and αVβ3 integrin could be detected on normal bone marrow B cell precursor populations, though αVβ5 was the more highly expressed integrin. In preliminary functional experiments, stimulation of CD19+/κ- cells with sCD23 induced cell proliferation whereas equivalent treatment of CD19+/κ+ cells did not. The data are consistent with the interpretation that αVβ5 integrin is expressed in B cell precursors but that its ability to sustain growth of these cells wanes as the cells mature towards a membrane immunoglobulin-positive state. The αVβ5-mediated proliferation was enhanced by the chemokine SDF-1 and PDGF but not by the cytokines IL-3, IL-4, IL-7 or IL-11. This effect was apparently restricted to earlier B cell precursors and was independent of levels of expression of both αVβ5 and CXCR4. Stimulation of SMS-SB cells by αVβ5 ligands provoked ERK phosphorylation and co-stimulation with SDF-1 promoted a more rapid and sustained ERK activation. PDGF induced a similar effect on αVβ5-mediated activation of ERKphosphorylation. These data suggest that ligation of αVβ5 by soluble, adhesion-independent stimuli activates ERK phosphorylation and this pathway can be modulated by inputs from G-protein-coupled and tyrosine kinase receptors. The murine pro-B cell line BAF03 also displayed αVβ5-mediated proliferation in response to human CD23. Preliminary experiments showed that human CD23 sustained growth of murine bone marrow B cell precursors. Therefore, these data suggest that murine B cells can also use αVβ5 integrin to sustain their growth and the studies described here in human cell lines could be translated into in vivo murine models. Further work is needed to confirm the proliferative response due to the αVβ5-CD23 interaction in normal B cell precursors in the bone marrow and to define the exact stage of development where this interaction is critical. In addition to its expression in the bone marrow B cell precursors, previous work has also demonstrated the expression of αVβ5 integrin in B cells from patients with ALL. Therefore, the αVβ5-CD23 interaction could have important implications not only in proliferation of normal B cell precursors but also in proliferation of neoplastic B cells. These data identify the αVβ5-CD23 interaction as a potentially important interaction during early B cell development, as αVβ5 expression and function is stage-specific, regulated by other molecules and can be demonstrated in both human and murine cell lines.

The exposure of cells to environmental stresses, including heat shock, heavy metals, alcohols or oxidative stress results in the cellular accumulation of a number of chaperones, commonly known as heat shock proteins (Hsps). Hsps maintains the correct folding and conformation of proteins and are vital in regulating the stability between protein synthesis and degradation. Hsps are not only fundamental in the cellular stress response but are also important in regulating numerous important normal functions, such as cell proliferation and apoptosis. The results presented in the thesis focus on Hsp90 in nematodes, its possible roles in acquired drug resistance and further characterisation of Brugia pahangi Hsp90. Hsp90 plays a key role in the cellular stress response by interacting with proteins after their native conformation has been altered by stress. Hsp90 is essential in all eukaryotes and plays a central role in multiple cellular processes. Since knock-out of hsp90 is lethal to most eukaryotes, inhibitors of Hsp90 have been widely used to study its function. The most widely used inhibitor is geldanamycin (GA). GA binds to the N-terminal/ATP binding site of Hsp90 which results in conformational changes and the degradation of client proteins. Although the gene encoding hsp90 has been characterized, little is known regarding its function in parasitic nematodes. Previous studies have shown that Caenorhabditis elegans Hsp90 is unique amongst eukaryotes because it fails to binds GA. This was suggested as an example of ‘adaptive evolution’ because C. elegans inhabits the same ecological niche of the soil as the Streptomyces species which produces GA. In contrast B. pahangi Hsp90 does bind to GA. The first objective of this project was to determine whether the resistance of C. elegans Hsp90 to GA is the norm or the exception amongst nematodes and to determine whether GA binding is associated with particular clades of nematodes. The results showed that the ability of Hsp90 to bind GA is associated with the life-cycle of the nematode. Free-living species or those that have a free-living larval stage in the soil do not bind GA, while those species which are obligate parasites (Trichinella and the filarial worms), or which are enclosed within a protective egg shell while in the environment (Ascarids), possess an Hsp90 that binds GA. These data support the adaptive evolution hypothesis. Further characterisation of B. pahangi Hsp90 showed that it was expressed in all life-cycle stages but in the pull-down assay, only adult B. pahangi Hsp90 bound to GA, not Hsp90 from L3 or Mf. The inability to detect L3 or Mf Hsp90 binding to GA beads could perhaps reflect a lack of sensitivity of the assay, or alternatively may suggest that Hsp90 from these stages is processed in such way that it fails to bind to GA. In addition, only a small amount (6.3%) of B. pahangi Hsp90 bound to GA after a series of pull-down assays. Whether these data represents an accurate reflection of the amount of Hsp90 that can bind to GA is not known, but if correct, the results suggest that it might be necessary to inhibit only a small percentage of total Hsp90 to kill the worm. Attempts were make to identify proteins that associate with Hsp90 by comparing the proteome of GA-treated adult B. pahangi with control worms. In this analysis, several proteins with structural functions were identified. These proteins were down-regulated after exposure to GA at 1.0 µM. Comparison of Hsp90 levels in B. pahangi and C. elegans showed that Hsp90 was expressed at relatively higher levels in B. pahangi (p<0.05) but there was no significant difference in the affinity of B. pahangi or C. elegans for Hsp90 binding to ATP beads. Results in Chapter 4 showed that the binding of Hsp90 to GA beads was competed by free GA or ATP. In addition, novobiocin, another Hsp90 inhibitor which binds at C-terminal domain of Hsp90 also inhibited binding of B. pahangi Hsp90 to GA beads, suggesting an interaction between the N-terminal and C-terminal of Hsp90. Hsp90 has also been shown to be involved in the acquisition of drug resistance in fungi. A previous study on fungi showed that the emergence of fluconazole resistance depended on high levels of Hsp90 and was abolished when Hsp90 expression was reduced. So, given the conservation of Hsp90 it is possible that Hsp90 may be involved in the acquired resistance of nematodes to anthelmintic. The analysis carried out did not produce any correlation between expression levels of Hsp90 and drug resistance in nematodes. RNAi experiments were carried out to reduce Hsp90 levels in C. elegans and to investigate whether Hsp90 may play a role in the tolerance of ivermectin-resistant C. elegans to drug. Results showed that reducing Hps90 levels altered the sensitivity of ivermectin-resistant C. elegans to drug.

Brachyspira hyodysenteriae is an anaerobic intestinal spirochaete and the aetiological agent of swine dysentery (SD). Throughout the UK and Europe, pathogenic and potential non-pathogenic isolates of B. hyodysenteriae have been recovered from pig herds, creating major obstacles for the detection and control of this economically important pathogen. Therefore, the main aim of this research was to compare one representative of virulent (P8544) and one representative of avirulent (P7455) strains of B. hyodysenteriae using genomic and proteomics approaches with a view to identify distinctive genes or proteins. The B. hyodysenteriae draft genomes of P8544 and P7455 consisted of a circular 3.0 Mb chromosome and a 31,469-34,822 bp circular plasmid that is also present in the only published B. hyodysenteriae genome, WA1. A considerable number of genes (~27-35) were identified in both the virulent and avirulent strains that shared high sequence homology with genes found in other spirochaetes, such as B. murdochii and B. intermedia, as well as in other species of bacteria; these may have been acquired via horizontal gene transfer. Comparative genomics of the two pathogenic genomes P8544 and WA1 versus the non-pathogenic genome P7455 revealed that the gene encoding for the methyltransferase type 11 (Bhyoa7455_20) was identified as being unique to the P7455 plasmid sequence and was successfully PCR amplified in a greater number of avirulent than virulent strains. However, as this was only just statistically significant (P=0.049), screening of a much larger strain set would clearly be required to support this gene as a suitable genetic marker to distinguish virulent and avirulent B. hyodysenteriae strains. Bacterial acquisition of iron in-vivo is crucial for successful colonisation and persistence in the host. A further aim of this study was to compare the growth phenotype of B. hyodysenteriae isolates P8544 and P7455 grown under iron-limiting conditions; such as would be found in-vivo in the large intestine of the host. Analysis of P8544 and P7455 growth rate in iron-sequestered media (containing 0.1 mM of the iron-chelator dipyridyl) demonstrated that both these isolates could replicate in this media although with an extended lag-phase of approximately 32-34 hrs; growth rate was on par with the iron-replete conditions. qRT-PCR analysis of eight putative iron-acquisition genes under iron-sequestered and iron-replete conditions revealed a difference in transcription for a number of ABC-transporter genes in P8544 and P7455, however, none of these were classified as statistically significant. Non-quantitative shotgun proteomic based approach was used to analyse outer-membrane protein (OMPs) expression of P8544 and P7455 under low-iron and iron-replete growth conditions and revealed alteration in the OM expression profiles between the isolates and conditions using KEGG analysis. The majority of expressed proteins under iron-replete conditions were categorized in membrane transport (11%) and carbohydrate metabolism (7%). Under iron-restriction the OM profile changed most obviously in a decreased percentage of proteins particularly assigned in the categories energy metabolism and membrane transport. The percentage of proteins assigned no predicted function increased by 19% under iron-limited conditions highlighting the fact that biological functions of the majority of these expressed proteins in such an environment remains to be determined. Two-dimensional gel-electrophoresis (2-DGE) of whole cell fraction indicated that the alkyl-hydrogen peroxide reductase protein (AhpC) in P7455 and the non-haem iron-containing ferritin (Bhyov8544_1528) in P8544 were significantly (P<0.05; 1.5-fold) more expressed under iron-restricted conditions than under iron-replete conditions. These data confirmed the importance of iron to virulent and avirulent B. hyodysenteriae. The so far identified significantly expressed proteins may serve as a potential biomarker for global diagnostic purposes for B. hyodysenteriae infections rather than a tool for differentiation for virulent and avirulent isolates. However, further work is required to prove if these candidates are expressed in-vivo and conserved in a wider panel of field isolates. In conclusion, this research has contributed to the scientific knowledge regarding B. hyodysenteriae stress responses induced by iron-starvation and has provided further insight into the genetic and proteomic make up of this spirochaete. This work should also aid future investigations concerning the biology and pathogenicity of this important and grossly understudied swine pathogen.