Feline chronic gingivostomatitis (FCGS) is an inflammatory disease of the oral cavity that causes severe pain and distress. No specific treatment methods are available and little is known about its aetiology. The aims of this study were:- 1) to identify the bacterial flora, including uncultivable and potentially novel species, in healthy cats and those with FCGS, using 16S rRNA gene sequencing in combination with conventional culture methods; 2) to investigate the viral status of cats with and without FCGS; 3) to assess the immune response by investigating the expression of cytokine and Toll-like receptor (TLR) genes in tissue biopsies from normal cats and those with FCGS; 4) to investigate the histopathological changes in tissue biopsies from normal cats and those with FCGS, 5) to assess putative risk factors for FCGS by the use of a questionnaire-based study. Oral swabs, mucosal biopsies and blood were collected and the location of the oral lesions was recorded. A total of 32 cats with FCGS and 16 normal cats were included in the study. Bacteria were identified from swabs by use of 16S rRNA gene sequencing and by conventional culture methods. Blood samples and swabs were used for diagnosis of infection with feline leukaemia virus (FeLV), feline immunodeficiency virus (FIV), feline herpes virus 1 (FHV-1), feline calicivirus (FCV) and for blood biochemistry and haematology. Gene expression levels for TLR2, TLR3, TLR4, TLR7 and TLR9, and cytokines IL-1β, IL-4, IL-6, IL-10, TNF-α and IFN-γ mRNA were determined using quantitative PCR in biopsy samples from healthy cats and cats with FCGS. Histopathological examination of the tissue biopsies was done using hematoxylin and eosin (H&E) staining. In the healthy group, 16S rRNA gene sequencing demonstrated that the most prevalent bacteria were part of the Proteobacteria and Bacteroidetes phyla, plus a group of uncultured bacteria. The most prevalent species in the healthy group were Xanthomonadaceae bacterium (6.2 % of clones analysed), Capnocytophaga canimorsus (5.4%), Capnocytophaga cynodegmi (4.8%), Bergeyella species (4.5%) and Pasteurella multocida subspecies septica (4.4%). Uncultured bacteria accounted for 29% of the clones analysed. In the FCGS group most of the identified species were part of the phylum Proteobacteria. The most prevalent species in the FCGS group were P. multocida subsp. multocida (14.1%) P. multocida subsp. septica (11.5%), Pseudomonas sp. (7.3%), Tannerella forsythia (6.6%) and Porphyromonas circumdentaria (5.6%). A variety of uncultured bacteria represented 7.7% of all analysed FCGS clones. The culture data showed the most prevalent bacteria in the healthy group were P. multocida subsp. septica (9.9%), and uncultured bacteria (30.5%). In the FCGS group the most prevalent isolates were P. multocida subsp septica and P. multocida subsp. multocida (both 9.9%). Uncultured bacteria accounted for 21.7% of all isolates. FCV was detected in 71% of cats with FCGS and in 13.3% of normal cats. FeLV antigen was detected in 33.3% of normal cats but not in any cats with FCGS. FIV antibodies were detected in 3.4% of cats with FCGS and in 33.3% of normal cats. FHV-1 was detected in 6.9% of cats with FCGS, but was not detected any of the normal cats. In the FCGS group a significant increase was seen in the expression of TLR2 and TLR7 genes as well as TNF-α, IFN-γ, IL-1β and IL-6 cytokine genes. The healthy cats and cats with FCGS in the study that were found to harbour T. forsythia and P. circumdentaria showed an increase in the expression of several TLR and cytokine genes when compared to the group of cats in which these bacterial species were absent. The most severely inflamed sites in the oral cavity of cats with FCGS included the tissue lateral to the palatoglossal folds and the maxillary attached gingiva. Histopathological analysis of the tissue from the palatoglossal folds showed two types of infiltrates:- 1) a combination of lymphocytes and plasma cells, most often seen in the milder inflamed tissue samples; 2) a predominantly plasmacytic infiltration, most often seen in the severely inflamed tissue samples. Preliminary data from a questionnaire-based epidemiological study showed that the presence of potential environmental stress factors such as no ability to roam outdoors and the presence of more than one cat in the household is significantly higher in cats with FCGS when compared to normal cats. This study highlights the possibility of a multifactorial aetiology for FCGS in which FCV, specific bacteria and stress factors may play an important role. Although species from the Bacteroidetes phylum appeared to be capable of eliciting an immune response, these were not the most prevalent species in the FCGS group. A shift could be seen in the composition of the bacterial flora when healthy cats and those with FCGS were compared.

Despite advances in treatment, peritoneal dialysis (PD)-associated peritonitis remains a major cause of morbidity and mortality in PD patients. Given that peritonitis can be the proximate cause of technique failure and cause ultrafiltration failure at a later time, it is important to understand the peritoneal immune response, microbiology and outcomes of these infections. Data presented in this thesis have shown that leukocytes are recruited to the peritoneal cavity, starting with a rapid accumulation of neutrophils, which are later replaced by a population of mononuclear cells, including monocytes/macrophages and T cells during acute peritonitis. Of note, Vγ9/Vδ2 T cells are also recruited to the peritoneal cavity in the early stage, which implies a significant role of Vγ9/Vδ2 T cells as early responders in acute peritonitis. In patients with acute peritonitis, the capacity of the causative pathogen to produce (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), together with the infiltration of activated Vγ9/Vδ2 T cells are important risk factors and possible predictors of patient outcomes from infection. By performing a detailed immunological and microbiological analysis in PD patients on the first day of peritonitis, our findings provide proof of concept that acute bacterial infections indeed leave characteristic disease-specific ‘immune fingerprints’ of diagnostic and prognostic value. Local fingerprints not only discriminated between episodes of culture-negative and culture-positive PD-associated peritonitis but also predicted infections caused by Gram− or Gram+ bacteria. HPMC play an important role in maintaining homeostasis of the peritoneal immunity. Our data revealed the regulation of Vγ9/Vδ2 T cells by HPMC and demonstrated that resting HPMC were potent suppressors of Vγ9/Vδ2 T-cell cytokine production and proliferation in the presence of HMB-PP. Collectively, these findings improve our insight into the complex cellular interactions in PD-associated peritonitis and peritoneal homeostasis, identify novel biomarkers of possible diagnostic and predictive value and highlight new avenues for therapeutic intervention.

Introduction: Varying degrees of periodontal disease affect the majority of the population. Severe forms of periodontitis have a considerable impact on oral health and quality of life. Periodontitis results from imbalances in the oral microbiome and the host immune response. The mainstay of periodontal treatment – removal of dental plaque – is only partially successful. B cells infiltrate the gingiva of periodontitis patients, but their role in pathology has not been well characterised. The overarching aim of this research was to better characterise the role of B cells in periodontitis. Periodontitis shares similarities in risk factors and aspects of immunopathology with rheumatoid arthritis. Epidemiological evidence suggests patients with rheumatoid arthritis are more likely to have periodontitis, which cannot be completely explained by shared risk factors. This has led to the hypothesis that the two diseases are immunologically linked, and that periodontitis may precede, and cause, rheumatoid arthritis. A further objective of this research was to investigate whether the autoimmunity characteristic of rheumatoid arthritis emerges in periodontitis. Results: B cell infiltrate in the gingiva of periodontitis patients was confirmed. Periodontitis patients were found to have elevated serum titers of anti-citrullinated peptide antibodies which were generally below the diagnostic threshold for rheumatoid arthritis, and were reduced following non-surgical periodontal treatment. In a murine model of periodontitis, subtle changes to B cell phenotype were observed in tissues regional to the oral cavity in mice with periodontitis, at an early stage of disease. Such changes included increased B cell expression of receptor activator of NfκB ligand in the gingiva, and increased proportions of GC B cells in the draining lymph nodes. Some of these trends were enhanced in mice with periodontitis exacerbated by interleukin-33 treatment. B cell-deficient mice were protected from the alveolar bone loss normally induced in the model of periodontitis. Conclusion: B cells form a substantial proportion of the inflammatory infiltrate in the gingiva of periodontitis patients. Treatment of periodontitis can reduce titers of anti-citrullinated peptide antibodies in patients, potentially reducing their risk of developing rheumatoid arthritis. Evidence from B cell-deficient mice suggests that B cells contribute to pathological alveolar bone loss. Therefore, B cells may be worthy of targeting therapeutically in periodontitis.

The extensive allelic variability observed in several genes related to the immune response and its significance in transplantation, disease association studies and diversity in human populations has led the scientific community to analyse these variants among individuals. This thesis is focussed on the development of a database and software applications for the investigation of several immune genes and the frequencies of their corresponding alleles in worldwide human populations. The approach presented in this thesis includes the design of a relational database, a web interface, the design of models for data exchange and the development of online searching mechanisms for the analysis of allele, haplotype and genotype frequencies. At present, the database contains data from more than 1000 populations covering more than four million unrelated individuals. The repertory of datasets available in the database encompasses different polymorphic regions such as Human Leukocyte Antigens (HLA), Killer-cell Immunoglobulin-like Receptors (KIR), Major histocompatibility complex Class I chain-related (MIC) genes and a number of cytokine gene polymorphisms. The work presented in this document has been shown to be a valuable resource for the medical and scientific societies. Acting as a primary source for the consultation of immune gene frequencies in worldwide populations, the database has been widely used in a variety of contexts by scientists, including histocompatibility, immunology, epidemiology, pharmacogenetics and population genetics among many others. In the last year (August 2010 to August 2011), the website was accessed by 15,784 distinct users from 2,758 cities in 136 countries and has been cited in 168 peer-reviewed publications demonstrating its wide international use.

Background: Salmonella bacteraemia is an important public health problem in children from sub Saharan Africa (SSA). Understanding what constitutes natural acquired immunity to Salmonella is crucial for the development of Salmonella vaccine. It was hypothesized that natural Salmonella exposure within the GIT and peripheral blood induces the generation of specific-antibodies and T cells and these might provide protection to subsequent Salmonella infection. Methods: Natural acquisition of antibody and T cell immunity to Salmonella was investigated in healthy and Salmonella infected Malawian children. Acquisition of typhoid vaccine induced T cell immunity in healthy adults from the United Kingdom (UK) was investigated to model natural immunizing events occurring within the gut associated lymphoid tissues (GALTs) following Salmonella infection. Acquisition of immunity was examined using immunological tools including the intra-cellular cytokine staining assay (ICS), serum bactericidal activity (SBA) assay, ELISA and ELISpot. Exposure to Salmonella was examined using microbiological tools including standard culture and real-time PCR. Principal findings: CD4+ T cells and IgG antibodies to Salmonella develops sequentially in under-five children. Acquisition of Salmonella-specific CD4+ T cells and antibodies coincides with the decline in S. Typhimurium bacteraemia cases in older children. As much as 47% of Malawian children (aged 6-18 months) are exposed to Salmonella at least once within the gastrointestinal tract (GIT). Natural Salmonella exposure within the GIT is associated with development of potentially protective SBA in children. Invasive Salmonella infection elicits an increase in generation of Salmonella-specific CD4+T cells, IgG and IgA antibody secreting cells (ASC). Oral Ty21a vaccination (model of natural Salmonella infection) did not elicit an increase in generation of both CD4+Cytokine+ and CD8+Cytokine+ T cells in the peripheral blood and gut mucosa compartments at day 11, and day 18 post vaccination. Conclusion: Young children (<2 years of age) are more vulnerable to invasive Salmonella infection. Salmonella exposure within the GIT and peripheral blood compartments tissues facilitates acquisition of robust immunity (mediated by antibodies and T cells) in children and these might provide protection to subsequent Salmonella infection. Public health interventions are urgently required in SSA including vaccination with cross-protective Salmonella vaccine, improvements in sanitation, access to clean and safe water and food hygiene.

The recruitment of peripheral blood lymphocytes (PBL) to sites of inflammation and their subsequent traffic into the lymphatic circulation is important in host defense. However, surprisingly little is known about their recruitment from the blood vasculature into inflamed tissue, and almost nothing about their egress from inflamed tissue via the lymphatic circulation. We showed that both human macrovascular and microvascular endothelial cells stimulated by TNF\(\alpha\) and IFN\(\gamma\), preferentially recruited memory T-lymphocytes (CD45RO positive cells) from a mixed pool of PBL. T-cells that had migrated across vascular endothelial cells subsequently utilised a combination of \(\beta\)1 and \(\beta\)2 integrins to traverse cytokine activated lymphatic endothelium. In addition we provide evidence that PGD2 was critical for the transmigration of lymphocytes through vascular endothelium. The process of trans-lymphatic migration was also significantly retarded in the presence of a function neutralising antibody against CCR7. Most importantly, we observed that memory T-cells showed a markedly enhanced capacity to migrate across lymphatic endothelium if they had first traversed a vascular endothelial cell barrier. We have shown that addition of exogenous PGD2 to isolated lymphocytes is able to restore the enhanced migration capacity of lymphocytes that have previously migrated through a vascular monolayer. The nature of the priming signal delivered by the process of migration across blood vessel endothelium remains to be fully identified, but is likely to be important in regulating the dynamics of an inflammatory response.

The liver has a unique connection with the immune system; harbouring vast numbers of lymphocytes, able to instigate secondary lymphoid organ-independent naive T cell activation, and promoting potent immune tolerance. We set out to determine the effect of this unique microenvironment on the biology of CD4+ T cells at three key interaction points: following migration into the parenchyma, after short-term hepatocyte contact, and at long-term tissue-residency. Modelling transmigration through hepatocytes revealed intrinsic, disease-specific cytokine responses in blood-derived CD4+ T cells, not discernible through static co-culture. However, short-term co-culture did induce activation-independent CD69 upregulation, reliant upon cell-cell contact. This phenotype mimicked the similar hepatic CD4+ CD69INT cells that we discovered in liver tissue. Unlike CD69HI cells which represented the tissue-resident memory T cells (TRM) of the liver, CD69INT cells were the most activated population, likely able to migrate to many liver and gut niches, and singularly able to produce IL-4 and IL-10. By contrast, CD69HI TRM displayed a resting phenotype, marked for more restricted movement, and produced the best multifunctional TH1 responses following stimulation. These data demonstrate the importance of studying migration, and provide detailed characterisation of CD69HI TRM and novel CD69INT cells, along with their proposed roles and generation pathways.

Invasive non-typhoidal Salmonellosis is a major cause of bloodstream infections in Sub-Saharan Africa, mainly caused by Salmonella enterica serovars Typhimurium and Enteritidis. Naturally shed outer membrane vesicles from Gram-negative bacteria are being explored to generate cost-effective vaccines against many infections, since antigens within vesicles maintain their natural conformation and orientation. Shedding can be enhanced through genetic modification and the resulting vesicles, Generalized Modules for Membrane Antigens (GMMA) offer potential as vaccines. We explored the potential of expressing a known immunogenic antigen of S. Typhimurium, OmpD, in GMMA derived from E. coli as a vaccine. Further, we showed that immunization with GMMA derived from S. Typhimurium (STm-GMMA) induced protection in mice. The response to STm-GMMA immunization included the generation of antibodies to two immunodominant antigens, lipopolysaccharide and porins. Strikingly, the IgG response towards these two antigens was induced with different rates during first week, with a dramatic induction of IgG targeting porins, and not LPS, in a T-cell independent manner. Nevertheless, the antibody response against both antigens persisted for over 200 days in sites including the bone marrow. Results from this thesis shows that STm-GMMA is both attractive as a vaccine and as a tool to facilitate investigations of B-cell responses.

This study examined the relationship between Mycobacterium tuberculosis global clades and patient origin. In the UK, the rate of tuberculosis is higher amongst patients who originated from the Indian subcontinent (ISC), where two dominant lineages are present, the Central Asian Strain (CAS) and the East African Indian (EAI) lineage. Mycobacterial interspersed units containing variable number of tandem repeats DNA fingerprinting of M. tuberculosis strains isolated from UK patients who originated from the ISC, as defined by novel software, identified that CAS was the most prevalent clade (39%) and EAI was the third most prevalent clade (15%). To further elucidate the relationship between host origin and strain lineage, two rigorous new models of infection were developed which assessed mycobacterial growth and host cell response. The monocyte-derived macrophage model was more appropriate for measuring cytototoxicity than the THP-1 cell model as in the absence of infection, 50% of THP-1 cells died compared to 2% of macrophages. CAS strains caused 1.5 fold more cell necrosis and their growth was four fold higher than EAI strains in the monocyte-derived macrophage model. Finally, the response of polarised monocyte-derived macrophages from Asian and Caucasian donors to different M. tuberculosis lineages was compared. CAS strains grew preferentially better in M2 macrophages from Asian donors. The prevalence of CAS in the Midlands is likely to be due to a combination of specific strain importation and increased ability of this strain to transmit to the population present in the Midlands.

Digital dermatitis (DD) is a superficial infectious dermatitis of the digital skin of cattle and sheep that can be very painful, causing severe lameness in affected animals. Bovine digital dermatitis (BDD) in dairy cattle has now been reported in most countries they are farmed, and DD in sheep, known as contagious ovine digital dermatitis (CODD) is rapidly emerging as a severe infectious foot disease since first reports from the UK in 1997. Spirochaetes, of the genus Treponema have frequently been found in large numbers in BDD lesions and are now considered the primary causative bacteria of BDD. Three treponeme phylogroups are consistently isolated from dairy cattle BDD in the UK and the USA, which are known as Treponema medium- like, Treponema phagedenis- like spirochaetes and Treponema pedis. Over the past 40 years research has focused on dairy cattle BDD and overlooked whether the disease exists in beef cattle herds in the UK, and whether the same aetiological agents are causal. There is also limited information on the causative bacteriological agents of CODD. Furthermore, no definitive transmission routes or infection reservoirs of DD in either cattle or sheep had thus far been delineated, with only a single study finding a potential reservoir site of DD treponemes in the dairy cattle gastrointestinal (GI) tract. Using molecular bacteriological studies it was found that CODD and beef cattle BDD, as in dairy cattle BDD, show a high association with the three DD treponeme phylogroups. All CODD and beef BDD lesions investigated had at least one of the three DD treponeme phylogroups present in the lesions and these treponemes were also isolated from a high proportion of lesions. No DD treponemes were detected in healthy sheep or beef cattle foot tissue. Upon 16S rRNA gene sequence analysis all isolates showed a high similarity, if not 100% identity, to representatives of each treponeme phylogroup isolated from dairy cattle BDD lesions, indicating a shared aetiology between DD in all three animals. Additionally, the same treponeme bacteria were detected and isolated from a new undefined foot disease in dairy goats in the UK indicating that cross-species transmission of DD may have occurred causing DD infection in a previously unaffected domestic livestock species. To understand potential transmission routes and infection reservoirs of DD, the host GI tract and hoof trimming equipment were investigated. Of the sheep gingival (n= 40) and rectal tissues (n= 40), 1/40 gingival tissues were positive for DD- associated treponemes (T. pedis), and 3/40 rectal tissues (one containing T. medium- like and two tissues containing T. pedis). No DD- associated treponeme DNA was amplified from beef cattle rectal tissues (n= 40), however 4/40 beef gingival tissues were positive for DD- associated treponemes (all containing T. phagedenis- like). A T. phagedenis- like DD treponeme was isolated from the rectal tissue of a CODD symptomatic sheep. Beef cattle (n= 41) and sheep (n= 79) faeces failed to amplify DD- associated Treponema DNA. Twenty two treponemes were isolated from sheep faeces; however, upon phylogenetic analysis these clustered with considered non-pathogenic treponemes, which interestingly exhibited farm specific diversity in their 16S rRNA gene. Trimming equipment was tested after being used to trim cattle and sheep hooves, and subsequently after disinfection of equipment. Of the blades used to trim DD symptomatic animals (n= 26, cattle and sheep combined), 25/26 were found to be positive for at least one of the DD Treponema phylotypes. This figure was reduced to 10/26 (38%) after disinfection of the blades. Following culture of a swab, an isolate belonging to the T. phagedenis- like spirochaetes was isolated from a knife sample after trimming a DD positive cow. Beef cattle sera from DD positive and negative farms were investigated to understand whether beef cattle’s perceived lower prevalence of BDD in the UK is due to a lack of exposure to treponemes, or a protective immune response. Beef cattle from DD positive farms appeared to produce a strong immunological response to treponemes, compared with DD negative farm animal sera. Therefore the perceived lower prevalence of DD in beef cattle does not appear to be due to a protective response in these animals, but more likely due to a lack of exposure to DD treponemes. In conclusion, these studies have produced vital information describing DD in beef cattle and sheep and their respective aetiological agents allowing for more appropriate treatments in the future. Additionally, given the two potential transmission routes delineated from the data, effective actions can be taken to prevent the spread of DD within current hosts and to limit emergence into yet unknown additional host species.