The role of IFN in acquisition of immunity against erythrocyte forms of P.c. chabaudi AS was studied. Inbred NIH mice given the construct 7 days before malaria infection, showed a significant delay in the onset and in the level of the recrudescent parasitaemia in comparison with controls. No differences, however, were observed in the recrudescent parasitaemia between the groups. NIH mice infected with malaria 3 days after or on the same day as the administration of the IFN construct, showed a primary peak of infection similar to controls, but the resolution of this patent parasitaemia occurred 1 or 2 days earlier in the experimental mice when compared with controls. In the same experiment, mice given the construct 10 days before malaria infection had a similar course of infection as controls. Simultaneous inoculation with two S. typhimurium constructs: IFN and TNF, 8 days before malaria infection resulted in a course of parasitaemia similar to that observed in mice given the IFN construct alone. On the other hand, inoculation of 'susceptible' inbred A/J mice with S. typhimurium/IFN 3 or 8 days before malaria infection had no effect on the course of the parasitaemia when compared with controls. The immune mechanisms involved in the better control of the malaria infection of NIH mice given S. typhimurium/ IFN, seem to be independent of nitric oxide (NO) production, since increased levels of the molecule were demonstrable around the peak of the primary parasitaemia in control groups but not in experimental mice. In the latter basal levels of serum NO were observed from the period after the S. typhimurium/ IFN inoculation until up to three days after the peak of the parasitaemia.

A growing problem with drug resistance in Human African Trypanosomiasis has necessitated the implementation of screening programmes to monitor for its spread. This thesis describes the study of several factors that can influence the selection and propagation of drug resistance in T. brucei. Human African Trypanosomiasis (HAT) is caused by T. brucei gambiense and T. brucei rhodesiense. The few drugs used for the treatment of the disease are either toxic, cause severe side effects or suffer from parasite resistance. The T. brucei P2 transporter, which is encoded by the gene TbAT1, mediates uptake of melaminophenyl arsenicals and diamidines. Reduced P2 uptake is associated with drug resistance. A number of point mutations found in a laboratory derived melarsoprol resistant T. brucei stock (STIB 777R) allowed development of a PCR/RFLP based molecular method to identify resistance alleles. By 1999, 20-30% of patients treated in Omugo, NW Uganda were failing to respond to melarsoprol. PCR/RFLP analysis indicated that mutant alleles accounted for 58.5% of those in circulation. Melarsoprol was withdrawn in 2001 and by 2003 mutant TbAT1 alleles accounted for only 14% of those in circulation in NW Uganda. The current study aimed to determine the incidence of the PCR/Sfa NI TbAT1 mutant alleles in 2006, some five years after melarsoprol had been withdrawn as first-line treatment. Successful molecular analysis of 91 of 132 (68.9%) T. b. gambiense field isolates from Omugo and Moyo in NW Uganda indicated the presence of only TbAT1 wild type alleles. Mutant alleles thus appear to have disappeared. This may be the result of parasite fitness cost following the withdrawal of melarsoprol as a stage II first-line drug from Omugo health centre, Arua, since 2001. This apparent instability of TbAT1 mutants in the field may be exploited for rational or alternating use of melarsoprol and eflornithine (DFMO) to ensure a longer life for eflornithine, delaying the onset of resistance. Insight into the overall population structure of the T. b. gambiense from Omugo, Arua (N=54) and Moyo (N=17) was obtained using mini/microsatellite marker analysis. Genetic diversity was observed to be more intra than inter regional. Multilocus genotype data analysis revealed the Omugo, Arua, population was genetically distinct from the Moyo population (Nei’s genetic distance=0.176). The evidence indicated surprisingly little genetic exchange with an excess in homozygosity (Fis >0) and alleles in linkage disequilibrium (P<0.05) within the Omugo, trypanosome population. This excess in homozygosity may be due to population sub-structuring, trypanosome inbreeding, or migration of patients. The latter is likely occurring from the neighbouring T. b. gambiense endemic disease focus in Southern Sudan. The findings suggested that the T. b. gambiense from Arua is not panmictic, clonal or epidemic but there is some level of genetic exchange. The possibility that T. b. gambiense can infect animals raises the prospect that wild or domestic animals may act as a reservoir and that a veterinary link to gambiense Human African Trypanosomiasis exists. Treatment of animals for babesiosis and trypanosomes with diminazene, uptake of which is mediated through TbAT1/P2 could select for P2-defective drug resistant trypanosomes, thereby threatening control of the human disease as well. Species detection by PCR for animal and human trypanosomes in dog isolates (N=190) from the tsetse fly endemic Jos Plataeu, Nigeria did not reveal T. b. gambiense, but multiple infections with T. brucei (95%), T. vivax (89%), and subspecies T. congolense forest (54%) and savannah (50%) were detected. The dogs were also infected with other parasites, including Babesia canis (22%) and Hepatozoon canis (16%). Multiple infections can make correct diagnosis difficult and the infections are likely to be missed by the less sensitive microscopy method. The trypanocidal action of the diamidine group of trypanocides, diminazene, pentamidine and furamidine (DB75) are principally mediated through the TbAT1/P2. In addition, pentamidine is taken up by two additional T. brucei transporters called High Affinity Pentamidine Transporter (HAPT1) and the Low Affinity Pentamidine Transporter (LAPT1). DB75 also has a secondary unknown route. Loss of TbAT1/P2 leads to significant resistance to DB75 and diminazene but not pentamidine. Identification of other markers of resistance is necessary to determine if other routes of drug entry do exist apart from P2 and whether these can be exploited for the delivery of new trypanocides into the trypanosomes. Adaptation of the T. brucei tbat1 knock-out cell line to higher concentrations of diminazene by in vitro selection for resistance led to loss of HAPT1. The resultant phenotype was similar to the previously characterised pentamidine resistant clone B48, but more resistant to diminazene and DB75. The adapted line was still capable of accumulating 1 µM radiolabelled diminazene suggesting both HAPT1 and LAPT1 as possible routes for diminazene uptake. Adaptation of the T. brucei tbat1 knock-out cell line to a high concentration of DB75 over the same 6 months period did not lead to increased resistance. Overall the project has confirmed an important role for tbat1/P2 in development of resistance to melarsoprol in the field. Importantly, it appears that removal of the selection pressure of melarsoprol leads to a loss of tbat1 alleles associated with resistance in a population of trypanosomes capable of genetic exchange in NW Uganda. Although evidence for a dog reservoir for T. b. gambiense in Nigeria was lacking in this study, a risk of selecting resistance in animals must remain high on any list of consideration. I have further shown that the diamidine drug, diminazene, used in veterinary medicine also appears to enter T. brucei via the HAPT1 transporter, as well as the P2 transporter. Loss of HAPT1 through selection with diminazene leads to high level pentamidine resistance, which could indicate a further risk in selection of human infectious trypanosomes also resistant to drugs like pentamidine.

Immunity to Salmonella enterica serovar Typhimurium (STm) is complex and requires both cell mediated and humoral immunity at different stages of infection. In infants in sub-Saharan Africa infection with non-typhoidal Salmonella (NTS), such as STm, can commonly cause fatal invasive disease. Evidence indicates that disease may be preventable by antibody, which makes vaccine development against these devastating infections a promising option. This work has explored the cell-mediated and humoral response to STm and its component antigens, their intrinsic properties, and capacity to act as protective immunogens in a mouse model. In particular, responses to surface exposed structures such as the outer membrane proteins (Omps) and the flagellar protein FliC, which are potent, immunodominant antigens and frequent targets of antibody, that may offer potential as vaccine candidates have been examined. Immunisation with soluble flagellin (sFliC) induces a potent Th2 response. Despite this, immunisation with sFliC results in accelerated clearance of STm after the first week of infection in an antibody independent, but T-bet-regulated manner. This suggests that the Th2 responses to flagellin are flexible since they can promote Th1 mediated clearance of STm. This moderate protection conferred by sFliC contrasts with the potent benefit conferred by porins. These proteins induce, and can mediate protection through a T-independent B1b cell population. In particular, antibody to OmpD is key for this protection. These results suggest that vaccines that induce protective antibody to STm may be more effective than vaccines that induce T cell-mediated protection, since they reduce bacterial numbers at the earliest stages of infection. Lastly, experiments using N. brasiliensis show that infectious history can impact on the host’s ability to control primary STm infection and the efficacy of antibody-mediated protection against infection. These projects further our understanding of the relationship between host and pathogen and the mechanisms used to control infection, but also identify the need to consider the impact of infectious history on the host’s capacity to implement protective immunity.

The autotransporters are a family of extracellular proteins, found in various Gram-negative bacteria, that have many different functions but appear to have a similar mechanism of export. In B. pertussis, the virulence-regulated proteins Pertactin, BrkA, Tcf, and Vag8 have structural homology at their C-termini (30-kDa) and the N-terminal of the mature proteins share structural characteristics such as RGD and SGXG motifs. Recently, another member of the B. pertussis autotransporter family, Bap-5 (Blackburn, 2000) (GenBank accession number AF081494) or BapC (GenBank accession number AJ277634.1) was identified. The present work has suggested that BapC, like BrkA, is a serum-resistance factor. B. pertussis brkA, bapC double and bapC single mutants were created, and showed greater sensitivity to killing by normal human serum than their wild-type strains but they were not as sensitive as a bvg mutant strain. Competition assays also showed an important role for BapC, like BrkA, in virulence of B. pertussis strains after intranasal infection in the mouse. Moreover, the brkA, bapC double and bapC single mutants were found to be more sensitive to the antimicrobial peptide, cecropin P1, than the parent strain. Nucleotide and amino acid analyses of the bapC region spanning the poly(C) and poly(G) tracts of a number of B. pertussis strains showed minor nucleotide and amino acid polymorphisms in some strains but it appeared that all had an ORF that would be able to produce some form of BapC.

The human bacterial pathogen, Streptococcus pneumoniae (the pneumococcus), has been shown to modulate different parts of the innate immune response of its host, however its ability to modulate the adaptive immune response remains largely uninvestigated. Furthermore, the importance of the adaptive arm of the immune system in responding to Streptococcus pneumoniae has only recently begun to be elucidated. I therefore investigated a potentially novel pneumococcal immunomodulatory mechanism involving the effect of the pneumococcal toxin, pneumolysin, upon the cells at the heart of the adaptive immune response; the CD4 T cell. I generated purified pneumolysin and a purified pneumolysin mutant called F433 to allow me to examine this potential effect. I found that pneumolysin inhibits in vitro antigen specific murine CD4 T cell proliferation and cytokine production and that this effect is not observed with the F433 mutant pneumolysin. Furthermore, I demonstrated that pneumolysin accomplished this inhibitory activity by inducing apoptosis of activated CD4 T cells and suggest that lipid rafts may be involved in this process since we also demonstrated that pneumolysin preferentially binds to lipid rafts. Finally I demonstrated that pneumolysin is able to inhibit the in vivo accumulation of T cells and also inhibits in vivo antibody production. I propose that the immunomodulatory mechanism I have described may play an important role during pneumococcal infection and that this warrants further investigation. I propose that detailed in vivo studies are required to demonstrate that this mechanism functions during infection and to elucidate the effect this has upon the course of infection.

Feline immunodeficiency virus (FIV) is an important veterinary pathogen with comparative significance because of its similarities to its human counterpart HIV. Since FIV is the only non-primate lentivirus which induces AIDS-like symptoms in its natural host, it serves as a valuable animal model for both prophylactic and therapeutic studies of HIV. It is accepted that the induction of neutralising antibodies (NAbs) is a key element in the control of lentiviral infection, since T-cell based vaccines alone failed to prevent infection in most experimental animal model systems. In this project a robust and reproducible in vitro neutralisation assay was developed and optimised, permitting the assessment of the NAb response in naturally infected cats and with the potential to evaluate candidate vaccines. It was demonstrated that, in general, primary FIV strains in the UK belong to subtype A, and therefore the development of a regional, subtype A-specific, FIV vaccine could be considered for use in the UK. The identification of a neutralisation resistant isolate of FIV led to the finding that a linear neutralisation determinant was located within the V5 region of Env and mutations in this region may lead to immune evasion in vivo. In addition, a second neutralisation determinant was identified in the C3/V4 region of Env. Finally, it was observed that a small proportion of naturally infected cats generated NAbs against FIV. Of these, only a very small proportion of the cats had antibodies with the potential to cross neutralise strains within the same subtype as the homologous isolate. Nonetheless, a plasma sample from a single cat was identified that neutralised all strains tested, including strains from different subtypes and geographical regions. It is likely that studies of the homologous isolate that induced the broad NAb response may be capable of inducing a similar broad response in vaccinated cats. Such a finding would have important implications for the design of potential novel lentiviral immunogens.

The post infectious paralytic autoimmune disease, Guillain-Barré syndrome (GBS), has been associated with the generation of cross-reactive auto-antibodies after Campylobacter jejuni infection. These auto-antibodies interact with both the ganglioside mimicking C. jejuni lipo-oligosaccharides (LOS) and endogenous gangliosides. This study sought to investigate novel interactions of the ganglioside mimicking LOS with immune system ganglioside specific receptors. In addition, studies investigated if such receptor recognition affects antigen trafficking or the immunostimulatory potency of the LOS, which could participate in auto-antibody generation. Results presented in this thesis demonstrate for the first time that certain members of the siglec receptor family are capable of recognising LOS from a GBS associated strain of C. jejuni. This interaction did not definitively result in enhanced, or altered, potency of ganglioside mimicking LOS in stimulating immune cells. Interestingly, ganglioside mimicry was shown to enhance phagocytosis of C. jejuni, however, in vivo differences in bacterial trafficking were not observed.

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is a formidable pathogen that uses a type-III secretion system to inject bacterial ‘effector’ proteins directly into host cells. Most effectors that are encoded within the locus of enterocyte effacement (LEE) have been studied extensively. This study aimed to characterise a selection of recently discovered non-LEE-encoded effectors using a variety of model systems. Firstly, a β-lactamase translocation assay was used to demonstrate translocation of novel effectors into host cells. The localisation of selected effectors was then investigated using mammalian cells and a yeast cell model. The effector EspM2 was shown to induce the formation of actin stress fibres in transfected HeLa cells and caused growth retardation when expressed in yeast. A number of NleG effectors also caused growth retardation and morphological changes when expressed in yeast. Growth retardation caused by the effector NleG8-2 was shown to be dependent on three conserved cysteine, aspartic acid and histidine residues. Transcriptomics and a high copy yeast gene suppression screen revealed that NleG8-2 may disrupt yeast physiology by affecting the secretory pathway. This study confirms that the effector repertoire of EHEC O157:H7 is much larger than previously imagined and provides insight into the function of selected novel effectors.

Enterohaemorrhagic E. coli (EHEC) are important gastrointestinal pathogens of humans. E. coli serotype O157:H7 is the EHEC most commonly associated with human illness. E. coli O157:H7 is carried asymptomatically by cattle which form an important reservoir for the bacterium. E. coli O157:H7 has been found to colonise at the terminal rectum of cattle in preference to other sites in the bovine gastrointestinal tract. The first objective of this work was to characterise the roles of bacterial secreted components responsible for key functions in the modulation of host defences against EHEC. Data presented here reaffirms the role of flagellin in the elicitation of a proinflammatory response in a cultured human epithelial cell line; however, the response of a bovine epithelial cell line to bacterial secreted products was not affected by the presence or absence of flagellin. A role in the modulation of the host response for the StcE protease was also investigated: although its role in interaction with the bovine host was not established, bovine secretory antibodies to StcE were detected in rectal mucosal scrapings from an E. coli O157:H7-challenged calf, suggesting that StcE is expressed and recognised in vivo. The second key objective was to isolate E. coli from the bovine intestinal tract in order to define the colonisation patterns of E. coli within the bovine intestinal tract and relate this to bacterial genotype and to provide bovine E. coli isolates to test for inhibitory activity against E. coli O157:H7 which may yield bacteria with potential as probiotic agents with a view to reducing the prevalence of EHEC in cattle. Genotypic analysis of bovine resident E. coli confirmed that these strains carry a variety of virulence factor-encoding genes; however, certain dominant genotypes were identified and the genomic structure of representative isolates was predicted by genomic microarray. EHEC-related genotypes were found to be positively associated with colonisation at the rectum, whereas non-EHEC genotypes were found to colonise multiple intestinal sites without showing any apparent site-specificity. The third and final objective of this analysis was to carry out genotypic analysis of Scottish EHEC strains in order to predict whether increased incidence of EHEC infection in Scotland may be related to the presence of EHEC strains carrying altered complement of virulence factor-encoding genes. The analysis of EHEC isolated in Scotland revealed that these strains exhibit a genomic profile which is largely typical of EHEC isolated elsewhere, although there were certain differences in the carriage of a certain genomic elements. The results presented here support the proposal that bacteriophages are the key mediators of genetic variability among E. coli isolates.