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The tsetse transmitted fatal disease of humans, sleeping sickness, is caused by two morphologically identical subspecies of the parasite T. brucei; T. b. rhodesiense and T. b. gambiense. Current distributions of the two forms of disease are not known to overlap in any area, and Uganda is the only country with transmission of both. The distribution of Rhodesian sleeping sickness in Uganda has expanded in recent years, with five districts newly affected since 1998. This movement has narrowed the gap between Rhodesian and Gambian sleeping sickness endemic areas, heightening concerns over a potential future overlap which would greatly complicate the diagnosis and treatment of the two diseases. An improved understanding of the social, environmental and climatic determinants of the distribution of Rhodesian sleeping sickness is required to allow more effective targeting of control measures and to prevent further spread and possible concurrence with Gambian sleeping sickness. The work presented in this thesis investigates the drivers of the distribution and spread of Rhodesian sleeping sickness in districts of central and eastern Uganda which form part of the recent disease focus extension. The spatial distribution of Rhodesian sleeping sickness was examined in Kaberamaido and Dokolo districts where the disease was first reported in 2004, using three different methodologies. A traditional one-step logistic regression analysis of disease prevalence was compared with a two-step hierarchical logistic regression analysis. The two-step method included the analysis of disease occurrence followed by the analysis of disease prevalence in areas with a high predicted probability of occurrence. These two methods were compared in terms of their predictive accuracy. The incorporation of a stochastic spatial effect to model the residual spatial autocorrelation was carried out using a Bayesian geostatistical approach. The geostatistical analysis was compared with the non-spatial models to assess the importance of spatial autocorrelation, to establish which method had the highest predictive accuracy and to establish which factors were the most significant in terms of the disease’s distribution. Links between Rhodesian sleeping sickness and landcover in Soroti district were also assessed using a matched case-control study design. Temporal trends in these relationships were observed using an annually stratified analysis to allow an exploration of the disease’s dispersion following its introduction to a previously unaffected area. This work expands on previous research that demonstrated the source of infection in this area to be the movement of untreated livestock from endemic areas through a local livestock market. With regards to the comparison of regression frameworks, the two-step regression compared favourably with the traditional one-step regression, but the Bayesian geostatistical analysis outperformed both in terms of predictive accuracy. Each of these regression methods highlighted the importance of distance to the closest livestock market on the distribution of Rhodesian sleeping sickness, indicating that the disease may have been introduced to this area via the movement of untreated cattle from endemic areas, despite the introduction of regulations requiring the treatment of livestock prior to sale. In addition, several other environmental and climatic variables were significantly associated with sleeping sickness occurrence and prevalence within the study area. The temporal stratification of the matched case-control analysis highlights the dispersion of sleeping sickness away from the point of introduction (livestock market) into more suitable areas; areas with higher proportions of seasonally flooding grassland, lower proportions of woodland and dense savannah and lower elevations. These findings relate to the habitat preferences of the predominant vector species in the study area; Glossina fuscipes fuscipes, which prefers riverine vegetation. The findings presented highlight the importance of the livestock reservoir as well as the climatic and environmental preferences of the tsetse fly vector for the introduction of Rhodesian sleeping sickness into previously unaffected areas, the subsequent spread of infection following an introduction and the equilibrium spatial distribution of the disease. By enhancing the knowledge base regarding the spatial determinants of the distribution of Rhodesian sleeping sickness within newly affected areas, future control efforts within Uganda may be better targeted to decrease prevalence and to prevent further spread of the disease.

Trypanosome alternative oxidase (TAO) is the sole terminal oxidase responsible for the aerobic respiration of the parasite T. b. brucei. Specific strains of this parasite cause the neglected tropical disease Human African trypanosomiasis (HAT), and thus TAO is an interesting target for the potential treatment of this disease. Inhibition of TAO with the natural product inhibitors colletochlorin B or ascofuranone has been shown to clear infections of T. b. brucei in mice at high concentrations. However, these natural product inhibitors contain undesirable chemical functionality and have poor physicochemical properties, preventing adequate drug exposure to effectively treat HAT. Robust protocols for the expression and purification of recombinant TAO were developed, which enabled the development of biochemical assays to identify inhibitors of TAO function. Single point inhibition screening of the Medicines Malaria Venture 'kinetoplastid collection' of 400 compounds identified a range of micro-molar inhibitors of TAO. A program of chemical optimisation was carried out around the natural product inhibitor colletochlorin B, with the aim to improve the physicochemical properties and retain inhibitory potency against TAO. The structure activity relationships generated over the course of this exploration identified a dependency on high lipophilicity to retain potent TAO inhibition. The TAO inhibitors synthesised were also assessed for parasite growth inhibition and mammalian cell cytotoxicity to correlate inhibition data with cellular efficacy, in collaboration with Novartis. The physicochemical properties of these novel compounds showed improvement over the natural product colletochlorin B and prompted further assessment of leading compounds in advanced parasite kill kinetic and parasite clearance assays at Novartis. The data generated in these assays for compounds synthesised in this thesis determined that TAO inhibition results in a trypanostatic response, and not a preferred trypanocidal response in T. b. brucei.

Trypanosomatids cause human diseases such as leishmaniasis and African trypanosomiasis. Trypanosomatids are protists from the order Trypanosomatida and include species of the genera Trypanosoma and Leishmania, which occupy a similar ecological niche. Both have digenic life-stages, alternating between an insect vector and a range of mammalian hosts. However, the strategies used to subvert the host immune system differ greatly as do the clinical outcome of infections between species. The genomes of both the host and the parasite instruct us about strategies the pathogens use to subvert the human immune system, and adaptations by the human host allowing us to better survive infections. We have applied unsupervised learning algorithms to aid visualization of amino acid sequence similarity and the potential for recombination events within Trypanosoma brucei's large repertoire of variant surface glycoproteins (VSGs). Methods developed here reveal five groups of VSGs within a single sequenced genome of T. brucei, indicating many likely recombination events occurring between VSGs of the same type, but not between those of different types. These tools and methods can be broadly applied to identify groups of non-coding regulatory sequences within other Trypanosomatid genomes. To aid in the detection, quantification, and species identification of leishmania DNA isolated from environmental or clinical specimens, we developed a set of quantitative-PCR primers and probes targeting a taxonomically and geographically broad spectrum of Leishmania species. This assay has been applied to DNA extracted from both human and canine hosts as well as the sand fly vector, demonstrating its flexibility and utility in a variety of research applications. Within the host genomes, fine mapping SNP analysis was performed to detect polymorphisms in a family study of subjects in a region of Northeast Brazil that is endemic for Leishmania infantum chagasi, the parasite causing visceral leishmaniasis. These studies identified associations between genetic loci and the development of visceral leishmaniasis, with a single polymorphism associated with an asymptomatic outcome after infection. The methods and results presented here have capitalized on the large amount of genomics data becoming available that will improve our understanding of both parasite and host genetics and their role in human disease.

African trypanosomes are protozoan blood parasites that infect both humans and livestock. BALB/c mice are highly susceptible to experimental infections by Trypanosoma congolense while C57BL/6 mice are relatively resistant, as measured by degree and pattern of parasitemia and survival time. Rapid death observed in highly susceptible BALB/c mice is due to a systemic inflammatory response syndrome (SIRS). A small subset of pathogenic, MHC class II-restricted CD4+ T cells, activated during the course of T. congolense infections, mediates early mortality in infected highly susceptible BALB/c mice via excessive synthesis of the cytokine IFN-gamma. Since these pathogenic T cells are matrixadherent, they could be distinguished from conventional Th1 cells. There is a possibility that this subpopulation of T cells has unique surface markers.<p> The complement system is highly activated in African trypanosomiasis, leading to persistent hypocomplementemia. Amplification of the alternative pathway of complement is faster in BALB/c mice than in C57BL/6 mice and the degradation of complement component C3b to complement component C3d, during the amplification of the alternative pathway of complement, proceeds faster in BALB/c than in C57BL/6 mice (Ogunremi et al., 1993). T. congolense-infected BALB/c mice have more immune complexes containing trypanosomal variant surface glycoprotein (VSG) than C57BL/6 mice in their plasma (Pan & Tabel, unpublished). T. congolense-infected BALB/c mice might have more VSG-C3d immune complexes than infected C57BL/6 mice. The receptor for complement component C3d is the cell surface molecule CR2, also referred to as CD21. It is known that CR2 is widely expressed on B lymphocytes and follicular dendritic cells. There is also some evidence that CR2 is expressed on a subpopulation of activated T cells. Binding of VSG-C3d immune complexes to the complement receptor CR2 might costimulate the CR2+ T cells to produce IFN-ã. I hypothesize that IFN-ã-producing T cells in T. congolense-infected BALB/c mice are CR2+ and that the CR2+ T cells increase in numbers in experimental murine T. congolense infections.<p> Kinetic studies were carried out by staining spleen cells of T. congolense-infected BALB/c mice for the presence of CR2 on T cells (CD3+ cells). Total numbers of spleen cells showed a 5-fold increase with progressive T. congolense infections. The total numbers of T cells in the spleen showed a 7-fold increase at day 8 post infection. The total numbers of CR2+ T cells in the spleen showed a 3 to 7-fold increase with progressive infection. Parallel studies on B lymphocytes (CD19+ cells) showed that absolute numbers of B cells in the spleen had a 5 to 6-fold increase with progressive infection. Absolute numbers of CR2+ B cells in the spleen showed a 4-fold increase at day 7 post infection. The total numbers of CR2+ cells in the spleen showed an increase while the mean numbers of CR2 molecules per cell showed a reduction with progressive infection.<p> These results show that CR2+ T cells in the spleen increase in numbers with progressive T. congolense infections in BALB/c mice. I suggest that CD4+CR2+ T cells might play a role in the pathogenesis of T. congolense infections.

African trypanosomes are protozoan blood parasites that infect both humans and livestock. BALB/c mice are highly susceptible to experimental infections by Trypanosoma congolense while C57BL/6 mice are relatively resistant, as measured by degree and pattern of parasitemia and survival time. Rapid death observed in highly susceptible BALB/c mice is due to a systemic inflammatory response syndrome (SIRS). A small subset of pathogenic, MHC class II-restricted CD4+ T cells, activated during the course of T. congolense infections, mediates early mortality in infected highly susceptible BALB/c mice via excessive synthesis of the cytokine IFN-gamma. Since these pathogenic T cells are matrixadherent, they could be distinguished from conventional Th1 cells. There is a possibility that this subpopulation of T cells has unique surface markers.<p> The complement system is highly activated in African trypanosomiasis, leading to persistent hypocomplementemia. Amplification of the alternative pathway of complement is faster in BALB/c mice than in C57BL/6 mice and the degradation of complement component C3b to complement component C3d, during the amplification of the alternative pathway of complement, proceeds faster in BALB/c than in C57BL/6 mice (Ogunremi et al., 1993). T. congolense-infected BALB/c mice have more immune complexes containing trypanosomal variant surface glycoprotein (VSG) than C57BL/6 mice in their plasma (Pan & Tabel, unpublished). T. congolense-infected BALB/c mice might have more VSG-C3d immune complexes than infected C57BL/6 mice. The receptor for complement component C3d is the cell surface molecule CR2, also referred to as CD21. It is known that CR2 is widely expressed on B lymphocytes and follicular dendritic cells. There is also some evidence that CR2 is expressed on a subpopulation of activated T cells. Binding of VSG-C3d immune complexes to the complement receptor CR2 might costimulate the CR2+ T cells to produce IFN-ã. I hypothesize that IFN-ã-producing T cells in T. congolense-infected BALB/c mice are CR2+ and that the CR2+ T cells increase in numbers in experimental murine T. congolense infections.<p> Kinetic studies were carried out by staining spleen cells of T. congolense-infected BALB/c mice for the presence of CR2 on T cells (CD3+ cells). Total numbers of spleen cells showed a 5-fold increase with progressive T. congolense infections. The total numbers of T cells in the spleen showed a 7-fold increase at day 8 post infection. The total numbers of CR2+ T cells in the spleen showed a 3 to 7-fold increase with progressive infection. Parallel studies on B lymphocytes (CD19+ cells) showed that absolute numbers of B cells in the spleen had a 5 to 6-fold increase with progressive infection. Absolute numbers of CR2+ B cells in the spleen showed a 4-fold increase at day 7 post infection. The total numbers of CR2+ cells in the spleen showed an increase while the mean numbers of CR2 molecules per cell showed a reduction with progressive infection.<p> These results show that CR2+ T cells in the spleen increase in numbers with progressive T. congolense infections in BALB/c mice. I suggest that CD4+CR2+ T cells might play a role in the pathogenesis of T. congolense infections.

The immunological responses in hosts infected with Trypanosoma lewisi were examined during the course of infection and after recovery. Peak antibody levels coincided with the time of parasite elimination, but remained significantly elevated for over one year after the end of the infection The antigen repertoire recognized by antibodies demonstrated that some were revealed only by sera taken during the infection, and other antigens were revealed for the first time only by post-recovery sera. Immunomodulatory protective and suppressive factors were demonstrated in the plasma of irradiated, infected rats. These factors were identified as parasite-derived exoantigens which are shed in vivo and in vitro; exoantigens are complexes of proteins, lipids and polysaccharides and are membrane-surface coat associated, as shown by phase-partitioning and surface-labeling studies. The suppressive activity of the exoantigens was dose-dependent, probably mediated by a suppressor substance(s) produced by macrophages that subsequently inhibits production of interleukin 2 by T helper cells.

No description available.

Thesis (Ph.D.)--Murdoch University, 2005. / Thesis submitted to the Division of Health Sciences. Bibliography: leaves 141-163.