Rosetting is an adhesion property of malaria parasites whereby infected erythrocytes bind to two or more uninfected erythrocytes, forming a so-called rosette. Rosetting of Plasmodium falciparum is associated with disease severity and high parasitaemia in sub-Saharan Africa, although currently the function of rosetting remains unknown. An early IFNg response elicited from the innate immune system is associated with resolution of malaria infection in mice. Published data suggests that optimal IFNg production may require contact between peripheral blood mononuclear cells and P. falciparum infected erythrocytes. The first part of this thesis investigates the hypothesis that rosetting is an immune evasion strategy to hide infected erythrocytes from detection by innate immune cells. Across five laboratory strains of P. falciparum rosetting was not associated with differential IFNg production when parasites were grown in group O blood. Reappraisal of the data with respect to blood group for one strain found that rosetting significantly reduced the IFNg response to parasites grown in group A blood (P=0.022, Wilcoxon signed-rank test), where it is known that rosettes are bigger and stronger. This is consistent with the hypothesis that rosetting is an immune evasion strategy and the first study to find evidence for a function of rosetting. Further work is needed in order to generalise this finding. The cytokine response to P. falciparum varies between people and this variation may be indicative of disease progression. In mice infected with malaria it is also apparent that parasite strain can determine the cytokine response of the host. It is unclear whether P. falciparum strains vary in their ability to induce cytokines. The second part of this thesis investigates variation in cytokine induction between P. falciparum strains. Across four laboratory strains of P. falciparum, IFNg production was significantly dependent on parasite strain (F3,178= 48.49, P<0.001). Production of GM-CSF, IL-1b, IL-6, IL-10 and TNFa significantly correlated with production of IFNg (P<0.001, Pearson correlation) and followed the same strain-dependent pattern. The ratio of pro-inflammatory cytokines to IL-10 was also dependent on parasite strain. These data provide strong evidence for P. falciparum strain-dependent cytokine responses which may be an important determinant of disease outcome. Phagocytosis by splenic macrophages is proposed to be the principle mechanism of parasitaemia control in malaria infection. CD36 mediated phagocytosis may by an important mechanism of non-opsonic parasite clearance. The final part of this thesis investigates the hypothesis that rosetting is an immune evasion strategy of P. falciparum in order to evade phagocytic clearance, in particular that mediated by CD36. Overall the data obtained were inconsistent. Phagocytosis was significantly reduced in rosetting versus non-rosetting parasites in some strains (e.g. R29; P=0.048, paired T test), whereas others showed no effect (e.g. Muz12; P=0.228, paired T test) or increased versus non-rosetting parasites (e.g. HB3, P=0.004, paired T test). The relationship between CD36 binding and phagocytosis was also unclear, and anti-CD36 antibody did not effectively block phagocytosis, suggesting the involvement of alternative mechanisms. Further experiments are needed to clarify these observations. Data presented in this thesis are suggestive that rosetting in non-group O blood may be an immune evasion strategy with regard to IFNg production by innate immune cells, mechanistically linking rosetting with enhanced parasitaemia and disease severity. Furthermore, parasite strain significantly affects cytokine production and may be a determinant of disease outcome. This thesis demonstrates the importance of continued research into the effect of parasite virulence on the immune response, with particular emphasis on rosetting.

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In this study, two plant species, Schefflera umbellifera (Araliaceae) and / Thesis (M.Sc.) - University of KwaZulu-Natal, Pietermaritzburg, 2007.

Every year over two hundred million cases of malaria occur worldwide causing human death and suffering often in the poorest countries. Most people who die from malaria are children under five years of age. Malaria is caused by parasites spread by mosquitoes when they feed on human blood. Currently prevention methods include insecticides and anti-malarial drugs. The problem with both is the increasing resistance towards them by mosquitoes and parasites, respectively. Therefore other approaches need to be investigated to find new solutions to this problem. One such research area is paratransgenesis, the genetic modification of symbiotic microorganisms in the mosquitoes to produce anti-malaria parasite molecules. One bacterium identified as a potential candidate for paratransgenesis is Thorsellia anophelis. When this study started, only one Thorsellia isolate existed in the world. The aim of this study was therefore to retrieve more Thorsellia isolates from Kenyan mosquito and water samples. The samples were screened by PCR followed by bacterial culturing of positive samples, which resulted in 38 new Thorsellia isolates confirmed by DNA sequencing. The isolation of new Thorsellia species enables further investigation of the potential for their use in paratransgenesis with the aim of contributing to the prevention of malaria transmission.

Malaria is still a significant public health problem in the Tropics, with an estimated 200 million cases a year and more than 1 million deaths, mostly in young children in sub-Saharan Africa. Plasmodium falciparum is the parasite responsible for the majority of the morbidity and mortality due to malaria. We know from the historical use of malaria to treat neurosyphilis that there were several different strains of P. falciparum, some of which were more pathogenic and had higher multiplication rates than others. High multiplication rates of P. falciparum isolates have been associated with severe disease in Thailand, but not in Kenya or Mali. In determining what differences exist between fast- and slow-growing malaria parasites, and understanding their relationship with clinical outcome, we may discover a way of targeting those parasites that cause most disease. This thesis describes a genetic analysis of the determinants of growth and pathogenicity in the rodent malaria parasite, Plasmodium chabaudi. The use of rodent malaria parasite strains for genetic analysis has several experimental, ethical and financial advantages over the use of human malaria parasites. In addition, rodent malaria parasite strains also vary significantly in their growth and pathogenicity, making them excellent candidates for a genetic analysis of these characteristics. The first section of this thesis is concerned with the characterisation of the growth, pathogenicity and transmissibility of two strains, DS and DK, of the rodent malaria parasite P. c. adami. The DS strain is fast-growing, pathogenic, non-selective in its invasion of red blood cells and a poor transmitter to mosquitoes. The DK strain is slow-growing, non-pathogenic, selective in its invasion of red blood cells and a good transmitter to mosquitoes. In the second section of this thesis is a detailed study of the growth characteristics of DS and DK in mixed infections, relative to their growth in single infections. Both sections provide information relevant for the main objective of this thesis, but also contribute to the body of work on pathogenicity and transmissibility, and pathogenicity and strain behaviour in mixed infections, which has been carried out in rodent malaria parasites to-date. The third section of the thesis contains the results of a genetic analysis of the difference in growth between P. c. adami strains DS and DK, using the Linkage Group Selection (LGS) technique. On several occasions, DS and DK were crossed in the mosquito vector and, following selection for fast growth in mice, the cross progeny were initially screened with genome-wide, quantitative AFLP markers. Markers specific to the slow-growing parent DK which were greatly reduced in intensity after selection were found on P. chabaudi chromosomes 6, 7 and 9. This result suggests that the difference in growth between the two strains is determined by multiple genetic loci. The selection on chromosomes 7 and 9 was then looked at in greater detail, using SNP-based markers quantified by Pyrosequencing™. It was found, consistently, that a region at one end of DS chromosome 9 was inherited as a single, non-recombining unit in cross progeny selected for fast growth. As this was the region most strongly selected against, it suggests that a gene (or genes) in this region has a major role in the determination of growth characteristics, and therefore pathogenicity, in P. c. adami. Narrowing down this region further, in order to identify the candidate gene(s), remains a key future objective.