I describe the isolation and characterisation of new sigma-viral isolates collected from Europe and North America. With these new isolates, I show that sigma virus has very low levels of viral genetic diversity across Europe and North America compared to other RNA viruses. I examine the susceptibility of <i>Drosophila melanogaster </i>to five of the viral isolates to investigate whether specificity exists in this system. The results suggest that there is little constraint on flies evolving resistance to all five viruses and that trade-offs between resistance against the five viruses are unlikely to explain why variation is susceptibility exists in wild populations of <i>Drosophila melanogaster</i>. Sigma-viruses increase the susceptibility of flies to the fungus <i>Beauveria bassiana</i> – that commonly infects insects in the wild. This could have profound effects in the wild where flies are constantly exposed to bacteria and fungus during feeding. One interpretation of the increased susceptibility of sigma-infected flies, is that the sigma virus is suppressing the Toll-pathway. However, I found no evidence for viral suppression of the Toll-pathway, nor did I find evidence that flies mount a Toll-dependent immune response against the sigma virus. This suggests that either <i>Drosophila</i> do not mount an immune response against the sigma virus, or that the immune response is controlled by other pathways. Finally, I describe the hypermutation of adenosines to guanosines in the genome of the sigma virus. The clustering of these mutations and the context in which thy occur indicates that they have been caused by ADAR–RNA editing enzymes that target double stranded RNA. This is the first evidence that ADARs target viruses outside of mammals, and it raises the possibility that ADARs could play a role in the antiviral defences of insects.

Theileria annulata is an economically important protozoan parasite (Api-complexa) which cycles between bovine and invertebrate tick hosts. Within the bovid, sporozoites invade leucocytes and develop into macroschizonts: macroschizonts divide, initially by binary fission, in synchrony with the newly transformed host cell, but later merogony takes place, producing merozoites which invade erythrocytes. These are subsequently ingested by the tick. The post-macroschizont life cycle stages are poorly defined, and this experimental project was aimed at investigating the basic biology of these stages at the molecular level. The polypeptide complement of the infected erythrocyte membrane has been investigated. The precise location of piroplasm polypeptides in the infected cells was not determined, but the experimental results indicate that several (most notably molecules of 122kDa, 98-100kDa & 77kDa) are associated with the erythrocyte membrane. Monoclonal antibodies have been raised against infected erythrocytes. By immunofluorescence microscopy, several antibodies recognise, mainly, either the outer perimeter of the piroplasm, vesicular structures (dots), or molecules located within the piroplasm. For the purpose of strain differentiation, a panel of these monoclonal antibodies distinguish between un-cloned preparations of Ankara, Hissar and Gharb stocks. The vast majority of the monoclonal antibodies (27 cloned lines) are stage specific and do not recognise slide preparations of macroschizonts or sporozoites. Merogony has been induced in vitro, in recently transformed macroschizont infected cell lines, and four anti-piroplasm monoclonal antibodies recognise preparations of merozoites. Immunoelectron microscopy results have shown that antibody 5E1 recognises the surface of heat induced merozoites. Western blot analysis suggests that the 30kDa and 120kDa polypeptides, recognised by antibody 5E1, also elicit an antibody response in the bovine host. These antigens are preliminary candidates for part of a molecular sub-unit vaccine against the disease (Tropical theileriosis) caused by T.annulata. The T.annulata gene, which encodes the epitope determining antibody 5E1 has been cloned from a lambda gtl 1 genomic expression library. A model system for investigating the molecular mechanisms of stage differentiation has been developed. Macroschizont merogony can be induced in recently infected cell lines by elevating culture temperatures. Similar heat-treatment, of the same cell line after prolonged passage, fails to result in the differentiation into merozoites. The epitope recognised by antibody 5E1 is stage specific to merozoite and piroplasm stages and preliminary Northern slot-blot analysis, with the cloned gene sequence, suggests that expression is regulated at the level of transcription.

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The Red Queen hypothesis proposes that antagonistic coevolution between parasites and their hosts is responsible for the evolutionary maintenance of sexual reproduction. In this thesis I examine various aspects of the Red Queen hypothesis using the Cladoceran crustacean Daphnia. A survey of parasite prevalence in North American populations of Daphnia pulex represents the first attempt to examine the role of parasites in the maintenance of breeding system variation in this species. Despite evidence of over- and underparasitism in some populations, parasite prevalences were generally very low suggesting that parasites are not a major source of selection in the populations studied. The Pluralist Approach to sex proposes that the effects of deleterious mutations and parasitism may interact. I established mutant lines of Daphnia magna using the chemical mutagen ENU and investigated the impact of the parasite Pasteuria ramosa on mutation load under different environmental conditions. I found that although parasite infection could exacerbate the effects of mutation load, this interaction was dependent on host environment and the implications of these findings for the general application of the Pluralist Approach are discussed. The impact of mixed strain infections on genotype-specific infection was also examined. In natural populations, hosts are likely to be exposed to a range of parasite genotypes and this may potentially affect the efficiency of the immune response. I found that the ability of certain P. ramosa strains to infect their hosts is affected by prior host exposure to different strains.

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<i>Nippostrongylus brasiliensis </i>is a GI nematode parasite of rodents and is an extensively applied laboratory model for defining the immune mechanisms that mediate worm expulsion. <i>N. brasiliensis </i>infection in the rat shares many similarities with <i>Trichostrongylus </i>infection in ruminants including, importantly, the functional proteins secreted or excreted by the nematode (ES proteins). Several of these ES proteins are potential candidates for vaccines. The aim of this project was to use the rat/<i>Nippostrongylus </i>parasite system as a model for vaccination with recombinant ES proteins. Prior to vaccination trials <i>N. brasiliensis</i> infections of varying levels were investigated and immunological assays were developed to allow the assessment of immune responses to infection and vaccination. Two proteins of interest, a Superoxide dismutase (SOD) and Acetylcholinesterase (AChE), were selected as vaccine candidates. Recombinant AChE was kindly provided for study by Prof Murray Selkirk. A cDNA encoding the SOD was amplified by PCR with a functional enzyme being obtained after expression in <i>E. coli</i>. Vaccination with these two recombinant enzymes was investigated to ascertain their protective capacity and thus their suitability as vaccine candidates. In a preliminary trial with AChE, vaccinated animals showed a 48% reduction in egg output compared to controls, this being associated with changes in antibody and RMCP II levels. The SOD did not induce any protection or a significant immune response. Further trials investigated how the route of administration and adjuvant might affect the protective capacity of these proteins, with protection varying between 0 and 38%. The relevance of these results to future vaccine trials in ruminants is discussed.

No description available.

No description available.

No description available.