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Previous studies have shown that cytotoxic CD8+ T cells are important mediators of immunity against the bovine intracellular protozoan parasite T. parva. The present study set out to determine the role of granule enzymes in mediating killing of parasitized cells, first by characterising the granzymes expressed by bovine lymphocytes and, second, by investigating their involvement in killing of target cells. Experiments using the perforin inhibitor concanamycin A confirmed that CD8+ T cell killing of T. parva-infected cells is dependent on granule exocytosis, a process that involves release of granzymes into the target cell, resulting in activation of apoptotic pathways. Analysis of the bovine genome sequence identified orthologues of granzymes A, B, H, K and M, as well as another gene O, most closely related to granzyme A. The genes were found within 3 loci in the genome. Using specific PCR assays, all of these granzymes were shown to be expressed in Theileria-specific CD8+ T cells. Further studies were undertaken to study the role of granzyme B in killing. DNA constructs encoding functional and non-functional forms of bovine granzyme B were produced and the proteins expressed in COS cells were used to establish an enzymatic assay to detect and quantify expression of functional granzyme B protein. Using this assay, the levels of killing of different T. parvaspecific CD8+ T cell clones were found to be significantly correlating with levels of granzyme B protein expression. Moreover, the granzyme B inhibitor III, Z-IETDFMK was shown to inhibit killing by CD8+ T cell clones.

The purpose of this thesis is to investigate associations between parasite infection and host life-history variation in the wild Soay sheep population of the islands of St Kilda, NW Scotland. Studying host-parasite interactions in wild animal populations is of interest because of the importance of heterogeneity in resource availability, genetics, and environmental conditions in determining resistance to parasites, with implications for human populations and wildlife conservation and management. However, very few studies are able to investigate these associations in a longitudinal manner, which is essential in order to understand how infection is associated with life-history variation across ages and environmental conditions. In this thesis, I investigate associations between parasite resistance and ageing and the importance of maternal effects on offspring parasite resistance. I also establish the shape of natural selection on parasite resistance, and associations between measures of parasite burden and antibody responses. The principle findings of the analyses presented in this thesis are: i) Adult sheep of both sexes show a decline in parasite resistance in old age which is consistent with senescence. Furthermore, the rate of decline in parasite resistance with age is accelerated in individuals that have experienced more stressful environmental conditions over their lifespan. ii) Aspects of maternal phenotype and lamb early life performance are significantly associated with parasite resistance in lambs. Some of these effects persist into adult life and may even affect late-life changes in parasite resistance with age. iii) Analysis of ageing in five female reproductive traits shows that the contributions of individual senescence, terminal effects, and selective disappearance vary across traits, and that therefore multiple traits should be studied in order to understand ageing more fully. Most strikingly, there was no evidence for significant senescence in the probability of producing twins. iv) The first estimate of the strength of natural selection on parasite resistance in a longitudinally-monitored population provided evidence for positive selection on parasite resistance in lambs but not adults. Selection in lambs also varied across environmental conditions, being stronger in years of more favourable conditions. v) Analysis of associations between estimates of parasite burden and antibody responses showed that an estimate of parasite burden was not correlated with either a general or parasite-specific antibody response. However, antibody responses were positively correlated, and there was some evidence for a genetic correlation between the two in lambs but not adults.

Immune responses are presumed to contribute to host fitness, either by fighting off infections or via immunopathology. Research in this thesis sought to relate the magnitude of a putative immune response to infection and host and parasite fitness. The experiments and field studies presented here all focus on the interactions between the freshwater crustacean, Daphnia magna and its sterilizing bacterial endoparasite, Pasteuria ramosa, using the number of circulating haemocytes as a measure of host immune activity. I found substantial genetic variation in Daphnia’s cellular response to P. ramosa, and that Daphnia genotypes that mount the strongest cellular responses are the most likely to get infected and suffer sterilization. Thus, a strong cellular response is associated with low, as opposed to high host fitness potential. There were also some host genotypes that mounted a weaker cellular response and did not go on to suffer infection, and some that lacked a cellular response and also never suffered infection with P. ramosa. These findings led to a heuristic two-stage model for infection, where the parasite has to (1) pass from the host gut to haemolymph and then (2) successfully overcome haemolymph-based immune effectors to reproduce and achieve fitness. I also demonstrate that both the magnitude of host cellular response and likelihood of infection increases with initial parasite dose in susceptible host genotypes, and that host cellular response is associated with likely infection under both host and parasite genetic variation. Parasitised Daphnia also have substantially more circulating haemocytes than their healthy counterparts in both the laboratory and in the wild, where there is substantial genetic and environmental variation. This is one of the very few examples of how an immune response designates low host and high parasite fitness potential in a wild system. Finally, using a mixture of field study and common garden experiment, I demonstrate evolution in parasite infection traits over the course of an epidemic in a wild population, and that this evolution is associated with a decline in host abundance.

Teladorsagia circumcincta is a major financial burden on the UK sheep farming industry. Disease control is becoming increasingly difficult due to the rapid emergence of anthelmintic resistance. This has prompted the search for alternative, sustainable control measures, including vaccination. Vaccine design would be aided by a thorough knowledge of the mechanisms involved in immunity to T.circumcincta. Most research has focussed on humoral and cellular responses to infection with this nematode. This thesis focuses on the impact of infection with regards to the proteins found locally within the abomasum. Using a well established infection model, proteomic analysis of lymph draining the abomasum was carried out by means of 2-dimensional electrophoresis (2-DE). The identity of many of the proteins in gastric lymph was revealed by means of MALDI-TOF analysis. The relative quantities of the lymph proteins were monitored over time using gel analysis software in both primary infection and immune challenged infection models. This study revealed a number of proteins of interest, including the acute phase proteins serum amyloid A, alpha-1 acid glycoprotein and haptoglobin, as well as the actin depolymerising protein, gelsolin. The effect of infection and immunity to T.circumcincta on these proteins was investigated further by means of biochemical assays, western blotting and real-time PCR. The impact of infection on the permeability of the abomasal mucosa will affect the resultant gastric lymph proteome. This “leak lesion” phenomenon is well documented in T.circumcincta infection but the underlying cause is unknown. Tight junction proteins in the abomasum were studied, using immunofluorescence techniques, in an attempt to define the role of these proteins in this important immunological/pathological event. The aim of this thesis is to contribute to the knowledge of innate immune responses and local pathology occurring within the abomasum during T.circumcincta infection.

Virulence (the harm to the host during infection) is the outcome of continuous coevolution between hosts and parasites. This thesis adds to a growing body of work on host-parasite interactions, and describes experiments that study the effects of variation in the genetic and the environmental contexts of infection. All of them focus on interaction between the planktonic freshwater crustacean Daphnia magna and a naturally occurring parasite, the spore-forming bacterium Pasteuria ramosa. I show that elevated minimum temperatures that facilitate parasite growth drive natural epidemics of this parasite. I also demonstrate that the expression of infection traits in P. ramosa is temperature-dependent in a genotype-specific manner [genotype-by-environment (GxE) interactions]. These GxE interactions could maintain polymorphism through environment-dependent selection. Next, I test if GxG interactions for infectivity can be altered by environmental variation (GxGxE interactions), and find that this trait is quite robust to thermal variation. Infectivity is also more important in determining parasite fitness relative to the production of transmission stages, highlighting the importance of considering natural infection routes, an aspect sometimes overlooked in studies of host-parasite systems. Another experiment under different food and temperature regimes showed evidence for environment-dependent virulence-transmission relationships, a fundamental component of virulence evolution models. Lastly, I show that variation in temperature does not increase the cost to the host of resisting infection.

Almost 2 billion people world-wide are infected with parasitic helminths. These complex multicellular eukaryotic organisms are capable of establishing long-term infections even in the face of an intact immune response. Typically, in these settings regulatory components of the immune response, such as Foxp3+ T regulatory cells (Tregs), become dominant, limiting protective effector responses towards the parasite. Helminths are thought to have evolved mechanisms, including release of immunomodulatory molecules termed excretory-secretory products (ES), to sway the balance between the regulatory and effector arms of the immune response to favour their persistence. In this thesis both the development of a protective immune response toward, and the potential manipulation of the immune response by, the rodent gastrointestinal nematode Heligmosomoides polygyrus have been studied. Firstly, the effects of H. polygyrus ES (HES) on bone-marrow derived dendritic cells (DCs) were analysed. Although HES did not alter the phenotype of the DC it was found to be able to suppress the ability of the DC to respond to inflammatory stimuli. This activity was lost when HES was heat-inactivated (hiHES). After adoptive transfer, HES-pulsed DCs were able to induce a HESspecific T helper (Th)2-type response even if co-treated with an inflammatory stimulus. Th2-type responses are protective against H. polygyrus infection. Surprisingly, the ability of HES to generate a Th2-response in a co-treatment situation was not related to its anti-inflammatory properties; DCs co-treated with hiHES and an inflammatory stimulus were able to drive an equivalent Th2-response to HES in this situation. Next, making use of mouse strains with different susceptibility phenotypes to primary H. polygyrus infection, potential mechanisms of resistance were characterised. Development of granulomas in the gut wall were found to be associated with reduced worm burdens. Furthermore, in highly susceptible C57BL/6 mice, production of IL-23 was shown to be counter-regulatory to this process, as mice on the same background but deficient in this cytokine have increased numbers of granulomas and dramatically enhanced resistance. Susceptibility to H. polygyrus was also considered at the level of epigenetic regulation. A protein that binds specifically to methylated DNA, methyl-CpG binding domain protein (MBD)2, was found to affect the proportion of Foxp3+ Tregs within the CD4+ T cell population in vivo. Additionally, in vitro induction of Foxp3 in response to TGF-β was enhanced in MBD2-/- CD4+ T cells. MBD2-/- mice had a trend towards increased worm burdens when infected with H. polygyrus, suggesting that the difference in proportion of Tregs may limit generation of an effector response. Finally, the ability of HES to directly affect the regulatory arm of the immune response was focussed upon. It was found that HES was able to induce Foxp3 expression in naïve peripheral T cells, and that this was mediated by stimulation of the TGF-β pathway. The TGF-β mimic was of parasite origin as a pan-vertebrate TGF-β antibody was unable to block its effects but sera from H. polygyrus infected animals was competent to do this. Activity of this type was not limited to HES as ES from the ovine helminth Haemonchus contortus was found to have the same property. These data imply that some helminth parasites have evolved mechanisms to support generation of Foxp3+ Tregs, thus favouring the regulatory arm of the immune response and hence their own persistence.

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 Drosophila melanogaster 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 Drosophila melanogaster. Sigma-viruses increase the susceptibility of flies to the fungus Beauveria bassiana – 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 Drosophila 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.

From an evolutionary perspective, natural selection is expected to maximize transmission to new hosts. If a live, mobile host often benefits parasite transmission, the question arises as to why malaria parasites are virulent? The favoured trade-off view of virulence evolution assumes that virulence arises as an unavoidable consequence of parasite resource exploitation within the host that is necessary to maximise parasite transmission. However, virulence is not always a simple function of parasite density and can arise as a result of immune-mediated virulence (immunopathology). This thesis explores how immunopathology contributes to virulence on the one hand, and parasite transmission on the other, in order to improve our understanding of parasite virulence evolution. In tackling this question, the role parasite genetic diversity plays in determining immunopathology induced during malaria infection was also addressed. Using the rodent malaria Plasmodium chabaudi chabaudi (P.c.c.) in C57BL/6 mice, I explored whether immune factors – in terms of specific host cytokines central to the protection-pathology balancing act of the immune response elicited against malaria parasites – help to determine the virulence induced during infection with genetically distinct parasites, and if so, what effect this may have on transmission-stage parasites. I showed that the cytokine milieu induced by P.c.c. parasites during primary infection varies with parasite genotype and that virulence can arise independent of parasite density, via immunopathology. Specifically, I showed propensity to induce the pro-inflammatory cytokine tumour necrosis factor [TNF]-a contributes to the virulence induced, regardless of P.c.c. clone. Importantly, I also showed that across P.c.c. genotype, TNF-a reduces the density of transmission-stage parasites. Thus, virulence is not always a simple function of parasite replication, having an immune-mediated component which acts to reduce transmission potential. The importance of parasite genotype in determining the degree of immunopathological virulence induced during malaria infection was revealed by studying the anti-inflammatory arm of the immune response. The extent to which the anti-inflammatory cytokines interleukin [IL]-10 or transforming growth factor [TGF]-b limited the immunopathology induced during P.c.c. infection depended on parasite clone. In addition, parasite genotype played a key role in determining how such anti-inflammatory manipulations affected the density of transmission-stage parasites; being detrimental, beneficial or incidental to parasite fitness, depending on P.c.c. clone. Although the general mechanisms of immune regulation are qualitatively unchanged across distinct P.c.c. clones, these data emphasize the importance of parasite genotype: distinct clones differ quantitatively in immune regulation, which contributes towards their distinct virulence and fitness schedules. Overall, I found that even within a parasite species – in this case P. chabaudi – the effect of immunopathology on the virulence-transmissibility relationship may be genetically variable and may not conform to that predicted by the trade-off hypothesis, having the potential to alter the costs and benefits of virulence, depending on parasite genotype. Thus, the host immune response may play a role shaping virulence evolution and defining the limit to malaria virulence in nature.

The work presented in this thesis is focused upon the community of endemic pathogens of African cattle in Sub-Saharan Africa, which has long constrained livestock production in these areas. The first aim of this work is to investigate whether the pathogen community as a whole shapes the ensuant epidemiology and morbidity which are currently attributed to any of its individual pathogens. The second aim is to determine if a greater understanding of the interactions present amongst genetically distinct parasites of the same species can be used to better explain epidemiological features that are at present poorly understood. Emphasis is placed on examining spatial variation in the epidemiology of Theileria parva, a tick-transmitted protozoan that causes East Coast Fever. To achieve these aims, this work examines field data collected from a large and comprehensive study conducted in south east Uganda. Through application of apposite statistical techniques and mathematical modelling, aspects of the complex relations amongst the pathogen community and their environment are explored. Evidence is presented that demonstrates the paramount role of the pathogen community as a whole in shaping the infection dynamics and pathogenicity of any of its individual components. By focusing on a single member of this pathogen community (Theileria parva), some of the influences of host, vector, geographical location, temporal dynamics and intra-species pathogen interactions are elucidated. Application of a polymorphic molecular marker to Theileria parva infected blood samples and the use of Cox proportional hazard analysis, show variability in the survival of infections in cattle in high and low tick challenge areas. Moreover infection survival, which plays a pivotal role in parasite transmission, is shown to be a function of the interactions established amongst genetically distinct co-infective parasites. In consequence, vector intensity alone is insufficient to develop reliable transmission models which can accurately predict the epidemiology of the parasite inside and outside enzootic belts. Finally, a theoretical model is developed which, based upon the field evidence obtained throughout this work, provides a possible explanation for the mechanics of T. parva survival in cattle. In summary, this thesis makes a case that consideration of both inter- and intra-species pathogen interactions, can greatly augment understanding of the epidemiology of these pathogen communities. An integrated approach to pathogen dynamics can better equip an integrated approach to control of important diseases of African cattle.

Expression of acquired immunity to gastrointestinal parasites usually breaks down during the periparturient period, which is characterised by an increased worm burden and nematode egg excretion. It is believed that this breakdown of immunity may have a nutritional basis, and that by reducing nutrient scarcity the lactating animal will be able to reduce her worm burden. Therefore, the aim of this thesis was to carry out four experiments to investigate the potential use of metabolisable protein as an alternative way to control gastrointestinal parasitism in periparturient animals. A lactating rat model was used to address this issue as lactating rats exhibit a breakdown of immunity to the gastrointestinal nematode Nippostrongylus brasiliensis. The first experiment (chapter two) aimed to verify that a reduction in worm burden is indeed related to changes in nutrient supply and not associated with changes in the gut environment. This was achieved by manipulating nutrient (litter) demand whilst nutrient supply was maintained constant. The results showed that the periparturient breakdown of immunity to N. brasiliensis (measured by a reduced worm burden) was sensitive to changes in nutrient demand and that these effects were independent of changes in the gut environment. The second experiment (Chapter Three) tested the effect of increased protein supply or reduced protein demand on the resistance to parasites in lactating rats whilst energy intake was kept constant. Under these conditions effects of protein supply could not be confounded with effects of any nutrient or energy intake. The results supported the view that under a restricted feeding regime, breakdown of immunity to N. brasiliensis (measured by a reduced number of eggs in the colon content) was sensitive to changes in protein scarcity. Following on from this, the next experiment (Chapter Four) assessed the effects of a gradual increase in protein supply on resistance and immune responses to N. brasiliensis in lactating rats. It was shown that as protein contents of the diets progressively increased, the number of worms and eggs present in the colon decreased. Although we found that differences in protein supply affected parasite burden, we found no affects of protein supply on local immune responses. This may have been due to the single sampling point used. Therefore, the objective of the last experiment (Chapter Five) was to assess temporal effects of increased protein supply on resistance and immune responses to N. brasiliensis. In agreement with previous experiments, the results showed that an increase in protein at times of protein scarcity improved resistance to N. brasiliensis, illustrated by a lower number of nematode eggs in the colon. The results also showed that local immune responses such as immunoglobulin levels (IgA, IgE & IgG2a), RMCP II levels and goblet cell counts were affected by differences in protein supply at various time points post secondary infection. The potential application of using a lactating rat as a suitable model to fully understand the underlying immunological basis of relaxation in immunity during the periparturient period and its sensitivity to nutrient scarcity is considered in the General Discussion (Chapter Six).

The rickettsial pathogen Ehrlichia ruminantium, transmitted by ticks of the genus Amblyomma, causes heartwater, an economically important, often fatal disease of domestic and wild ruminants in sub-Saharan Africa and in the Caribbean. The studies described in this thesis have contributed to understanding several aspects of heartwater. First, a real-time PCR method was developed in order to study the kinetics of infection with E. ruminantium in the mammalian host. The assay was validated for specificity and sensitivity and was used to estimate numbers of the organisms in the blood of infected sheep. However, organisms were only detected during the clinical phase of infection, indicating that the way in which it was applied did not provide sufficient sensitivity to follow the early stages of infection. This PCR assay was then used, together with transcription and proteomic analyses, to investigate differential gene expression of E. ruminantium in the arthropod and mammalian hosts, in order to identify genes that may allow the organisms to successfully adapt to different environments. These studies used in vitro tick and mammalian cell culture systems, as well as tissues from infected A. variegatum ticks, and initially focused on the map1 multigene family. Although transcripts for most of the map1 paralogs were detected in organisms grown in vitro, in both mammalian and tick cells, only transcripts from map1 and map1-1 were detected in infected ticks. Moreover, map1-1 transcripts were more abundant in midguts than in salivary glands whereas map1 transcripts were most abundant in salivary glands and were expressed at higher levels following several days of tick feeding on a mammalian host. Because of the quantities of material required, proteomic analysis was only possible using in vitro-cultured organisms. Comparison of proteins encoded by the map1 cluster in E. ruminantium grown in tick or bovine endothelial cell cultures, using 2D gels and MALDI-TOF analysis, revealed that different proteins predominated in the corresponding spots in 2D gels from the different cultures; products of the map1-1 gene were abundant in tick cells, while products of map1 were abundant in endothelial cells. The detection of higher levels of map1 transcripts in salivary glands than in midguts of infected ticks, together with the presence of abundant MAP1 protein in organisms grown in mammalian but not in tick cell lines, suggest that expression of this protein may be associated with infectivity for mammalian cells. In contrast, map1-1 transcripts were abundant both in midguts of infected ticks and in tick cell lines, and the protein was expressed at high levels in infected tick cell cultures. Since both of these stages have low infectivity for sheep, these results suggest that the MAP1-1 protein may play an important role within the vector, possibly associated with colonisation and replication of E. ruminantium in the tick midgut. Collectively these findings suggest that this multigene family is involved in functions of biological relevance in different stages of the life cycle of E. ruminantium. Lastly the suppression subtractive hybridisation (SSH) technique was applied to RNA extracted from E. ruminantium-infected endothelial and tick cell cultures in an attempt to sample a large portion of the E. ruminantium genome for differentially expressed genes; although not resulting in identification of any differentially transcribed genes in the present study, this method was shown to work in principle.

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.

T. lestoquardi is an important tick-borne parasite that infects leukocytes of sheep and goats. The disease is mainly caused by the schizont stage which induces transformation of the infected cell to a state of uncontrolled proliferation. The purpose of this study was to identify schizont secreted proteins, which are likely to include molecules targeted by the immune response as well as those associated with the transformation process. An efficient method for the lysis and purification of schizonts from infected cells was established. This involved use of complement treatment to release schizonts from infected cells and an optimal density of Nycoprep gradient medium to separate schizonts from host cell components. An optimised method for metabolic labelling of schizont-infected cells was also established. Putative secreted proteins were detected from metabolically labelled schizont culture supernatants by SDS-PAGE and autoradiographic analysis. Using two-dimensional electrophoresis, mass spectrometry, including MALDI­TOF and Q-TOF, and N-terminal sequencing analysis, three putative secreted proteins of parasite origin were identified, namely Theileria mhsp70 (TLS3), hsp70 (TLS15) and inorganic pyrophosphatase (TLS16). Seven host-derived proteins were also identified, namely mhsp70 (TLSl), hsp60 (TLS2), mitochondrial ribosomal protein L12 (MRPL12, TLS4), ATP synthase-d (TLS5), ATP synthase 6(TLSll), PDH-Elb (TLS12) and A TP-dependent proteinase SP-22 (TLS13). Further investigation of the relationship of the host-derived proteins with the schizont using dual-labelling and confocal microscopy analysis provided evidence that the host-derived protein, MRPL12, is associated with intracellular schizonts with some co-localising with schizont mitochondria. The analysis also revealed that some host mitochondria are closely associated with the schizonts of T. lestoquardi.

The isolated and un-managed Soay sheep population of Hirta, St. Kilda, which has been monitored intensively since 1985, is a model system for host-parasite studies. In hosts that died in the 2001/2 population crash, hitherto unknown and unexpected trends in the abundance of the main parasite species with host age were revealed. Specifically, previous studies in this system have failed to identify large Trichostrongylus axei and T. vitrinus burdens in the abomasum and small intestine respectively, which declined with host age, whereas Teladorsagia circumcincta burdens actually increased over the first few host age classes. Also male hosts had significantly higher burdens of Trichostrongylus species than females, with this genus making up a higher proportion of the strongyle-egg producing female adult nematode community in male hosts. These findings raise questions concerning previous interpretations of the main strongyle species contributing to strongyle egg counts. In living hosts sampled in late summer, the inventory of parasite species known to infect the sheep was increased by 40% with the identification of thirteen species of protozoa (Cryptosporidium parvum, Giardia duodenalis and eleven species of Eimeria) most of which have intracellular phases in their life cycles and likely, therefore, to exert contrasting selection on their hosts compared with extracellular helminth parasites. In general, protozoan burdens declined with host age and were higher in males than females; different species’ burdens appeared to lag the host population dynamics to different extents and only C. parvum varied positively with host population density. In living hosts sampled in late summer, simultaneous measures of strongyle, protozoan and ked (Melophagus ovina) intensity for individual hosts each explained independent variation in host body weight. No associations were detected between parasite diversity or intensity and the probability of survival through the 2001/2 population crash, possibly because of low statistical power.

Nippostrongylus brasiliensis is a GI nematode parasite of rodents and is an extensively applied laboratory model for defining the immune mechanisms that mediate worm expulsion. N. brasiliensis infection in the rat shares many similarities with Trichostrongylus 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/Nippostrongylus parasite system as a model for vaccination with recombinant ES proteins. Prior to vaccination trials N. brasiliensis 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 E. coli. 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.

The aim of this thesis was to investigate the two hypotheses that have been put forward to account for the reduction in the level of parasitism observed in sheep that consume condensed tannins. For this purpose in vitro (larval development/viability assay) and in vivo experiments were performed. In the first two experiments, a model condensed tannin extract (Quebracho) was given as a drench to parasitised sheep to test for the direct effect of condensed tannins towards established adult nematodes. Experiment 1 aimed to provide evidence for a direct anthlemintic effect of condensed tannins towards an established Trichostrongylus population. For this purpose the extract was administered to sheep four weeks after their initial infection. There was clear evidence for a direct anthelmintic effect of condensed tannins towards adult T.colubriformis, as demonstrated by reduced faecal egg counts and worm burdens. As T.colubriformis is an intestinal ovine nematode, the susceptibility of other intestinal and abomasal ovine nematodes to condensed tannins was investigated in vitro and in vivo. In vitro larval development/viability assays were performed to test the effects of different concentrations of Quebracho extract on pre-parasitic larval stages of one intestinal (Trichostrongylus vitrinus) and two abomasal nematodes (Teladorsagia circumcincta and Haemonchus contortus) (Experiment 2a). The results from these assays showed that larvae from all species were able to develop to infective larvae, irrespectively the concentration of Quebracho extract. However, the viability of infective larvae of all species decreased as the concentration of condensed tannins increased. Concurrently, Experiment 2b was designed to test whether condensed tannins had a direct anthelmintic effect towards adult populations of either T. circumcincta or H. contortus, or towards a mixed intestinal population of Nematodirus battus and T.colubriformis. Both ovine intestinal species were susceptible to the presence of condensed tannins in the gastrointestinal tract of sheep, as demonstrated from reduced faecal egg counts and worm burdens.

The gastrointestinal parasite of dogs and their related species, Toxocara canis, is a prime example of a zoonotic parasite. It is the principal agent of visceral larva migrans and also a cause of ocular larval migrans. As a result of the close association that dogs have with man, the parasite enjoys a world-wide distribution. Infections can be contracted from contaminated soil and handling of infected dogs, and is particularly prevalent amongst professions that have a close association with dogs, such as hydatid control officers in New Zealand. Once inside the non-canid host, the parasite enters a state of arrested development, in which it neither grows nor differentiates. In this state the parasite releases up to 1% of its body weight in excretory/secretory antigens per day. Assuming that this high protein production was in some way linked to immune evasion, a modest EST project was undertaken using a cDNA library generated from infective larvae. The hypothesis behind this approach was that the high protein output demonstrated by these parasites would be mirrored at the mRNA level. In total 266 clones were sequenced, the majority of which were from the both the 5' and 3' ends of the transcripts. Homologues for these genes were sought by similarity searching against the GenBank protein and the dbEST nucleotide databases. Cluster analysis of the clones identified 129 distinct gene products, all but three of which represented new genes. The majority of the genes (96) were represented by single clones, although 8 transcripts were present at high frequencies, each composing >2% of all the clones sequenced. These high abundance transcripts include a mucin and a novel C-type lectin, which together comprise the two major excretory/secretory antigens released by the parasite. Four highly expressed novel transcripts were found, termed ant genes (abundant novel transcripts). Together these genes represented 18% of all the cDNA clones isolated, but no similar sequences occur in the C. elegans genome. While the coding regions of these genes are dissimilar, they exhibit a remarkable level of similarity in their 3' UTR at the nucleotide level. The discovery of these abundant parasite specific genes, of newly-identified lectins and mucins, as well as a range of conserved and novel proteins, provide defined candidates for future analysis of the molecular basis of immune evasion by Toxocara.

Causes and consequences of plasticity in parasite life histories were investigated using a gastrointestinal nematode species, Strongyloides ratti, a natural parasite of rats. Empirical work focused on three putative instances of adaptive and non-adaptive plasticity in the life history of S. ratti: host-specificity, facultative sexuality, and immune-dependent maturation time. Host-specificity (the differential success of a parasite on alternative host types) represents (non-adaptive) plasticity in fitness and is commonly viewed as an unavoidable outcome of parasite specialisation - an intuitive conclusion that has rarely been questioned. While the lifetime reproductive success of two S. ratti lines was unaffected by host (rat) genotype, the frequency and timing of sampling was crucial in quantifying host-specificity accurately. Proximate processes generating the differential performance in S. ratti in rats and mice were then characterised and quantified. Reduced parasite fitness in mice resulted from lower parasite establishment, more rapid expulsion and suppressed fecundity. Differences in the efficacy of thymus-dependent (T-dependent) immunity between host species were insufficient to explain this variation in parasite fitness. Experimental natural selection and reciprocal fitness assays were used to discriminate between alternative models and host-specific specialisation. Selection failed to modify host-specificity suggesting either a lack of genetic variation among parasites or the action of unidentified factors underlying the different performance of S. ratti in rats and mice. The S. ratti life-cycle incorporates a facultative sexual phase and the frequency of sex depends on the strength of host acquired immunity. Immunisation of rats with infective larvae of other nematode species or with mammalian antigens reduced the reproductive success of parasites but only immunity acquired against S. ratti induced a facultative increase in the frequency of sexual reproduction indicating antigen-specificity of this plasticity.

This study sought to identify antigens involved in protective immune responses to Cowdria by Western blotting using immune sera and surface labelling of elementary body (EB) proteins using biotin to identify which of these are outer membrane proteins. Antigens which could be considered as potential vaccine candidates were identified. Immunisation of goats with live Cowdria or with inactivated elementary bodies (IEBs) leads to development of antibodies to at least six antigenic components of the EB of 24kDa, 27kDa, 31kDa, 58kDa and 66kDa. In contrast immunisation of goats with detergent extracted soluble antigens stimulated production of antibodies to only four antigens of 24kDa, 27kDa, 28kDa and 31kDa. Six surface exposed antigens of the Cowdria elementary body were identified by biotin labelling, with molecular masses of 21kDa, 28kDa, 31kDa, 62kDa, 74kDa and 115kDa and are therefore considered outer membrane proteins. These proteins reacted with antibodies in sera raised by immunisation of goats with live or inactivated EBs. The 58kDa heat shock protein (GroEL) of C. ruminantium is an immunodominant antigen. The immune responses to 58kDa antigen expressed as a recombinant in E. coli were investigated by immunisation of mice and sheep. The immunised animals stimulated specific antibody which reacted with the native homologue on the EB 58kDa. Immunisation with recombinant 58kDa heat shock protein of C. ruminantium led to development a of specific antibody response of the IgG₁ isotype. Challenge of immunised sheep showed a highly significant reduction (p<0.005) in infection rates of brain capillary endothelial cells in the immunised group compared to control animals. However the incubation period and clinical outcome were not significantly different in controls and immunised sheep. Mice immunised with recombinant 58kDa were partially protected against virulent homologous challenge. The lack of protection in sheep was attributed to failure of the antigen to induce a detectable lymphocyte response.

Pasteurella haemolytica serotypes A1 and A2 and Pasteurella trehalosi serotype T10 were cultured in "in-vivo" fluids (ruminant tracheobronchial washings and serum), under defined iron-restrictive conditions, and compared for changes in cellular composition. In analyses by SDS PAGE and Western blotting, envelope and cell contents of the bacteria showed differences in protein content and changes in antigens recognised by convalescent antiserum. Capsule size was not influenced by growth medium. Lipopolysaccharide was detected by PAGE in all the fluids except bovine tracheobronchial washings. However, a Limulus amoebocyte lysate assay did detect trace amounts of endotoxin. Leukotoxin activity was only detected in broth and iron-restricted cultures and not in any "in vivo" fluids. Neutralisation of the toxin with homologous and heterologous convalescent antiserum showed little cross-reactivity in the neutralisation capacity against A1 leukotoxin but heterologous antiserum was effective with leukotoxin of the other serotypes. Using the same fluids to monitor long term culture, all serotypes showed the capacity for extended survival and resuscitation with the addition of nutrients. All three strains survived better in ruminant than in other species' fluids. In non-ruminant fluids and natural water viability was detected only at low temperatures. Morphological changes in both colonial and microscopic appearances were apparent during long term survival. These results imply that this organism possesses some starvation survival mechanisms. Immunomagnetic beads with bound antibody specific for the three serotypes proved a useful reagent for the isolation of target organisms from host tissues and fluids. Differing protein profiles from those seen in bacteria grown in-vitro were demonstrated. Western blotting unexpectedly failed to identify many antigens when recovered whole cells were compared to those grown in laboratory culture, indicating that perhaps mechanisms were present which helped to evade an immune response. Superoxide dismutases were detected in all three strains. Level of enzyme activity, electrophoretic mobility on native PAGE, and metal-active sites were shown to differ.

Two T. evansi Ag-ELISAs based on different monoclonal antibodies (2G6 Ag-ELISA and Tr7 Ag-ELISA) were evaluated using buffaloes in Southeast Asia, where T. evansi is endemic. The repeatability and robustness of the two Ag-ELISAs were shown to be high. Profiles of antigenaemia varied between individual buffaloes and between the two Ag-ELISAs. Antigen and antibody responses were first detected 7 to 42 days after infection, but in some buffaloes responses fluctuated below cut-off values during infection, whilst in other buffaloes antigen and antibody responses persisted after trypanocidal drug treatment. With the naturally-infected buffaloes, the diagnostic sensitivity estimate of the Tr7 Ag-ELISA (81%) was significantly higher than that of the 2G6 Ag-ELISA (71%), and the IgG ELISA sensitivity (89%) was significantly higher than either the IgM ELISA or CATT sensitivities (78%). In Central Java, 2387 buffaloes were blood sampled in 59 villages, and estimates of test prevalence were 4% with the MHCT, 9% with MI, 58% with the 2G6 Ag-ELISA and 70% with the Tr7 Ag-ELISA, but prevalence values differed between districts and between villages. True incidence rates per animal-year at risk were 0.44 with the Tr7 Ag-ELISA and 0.22 with the 2G6 Ag-ELISA. of 239 market buffaloes sampled, 10% were parasitaemic, 39% antigenaemic, 56% positive by IgG ELISA and 47% positive by CATT, representing an important source of T. evansi. The T. evansi Ag-ELISAs and antibody-detection tests used in this study have many advantages as screening tests over commonly used parasitological tests, in terms of their diagnostic sensitivity and ability to rapidly test large numbers of samples. The two T. evansi Ag-ELISAs could be applied in high prevalence areas, whilst antibody-detection tests (in particular, the IgG ELISA or CATT) would be more appropriate to test buffaloes in low prevalence areas or to confirm the negative-status of buffaloes prior to movement within Indonesia or export. Future work should aim to improve the specificities of the Ag-ELISAs, which were low in this study in contrast to previous reports.

A description of the isolation of new Cowdria isolates by different methods and from different vectors and geographical locations in Kenya is given. These included Amblyomma variegatum, A. gemma and A. lepidum. Isolates from the later two species were also by feeding adults moulted from nymphs collected in the field and is the first report on transtadial transmission by A. gemma ticks. A spectrum of virulence ranging from highly virulent to mildly virulent for sheep was found among the Cowdria isolates. The majority of isolates were highly virulent. There was a range of mouse infectivity among the isolates from inapparent to lethal. The Asembo and Baragoi isolates were pathogenic and lethal, the Kiswani, was infective and non pathogenic for Balb-C mice while the other 8 were avirulent or refractile to mice inducing only antibody production in various proportions of mice. There was a difference in the infectivity for neutrophils both in the frequency of infected cultures and in their level of infection. The different isolates were classified as of low infectivity where even the few positive cultures rarely reached 1% infection rate, medium infectivity if a good number of cultures regularly attained 1% infected neutrophils, or high infectivity if a large proportion of the cultures which became positive regularly attained 1% infected neutrophils and at least some of then attained more than 10% infected neutrophils. The isolates also had a range of infectivity for the brain endothelial cells, from no detectable colonies to greater than 16% infected endothelial cells in individual animals. The author concludes that the agent of heartwater is endemically widespread in many districts in Kenya and poses a potential threat of outbreaks to areas newly invaded by vector ticks and also to areas where immunity due to the local agent may not protect against an invading one. The author recommends that the Baragoi or Marigat isolate should be adopted for possible vaccine development in Kenya.

This thesis sets out to understand interactions between T cells and T. annulata infected cells, both in vitro and in vivo and the consequences for the generation of immunity. In vitro stimulation of peripheral blood T cells from naive animals by IC caused the cells to proliferate, peaking 5 days post stimulation. Phenotypic analysis showed that CD25 and MHC class II were expressed upon the surface of all T cells (CD4, CD8 and γδT cells) within 24hrs of stimulation, reaching a peak at 48hrs and remaining stably expressed for up to 7 days post activation. The parasite infected cells could activate both "memory" and "naive" CD4 T cells, with little change in the CD45RB isoforms during activation. Activation of T cells was contact dependent. T annulata infected cells can therefore cause the activation of the majority of T cells from naive animals irrespective of memory status and , presumably, antigen specificity. The cytokines produced by IC stimulated T cells 1-7 days post stimulation were assessed by reverse transcription polymerase chain reaction (RT-PCR) using primers for IL2, IL2 receptor (IL2R), IL4 and interferon gamma (IFNγ). None of these cytokines were found to be expressed by IC. T cells within PBM expressed mRNA for IL2, IL2R, IL4 and IFNγ 24-48 hours post IC stimulation. IL2 and its receptor were still expressed at day 5 (peak proliferation), and waned by day 7. IFNγ was expressed by all tested animals' cells at all timepoints, while IL4 was intermittently found at day 5 and was always absent at day 7. IL4 was only expressed by CD4 T cells, while IL2/IL2R/IFNγ was expressed by all T cell types. The presence of CD4 cells was required for IL2 and IL2R expression by non CD4 T cells. In summary, this thesis has shown that T. annulata infected cells possess an innate ability to activate naive T cells. Although all T cell types are activated in PBM, this is dependent upon cytokine release by CD4 cells, subsequently leading to a type 1 response. In vivo, a similar mechanism leads to activation of DLN T cells primarily by IC. Such interactions do not lead to the induction of an antigen specific immune response, but to the loss of GC and suppression of further T cell activation.

Trichostrongylus vitrinus is one of the principal causative nematodes of ovine parasitic gastro-enteritis within Scotland and infests the proximal small intestine of the sheep. At present, control is achieved mainly by the administration of anthelmintic drugs, but with the increasing emergence of anthelmintic resistance, much research is now centred on vaccine development. Recent evidence has suggested that the excretory-secretory components (ES) from parasitic nematodes may be an important source of host-protective antigens. The overall aim of the present study was to characterise the nature and properties of T. vitrinus ES components. The initial part of the work involved the partial characterisation of two of the enzymes, acetylcholinesterase (AChE) and proteinases, excreted and secreted during the in vitro culturing of adult T. vitrinus. These enzymes were defined on the basis of their substrate specificity, molecular size, pH optima and inhibitor sensitivity. Attempts were also made to isolate cDNA fragments encoding AChE from an adult T. vitrinus cDNA pool, using the polymerase chain reaction (PCR) and degenerate oligonucleotide primers directed towards highly conserved regions of AChEs that had previously been identified by comparison of known polypeptide sequences from a number of higher eukaryotic organisms. No adult T. vitrinus cDNA fragments encoding AChE were amplified, suggesting that T. vitrinus AChE(s) is/are distinctly different to AChEs from higher eukaryotes, at least at the level of nucleic acid sequence. Subsequent research focused on the molecular characterisation of adult T. vitrinus ES. Immunoscreening of an adult T. vitrinus cDNA lambda gt11 library with antiserum raised against adult T. vitrinus ES, resulted in the isolation of ten immunopositive clones. Their inserts were sequenced and the results were analysed using computer databases. Three of the clones were identified as harbouring inserts that encoded proteins that shared significant homology to myosin heavy chain, vitellogenin and serine proteinase inhibitor (serpin) respectively. The other seven clones contained inserts that showed no significant homology to any of the sequences present in the computer databases.

Tropical theileriosis, caused by the haemoprotozoan parasite Theileria annulata, is one of the major threats to cattle health in Tunisia. The aim of the study described in this thesis was to assess the extent of diversity in T. annulata parasite population from within a single country in order to provide a better understanding of the epidemiology of the disease and biology of the parasite. The work also provided useful background information to vaccine development studies in Tunisia. Different T. annulata stocks, isolated from different bioclimatic zones in Tunisia were characterised using monoclonal antibodies (MAbs), isoenzyme and DNA analyses and compared with each other and with a number of recognised laboratory stocks from other theileriosis endemic areas of the world. The study comprises seven chapters. In the first, an introduction to the literature describing the parasite T. annulata and the disease it causes are presented and the epidemiology of tropical theileriosis is discussed with particular regard to the situation in Tunisia. Finally an overview on diversity in protozoan parasites, including Theileria, is given to emphasise the rationale behind the present study. The next chapter details how the biological material, including 51 field isolates, was generated for the three parasite life-cycle stages, sporozoites, piroplasms and schizonts. In the third chapter the production of antischizont MAbs and their use as immunological makers to reveal differences between stocks of T. annulata is described. The fourth chapter demonstrates the use of a biochemical marker, glucose phosphate isomerase isoenzyme to study diversity in T. annulata isolated in Tunisia.

The excretions, secretions and surface components of a parasite are by their nature centrally involved in the host/parasite interaction. Rats, like cattle, are capable of developing resistance to fasciolosis after primary infection and are therefore considered a suitable laboratory model for cattle. The objective of this study was to characterise the excretory/secretory (ES) and surface components of Fasciola hepatica as it develops in the rat, and to identify those components involved in the host/parasite interaction that may have diagnostic and/or protective value. Three trials were conducted during the study in order to produce supplies of rat antiserum which was protective against F. hepatica, Rats were infected with either 10 (first trial) or 20 (second and third trials) F. hepatica metacercariae as information from the literature indicated that these doses were adequate to stimulate the production of protective antibody levels. The rat sera from the three trials were checked for the presence of protective antibodies by passive protection studies. Only in the latter two trials was the level of protection conferred on recipient statistically significant. The probable causes for the lack of significant protection in trial 1 are discussed. The silver stained protein profiles of ES from newly excysted (D0) flukes and one-day old (D1) flukes were characteristic and were similar to each other. The ES of parenchymal 14-day old (D14) and adult (D56) flukes were markedly different from D0 and D1 flukes but similar to each other. The silver stained total ES protein profiles of the developing flukes were very different from the total biosynthetically (³⁵S-methionine) radio-labelled ES protein profiles. Possible reasons for this are discussed. However, as with the total silver stained ES protein profiles there were clear changes in the profiles of the biosynthetically radio-labelled ES as the flukes developed. It is suggested that these differences in the ES products may reflect the changing environment and activities of the flukes. The possible functions of the changing ES products are discussed.

In part one, epidemiology of fasciolosis in eastern Nepal, a 19 months field survey on the epidemiology of fasciolosis is described. Four Lymnaea spp; L. auricularia race rufescens, L. auricularia sensu stricto, L. viridis and L. luteola were identified. L. auricularia race rufescens was the predominant species. The snail main habitats were spring or stream fed rice-fields, irrigation channels, ponds and road-side pools. The monsoon rains and rice cultivation practices contributed to the creation and expansion of the habitats. The snail population density was high during the dry period and declined with the onset of the monsoon. Snail egg masses and young snails were observed throughout the year. Mature Fasciola spp, infections were found in the hills from May to February and throughout the year in the Terai. In part two, experimental studies on pathogenesis of fasciolosis with special reference to its effects on productivity of ruminants is described. Monitoring included clinical, parasitological, haematological, biochemical and pathological observations. Pilot comparative studies in Scottish Blackface and Suffolk cross sheep conducted in Edinburgh indicated that F. gigantica was more pathogenic than F. hepatica. In another pilot experiment in Nepal using local Baruwal sheep, it was also found that very low infections with F. gigantica caused measurable production losses. Pathogenesis in goats was investigated using Nepalese hill goats. Infection caused production losses including weight loss. Burdens of more than 1.3 flukes/kg of initial liveweight produced clinical chronic fasciolosis. In part three, the relative merits of the current methodologies for speciation and differentiation of Fasciola spp. are reviewed. The development of species-specific (MHFh and MHFg) and cross-reactive (MHFx1 and MHFx2) DNA probes for the identification of Fasciola spp. are described. If used in conjunction these probes clearly differentiate F. hepatica and F. gigantica.

The work described in this thesis examined 44 stocks of T.evansi, 41 of which were isolated from camels in various areas of Kenya. These trypanosomes had a mean length of 25.5 ± 2.8 μm a mean pre-patent period of 4.7 ± 3.4 days and fulfilled the classical criteria of this species in regard to these two parameters. Initially, the sensitivity of these trypanosome stocks was determined to four trypanocidal drugs active against T.evansi. The 44 stocks exhibited resistance to Berenil (57%), Samorin (7%), suramin (22%) and Trypacide (18%). It was noted that although Berenil is not recommended for use in camels due to its toxicity, more than half of the stocks were resistant to this drug. The malic enzyme patterns of these stocks were determined to investigate possible correlation with resistance to the trypanocides used. Most of the stocks (66%) possessed malic isoenzyme pattern II (66%), although patterns X (23%), IV (7%) and VII (5&37) were also observed. Malic enzyme patterns IV and X had not been identified previously in this trypanosome species. No linkage was found between any of these patterns and resistance to the four drugs. Further work concentrated on three trypanosome stocks which were highly resistant, of intermediate resistance or sensitive to the action of suramin. Investigations on the effect of trypanosome inoculum and timing of suramin administration in obtaining cures in T.evansi infected mice were carried out. The time of administration after infection was shown to be an important factor that influenced the outcome of suramin treatment. Mice which were treated immediately after infection had higher cure rates than those treated at the onset of parasitaemia. This was observed particularly in a trypanosome stock normally resistant to suramin at a dose rate of 160 mg/kg; if treatment was administered immediately after infection, most of the infected mice were cured. Except in the highly resistant stock, the role of trypanosome inoculum was not as important as timing of treatment in determining cures.

The relationship between the feeding of Amblyomma variegatum ticks and Dermatophilus congolensis infections has been studied under laboratory conditions. The local effects of hypersensitive or inflammatory reactions to larval and nymphal A.variegatum on subsequent D.congolensis infections were investigated using rabbits and sheep respectively. Multiple or single infestations of ticks were used to produce hypersensitivity or inflammatory reactions respectively. These reactions were confirmed by histological assessment of the tick attachment sites. Identical titrated doses of D.congolensis were applied to the tick attachment sites after the ticks had detached, a control titration was set up on skin with no previous exposure to ticks. The progression of the resulting lesions was assessed using a non-parametric ranking system. There was no significant difference (P > 0.05) between the severity or duration of the three groups of dermatophilosis lesions, either on the sheep or the rabbits. Therefore it was concluded that the local effects of the feeding of immature instars of this tick do not affect the pathogenesis of subsequent D.congolensis infections. The local effect of hypersensitive or inflammatory reactions to A.variegatum nymphs on simultaneous D.congolensis infections on rabbits was also studied. There was an increase in the initial severity of the dermatophilosis lesions and a positive correlation between inflammatory tick attachment sites and dermatophilosis foci. However, the local effects of the feed of nymphal A.variegatum did not result in the development of chronic dermatophilosis lesions. The systemic effect of adult and nymphal A.variegatum on simultaneous dermatophilosis lesions was compared, using sheep as the experimental hosts. Dermatophilosis congolensis infections on sheep infested with adult A.variegatum developed into chronic lesions which persisted for several months. Serological and skin tests revealed significantly (P < 0.01) reduced humoral and cellular immune responses in sheep infested with adult A.variegatum compared with sheep infested with nymphs or control sheep not exposed to ticks.

The objective of this work has been to investigate the frequency of cross-fertilisation between gametes of genetically distinct clones of the human malaria parasite Plasmodium falciparum. Previous genetic experiments involving both rodent malaria parasites in vivo and human malaria parasites in vitro have demonstrated higher than expected numbers of recombinants among the progeny of crosses. It has been suggested that this could be due to a favouring of cross-fertilisation over self-fertilisation in the mosquito phase of the life-cycle. The work has involved examining the genotypes of individual oocysts (derived from individual zygotes) resulting from mixed infection of two clones in mosquitoes. In preliminary work using the mouse malaria parasite P. yoelii nigeriensis attempts were made to examine the chromosomes of single oocysts using pulsed field gradient gel electrophoresis. However there was insufficient DNA in an oocyst to allow chromosomes to be visualised using this technique. The bulk of the work has been concerned with P. falciparum. In the first stage oligonucleotide primers suitable for use in the polymerase chain reaction (PCR) were designed to allow amplification of repetitive regions of two polymorphic antigen genes, denoted MSP1 and MSP2. The two clones of P. falciparum used in the crossing experiments possessed a different allele of each gene. These alleles were found to be recognisable as size differences of the PCR-amplified fragments on agarose gels. Gametocytes of the two clones were grown in vitro. Mixtures of gametocytes of each clone were made and fed to Anopheles stephensi or A. gambiae mosquitoes through membrane feeders. 9 to 10 days later the mosquitoes were dissected and their midguts were examined for the presence of oocysts. Individual oocysts were dissected from the midguts and the DNA extracted from them.

Allergens from somatic extracts of adult Nippostrongylus brasilinesis worms were characteriszed by SDS-PAGE and Western blotting. Seven allergens, with molecular weight ranging from 14,000 - 69,000 were identified after blotting with hyperimmune serum (HIS) from rat immunized by several infections with N. brasiliensis. Monoclonal mouse anti rat - IgE was used as a specific probe. The major 14,000-17,000 MW allergens, partially purified by electrolution, retained activity by passive cutaneous anaphylaxis (PCA). Other immunoglobulin isotypes, notably IgC₁ also bound to allergens on Western blot. The specificities of other immunoglobulin isotypes and IgE were compared by sequential incubation of blotted allergens with IgE - depleted HIS and with affinity-purified serum IgE. Despite retaining biological activity by PCA, the affinity-purified IgE proved to be insufficiently specific for use on Western blots. The range of allergens detected and the kinetics of appearance of parasite-specific IgE differed between LOU. Hooded Lister, August and F334 undergoing primary infection with N. brasiliensis. Whereas August rats responded uniformly, there were variations in the timing and specificity of IgE responses between individual rats within the other strains. Nevertheless, it was possible to classify LOU and Wister rats as early and F334 rats as late responders. Internalization of IgE within the cytoplasm of intestinal mucosal mast cells (MMC) of rodents infected with intestinal nematodes, and the absence of this isotype from the cytoplasm of connective tissue mast cells (CTMC) suggested that the MMC granule protease, rat mast cell protease II (RMCP II), might fail to catabolize IgE. However, when compared with the granule protease RMCP I from CTMC. RMCP II was more efficient in catabolizing the heavy chains of both IgE and IGG_2a. The light chains were apparently resistant to digestion. The presence of IgE-bearing cell populations in bone marrow, peripheral blood, and peritoneal cavity, was monitored by flow cytometry in normal Wister rats and during the course of infection with N. brasiliensis. The proportions of IgE-bearing cells in the peritoneum and bone marrow increased on day 10 and, in peripheral blood on day 15. The IgE-bearing cells in each compartment were of mixed phenotype.

Onchocerciasis or river blindness is a major cause of infectious blindness in the world. In severely affected areas, half of the adult population may be blinded by the disease. The human parasite, O.volvulus, is very closely related to the cattle parasite Onchocerca gibsoni which provides an abundant source of biological material for analysis. Thus, O.gibsoni offers a model system for biochemical analysis and drug screening. In these organisms, as in other eukaryotes, microtubules are essential, multifunctional, subcellular components. They are involved in chromosome segregation, cell architecture, motility, intracellular transport, and secretion. The major protein of microtubules is tubulin, a heterodimer of two distinct polypeptides designated α and β. Each subunit has a molecular weight of about 50kD and is encoded by a distinct set of genes. Tubulins are highly conserved proteins and the number of genes coding for both α and β-tubulin vary dramatically between different species. This major structural protein of eukaryotic cells has particular importance in helminthic parasites as it is a target for anthelmintic benzimidazoles, which directly bind to tubulin and inhibit the assembly of microtubules. The effectiveness of such drugs depends on differences in the structure of the tubulin molecules from the parasite and its host. In this context, a genomic library from O.gibsoni has been constructed, screened with a β-tubulin gene from Plasmodium falciparum and the gene isolated. The chromosomal copy of the gene has been completely cloned and sequenced. This has revealed the gene to possess a very complex structure compared to the organisation of all the known β-tubulin genes as the coding region is interrupted by 11 introns. The deduced polypeptide is 444 amino acids long, and its sequence is highly conserved. The position of some introns appear to demarcate functional domains within the protein. The results that have emerged from this thesis are a step forward not only in the rational design of drugs, but also offer an insight into the biology and evolution of an Onchocerca gene and its product.

The literature relating to the development of the technique of arthropod tissue culture, its application to biological research, and the cultivation of trypanosomes is reviewed. Cultivation of tissues and organs from Glossina morsitans morsitans was investigated using several culture media. Of the media tested, modified Trager's medium was found to be the best for the growth and maintenance of tissues. Successful cultures of developing adult tissues from ticks, Rhipicephalus appendiculatus, R.bursa and Amblyomma hebraeum and from partially engorged adult Ixodes ricinus were obtained in VP4 medium. The greatest success in the cultivation of Trypanosoma brucei and T.congolense in association with Glossina tissues was achieved in the presence of complete alimentary tract of pupae older than 21 days in 15 -20a1 hanging -drops of modified Trager's medium incubated at 28 ?C. Growth of the trypanosomes also occurred in cultures in which the flagellates were separated from the insect tissues by a semi -permeable membrane. No multiplication of the parasites was obseived in (a) culture medium alone, (b) medium containing extracts of alimentary tract and (c) medium in which alimentary canal had been cultured for 3 or 4 days. In all the cultures in which multiplication of the trypanosomes occurred, it was accompanied by morphological transformation. Bloodstream trypomastigotes in the inoculum changed into long slender forms similar to those seen in the midgut of the tsetse fly. Infective stages were not observed. Complete digestive tract from G.morsitans submorsitans, G.austeni and G.palpalis was as suitable as that from G.m.morsitans for the cultivation of the trypanosomes and the same applied to the alimentary tract of the non- haematophagous fly Sarcophaga, a dipteran closely related to Glossina. On the other hand, cultures of comparable tissues from mosquitoes, Aedes aegypti and ticks, R.appendiculatus failed to support the growth of trypanosomes. Fly transmissible as well as laboratory adapted strains of T.brucei and T.congolense could be cultivated in tsetse organ cultures. Negative results were obtained, however, with laboratory adapted strains of T.gambiense and T.vivax and with the monomorphic T.evansi which is not cyclically transmitted in nature by tsetse flies. Stercorarian species, Trypanosoma musculi, T.lewisi, T.mzilophagium and T.theileri were also grown in the presence of tsetse alimentary tract. All these species transformed into and multiplied in forms resembling those formed in the hindgut and rectum of their arthropod vectors. Unlike the cultures of the salivarian trypanosomes, those of T.musculi and T.lewisi remained infective to laboratory rodents for many days. Amino acid analyses of haemolymph of pupae, pre- emerged and adult G.morsitans revealed that of the 21 amino acids, proline was present in highest concentrations in all the stages examined. There were also considerable differences in the concentrations of the individual amino acids in these three stages. The results of the amino acid analyses formed bases for the design of culture media which were capable of supporting growth of T.brucei and T.congolense.

This work is a study of both active and passively acquired immunity to Taenia taeniaeformis infection. The role of humoral components in serum as demonstrated by passive transfer of immunity and both active immunity following infection or immunisation were investigated. The antigens derived from larval T. taeniaeformis for immunisation studies were also used to raise immune sera, to monitor experimental infections, and to absorb out protective antibodies. Netacestodes retrieved from mice were maintained in a defined medium for between 10 and 23 days. The tegumental integrity of the metacestodes during maintenance was assessed by dye uptake and transmission electron microscopy (TENT). The medium in which the larvae had been held was used as a source of excretory-secretory antigen (ESA). Somatic antigen (SA) was derived from a saline extract of homogenised larval somata. Anti sera raised against both ESA or SA preparations, were used to detect antigens in the used medium by the enzyme linked immunosorbent assay (ELISA). After verifying that the ELISA technique could quantitate antigens, and that the assay was not affected by the presence of serum in the medium, the effect-of several factors on antigenic activity was investigated. The antigenic activity of the used medium did not appear to be unduly affected by the age of the larvae, by sealing the maintenance vessels, by variations in the integrity of the larval tegument or by the absence or presence of serum during maintenance. However, the beneficial effect of including serum in the maintenance was reflected in the dye uptake and MM results. The aim of studying these factors was to determine the optimal conditions under which the larvae would release 7nax4ma1 amounts of ESA. ELISA was also used to monitor the humoral response of mice and rats experimentally infected with TT taeniaeformis against either ESA or SA as the detecting antigens in the assay. It was found that ESA was a more sensitive indicator of the infection than SA. The ability of ESA and SA to protect rats against infection was investigated. Both these crude antigenic complexes seemed to possess similar immunising capacities. An association was found between the reactivity of these antigens in vitro by ELISA and their immunising or protective activity in vivo, which suggests that such antigens may provide a useful measure of the levels of protective antibody in the serum of the host. Homologous and heterologous immune sera were obtained by immunising rats and sheep respectively with ESA or SA. The immune sera obtained in this manner was protective in passive transfer experiments. This appears to be the first time that serum from animals immunised with ESA has been demonstrated to confer protection against a helminth infection. Immune serum from experimental infections of six weeks duration conferred almost absolute protection, confirming previous published reports. The highly protective immune globulins from active infections was absorbed with ESA, SA or live larvae. The absorption had a measure of success when assessed by in vitro estimation. However, when used in passive transfer, the absorbed globulins retained their protective ability. Several possible reasons were adduced for this apparent failure. Thus there may have been insufficient concentration of antigen to remove all the protective antibodies from the highly active serum. Alternatively the protective antibodies produced in an active infection may not be induced exclusively by the soluble components in ESA or SA, but possibly by both of these and even other antigens. Either of these suppositions would account for the partial efficacy of absorption found in vitro.

A study was made of Nematodirus battus and Trichostrongylus colubriformis infections in New Zealand White rabbits. Experimental studies with N. battus were not continued because of the unsuitability of the parasite in the host. The distribution and development of T. colubriformis in the intestinal environment of N.Z.W. rabbits was studied at various dose levels. The clinical symptoms and pathology of infected animals was studied. The nature of the changes in gut histology and the architecture of villi was established at two sites in the small intestine by examining paraffin sections and studying the surface architecture of villi. The structure of epithelial cells was examined with the electron microscope and the cellular changes were correlated with observations made on enzyme activity using histochemical techniques. The general picture during infection by large doses of T. colubriformis was that of the intestinal malabsorption syndrome. The development of challenge infections given on days 12 and 20 of a primary infection was studied on day 8 post challenge infection. The dose level at which no marked clinical, pathological or immunological changes occurred was used as the dose for the anthelmintic selection study. Rabbits were dosed with a rat strain T. colubriformis and the anthelmintic thiabendazole was used as a selection pressure at 45 mg/body weight against the adult parasite population. After three generations of selection pressure the anthelmintic selected and non-anthelmintic selected povalations were compared in an anthelmintic titration. Following this experiment it was recognised that an anthelmintic resistant population was present. The criterion of resistance was established by studying uterine e-9 counts and the cell development stages in the two populations in anthelmintic treated and untreated rabbits. It was also noted that the response of sheep and rabbit strain T. colubriformis to the anthelmintic was different, the latter was less responsive to the drug. The role of the host in the modification of the parasite phenotype was considered.

TheUeria an ,plat, infection is a tick -borne protozoal disease of cattle. The high mortality and lack of any effective treatment make this disease one of great economic importance. The success achieved in adaptation of the parasite to culture in vitro facilitated investigation of the disease and the consequent attenuation of the parasite led the production of experimental vaccines. The literature relevant to the aspects studied was reviewed. Methods of isolation and adaptation of strains of the parasite in tissue culture were compared. A simple and new method of isolation from whole blood was introduced. The first isolation of a very virulent strain in tissue culture was achieved. Growth rates of five strains of T. annulate in vitro showed an inverse relationship between rate of growth and virulence of strain. Several growth media and supplements were compared and the most satisfactory were shown to be Eagle's MEM with calf serum, lactalbumin hydrolysate and yeast extract. Growth of T. annulata- infected cells in various conditions was measured and factors affecting it were investigated. Of these, pH of the medium and percentage of viable cells in suspension were very important. For morphological studies, from several methods of preparation of smears and staining, a modified method of linear smears was adopted. The morphology of the macroschizont and nuclear particles in the host cells was studied. Various factors including developmental changes of the macroschizont were demonstrated. Attempts to infect normal bovine cells with schizonts in vitro using infected cells and freed parasites and to infect calves with separated schizont particles were unsuccessful. Establishment of cell lines from normal bovine leucocytes and lymphoid cells using mitogens failed. The amounts of glucose uptaken and lactate produced in the culture of schizont -infected cells were measured and these correlated with cell growth. A surplus of glucose in growth medium did not affect its utilisation by the infected cells. The methods for cryopreservation of T. annulata- infected cells were investigated. The cryoprotectants used, the methods of freezing and thawing, the duration of viability of the infected cells in low temperature storage and re- establishment of the cells after preservation at low temperatures were studied.

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.

Fresh specimens were collected from natural infections and from experimentally infected animals. Collections were made in the United Kingdom and in Nigeria and a few specimens were also obtained from other countries abroad. Materials from the specimens was compared by staining, serological techniques and enzyme electrophoresis. Staining ova by the Ziehl-Neeleen method was not successful in differentiating ova of any of the species including T. saginatue and T. solium. Serological techniques that were tried included oval precipitation, agar gel diffusion precipitation, fluorescent antibody, immunoeleotrophoresis and the enzyme-linked immunosorbent assay (ELISA). Strobilate, oval and, in some instances, larval cyst antigens, were used in tests against antisera raised in rabbits and other species. With antisera that had been absorbed by one or two heterologoue antigens, some success was achieved in agar gel diffusion precipitation tests and with ELISA in differentiating strobilate tissue, but serological techniques were not generally adequate for this purpose. Success in differentiating taeniid material was achieved by enzyme electrophoresis on thin layer starch gels. Of the many enzymes tested, five were found to display inter-species zymogram differences in the pattern and mobility of their multiple forms: adenylate kinase, glutamate dehydrogenase, malate dehydrogenase, hexokinase and glucose phosphate isomerase, of which the latter two were the most useful for differentiation. No differences were seen between larval and adult material except for the presence of host enzymes in the former which were easily identified by comparison with host tissue extracts. No differences were seen between immature, mature and gravid proglottides and no individual variation was seen between worm specimens. Anthelmintic treatment did not affect worm enzyme patterns. Taeniid species were compared with one another and also with other tapeworm species. The mobility and pattern of the enzyme forms in zymograms differed more between unrelated than between related species. With glucose phosphate isomerase it was possible to identify most of the taeniid species. When this enzyme was preserved in a solution containing enzyme stabilisers, it stored well over a long period especially in liquid nitrogen or, freeze dried and kept at -20 °C. Dilution of samples did not distort zymogram patterns and crude extracts could be identified using the mobility of the slowest moving band as the criterion. This method was used successfully for a survey in Nigeria. Identification was by comparison with a known species. The other enzymes were useful for confirming the diagnosis. Strain variation between E. granulosus of horse and sheep origin was seen in specimens collected from Belgium and different parts of the United Kingdom. Cysts from oxen were identical to those from sheep, but indirect comparison with cysts from Nigerian camels showed these to be different from both the equine and ovine strains.

The Niallophaga, a suborder of the Pthiraptera, are a group of obligate ectoparasites living on birds and mammals which present interesting problems of evolution and phylogeny.+ The present distribution of the avian Niallophaga suggests that these insects became parasitic on the birds early in the evolution of the latter class and that they evolved with their hosts. In a group of related host species, each species may have allopatric species of a number of sympatric genera of Niallophaga common to the host group (VI: tables 1+, 5, 6), and in addition, sympatric species of one or more of these genera. In many cases, therefore, a single host species may have a considerable number of genera and species of Mallophaga (1 :279). The problem is to find an explanation of the presence of often closely related genera and species in what is the equivalent of a restricted and isolated geographical area. A study of the morphology of the Mallophaga and their present distribution both on a single host individual and throughout the class Aves makes it possible to deduce some of the factors which may have influenced speciation in this group of ectoparasites and to compare these factors with those influencing groups of free -living animals. Apart from the intrinsic interest of evolutionary problems in a group of ectoparasites, it is necessary when attempting to formulate a natural classification of the Mallophaga to have some understanding of the possible steps in the evolution of the group.

Following a general introduction, the extensive literature on fascioliasis is reviewed. The distribution, importance, pathogenesis and pathology of the disease are considered, with special emphasis on Fasciola gigantica infections. Field studies are reported on the epidemiology and control of F. ßiganti.ca in East Africa and a survey described of infections with Fasciola spp. in African wild mammals. This survey was undertaken in order to assess the importance of wild mammals in the epidemiology of this disease, in view of the increasing development of game parks and ranching schemes. The techniques used in the experimental studies are outlined with emphasis on those modified during this work. The techniques described include the cultural methods by which the infective agents were obtained, with special reference to the large scale production of metacercariae of F. gigantica and a detailed discussion of the problems which had to be overcome before this was possible. Other techniques described include those used in haematological examinations, faecal egg counts, liver function tests, serum enzyme assays and other biochemical studies. The difficulties which had to be overcome to obtain and maintain experimental cattle and sheep free of intercurrent diseases in Kenya are discussed. Observations are described which were made in the course of several experiments using a total of 35 sheep and 46 cattle in Edinburgh and in Kenya. In Edinburgh the experiments comprised studies on the pathogenesis of acute and chronic fascioliasis (Fasciola hepatica) in sheep. In Kenya chronic fascioliasis (F. gigantica) was studied in sheep with special emphasis on the economic importance of this condition. It was found that the infected animals gained weight more slowly than the uninfected controls and that this arose mainly from differences in the amount of body fat. Several experiments were carried out in Kenya on cattle infected with single doses of 500, 1000 or 2000 metacercariae of F. gigantica or with two doses of 500 metacercariae ten weeks apart. The pathogenesis of the disease and the parasitological findings in these animals are described in detail. Other studies described include observations on long term chronic fascioliasis in cattle. The gross pathology of P. giçantica infections in cattle from 2 -51 weeks after infection is also described. The main finding from these studies is that F. gigantica appeared to be less pathogenic for cattle than reported by earlier workers. There was no evidence of an "acquired self- cure" under the conditions of these experiments, which would support the suggestion that resistance to F. sigantica infections in cattle may be merely related to the fibrosity of the liver parenchyma. Further support was found for this suggestion from the study of the leucocyte levels and other observations. Evidence was found that F. gigantica has a relatively short life in cattle. The dissertation ends with a discussion of the results from the experimental infections.

A preliminary quantitative study on the population growth rate of Theileria parva in the bovine host failed to confirm the constant growth rate independent of size of infective dose reported by earlier workers. Experiments in methodology were performed therefore to standardize the techniques for obtaining (a) accurate estimates of infection rates from biopsy smears, (b) representative samples from a superficial lymph node source. A further experiment was carried out to establish that such samples were representative of the total parasitic biomass. These techniques were applied in a wider ranging quantitative study using four infective doses at ten fold intervals for infecting animals. The resultant growth rates were again dependent on the size of the infective dose. A definitive experiment, using five infective doses at ten fold intervals confirmed the divergence of growth rates. It was also shown that the severity of the clinical reaction, and the survival time was dependent on the size of the infective dose. The implications of these results in relation to immunization of cattle against T. parva are discussed. The standardized methods were applied also in a chemoprophylactic study to observe the growth rates of T. parva as affected by different regimens of tetracycline. Other possible applications of these standardized methods are discussed.