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Lernaeocera branchialis (L., 1767) is a parasitic copepod that parasitises a range of gadoids by anchoring in the proximity of the branchial chamber of its host, deriving nutrition from the blood of its host and causing serious pathogenic effects. This study investigates the taxonomy of the juvenile free-swimming stages and host location behaviour in the pre-metamorphosed adult female. The large size and distinctive appearance of the metamorphosed adult female stage, coupled with the wide exploitation and commercial importance of one of its principle final gadoid hosts, the cod (Gadus morhua L.), means that this species has long been recognised in the scientific literature, and here the extensive literature concerning this potentially important and damaging pathogen is re-examined to provide an up to date overview, which includes both aquaculture and wild fisheries perspectives. Due to disagreements between several descriptions of the L. branchialis juvenile stages, and because the majority of the descriptions are over 60 years old, the juvenile free-swimming stages are re-described, using current terminology and a combination of both light and confocal microscopy. The time of hatching and moults in these stages is also examined. Techniques for the automated creation of taxonomic drawings from confocal images using computer software are investigated and the possibilities and implications of this technique are discussed. The method of host location in L. branchialis is unknown but is likely to involve a variety of mechanisms, possibly including chemo-reception, mechano-reception and the use of physical phenomena in the water column, such as haloclines and thermoclines, to search for fish hosts. In this study the role of host-associated chemical cues in host location by adult female L. branchialis is investigated by analysing the parasites behavioural responses to a range of host-derived cues, in both a choice chamber and a 3D tracking arena. To analyse the data from the experiments, specialised computer software (“Paratrack”) was developed to digitise the paths of the parasites’ movements, and calculate a variety of behavioural parameters, allowing behaviour patterns to be identified and compared. The results show that L. branchialis responds to host-associated chemical cues in a similar way to many copepods in the presence of chemical cues. Of the different cues tested, gadoid conditioned water appears to be most attractive to the parasites, although the wide variation in behavioural responses may indicate that other mechanisms are also required for host location. The different behavioural responses of parasites to whiting (Merlangius merlangus L.) and cod (Gadus morhua) conditioned water, which are both definitive hosts, provide some evidence for sub-speciation in L. branchialis. The role of chemical cues in host location of L. branchialis, and the relative importance of chemical and physical cues in host location are discussed. As well as demonstrating several techniques, which show potential for further development, this work has improved our knowledge of the biology and life-cycle of L. branchialis. Further study of this, and other areas of L. branchialis biology and its host-parasite interactions, should assist the development of contingency plans for the effective management and control of this widespread and potentially devastating pathogen.

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.

Newly released eggs of the parasitic wonn Schistosoma mansoni either pass through the gut wall to escape from the host or are washed away in the host's bloodstream. In the latter scenario most eggs become lodged in the host's liver, where they become the focus of a granulomatous response which can have severe pathological consequences. In this study, the S. mansoni soluble egg proteome is described and characterised for the first time. Mature eggs were separated from immature eggs and then fractionated into their morphological components: the miracidia, the hatch fluid (which bathes the miracidia) and the egg-secreted proteins. Each egg preparation was subjected to two-dimensional electrophoresis and tandem mass spectrometry. Developmental proteomic changes were then described in terms of the egg's morphology so insights into the egg's natural history were gained. For example, acquisition of aerobic respiratory enzymes by the miracidium was seen, but nevertheless the miracidium still favours the use of energy-efficient heat shock proteins. Western blotting was used to show that the immature egg adopts the ubiquitin-proteasome pathway to degrade its nutritive vitelline cells. The hatch fluid contains host proteins but it also has a defensive role, although its most abundant constituent (a large, acidic glycoprotein) is of unknown function. The egg-secreted proteins consist of different variants of just four proteins, one of which has a pro-protein convertase domain and another of which appears to be a general purpose binding protein. A protocol is devised to purify each variant, so further functional studies into the individual secreted proteins can be carried out in the future. The secreted proteins induce a profound proliferative response in lymphocytes from acutely infected mice, indicating that they may work by activating granuloma T cells to secrete pro-proteases that are subsequently activated, enabling the egg to cross the gut wall.

How host and habitat heterogeneity affects the distribution of ticks in the Scottish upland environment was examined, and the prevalence of two important tick-borne diseases in questing, field-collected ticks was investigated.  This data was used to test the predictions of an influential model of louping-ill virus. Both hosts and habitat were found to be important predictors of Ixodes ricinus nymph abundance.  In general, the highest nymph numbers were collected from areas of heather habitat, and lowest numbers from areas of boggy ground.  Nymph numbers increased with increasing red deer density, but were negatively associated with increasing both mountain hare and red grouse density. A total of 1063 field collected, questing ticks were individually tested for louping-ill virus, of which 621 were also tested for Borrelia burgdorferi sensu lato.  Louping-ill RNA was detected in 7.8 % of all ticks tested.  The percentage of ticks positive for louping-ill virus did not vary with tick stage, collection site, month, or vegetation type, and was not correlated with host density of host seroprevalence.  B. burgdorferi was detected in 1.4 % of the ticks.  No simultaneous infections of louping ill virus and Borrelia spp. were detected. Field data on tick abundance and pathogen prevalence collected during this study was used to challenge an influential model of louping-ill virus dynamics.  The model proved relatively successful at predicting tick abundance, but underestimated the extent of louping-ill virus.  The single patch model was adapted to form a two-patch modelling allowing red deer movement between the two patches.  Allowing the movement of deer between two patches with different host densities shifted the threshold for louping-ill virus persistence, allowing its persistence in almost all red deer-grouse-hare scenarios.

This thesis investigates how host metapopulation processes (local population size and dispersal) affect the prevalence and persistence of ectoparasites of water voles (Arvicola terrestris). Water voles have been found to persist as metapopulations in the study areas in northern Scotland (fragmented populations with frequent extinction and colonisation and linked by dispersal). The ectoparasites examined were: 1) three tick species: Ixodes trianguliceps, Ixodes ricinus and Ixodes apronophorus; 2) four flea species: Megabothris walkeri, Peromyscopsylla spectabilis, Ctenopthalmus nobilis and Histrichopsylla talpae; 3) two species of mites: Laelaps muris and Hyperlaelaps amphibia. The prevalence and persistence of these parasites were examined over three years in 155 vole sub-populations which varied in density and isolation from each other. The effect of connectivity between vole sub-populations (where the number of immigrants a patch receives can be indexed by both the size of populations in the surrounding area and distance to these populations) on the spatial population dynamics of two tick species: a specialist small mammal tick (I. trianguliceps) and the sheep tick (I. ricinus) were examined. Connectivity had positive effects on the prevalence and burden of I. trianguliceps but not on I. ricinus. The relative effect of local (vole population size/density) versus metapopulation processes (connectivity) on the burden and prevalence of fleas was investigated. Local population dynamics had a weak influence on flea burden and prevalence (no or negative effect) whereas number of infected voles in neighbouring populations increased flea burden and prevalence. Extinction thresholds, taken as connectivity or proportion of habitat loss/“disinfection” below which a parasite cannot persist, were examined and compared for all parasites. Overall, the findings of this study show that host spatial structure and distance dependent dispersal (heterogeneous mixing) are important factors that affect parasite persistence. Therefore, these factors should be considered when planning conservation and disease control programmes.

Haematophagous, metazoan ectoparasites and protozoan blood parasites of intertidal fishes from Wales and South Africa formed the main focus of this study. The ectoparasites examined were largely juvenile isopods of the genus Gnathia, and specimens ofthe leech, Zeylanico~della arugamensis. These taxonomically distinct ectoparasites were suspected vectors of the blood protozoans, and the latter were mostly apicomple{(ans of the genus Haemogregarina and euglenozoans ofthe genus Trypanosoma. Knowledge of digestive tract anatomy in juvenile gnathiids is essential for assessing their role as potential vectors and this system was examined using histological, microscopical and computer-based techniques. Features that had been unreported, or insufficiently recorded, previously' included several structures within the dorsal afid ventral stomach chambers, a typhlosolelike formation in the anterior hindgut, haemozoin-like deposits in the digestive caeca of Gnathia africana and Gnathia pantherina, and an extensive gut flora. Connections between the stomach, anterior hindgut and digestive caeca were identified, salivary gland ducts were followed towards the mouthparts, and the digestive cycles of Gnathia maxillaris and G. africana juveniles were examined over a period of 30 days. The pathology associated with the attachment sites of G. africana and Gnathia sp A to teleosts, and with G. pantherina to an elasmobranch, was found to be considerable in the case of G. pantherina, while Gnathia sp A prompted the death ofits host unde: experimental conditions. Oocyst stages, presumed to be those ofHaemogregarina bigemina, were ,' . located in sections through the digestive caeca of G. africana, confirming the vector status ofthe isopod. Development stages of a new species, Haemogregarina curvata, were observed in the leech, Z. arugamensis, and within peripheral blood smears from several fishes. The life cycle oftrypanosomes found in Z. arugamensis and in fishes was also established, although interpreting the morphometries ofthese flagellates was challenging and speciation proved difficult. Identical molecular sequences oftrypanosomes were derived from separate leech samples and these were close to published sequences for marine fish trypanosomes. Thus, both gnathiids and leeches were likely vectors ofthe fish blood protozoans. Finally, probable dual transmission of a haemogregarine and trypanosomes by a leech was illustrated, a rare event, reported infrequently in the literature.

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.

Proliferative kidney disease (PKD) poses a high financial burden upon the freshwater salmonid aquaculture industry of Europe and North America. The alternate hosts of the causative agent, Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea), have been identified as freshwater bryozoans (Bryozoa: Phylactolaemata) within which spores capable of infecting salmonid fish develop. Currently, control of PKD relies upon complex management practices, with no licensed prophylaxis or treatment available. Assessment of the nutritional preferences of phylactolaemate bryozoans allowed development of an optimised laboratory culture system. Following laboratory maintenance, bryozoans collected from PKD-endemic sites were found to be infected with the malacosporean parasites T. bryosalmonae and Buddenbrockia plumatellae. Subsequent parasitic development was observed using light-, electron- and confocal-microscopy techniques. Methods of challenging rainbow trout with T. bryosalmonae spores were developed, with the minimum infective dose established. The presence of Thomsen-Friedenreich and Tn epitopes within the parasite was investigated, and experimental vaccine preparations based on either these specificities or T. bryosalmonae-infected bryozoans were efficacy tested in rainbow trout. In addition, salinomycin and amprolium were tested as prospective chemotherapeutants for PKD. Further insights into the development and subsequent release of malacosporean spores within their invertebrate hosts have been revealed. Long-term maintenance of T. bryosalmonae allowed controlled infection of rainbow trout previously vaccinated with experimental preparations. Findings of the project could potentially be utilised in future research into the development of control methods for PKD.

This thesis examines the parasite dynamics and the mechanisms affecting parasite load and transmission focalising on the role played by host and habitat heterogeneities. This study is based on the gastrointestinal nematode Heligmosomoides polygyrus and the small mammal yellow necked mouse and uses data gathered from experimental field manipulations of parasites intensities and data gathered from trapping monitoring. Initially the parasite community of yellow-necked mouse (Apodemus flavicollis) was explored in North-Eastern Italian Alps with the aim to describe the major patterns and identify the factors affecting parasite community structure. Despite the observed spatial variability it has been found that differences within the host population such age and secondly sex and breeding conditions, were the major factors acting on parasite occurrence and intensity. Habitat differences had a less apparent effect on parasite community structure. The consequences of H. polygyrus infection on other parasite species infections have been analysed, in specific the infestation of the tick Ixodes ricinus in populations of A. flavicollis. H. polygyrus load and tick infestation were monitored as well as were carried out field manipulations of H. polygyrus intensity and were monitored changes in tick infestation. It has been found that H. polygyrus load was negatively related to I. ricinus infestations. Host factors mediated the H. polygyrus-I. ricinus interaction such that young and non-breeding mice exhibited higher I. ricinus to H. polygyrus intensity respect breeding adults. The role of host sex on parasite abundance was then investigated carrying out a field experiment where the H. polygyrus intensity were manipulated in relation to mice gender. In specific, H. polygyrus was removed alternately from either sexes and the parasite load was analysed in the untreated sex. It was found that males mice were responsible to drive parasite transmission in the host population and this was observed in absence of sex-bias in parasite infection, suggesting that this pattern was not a mere consequence of quantitative differences in parasite loads between sexes. To disentangle the possible mechanism causing this sex bias in parasite transmission mathematical simulations based on parameters obtained for the field experiment were used. Two non mutually exclusive hypotheses causing sex bias in parasite transmission were tested: a- males immune response is less efficient and this causes the development of more successful parasite infective stages or b-males behaviours allow them to be more efficient is spreading in more exposed areas parasite infective stages. Multi-host models were developed and simulations were compared with field results. While it was not disentangled the most dominant mechanism causing sex bias in parasite transmission this study underlined the importance of host sexes in affecting parasite dynamics and host-parasite interaction. In conclusion this thesis highlighted the importance of considering host and environmental differences when investigating host parasite interactions. This finding could be extremely important when planning measured of disease control or to avoid disease outbreak. Controlling target group of individuals host could avoid economical losses and a more effective measure of intervention.

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.

This study examines, from a modelling point of view, the dynamics of infectious diseases in wildlife caused by macroparasites and by tick-borne infections. The overall aim was to investigate the important role played by parasite aggregation in the dynamics of both systems. For macroparasites we first developed some deterministic models that incorporate explicit mechanisms for generating aggregation in parasite distribution, specifically multiple infections and host heterogeneity. We explored the role of aggregation in host regulation and in determining a threshold value for parasite establishment. A large aggregation makes it more difficult for parasites both to regulate hosts, and to get established in a population at carrying capacity. Furthermore, the stabilization yielded by aggregation strongly depends on the mechanism that produces the aggregation. We then introduced some uncertainties into the host-macroparasite system, presenting an individual-based stochastic model that incorporated the same assumptions as the deterministic model. Stochastic simulations, using parameter values based on some real case studies, preserved many features of the deterministic model, like the average value of the variables and the approximate length of the cycles. An important difference is that, even when deterministic models yield damped oscillations, stochastic simulations yield apparently sustained oscillations. The amplitude of such oscillations may be so large as to threaten the parasites’ persistence. With respect to tick-borne diseases we presented a general model framework that incorporated both viraemic and non-viraemic routes of infections. We compute the threshold for disease persistence and study its dependence on the parameters and on host densities. The effects of tick aggregation and correlation between different tick stages on the host have both an important effect on infection persistence, if non-viraemic transmission occurred. In the case of Lyme Disease and Tick-borne Encephalitis (TBE) in Trentino (northern Italy) we showed some numerical results, using parameter estimates based on a detailed field study, and explored the effects of uncertainty on the endemic equilibrium of both diseases assuming only viraemic transmission for Lyme Disease while for TBE we permitted only non-viraemic transmission through co-feeding ticks. In conclusion we have examined the patterns and changes of aggregation in a number of contrasting systems and believe that these studies highlight both the importance of considering heterogeneities in modelling host-parasite interactions and, more specifically, modelling the biological mechanisms that produce aggregation in parasite distributions.

Blood sucking insects have defence strategies against infection. In the stable fly, Stomoxys calcitrans, one of the defensive mechanisms is a lectin which is blood meal stimulated. While attempting to clone and sequence the lectin from the reservoir zone of the midgut tissue of S. calcitrans, a trypsin was obtained. The trypsin is blood meal induced, and is midgut tissue specific. The cDNA of the mature protein has 260 amino acid residues and has a structural similarity with a trypsin - like proteinase Try29F of the fruit fly Drosophila melanogaster (57% sequence identity); with trypsin 7 precursor of African malaria mosquito Anopheles gambiae and trypsin 3Al precursor of yellow fever mosquito Aedes aegypti (both 55%). The protein has a relative molecular weight of 28 kDa. It has both the serine and the histidine active site signatures; with its catalytic domain between 60 to 240 amino acids. It has an N-glycosylation site (NES) at position 163 to 166 with a significant probability of occurrence (P = 0.005). The cloned trypsin was extracted from the midgut of 8 - 9 day old flies which have been starved for 24 hours prior to dissection. The extract from which the trypsin was purified, was capable of agglutinating rabbit red blood cells an activity that was strongly inhibited by N-acetyl-D-glucosamine. The molecule was purified by affinity chromatography designed to purify lectins and the subsequent isolation of lectin using SDS-PAGE. The trypsin molecule suggests the presence of a trypsin - lectin dimer in S. calcitrans midgut, and in the light of work on tsetse flies, this possibly helps to explain why Stomoxys is not a cyclical vector of trypanosomiasis despite being sympatric with Glossina. The study has also shown that parasites have adaptive evolutionary changes which help them to evade the defence mechanisms of vectors. The study presents three groups of newly isolated trypanosomes of T godfreyi types similar to the known T godfreyi by 96 & 95.6% sequence identity; the T simiae Tsavo by 95% and T vivax type by 86.2%. This work has also shown that different tsetse species respond differently to infection even if they share the same habitat and hosts. The patterns of infection in four different species investigated, ( Glossina swynnertoni Austen, G. morsitans morsitans Westwood, G. brevipalpis Newstead and G. pallidipes Austen) varies from species to species and from one place to another and also are conserved within species.

The work described is primarily concerned with measuring the induced AC electrokinetic properties of the transmissive stages of two water-and food-borne intestinal pathogens, namely oocysts of Cyclospora and eggs of Ascaris, in order to demonstrate the applicability of electrorotation for determining the viability of the organisms. An electrode design that maximises the space over which a uniform electric field is applied to the electrorotation chamber is described. This is important when investigating organisms that are few in number, or for investigating large, mixed populations. It was found that Cyclospora viability and sporulation state could be determined using electrorotation. Since there is no vital dye assay for this emerging parasite, and its life-cycle is not fully understood, these are important findings for the water and food industries. Modelling of the electrorotational response was found to be in good agreement with the experimental data. Limited electrorotational spectra from oocysts recovered from baboons showed slight differences when compared with those of human source. The first electrorotational studies of a complex biological organism, namely the eggs of the nematode Ascaris, which poses a global health problem, are presented. It was found that unfertilised and fertilised eggs rotate in opposite directions, allowing for rapid visual determination of mixed populations. Different stages in the development of fertilised Ascaris eggs were also observed by electrorotation. Modelling of the electrorotational response of the Ascaris eggs was found to be in reasonable agreement with the experimental data. The work presented here shows that the electrorotation technique can be used for the rapid determination of the viability and sporulation state of oocysts of C. cayetanensis, and the fertilisation state of eggs of A. suum. As current viability assays are complicated and time consuming, requiring trained personnel, the electrorotation technique offers many advantages to the food and water industries, especially as it can be automated.

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 ability of parasites to manipulate their hosts is the major empirical theme of this thesis, in particular the problem of cooperation among parasites that host manipulation entails. Contributing to host manipulation is likely to bear a cost to the individual, while the benefits will be felt by every conspecific parasite (and others) within the host. I develop in Chapter Two a game-theoretical model of parasite-induced host manipulation, which focuses on co-operative interactions between individuals and a larger group. In Chapters Three and Four I consider two macroparasitic case-studies. Chapter Five marks the beginning of the macroparasitic half of the thesis, with a study of the cestode Ligula intestinalis in its intermediate host, the Roach (Rutilus rutilus). Chapter six uses a similar mix of statistical and analytic approaches to investigate the interaction between the manipulative trematode Microphallus papillorobustus and its intermediate gammarid host, with a focus on the potential costs of host manipulation. Chapters Five and Six introduce aggregation as respectively a side-effect and a cost of manipulation. In Chapter Seven, I add that, should aggregation be costly to established worms, the probability of subsequent recruits may itself fall under the influence of established parasites. Using the case study of schistosome worms in their definitive mammalian hosts, I develop a stochastic-simulation model to explore the potential for adaptive population regulation in parasites. In Chapter Eight, I review the progress made in this thesis, with an emphasis on developing a synthesis between models of virulence and of host manipulation.

The natural resistance associated macrophage protein gene (Nramp1) influences susceptibility to several intracellular infections. To investigate the mechanism of this effect, congenic Nramp1 wild type (infection resistant) and mutant (infection susceptible) mice were infected with Leishmania donovani. The Nramp1 mutant mice developed more severe infection. Quantitative RT-PCR and fluorescence microscopy demonstrated that the mutant mice had attenuated hepatic chemokine and pro-inflammatory cytokine innate immune responses, reduced hepatic cellular infiltrate, reduced hepatic apoptosis and abnormal accumulation of cellular iron. This validated methodology was used to investigate the function of Nramp1 in cerebral infection. Following Toxoplasma gondii infection, mutant mice had significantly greater mortality. Laboratory analysis demonstrated for the first time that the naturally occurring infection-susceptible mutation in Nramp1 attenuates brain expression of chemokines, pro-inflammatory and immuno-regulatory cytokines in response to infection. During this research, it was noted that Nramp1 genotype also affected the behavioural stress response and this was investigated using restraint stress and quantitative in situ hybridisation. Compared with wild type mice, Nramp1 mutant mice had larger adrenal glands and higher circulating corticosterone concentrations but their hypothalamic, adrenal and behavioural stress responses following restraint stress were reduced. This abnormal stress response in Nramp1 mutant mice was associated with restraint stress increasing susceptibility to T. gondii infection in aged but not young mice. Immunochemical studies suggested that the Nramp1 protein was expressed on cerebral neurones as well as cells of the monocyte/macrophage lineage.

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.

The aim of the study was to investigate the role of helminth parasites in the population ecology of the wood mouse Apodemus sylvaticus in the field. Specifically, the study determined the species richness, prevalence and intensity of helminths in wood mice in deciduous woodland at Balmedie, Aberdeenshire, North East Scotland and evaluated faecal egg counting technique for detecting and estimating worm burdens of helminths in wood mice. In a capture-mark-recapture field experiment both the prevalence and intensity of helminths were manipulated by administering the anthelmintics Levamisole and Praziquantel to wood mice in deciduous woodland (Foveran and Auchmacoy, Aberdeenshire). Wood mice captured in Balmedie woodland harboured 3 species of nematode, Heligmosomoides polygyrus, Syphacia stroma and Trichuris muris, the digenean Brachylaemus recurvum and 2 species of cestode Skrjabinotaenia labota and a larval strobilocercus of Taenia taeniaeformis. Frequency distributions of all helminths other than T. muris and T. taeniaeformis, displayed over-dispersion. The prevalence and intensity of infection of the helminths showed a clear pattern of seasonal variation. There were no significant differences in the prevalence and intensity of infection of helminths between male and female wood mice. The intensity of infection of H. polygyrus and S. stroma was higher in heavier (older) mice. Wood mice underwent seasonal variation in density at both Foveran and Auchmacoy woodlands. The density of mice was greatest in late autumn/early winter and declined through the spring, being lowest in late spring / early summer. The density increased throughout summer and was highest in later summer / early autumn at Auchmacoy and in early winter at Foveran. At each site, the population density was similar in control and heated grids. Adult male and female wood mice showed a clear pattern of seasonal variation in mean body weight at both study sites. The mean body weights were highest in summer, declined through autumn being lowest in late autumn and early winter. There were no significant seasonal (breeding vs non-breeding) and treatment (control vs treatment) effects on the mean body weights of adult male and female wood mice.

The present study investigated aspects of the biology of the cestode, Proteocephalus filicollis from the three-spined stickleback Gasterosteus aculeatus from Airthrey Loch, Scotland. The population biology study demonstrated that the parasite has an annual cycle of recruitment, which occur mostly in late summer and early autumn. The cestode did not show preference for any sex of the host. Maturation of the cestode also showed a seasonal cycle with the majority of worms maturing in late spring and early summer, but this period may be extended in different generations. Proteocephalus filicollis was overdispersed throughout the year in all sizes of fish, moreover variance to mean ratio always exceeded unity. No severe pathology was observed due to attachment of the worm to the intestine of the fish. The worm population in different sections of the intestine varied according to season and maturity stage. The P. filicollis migrate from the rectum to the anterior intestine as they mature and it is suggested that growth and maturation of the worm is a major stimulus for this migration. Proteocephalus filicollis has a high fecundity as indicated by the higher number of eggs per mm of gravid portion of the strobila and high fertility. Infrapopulation size did not show any relationship with length of worm, percentage gravid portion, number of gravid segments or mean length of gravid segments. Numbers of eggs are correlated to length of the worm, but not to infrapopulation size. Numbers of eggs per mm of the gravid portion are not correlated to length of worm or infrapopulation size. Acanthocyclops robustus was used as an experimental intermediate host. 15-16°C was the optimum experimental temperature for growth and a fully developed larva was formed in 23-27 days at this temperature. No growth was observed at 4°C, growth was slow at 10°C, but rapid at 21-22°C. The eggs are infective for 25 days at 4°C, 10°C and 15-16°C, but for only 15 days at 21-22°C. Prevalence and mortality of copepods are significantly correlated to their exposure time to parasite eggs, but mean intensity of infection did not show any relationship to the exposure time to the eggs. Ultrastructural studies demonstrated that a mature egg is surrounded by at least four embryonic envelopes, the capsule, the outer envelope, the inner envelope, and the oncospheral membrane. All these envelopes originate differently and undergo definite changes during their development.

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 examined the physiological, behavioural and pathological effects of sea lice, Lepeophtheirus salmonis (Krøyer, 1837), on wild sea trout, Salmo trutta L., and experimentally infected sea trout and Atlantic salmon, Salmo salar L. to determine how, and to what extent, sea lice cause mortality of their infected hosts. A feeding hierarchy was established in groups of sea lice infected and uninfected Atlantic salmon as a physiological stressor. Results indicated that chalimus did not stimulate physiological changes leading to marine mortality and the intensity of the sea lice infection on a fish was independent of that individual's food consumption. However, preadult stages caused appetite suppression, severe skin lesions and changes in the blood biochemistry of the fish with subsequent recovery to the levels of uninfected fish as the parasite moulted through to the adult stages of the lifecycle. The effects of sea lice on sea trout at either 2 or 6 weeks after sea water transfer were assessed. Both infection intensity and developmental rate were not significantly different between the groups, but a trend of fewer lice on the fish infected 6 weeks after sea water transfer was recorded. Fewer of the fish infected 2 weeks after sea water transfer had resumed feeding by the end of the experiment, leading to a loss of body condition, and suffered more severe damage to the skin and detrimental changes in the physiological integrity as a consequence of feeding preadult lice. The mortal impact of sea lice infection was significantly enhanced in the fish infected 2 weeks after sea water transfer. The findings from this thesis have shown that preadult sea lice can cause mortality of wild and experimentally infected sea trout and Atlantic salmon, particularly if infected at the time of sea water transfer, but under certain conditions sea lice infected hosts can recover from the detrimental effects of both chalimus and mobile stages.

The parasitic nematode, Teladorsagia (Ostertagia) circumcincta is the primary cause of Parasitic Gastro-Enteritis (PGE) in lambs in Britain. Control of this parasite has largely depended on the use of broad spectrum anthelmintic drugs since their inception three decades ago. Widespread and unconstrained use of anthelmintics has resulted in selection for resistant strains of nematode, particularly within the T. circumcincta species. Control of PGE now involves optimizing parasite control whilst preserving the susceptibility of the parasites to the anti-parasitic drugs. Two aspects of the epidemiology of T. circumcincta are investigated in this thesis. First, the effect of temperature on the development and survival of the free-living stages is investigated. The conventional nematode development models are replaced by more sophisticated and biologically meaningful methods of describing temperature-dependent development rate phenomena in nematodes. The effect of geographical, temporal and developmental variation on the population dynamics of T. circumcincta are explored to determine possible sources of observed variability in infection levels in the field. Next, a suite of models generic to most direct life cycle parasites undergoing intensive drug therapy, is constructed and analysed. Provision is made within these models to explore the impact of important life history events such as refugia and immigration on the evolution of resistance. A novel technique in resistance control involving overwhelming a resistant strain of nematode with a susceptible strain is modelled and suggestions made for the practical implementation of such a method.

In recent years the bewildering diversity of myosin types and function has become apparent. Over ten different classes have been identified in a wide variety of organisms - ranging from protozoans to vertebrates and even higher plants. The intriguing question is - what do they all do? C. elegans, "the worm", is a relatively simple organism, both anatomically and genetically and has been studied extensively with respect to development, cell lineage and genetics. However only myosins of the conventional type (muscle or "class II" myosins) had been identified prior to the work described here. The first novel myosin from C. elegans has been cloned and fully sequenced. The predicted amino-acid sequence shows that this myosin contains the conserved motor or "head" domain responsible for actin-activated ATPase activity. This is followed by two motifs thought to be capable of interaction with members of the calmodulin class of Ca²⁺ binding proteins and a tail with a general positive charge. The sequence of the entire protein, along with expression and immunolocalisation pattern, suggests that it may be the nematode homologue of a recently discovered unconventional myosin from the rat, myr4. A multiple alignment of the 86 available conserved motor domain sequences has enabled the construction of an unrooted phylogenetic tree, which indicates that the myosins fall into 13 classes. C. elegans Myosin IA is a member of the class I myosins. The conservation of residues within this alignment has been explored further by scrutinising their position within the available myosin crystal structures. From this important residues involved in myosin function can be identified. Furthermore, the alignment allows the positioning of residues from myosins, whose structure is unknown, within the framework provided by the existing crystal structures - enabling, for example, the interpretation of existing mutations in unconventional myosins.

Dinoflagellate parasites of the genus Hematodinium are associated with heavy mortality of the Norway lobster (Nephrops norvegicus) off the west coast of Scotland. The Syndinean dinoflagellate has been isolated from N. norvegicus and has been successfully cultured axenically in vitro. Twelve isolates have been serially cultivated in a medium of 10% fetal calf serum in a balanced Nephrops saline with added antibiotics at 8-10°C. In this medium the parasite undergoes developmental changes that are believed to represent stages in the life cycle of the parasite in vivo. This is the first complete life cycle in vitro to be described for a Syndinean dinoflagellate. Flagellate dinospores arise in vitro from circulating sporogenic parasite forms - sporoblasts removed in the haemolymph from infected lobsters. Sporogenic parasites are recognised by the presence in the cytoplasm of structures not found in the trophont. These are (1) trichocysts and (2) flagellar hairs within swollen endoplasmic reticulum cisternae. Dinospores are of two types - a larger macrospore and a smaller, more active microspore. Individual isolates produce one or the other, not both. Condensation of chromosomes in the nucleus is more pronounced in the microspore than in the macrospore. Both spore types germinate after 18-62 days in the culture medium to produce the main multiplicative stage of the parasite in vitro - the multinucleate filamentous trophont. No fusion of flagellates has been observed and each type of spore can germinate independently of the presence of the other, indicating that the spores are not gametes. The filamentous trophonts correspond to the only form of the type species of the genus, Hematodinium perezi, found circulating in the blood of infected crabs by Chatton and Poisson (1931). (DXN004,487)

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.

This study investigated the epidemiology and taxonomy of Diplostomuni species in the deeper parts of the eye (DPE) of roach Rutilus rutilus (L. ), perch Perca fluviatilis L., ruffe Gymnocephalus cernua (L. ) and gwyniad Coregonus lavaretus (L. ) at Llyn Tegid, North Wales, and in the cranium of catfish Clarias gariepinus (Burchell, 1822) and the vitreous humour of Oreochromis species at Mindu dam and river Ruvu, Tanzania. At Llyn Tegid, two species of Diplostomum metacercariae, large and small forms, coexisted in DPE of perch, ruffe and gwyniad but only one, large forms, occurred in roach. Large forms were distinguished by their large size, oval body tapering at both ends and pseudosuckers at the level of the oral sucker. Small forms were distinguished by their smaller size, oval body with parallel sides and pseudosuckers occurring below the level of the oral sucker. The taxonomy of the two forms was not resolved even after obtaining adults from chickens. Tentatively, small forms keyed close to D. gasterostei Williams, 1966 and large forms to D. volvens Nordmann, 1832. Inadequate identification manuals and remarkable similarity between Diplostonlum species confounded identification. Mean intensity of metacercariae was highest in ruffe and lowest in perch and increased with host size in all hosts. Factors responsible for differences in mean intensity between hosts were discussed. There was no seasonality of occurrence. Recruitment occurred from May - November correlating with water temperatures. Immature metacercariae occurred throughout the period of investigation. Metacercariae were overdispersed in all three hosts. At Mindu and Ruvu two species were present, D. sp. X (1,2) in the cranium of catfish and D. sp. Y in the vitreous humour of Oreochromis sp. D. sp. X (1,2) were distinguished by their elongate body, oval calcareous corpuscles and long hindbody. D. sp. Y were distinguished by a vestigial ventral sucker, glandular Brandes organ, short hindbody and spherical calcareous corpuscles. D. sp. X (1,2) were identified as D. mashonense Beverley - Burton, 1963. Adults cultured in chickens confirmed identification. D. sp. X (1,2) developed to ovigerous adults within 24h. Percent recovery of adults from chickens declined with days p. i. Miracidia developed in eggs in six days. D. sp. Y is considered an undescribed taxon. Prevalence and mean intensity of D. sp. Y in Oreochromis sp. were low and did not fluctuate seasonally. Prevalence of D. mashonense in catfish was similar at Mindu and Ruvu and exhibited no seasonal fluctuations. Mean intensity of D. mashonense in catfish of Mindu fluctuated seasonally, increased with host size and was higher than in catfish from Ruvu. Causes of high intensity and seasonal changes at Mindu were discussed. D. mashonense was overdispersed in catfish at both localities but only data from Mindu fitted the negative binomial model. Small samples and the dynamism of forces creating overdispersion were considered responsible for the situation at Ruvu. Using SEM ciliated-pit and dome-shaped papillae were observed on the surface of metacercariae and adults of D. mashonense . Ciliated-pit papillae occurred only on metacercariae. Dome-shaped papillae, in adults and metacercariae, were aggregated on suckers, excretory and genital atria. Spines were multipointed in adults but single pointed in metacercariae.

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.

Infestation of commercially reared salmonids with the salmon louse Lepeophtheirus salmonis, is a large problem for the fish farming industries in Scotland, Ireland and Norway. Lice infestations cause high fish mortalities and losses in revenue for the fish farmer. Conventional treatment of lice infestation involves the use of organophosphate insecticides which are limited in their efficacy against lice, as well as being highly toxic to the flora and fauna which surround the sea cages. We have investigated the possibility of producing a recombinant vaccine against L. salmonis based on immunisation of farmed salmonids with 'concealed' antigens from lice. Murine monoclonal antibodies were raised against L. salmonis antigens using standard cell fusion techniques. The antibodies were screened by immunohistochemistry in order to establish the location of the recognised antigen in L. salmonis sections. We used the monoclonal antibodies to screen L. salmonis DNA libraries constructed in gt11 and ZAP in order to isolate the DNA coding for each recognised antigen. A monoclonal antibody was produced which showed strong immunoreactivity with cuticle-associated areas in lice sections. This antibody was subsequently shown to recognise the enzyme chitinase from L. salmonis. We have expressed recombinant proteins from the isolated phage which have been used in immunisation trials on the Atlantic salmon (Salmo salar). Lice feeding on fish immunised with three of the recombinant antigens, displayed abnormal egg development and an overall decrease in egg number. We are currently retesting these antigens in a larger immunisation trial. We have also used polymerase chain reaction technology to look for known genes in L. salmonis and have identified a member of the wnt gene family, wnt-5a.