Analysis of Faecal Egg Count (FEC) and Faecal Egg Count Reduction Test (FECRT) datasets is frequently complicated by a high degree of variability between observations and relatively small sample sizes. In this thesis, statistical issues pertaining to the analysis of FEC and FECRT data are examined, and improved methods of analysis using Bayesian Markov chain Monte Carlo (MCMC) are developed. Simulated data were used to compare the accuracy of MCMC methods to existing maximum likelihood methods. The potential consequences of model selection based on empirical fit were also examined by comparing inference made from simulated data using different distributional assumptions. The novel methods were then applied to FEC data obtained from sheep and horses. Several syntactic variations of FECRT models were also developed, incorporating various different distributional assumptions including meta-population models. The inference made from simulated data and FECRT data taken from horses was compared to that made using the currently most widely used methods. Multi-level hierarchical models were then used to partition the source of the observed variability in FEC using data intensively sampled from a small group of horses. The MCMC methods out-performed other methods for analysis of simulated FEC and FECRT datasets, particularly in terms of the usefulness of 95% confidence intervals produced. There was no consistent difference in model fit to the gamma-Poisson or lognormal-Poison distributions from the available data. However there was evidence for the existence of bi-modality in the datasets. Although the majority of the observed variation in equine FEC is likely a consequence of variability between animals, a considerable proportion of the variability is due to the variability in true FEC between faecal piles and the aggregation of eggs on a local scale within faeces. The methods currently used for analysis of FEC and FECRT data perform poorly compared to MCMC methods, and produce 95% confidence intervals which are unreliable for datasets likely to be encountered in clinical parasitology. MCMC analysis is therefore to be preferred for these types of data, and also allows multiple samples taken from each animal to be incorporated into the analysis. Analysing the statistical processes underlying FEC data also revealed simple methods of reducing the observed variability, such as increasing the size of individual samples of faeces. Modelling the variability structure of FEC data, and use of the inferred parameter values in precision analysis and power analysis calculations, allows the usefulness of a study to be quantified before the data are collected. Given the difficulties with analysing FEC and FECRT data demonstrated, it is essential that such consideration of the statistical issues pertaining to the collection and analysis of such data is made for future parasitological studies.

The kinetics of the host's immune responses to challenge infection were studied and identified clear patterns in plasma IgA activity, peripheral eosinophil counts, faecal egg counts and plasma pepsinogen concentrations but not in plasma IgG activity. It was determined that when used in parallel and when tested at multiple time points, these parameters have much greater potential as markers of resistance than when used individually or more importantly if only assessed on a single occasion. Further work investigated the recognition of stage specific parasite antigens by host plasma IgA by Western blotting. After adjusting for differences in the activity of IgA in each plasma sample the work in this thesis identified that preferential recognition of a different set of antigens was associated with resistance in the group of experimentally challenged animals compared to previous publications. Additionally, and for the first time this investigation was also carried out on naturally infected animals. There was little correlation in the patterns of antigen recognition between the experimentally challenged and naturally infected animals. Finally, the role of MHC was investigated and it was determined that MHC heterozygotes produced significantly more plasma IgA then MHC homozygotes but did not harbour significantly shorter worms. The analysis also confirmed in naturally infected sheep that there was no obvious relationship between MHC polymorphism and antigen recognition. The results suggested that resistance was due to the recognition of several molecules rather than a single molecule. The work detailed in this thesis has further increased our understanding of the complex host/parasite relationship and has confirmed that selective breeding using the various phenotypic and genetic markers studied is possible. However, this will only be viable if the tests involved in assessing these traits become cheaper and easier to perform, especially if they are to be carried out by the farmer, on the farm.

The pain transmission system is inherently plastic in nature; plasticity of nociceptive processing in the dorsal hom of the spinal cord is believed to contribute to clinical states of post-injury pain hypersensitivity. Both enhancement and tachyphylaxis of nociceptive processing have been reported previously following repeated carrageenan-induced inflammation. The present study aimed to investigate central mechanisms involved in the transformation of pain transmission from 'physiological' to 'pathophysiological' in adult rats, using a model of mild intraplantar inflammation induced by intraplantar administration of carrageenan at doses markedly lower than those standardly used in research into central mechanisms of inflammatory pain transmission. Changes in plantar inflammation, thermal and mechanical sensitivity were assessed following intraplantar injection of repeated doses of carrageenan (0.5%, corresponding to a dose of 0.25 mg and 0.1 %, corresponding to a dose of 0.05 mg), administered at weekly (0.5% and 0.1 %) and daily (0.1%) intervals. Expression of mRNA of key genes implicated in plasticity of central spinal pain transmission in laminae I, II and V of the dorsal hom of the lumbar spinal cord (laminae involved in central nociceptive transmission) was investigated using in-situ hybridisation techniques. The genes investigated were calcium calmodulin kinase IIa (CaMKIla), a key intracellular molecule instantaneously activated by neuronal stimulation; alterations in CaMKIla expression can rapidly induce nociceptive plasticity through modulation of many excitatory and inhibitory nociceptive mediators; the cyclooxygenase enzymes COX-1 and COX-2, which catalyse prostaglandin synthesis and are implicated in the modulation of the central nociceptive response to inflammatory injury; the immediate early genes zif11268, junD and tissue plasminogen activator (tPA), which have been implicated in the induction and maintenance of neuronal plasticity in higher centres, and the precursors for the inhibitory neurotransmitter molecules y-amino butyric acid (GABA), enkephalin and dynorphin. A method for organotypic culture of neonatal spinal cord was developed and characterised with the aim of providing a useful technique for more detailed study of the molecular basis of nociceptive plasticity. Mild inflammatory injury induced by 0.5% and 0.1% carrageenan treatment induced consistent hyperalgesic behaviour, which did not change following weekly repeated injection. Temporary attenuation of hyperalgesia developed following daily repetitive administration of 0.1 % carrageenan, but hyperalgesia returned when this repetitive inflammatory stimulation was maintained. Preliminary studies on the role of NMDA receptors, opioid receptors and a 2A adrenoreceptors in the mediation of this tachyphylaxis suggest that these receptor systems did not playa major role in the observed tachyphylaxis In-situ hybridization studies did not identify changes in gene expression induced by repetitive carrageenan treatment in lamina V. In laminae I1II, changes were observed in expression of certain genes (notably CaMKIla, COX-2 and proenkephalin), but not of immediate early genes, GAD 67 or prodynorphin. Hyperalgesia associated with weekly carrageenan treatment correlated closely with significantly enhanced transcription of CaMKIla mRNA in laminae IIII; moreover, tachyphylaxis of hyperalgesic behaviour correlated with attenuation of CaMKIla upregulation. Since increased expression of CaMKIla, leading to regulation of expression of a range of kinase-dependent receptors and intracellular mediators, is a hallmark of neuronal plasticity in higher centers, this suggests that central plasticity of nociceptive transmission in the dorsal hom could have contributed to the development of hyperalgesia following carrageenan treatment. Weekly administration of carrageenan also consistently induced significant upregulation of COX-2 and proenkephalin mRNA expression in laminae I1II, suggesting that ultimate modulation of pain sensation following inflammatory injury is determined by the interaction of excitatory and inhibitory transmitter pathways. COX-I, prodynorphin and GAD 67 mRNA expression were not significantly changed in relation to the intensity of inflammatory injury or in relation to changes in nociceptive responses. This would suggest that these mediators did not play a key role in the modulation of spinal nociceptive transmission associated with mild inflammatory injury. With the possible exception of CaMKIla, changes in gene expression did not correlate closely with plasticity of nociceptive behaviour induced by daily repeated carrageenan treatment. 200 !lm transverse slices of postnatal spinal cord were cultured successfully for up to 5 days using a simple interface culture system. Histochemical and immunocytochemical assays indicated that the architecture of organotypically cultured spinal cord closely resembled that observed in-vivo. This study presents a new approach to the investigation of neuronal plasticity associated with tissue injury and inflammation. Different mechanisms underlying plasticity of nociceptive responses may be induced by induced by high intensity as opposed to lowintensity injury. The observation of tachyphylaxis of hyperalgesia following daily repeated carrageenan treatment may represent engagement of endogenous 'anti-hyperalgesic' mechanisms. Further investigation of the molecular basis of endogenous 'antihyperalgesia', facilitated by organotypic slice culture techniques, may identify new targets for the treatment and prevention of persistent pathological nociceptive transmission following inflammatory injury.

No description available.

Cytokines are small proteins or glycoproteins that mediate cellular growth and differentiation and regulate immune responses. Upon encounter with antigen, CD4+ T cells are able to influence the character of the immune response elicited through the expression of distinct types of cytokines. Thl cytokines, especially IFN-γ but also TNF-β and IL-2, constitute one such pattern of expression, promoting cell mediated immune responses. In a broader sense, interleukin-12 and interleukin-18 can also be classified as type I cytokines in as much as they are able to shift the CD4+ cytokine expression pattern to a Thl phenotype and specifically stimulate IFN-γ production not only by T helper cells, but also by cytotoxic T cells and natural killer cells. The purpose of the present project was to develop equine Thl cytokines, making them available for dissecting the equine immune system, particularly in what concerns to defence mechanisms against infectious micro-organisms. Following the trend in human medicine, the cytokines produced will be useful for the development of new therapeuticals and prophylactics to be used in the control and prevention of infectious diseases of the horse. For this effect we have initiated studies on equine interferon-gamma and related cytokines and obtained the following results. Equine interferon-gamma was cloned and produced in a variety of heterologous protein-expression systems. The biological activity of recombinant equine interferon-gamma was assessed in vitro. Polyclonal serum preparations against equine interferon-gamma, obtained by immunization with recombinant protein, were recovered and characterised. Also described is the cloning of the interferon-gamma inducing cytokines equine interleukin-12 and equine interleukin-18. The potential use of the cloned equine cytokine genes as vaccine adjuvants was evaluated by DNA co-administration with plasmids encoding the equine influenza virus antigens haemagglutinin and nucleoprotein, followed by viral challenge.

The aims of this study were to investigate aspects of the aural microenvironment in dogs. The aural microflora, histological features of the aural integument, and biochemical components of cerumen from healthy canine ears and those with otitis externa were studied. Interactions between canine cerumen and one member of the resident aural flora, Malassezia pachydermatis were also investigated. Gram-positive, coagulase-negative cocci, and M.pachydermatis were the most common resident flora found in 52 healthy canine external ears. Microscopic examination of cytological smears from cerumen and microbiological culture indicated that these organisms were present in low numbers. Gram-positive coagulase positive staphylococci, Gram-negative rods, and M.pachydermatis were isolated most frequently from 27 canine ears affected by otitis externa. Eighty percent of these inflamed ears were associated with microbial overgrowth. The numbers of microorganisms found in cerumen cytological smears appear to be correlated to the growth density of microbial colonies on culture plates. In an anatomical survey of the external ear canal, 40 canine ears were examined. The average length of the cartilaginous part of these canals was 5.3 cm. The diameter at the most proximal end of the annular cartilage averaged 0.5 cm; at the proximal end of the auricular cartilage the mean diameter was 0.7 cm; at the distal extremity of the ear canal, the average diameter was 4.8 cm. Morphometric stereology was used to evaluate histological features of 28 clinically normal and 15 otitic canine ears at four anatomical levels. Marked variation was found in the distribution of sebaceous and apocrine glands in the aural integument in healthy ears and those with otitis.

Tropical theileriosis, caused by the tick-transmitted protozoan Theileria annulata, is a major disease of cattle in many regions of the developing world. Current research is directed towards developing a sub-unit vaccine, and it is therefore important that genetic diversity in field populations of the parasite is investigated and quantified. The recently completed genome sequence provided an opportunity to develop a panel of genetic markers for population studies and also enabled the identification of novel antigen genes. The genome was bioinformatically screened to identify micro- and mini-satellite loci, several of which were PCR amplified from a series of diverse parasite stocks in order to characterise their polymorphism and to determine their species-specificity. A panel of ten markers were selected for population genetic studies and were used to genotype laboratorymaintained cell lines and clonal stocks of T. annulata isolated from different countries. Cell lines comprised a multiplicity of genotypes, while clonal stocks showed evidence of a single haploid genome. Preliminary population genetic analysis revealed a large amount of genotypic diversity both between and within countries and indicated that the parasite population is geographically sub-structured. Comparison of a limited number of stocks isolated in different countries demonstrated that genetic differentiation between populations positively correlates with intervening physical distance. A low standard index of association (IS A) suggested that the population in Tunisia is in linkage equilibrium, indicating that the parasite possesses a panmictic (randomly mating) population structure. To confirm these findings, a large number of field isolates from Tunisia and Turkey were analysed (n = 305). This supported the earlier finding that geographical sub-structuring separates panmictic populations and an almost identical amount of genetic differentiation between countries was evident (FST = 0.05). Limited linkage disequilibrium was observed in some populations and this was attributed to several factors including inbreeding and the Wahlund effect, caused by putatively immigrant sub-populations. A similar multiplicity of infection was demonstrated in vaccinated and unvaccinated animals and the immunising genotype did not appear to establish in the field population. Multiplicity of infection was instead shown to positively correlate with the host age in several sampling locations. The genome of T. annulata was compared with that of T. parva to identify gene families under the influence of positive selection using mean family inter-genomic nonsynonymous to synonymous substitution rates (dNdS). Codon usage between the species and between several life-cycle stages within T. annulata was shown to be virtually invariant and independent of the dNdS distribution. In addition to a subset of merozoite genes, which were predicted to be antigens on the basis of their motif signature, a subtelomeric gene family (SVSP) and a family of parasite-encoded host nuclear genes (TashATs) showed evidence of positive selection between the species. An allelesequencing approach was taken to verify these predictions which indicated that, in general, the TashAT genes are under the effect of purifying selection while two SVSP genes were shown to be highly variable, however there was no firm evidence of positive selection. One of the merozoite antigen candidates showed evidence of both positive immune selection and balancing selection. Consequently, further studies are indicated to assess whether this gene has value as a vaccine candidate.

The measurement of acute phase proteins is used by human clinicians to give valuable infonnation about a patient's inflammatory response, both when monitoring clinical disease and when assessing the effect of therapy. Levels of soluble receptors for the cytokine, tumour necrosis factor, also increase as a result of inflammatory stimuli and are useful prognostic markers over the asymptomatic phase of human immunodeficiency virus infection. The aim of the work presented in this thesis was to detennine whether these markers are of value when investigating feline disease. Reference ranges for two acute phase proteins, aI-acid glycoprotein (AGP) and haptoglobin were detennined by measuring their concentrations in serum samples from healthy cats. Analysis of samples from cats with feline infectious peritonitis (FIP) and from cats suffering from conditions with a similar clinical presentation revealed that measurement of AGP can be a useful adjunct to other laboratory tests when reaching a diagnosis. In contrast, measurement of haptoglobin was not found to be of value. Despite increases in the levels of pro-inflammatory cytokines in samples taken from cats during the asymptomatic phase of feline immunodeficiency virus (FIV) infection, no changes were detected in the levels of AGP and haptoglobin. It was concluded that these acute phase proteins are of no benefit as prognostic markers in FlY. The L929 bioassay was used to investigate anti-TNF-a activity in cell culture fluids from feline splenic cells. Cytotoxic activity was demonstrated in very few of the samples whilst anti-cytotoxic activity was detected in the majority of samples. This anti-cytotoxic activity was attributed to the presence of feline soluble TNF receptor type 1 (sTNFR-I) binding to and inhibiting the effects of TNF-a. This was not confinned because of the lack of specific neutralising antibody. Subsequent work was therefore directed towards the development of immune-based species-specific assays for feline soluble TNF receptors (sTNFRs). The polymerase chain reaction was used to amplify the sequences coding for feline sTNFRs. Most of the extracellular domain of feline TNFR-l and part of the intracellular domain of feline TNFR-2 were cloned and sequenced using this technique. The amplified regions demonstrated 85% and 77% homology at the nucleic acid level and 83% and 67% homology at the amino acid level to the corresponding regions of the human sequences for TNFR-l and 2 respectively. Feline sTNFR-l was expressed as a glutathione-S-transferase fusion protein. After purification, concentration and electrophoresis, the appropriate protein band was excised and used to inoculate a sheep. Antiserum taken from the sheep post-inoculation recognised the expressed protein by western blotting, but results were inconsistent and analysis of the antiserum was hampered by the very small amounts of expressed protein available. Two peptides were synthesised based on regions of antigenicity in feline sTNFR-l and were used to inoculate sheep. Antiserum to peptide A showed a strong reaction against peptide A in an ELISA and gave a positive result when used as the primary antibody to stain healthy feline liver tissue. In conclusion, both antiserum to expressed feline sTNFR-l and anti-peptide antibody based on a region of feline sTNFR-l have been raised in sheep and are available for the development of an assay for this protein. Further expression of feline TNFR-l will be required before these antisera can be analysed fully.

The parasitic nematodes Teladorsagia (Ostertagia) circumcincta and Haemmonchus contortus are two of the most important pathogens of sheep and goats worldwide. The purpose of the work described in this thesis was to identify the mechanism of resistance to these parasites in young lambs. Lambs infected with T. circumcincta are incapable of controlling their worm burdens. However, it appears that some are capable of controlling the growth and therefore the fecundity of adult female worms. Work described in chapter three shows that the most important mechanism controlling the growth and fecundity of this parasite is the local IgA response. 933 lambs were studied over 5 years. Faecal egg counts were performed on these lambs and 485 of these lambs were slaughtered and the average female worm lengths determined. Analysis showed a highly significant effect of parasite specific IgA on worm length. Those lambs with higher IgA response to fourth-stage larvae had on average shorter worms. This response was heritable. Thus genetic resistance to T. circumcincta acts by reducing worm fecundity and works through a parasite-specific IgA response. In addition, this response is sex related with male lambs having the poorest response and females the best. Not only is the quantity of IgA important in determining host resistance, but also the specificity. Chapter four details work done in investigating the antigen specificity of the IgA response to T. circumcincta.

Canine herpesvirus 1 (CHV-1) is as yet the only described canine herpesvirus. Only limited information on this virus, in terms of both its molecular biology and pathogenesis, was available at the start of this project. Degenerate primer polymerase chain reaction was used to amplify a small part of the glycoprotein B (gB) gene of CHV-1. This product was cloned and sequenced in order to provide some initial sequence information on the virus, thus assisting in its definitive classification and permitting further study using molecular biology techniques. Conventional primers were then designed from the known gB sequence and used to test a variety of canine tissues for the presence of viral genome. Viral sequences were found in a number of tissues, and it would appear that on the basis of these experiments, the prevalence of CHV-1 is much higher than previously reported. The molecular biology techniques used in the first part of this project were then applied to the study of canine lymphoma, one of the most common neoplasms affecting the dog. Following the success of the degenerate primer system in amplifying a portion of the gB gene of the known canine herpesvirus, a degenerate primary system was designed that was capable of amplifying part of the gB gene of any known gammaherpesvirus. This system was used to test tissues from a number of cases of canine lymphoma for the presence of gammaherpesvirus sequences. Representation difference analysis was also used to analyse cases of canine lymphoma for genetic differences from normal tissue. A number of differences were identified using this technique; their significance, and avenues of further study, are discussed.