Lymphatic filariasis is a long term chronic infection characterised by a Th2 dominated immune response and suppressed Ag-specific proliferation. This immunological hyporesponsiveness is most profound amongst individuals with circulating microfilariae (mf) suggesting an important role for mf in generating proliferative suppression. The use of single life cycle stage infections in murine models of filariasis has facilitated the study of stage specific mechanisms of immunomodulation. Intravenous infection of BALB/c mice with <i>B. pahangi</i> mf or L3 (the third stage larvae) leads to development of differentially polarised immune responses. At 12 d.p.i. splenocytes from L3 infected animals produce Ag-specific IL-4, IL-5 and IL-10 and show strong Ag-driven proliferative responses. In contrast splenocytes from mf-infected animals show a cytokine profile dominated by IFN-gamma and suppression of Ag-specific proliferation. After 96 hrs of Ag-stimulated culture splenocytes from mf-infected animals proliferate at levels below background indicating that an active form of suppression is operable <i>in vitro</i>. A lack of IL-2 does not account for the defective proliferative response as addition of recombinant IL-2 failed to restore Ag-specific proliferation. Splenocytes from mf-infected animals produce high levels of NO in Ag-stimulated culture which correlates inversely with their proliferative responses. No such accumulation of nitrite is seen in cultures of cells from L3 infected animals. The proliferative defect is dependent upon inducible nitric oxide synthase (iNOS) activity as inhibition of iNOS activity with either L-NMMA or AMG restored Ag-specific proliferation.

Gastrointestinal parasites infect all grazing livestock and are a major cause of economic loss. Each year in the UK, gastrointestinal parasites cost the sheep industry an estimated £85 million. Current control strategies are based on anthelmintic treatment; however parasite resistance to anthelmintic compounds is becoming an ever-increasing problem worldwide. Thus alternative control measures are now sought. This thesis comprises a number of studies which aim to investigate the genetic control of several aspects of the host response, and thus the application of such knowledge to develop alternative control strategies for gastrointestinal parasites in a commercial sheep population. Analysis of data from 6-month old Scottish Blackface lambs exposed to a mixed, natural nematode infection demonstrated that the indicator traits, faecal egg counts (FEC), immunoglobulin A activity, eosinophil count, plasma pepsinogen activity and fructosamine concentration, investigated at 6 months of age were highly heritable and strongly correlated with the worm development traits. For example at a mean age of 22 weeks the heritabilities (± SE) for fructosamine concentration, IgA activity, eosinophil count and pepsinogen activity were 0.39 ± 0.16, 0.57 ± 0.15, 0.35 ± 0.15 and 0.56 ± 0.16 respectively. Strong negative genetic correlations «-0.6) were often observed between worm development traits and eosinophil count, IgA activity and pepsinogen activity. A substantial genetic correlation was also observed between fructosamine concentration and worm length (0.67). However when such correlations were investigated across the 6-month time-period, the genetic correlations changed systematically and dramatically over time. For example, for all worm development traits, genetic correlations with eosinophil count were initially positive and moderate to strong, and then declined dramatically eventually becoming moderate to strong and negative at 5 months of age. These results provide an insight into the evolution of the genetic basis of the host parasite interaction at a time when the host immune response is developing, and help to define optimal measurement ages for selection purposes. Two quantitative trait loci (QTL) studies were carried out on populations comprising different breeds and population structure; firstly a purebred Scottish Blackface flock and secondly a wide-breed cross flock developed from aresistant breed, Gulf Coast Native, and a susceptible breed, Suffolk. Both studies identified OTL associated with parasitic resistance traits, and although there is no concordance between the results, this is possibly due to the animals being infected with different nematode species. In the Blackface study OTL associated with specific IgA activity were identified in chromosomes 3 and 20, in regions close to IFNG (chromosome 3) and the MHC (chromosome 20). OTL associated with Nematodirus FEC were identified on chromosomes 2, 3 and 14 and OTL associated with non-Nematodirus Strongyle FEC were identified on chromosomes 3 and 20. In the Suffolk x Gulf Coast Native study OTL associated with packed cell volume (PCV) were identified on chromosomes 1, 9 and 19 and with FEC on chromosomes 1, 6 and 19. OTL such as those identified in this thesis could be utilised in a marker assisted selection scheme to increase resistance to parasitic infection. In the final study interactions between different parasite species within the host animal were investigated. Significant interactions were observed between Cooperia and Te/adorsagia circumcincta, and T. circumcincta and Trichostrongylus vitrinus. Additionally Cooperia had a greater effect on FEC than T. circumcincta. The results from this study indicate that complex multi-parasitic relationships exist and hence when developing new control strategies it is essential to consider this background multi-parasitic infection and not simply focus on a specific species. In conclusion this thesis provides evidence that many aspects of the host response are under some level of genetic control. Highly heritable indicator traits have been identified along with OTL associated with resistance traits, both of which could be utilised as selection criteria to increase the response to selection for resistance to gastrointestinal parasites within a commercial sheep population. 14

A mathematical approach was used to model seasonal variation of cyathostomin faecal egg output, and in simulating anthelmintic control strategy. The model is based on parameters of biological development of cyathostomins and climatic data. A good fit of the model prediction to the field data was obtained after some parameter adjustments. The development rate of ingested larvae to egg laying adults, survival time of adults and the assumption made in modelling the peak pasture larval availability were the main driving forces for the model prediction to fit to the observed data. The apparent fit of the model prediction to the field data obtained after parameter adjustment generally indicated some major differences between donkeys and horse in their reaction to the parasite and/or between cyathostomins of donkeys and horses. The results of the stimulation of the effect of various protocols for the timing and frequency of anthelmintic treatment on the adult cyathostomin worm burden have shown that treating donkeys only once in a year or a combination of once in a year followed by every two or even four years can substantially reduces and maintains the parasite burden far below the pre-treatment level for many years. Generally the study made has revealed that the non-strongyle gastrointestinal parasites of donkeys are highly prevalent and have a high pathogenic potential, and the findings of cestodes and trematodes are not accidental or unusual, as previously suggested.

Recognition, alleviation and control of pain are central to ensuring good welfare in food producing animals. An intervention trial was conducted on 117 dairy cows with naturally-occurring, mild or moderate, clinical mastitis in a single quarter in order to investigate the effect of the administration of the non-steroidal anti-inflammatory drug, meloxicam (Metacam, Boehringer Ingelheim Animal Health GmbH) used in conjunction with an intramammary antibiotic infusion of cefquinome (Cephaguard LC Intramammary, Intervet UK Limited, Milton Keynes). Clinical and laboratory parameters were investigated to assess their usefulness as objective markers of pain. Measuring the cows’ response to a mechanical stimulus assessed altered pain processing. Heart rates, respiratory rates and rectal temperatures of cows were higher in moderate cases compared to milk cases of clinical mastitis (p<0.001). The hindleg stance of cows, as measured by hock-to-hock distance, with clinical mastitis was greater than in normal cows (p<0.001). Alterations in the response to mechanical stimulation were recorded in cows with both mild and moderate clinical mastitis and treatment with meloxicam was shown to attenuate the mechanical alterations (p=0.04). There was however, no difference in cows that received one, compared to three doses of meloxicam (p>0.05). On bacteriological examination to the recruited cases, 30% of isolates yielded S uberis, 24% yielded no growth, 22% E coli, 11% Staph aureus, six per cent other microbiological species, four per cent A pyogenes and three per cent S dysgalactiae. When compared with mild cases of mastitis the moderate cases had a greater proportion of isolations of S uberis (p<0.01) and a smaller proportion of no growths (p<0.01). There was no effect of mastitis on the fat percentage of milk, as judged by the comparison of affected quarters to diagonally opposite quarters and to normal cows. The protein content of milk increased with mastitis and the lactose content decreased. The most common somatic cell type in quarters affected with mild and moderate mastitis on the day of recruitment to the study were polymorphonuclear cells, whereas large mononuclear cells were the predominant cell type in normal animals. The concentration of haptoglobin in milk from the affected quarters of cows with moderate mastitis was greater (p<0.01) than that in milk from affected quarter of cows with mild mastitis. Mammary-associated serum amyloid A concentrations were higher in the affected quarter of cases of milk and moderate clinical mastitis than in normal cows (p<0.001). Concentrations of the inflammatory mediator, prostaglandin E2 in milk were lower in mild cases of clinical mastitis compared to moderate cases (p<0.05). In conclusion, physiological and laboratory parameters may serve as indicators of clinical mastitis, help differentiate between different severities of clinical mastitis and, therefore, aid in appropriate targeting of therapy.

The effects of thermal environments from 20oC. to 40oC. at high and low humidities on the physiological reactions of young calves.

This research applied the psychometric approaches of proxy human chronic pain and HRQL instrument development to the development of an instrument to measure chronic pain in the dog, using the owner to provide a proxy report. The development of the instrument followed established steps designed to ensure an instrument’s validity: the identification of all domains relevant to the measurement of interest; generation of a pool of potential instrument items; selection of instrument items from the item pool, and validation of that selection; design and pre-testing of the prototype instrument; field-testing of the instrument to establish its psychometric properties. Domain identification was carried out through interviews with 47 owners of dogs suffering chronic pain. Potential items (descriptive terms) were generated using questionnaires completed by 165 dog owners. These domains and the items selected were subsequently validated by 12 veterinary practitioners and by 10 owners of dogs suffering chronic pain. The validated list of items was incorporated into a structured questionnaire, and this instrument was pre-tested using 26 dog owners. The finished instrument was then field-tested using the owners of 155 dogs who completed a total of 390 questionnaires prior to and during treatment at the University of Glasgow Small Animal Hospital and at a local Veterinary Practice, a majority of which dogs were suffering from chronic degenerative joint disease (DJD). Factor analysis of the instrument responses for dogs suffering DJD revealed an interpretable 12-factor model, in which factors were interpreted as domains of canine HRQL: ‘vitality’, ‘physical limitation’, ‘lethargy’, ‘anxiety’, ‘aggression’, ‘emotional upset’, ‘appetite’, ‘consistency of behaviour’, ‘mental disturbance’, ‘attention-seeking’, ‘sadness’ and ‘acceptance’. This analysis provided evidence for the construct (factorial) validity of the instrument, since responses to instrument items revealed an underlying structure that reflected the construct upon which the instrument was developed. Scores were calculated for each of the 12 domains of HRQL identified by the factor analysis, and these were able to discriminate between dogs with chronic pain and healthy dogs on >86% of occasions. Profiles of HRQL scores obtained for dogs with chronic pain were compared with those obtained for healthy dogs in a control group, and differences in these profiles were observed. An examination of changes in HRQL domain scores over time for individual dogs revealed that these scores tended to reflect clinical change in those individuals. The process described here offers a novel approach to the development of chronic pain and HRQL instruments for a range of animal species, and may have relevance for human chronic pain and HRQL instrument development.

The area of the skull incorporating the tympanic bulla (TB) and temporomandibular joint (TMJ) is significant clinically in the dog, cat, and more recently the rabbit. Diagnostic imaging is important in the assessment of disease of these structures but there is a relative lack of comparative anatomical information relating to the normal that may be used to understand the abnormal features encountered when using currently available diagnostic imaging modalities. A review of conventional radiography demonstrated that views for imaging the canine and feline TB could be extrapolated for use in the rabbit but the same did not apply to the TMJ. Plastinated multiplanar anatomical sections proved useful for the identification of anatomical features on corresponding tomographic images. Ultrasound imaging of this region has not been widely reported but allowed evaluation of the TB in all three species, although the information obtained regarding the TMJ was limited. Directly acquired computed tomography (CT) and Magnetic Resonance (MR) images were of better quality than previous publications due to technological advances in the equipment available. Directly acquired images were still better than reconstructed ones and reduced image acquisition times are likely to make this viable in clincal cases. CT produced optimal imaging of the TB but only allowed assessment of the bony elements of the TMJ. Little information was obtained regarding the normal TB using MR imaging due to the indistinguishable signal voids produced by the bone wall and gas lumen. However, T1 weighted sequences allowed identification of intra-articular TMJ soft tissue structures in the dog and rabbit. While opening the mouth altered the areas of the TMJ examined using each modality, it did not improve visualisation of the intra-articular structures. The introduction of fluid into the middle ear cavity of dog, cat and rabbit cadavers aided identification of the TB and acted as a model of one of the major features of acute otitis media, or inflammation of the middle ear cavity. CT was most accurate at identifying middle ear material in cadavers and clinical cases, while ultrasound produced better results than radiography in cadavers but not clinical cases. These imaging modalities also proved useful in the characterisation of the unexpected anatomical anomalies that were encountered during the study. The results of this study indicate that the optimal imaging technique will vary with the species and area being examined, and that extrapolation between species is not always appropriate. Continual improvements in technology and image quality make studies such as this necessary to allow selection of the most appropriate single or combination of imaging techniques and to obtain the maximum amount of information from the resulting images.

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.