Parasitic nematode infections worldwide cause a significant impact on human health, as well as economic and welfare losses to the animal and agriculture industries. The principal method of control for parasitic nematode infections is currently limited to repeated treatments with anthelmintic drugs, but widespread resistance to all major classes of these drugs is a growing problem. As a result, there is an urgent need for alternative methods of controlling these infections, and the development of molecular vaccines and novel drugs represent two possible approaches. However, both these approaches require a deeper understanding of gene function in order to identify suitable control targets. This project examines RNA interference (RNAi) in the parasitic nematode Haemonchus contortus to determine if this could be developed as a functional tool and advance the discovery of novel control targets for parasitic nematodes. RNAi has proven less effective in parasitic nematodes than in the free-living model nematode Caenorhabditis elegans and it is unclear why this is so. This project examined the reliability of RNAi in H. contortus, and several genes were successfully silenced using RNAi. Further analysis of RNAi susceptible genes revealed that RNAi silencing appears to be related to the site of expression of the target gene; genes expressed in tissues which are accessible to the environment such as intestine, excretory cell and amphids were silenced by RNAi. Upstream promoter regions of RNAi susceptible genes were examined for the presence of motifs which may regulate spatial gene expression, an approach that could be used to predict gene susceptibility to RNAi. RNAi treated larvae were subsequently used to infect sheep in the first in vivo RNAi study, resulting in a significant impact on worm survival in vivo. In addition, several components of the RNAi pathway in H. contortus were characterised in this project, demonstrating the presence of a functional RNAi pathway that is capable of reliably silencing genes. In conclusion, the findings presented in this project suggest that RNAi may be used in the future to evaluate the function of a novel vaccine or drug target for controlling H. contortus infections in sheep.

Master of Science / Department of Clinical Sciences / Walter C. Renberg / Objective: To compare the in vitro elution characteristics of six common analgesic medications from a commercially available absorbable gelatin sponge. Study Design: Experimental study. Methods: Gelatin sponges were loaded with various analgesic medications, including two opioids, preservative-free morphine and fentanyl, two local anesthestics, bupivacaine and lidocaine, and two α2-adrenergic agonists, dexmedetomidine and xylazine. The loaded sponges were placed in dishes containing phosphate-buffered saline (PBS) and maintained at 37° C with constant agitation. Concentrations of each drug were determined at various time points up to 24 hours post-inoculation using high-pressure liquid chromatography. Two phases were conducted, utilizing undried loaded sponges (phase one) and dried loaded sponges (phase two). Results: In both phases, all analgesic medications eluted from the gelatin sponge and equilibrated rapidly with the PBS, achieving maximal concentration within 30 minutes. In phase two, the rapid nature of the release was captured by increasing sampling within the initial 30 minutes. Results were consistent for each medication with minimal variation. Steady state concentrations were significantly higher in phase two with four out of six medications. Conclusions: Analgesic medication elution from an absorbable gelatin sponge was rapid and consistent. Drying the loaded sponge prior to use will likely substantially increase the amount of medication eluted but not prolong release. Clinical Relevance: The rapid release of analgesic medications from the gelatin sponge makes a prolonged analgesic effect unlikely without further modification. Toxicity may be a concern. Further study is required to investigate efficacy in vivo, safe dosing regimens and prolongation of duration of action.

Call number: LD2668 .T4 1957 A58 / Master of Science

Call number: LD2668 .T4 1960 A23

Thesis (M.Tech.: Homoeopathy) -Dept. of Homoeopathy, Durban Institute of Technology, 2003 xi, 59 leaves / The purpose of this placebo-controlled study was to evaluate the effect of a Homoeopathic complex (Aconitum napelus 30CH, Arnica montana 30CH and China officinalis 30CH) on broiler chickens being transported to the abattoir.

Musculoskeletal conditions and exercise-induced pulmonary haemorrhage are commonly diagnosed in Thoroughbred racehorses worldwide, and have serious consequences for racehorse welfare and the racing economy. Despite increasing interest in the study of genetic susceptibility to disease from the veterinary research community as a whole over past decades, the Thoroughbred has been largely ignored as a study group. The availability of software capable of complex genetic analyses using large, unbalanced pedigrees has made the study of genetic susceptibility to disease a realistic prospect for veterinary researchers. This study aimed to complete preliminary analyses of the genetics of a number of important musculoskeletal conditions, and of exercise-induced pulmonary haemorrhage, in two different Thoroughbred populations. Multivariable regression analyses were performed to identify important environmental risk factors for each condition in each population, and heritability analyses were conducted. Genetic correlations between disease conditions were also investigated. Fracture, tendon injury, suspensory ligament injury, osteoarthritis and EIPH/epistaxis were found to be heritable traits in the Hong Kong population. Distal limb fracture, SDFT injury and epistaxis were also found to be heritable in the UK Thoroughbred population. Most heritability estimates were small or moderate in magnitude. Selective breeding strategies that identify those animals with low genetic risk could play a part in future efforts to reduce the incidence of these conditions, in conjunction with favourable environmental manipulations based on research evidence. Due to low heritability, most of the conditions studied here would reduce in incidence slowly if selective breeding were implemented, thus strategic environmental manipulations would be warranted alongside such longer-term efforts to provide effective incidence reductions. A number of conditions were found to be positively genetically correlated, suggesting that risk reduction through breeding could reduce the risk of multiple diseases concurrently. For example, fracture and osteoarthritis were found to be positively genetically correlated (0.85 – 0.89) in the Hong Kong racehorse population. However, using the Hong Kong Thoroughbred population dataset, EIPH/epistaxis and tendon injury were negatively genetically correlated, which suggests that reduction in genetic risk of one of these may lead to increased genetic risk of the other. iii Measures of the durability and performance of racehorses were investigated to assess whether they were heritable traits in the UK and Hong Kong racehorse populations, and to assess their relationship to the disease conditions studied. Selection based on more holistic measures of horse health and longevity such as ‘career length’ could be a more attractive prospect for stakeholders, as this could forego the need to select for many different traits individually. Career length, number of starts over the career, and the level of earnings were all heritable traits in both populations. These holistic traits were found to have variable relationships with the disease conditions studied in each population. These analyses are the first to assess the genetic contribution to risk for many important diseases in the Thoroughbred. They provide a starting point from which further investigations into the applicability of genetic manipulations could yield realistic and achievable tools for racing stakeholders to use to ‘improve’ the breed in future.

Physical inactivity is a major contributor to non-communicable diseases and many adults and children are insufficiently active to maintain good health. The proportion of children who meet the United Kingdom recommendations for physical activity (at least 60 minutes of moderate-vigorous intensity physical activity each day) has been reported to be as low as 3% for boys and 2% for girls. Systematic reviews on interventions to promote physical activity in childhood have shown that although physical activity is modifiable to some degree most interventions have had only modest and short-term impacts on physical activity. Therefore, novel approaches to physical activity promotion in childhood are required. Dog ownership is a significant societal factor that may be used to encourage and sustain health behaviour change at individual and population levels. A number of observational studies have reported that dog ownership and/or dog walking are associated with increased levels of physical activity. However, evidence is lacking as to whether and how interventions with families and their dogs can be used to promote physical activity. Therefore, the major aim of this thesis was to assess the feasibility, acceptability and potential efficacy of a theory-driven, family-based, dog walking intervention for 9–11 year old children and their families. However, prior to this it was essential to develop ActiGraph cut-points for measuring physical activity intensity in dogs. The ability to measure the intensity of dog physical activity accurately was important as it allows for the effectiveness of dog walking interventions to be tested, therefore another aim of this thesis was to calibrate and cross-validate ActiGraph cut points that can be used to describe physical activity in dogs by intensity. Similarly, no studies have been published previously that assess which factors are related to dog physical activity when measured using ActiGraph accelerometry. It was therefore desirable to explore whether body condition score, breed, age, and neutered status are associated with ActiGraph measured dog physical activity. Furthermore, no published studies have described the spontaneous changes in dog physical activity during substantial weight loss; therefore, another aim of this thesis was to explore changes in physical activity in dogs during a 6 month calorie controlled weight loss programme. Using Receiver Operating Curve analyses Chapter 2 showed that the ActiGraph GT3X can accurately measure the amount of time a dog spends sedentary, in light-moderate intensity physical activity and in vigorous intensity physical activity. The sensitivity and specificity of the cut-points developed when using both the integrated axes and vertical axis accelerometry data were high. Agreement between the accelerometer data and direct observation in the cross-validation subset was also ‘very good’ (as measured by Cohen’s Kappa). This indicates that the ActiGraph GT3X accelerometer is accurate when measuring the intensity of physical activity in dogs, facilitating the use of the ActiGraph GT3X to describe the frequency, intensity and duration of dog physical activity in Chapters 3-6 of this thesis. Chapter 3 shows that, in a sample of dogs of varying breed and body condition scores, obese dogs spend significantly less time in ActiGraph measured vigorous intensity physical activity than ideal weight dogs (6 ± 3minutes/day versus 20 ± 14 minutes/day). Chapter 4 focussed on the factors related with physical activity in the two most commonly registered dog breeds in the United Kingdom, Labrador Retrievers and Cocker Spaniels. Five potential correlates (age, sex, breed, neuter status, body condition score) were tested with associations with ActiGraph measured physical activity. Age and breed were associated with total volume of physical activity, light-moderate intensity physical activity and sedentary behaviour in the final models and age was also associated with vigorous intensity physical activity. Unlike Chapter 3 body condition score was not related with any physical activity variables. Chapter 5 explored the changes in physical activity and sedentary time during weight loss in dogs enrolled in a 6 month calorie controlled weight loss programme. Despite an average weight loss of 15% body weight from baseline there was no marked increase in any ActiGraph measured physical activity variable. Chapter 6 describes the results of the Children Parents and Pets Exercising Together (CPET) Study. CPET was the first exploratory randomised controlled trial to develop and evaluate an intervention aimed at dog-based physical activity promotion in children, their parents and pet dogs. The results show that the CPET intervention was both feasible and acceptable to study participants. Eighty-nine percent of families enrolled in CPET were retained at follow up. Ninety-five percent of intervention sessions were delivered and ActiGraph measured physical activity data were collected for 100% of children, 96% of parents and 96% of dogs at baseline, and 100% of children, 96% of parents and 96% of dogs available at follow up. Despite the apparent feasibility and acceptability of CPET there was no significant change in the primary outcome measure (child physical activity) or the majority of the secondary measures. This thesis shows that the ActiGraph GT3X accelerometer is capable of accurately measuring the intensity of dog physical activity. It also shows that obesity may be related to lower levels of objectively measured vigorous intensity physical activity and the physical activity levels in dogs decline with age and vary by breed. However, it appears that physical activity levels do not increase spontaneously as dogs lose substantial amounts of body weight. Using pet dogs as the agent of lifestyle change in physical activity interventions in children and their parents is both feasible and acceptable; however, the lack of any apparent increase in child physical activity suggests that the intervention may need to be modified in a future, more definitive trial. In summary, the findings of this thesis have important implications for the measurement of physical activity intensity in dogs, the understanding of factors associated with dog physical activity and for the development of dog-walking interventions in children and their parents.

The molecular evolution of feline calicivirus (FCV) was studied in cell culture and in persistently infected cats. Sequence analysis of the 5' hypervariable region of the FCV capsid (5'HVR; located at the 5'end of variable region E), a region known to contain linear neutralising B cell epitopes, showed FCV existed as a quasispecies which evolved at the nucleotide and amino acid level during persistent infection. Quasispecies heterogeneity tended to decrease during the course of persistence. Sequential isolates from a cat showed marked antigenic variation during the course of persistent infection. Sequential passage of FCV in cell culture was also associated with sequence evolution of the 5'HVR. However, these isolates showed no change in antigenicity suggesting that individual substitutions observed in viruses from cats, but not in viruses from cell culture, may be responsible for changes in antigenicity. Alternatively, the observed antigenic changes may be associated with mutations elsewhere in the genome. In order to identify regions of 'the FCV capsid protein containing linear B-cell epitopes, two approachesw ere used.F irstly, an expressionl ibrary containingr andom, short (100- 300bp) fragments of an FCV capsid gene was constructed. This library was screened using polyclonal antisera from a cat that had been challenged experimentally with FCV to identify immunoreactive clones containing B-cell epitopes. Initial screening identified five clones that reacted positively to feline antisera in immunoblots. FCV derived sequencefr om thesec lones all mappedt o the 5'HVR suggestingt his region containst he immunodominant linear epitopes of the capsid. The second approach used to identify B-cell epitopes was to map more accurately the epitope of a neutralising monoclonal antibody (IG9) which had already been shown to lie in a 37 amino acid region of the 5'HVR (Milton et al. (1992), Journal of General Virology 73,2435-2439). Replication of plaque purified IG9-sensitive parent virus in sub-neutralising concentrations of IG9 led to the generation of a neutralisation resistant escape mutant. Sequence analysis of this mutant and the parent virus revealed a single non-synonymous nucleotide substitution within the 5'HVR suggesting this residue is critical to the correct formation of the IG9 epitope. A method to type FCVs based on sequence analysis of the 5'HVR was established. Most isolates appeared relatively homogenous. However, some isolates, both from vaccines and vaccine failures, appeared to contain more than one FCV. Comparison of 5'HVR sequencesfr om different isolatess howed that most isolates were either 0-5.3% different (related isolates) or 20.7-42.7% different (unrelated isolates). The majority of the relatedi solatess hareda n epidemiologicall ink, implying they representedis olatest hat originated from a common source. Comparisons of sequences obtained from vaccine failures and vaccine virus fell into two similar categories; those with closely related sequences(0 .0-5.3%) implying a role for the vaccinei n diseasea nd those with divergent sequences(2 1.3-38.7%)i mplying field virus causedt he disease. These results were compared with those obtained by using a serotyping method based on virus neutralisation (VN) which exploits differences in antigenicity between most FCVs gene (Dawson et al. (1993), Veterinary Record 132,346-350). VN and sequence analysis gave the same typing result in 65-73% of individual cases. Based on these results and the difficulty of interpreting VNs, we suggest that molecular based sequence analysis may be more suitable to the epidemiological investigation of FCV related disease particularly in the case of vaccine reactions.

Streptococcus uberis is an important bovine mastitis pathogen, which places a significant financial burden upon the dairy industry. Determining the genetic diversity of a collection of field isolates and the mechanisms by which S. uberis colonises the host were the general aims of this project, in particular the determination of the basis for bacterial persistence despite antibacterial therapy. Multi-locus sequence typing identified high levels of recombination within the population, but also a single dominant clonal complex which comprised nearly all sequence types which were isolated from more than one animal. The dominant clonal complex also comprised isolates, derived, however, from both persistent and non-persistent infections, but RAPD typing demonstrated that these isolates can differ in genetic composition elsewhere in the genome. Whole genome sequencing of additional S. uberis isolates confirmed that despite significant homology between much of these genomes, novel genetic material was commonly obtained by phage insertion and horizontal gene transfer. Isolates with identical housekeeping sequences are thus highly likely to differ in their virulence gene repertoires. In this study, the potential for differentiating S. uberis isolates based instead upon protein profiles derived from mass spectrometry of disrupted whole cells was therefore also explored. Differentiation between small numbers of isolates was achieved after optimisation of this protocol, however, discriminatory ability and reproducibility were somewhat compromised when the technique was scaled up to analyse 50 Italian isolates. During the period of study, profile differences between persistent and non-persistent isolates could not be explored. Basic methods were thus also utilised in an attempt to identify factors which promoted bacterial survival in vitro; and a defined medium, representative of the udder environment, was optimised for this purpose. The use of this medium permitted the demonstration that S. uberis was reliant upon magnesium and manganese for proliferation and that, interestingly, the absence of iron did not inhibit bacterial growth. It was also shown that S. uberis had the ability to directly utilise casein, identifying a potential alternative pathway for the acquisition of essential nutrients from nutritionally-limited environments. It was also observed that to a limited extent S. uberis seemed to produce a siderophore. Although this remains to be confirmed, it may correlate with the observation that iron, although not essential for proliferation, improved the growth rate of the bacterium. It was also notable that most novel genes, identified from S. uberis genome sequences, exhibited functions for nutrient metabolism, demonstrating that flexibility in nutrient acquisition is central to the ability of the bacteria to adapt, permitting survival in vastly different environments. The use of the defined medium also demonstrated that S. uberis was able to form biofilms; this ability being variable depending on the growth conditions used and the isolate studied. Most significantly, under conditions representative of the mammary gland, there was an apparent trend for high levels of biofilm formation to correlate with isolates from persistent infections. Biofilm formation by Staphylococcus aureus is considered to be pivotal to the development of chronic mastitis, thus, biofilms may similarly play a role in S. uberis persistence. In an attempt to identify the molecular basis for S. uberis biofilm formation, genes with homology to those of the intercellular adhesion (ica) operon, well described for their involvement in Staphylococcus epidermidis and S. aureus biofilm formation, were identified in the genome sequence of S. uberis 0140J. A targeted mutagenesis protocol was optimised to ‘knock out’ these genes and observe the subsequent effects of these mutations on biofilm formation. During the course of this study, two of these potential biofilm genes (hasA and SUB 0809) were deleted from the S. uberis 0140J chromosome. Surprisingly, deletion of these genes did not retard subsequent biofilm formation, but instead biofilm formation was dramatically improved in the mutant strains. Characterisation of mastitis-causing S. uberis strains and a detailed understanding of the pathogenicity of the organism are required to further the development of a successful vaccine. The research presented in this thesis has increased the knowledge of these important research objectives and optimised techniques which will allow further advancement of knowledge in this field.

Human African trypanosomiasis (HAT) is a parasitic disease caused by the protozoan parasites T. b. rhodesiense and T. b. gambiense. The disease is currently endemic in 36 sub-Saharan countries with an estimated 60 million people at risk from the infection. The disease progresses through two stages; an early or haemolymphatic stage where the parasites are confined to the peripheral compartment and a late or encephalitic stage where the parasites penetrate the blood-brain barrier (BBB) and invade the CNS. Without treatment the disease is invariably fatal but at present chemotherapy is reliant on a small handful of drugs. Pentamidine and suramin are available for the treatment of the early stage of the disease while the CNS stage of the disease is treated with a combination of nifurtimox and eflornithine known as NECT therapy or melarsoprol. NECT therapy is only effective in the treatment of T. b. gambiense infections meaning treatment of T. b. rhodesiense infections is completely dependant on the trivalent arsenical melarsoprol. Melarsoprol is an extremely toxic compound, the administration of which is very painful and associated with numerous adverse reactions. The most series of which is a post treatment reactive encephalopy (PTRE). The PTRE occurs in up to 10% of all patients given melarsoprol of which 50% die as a result of the complication. This gives melarsoprol an overall fatality rate of 5% which is unacceptably high. There is therefore an urgent need for new trypanocides, which are safe and easily administrable. To improve the physiochemical and pharmacokinetic properties of melarsoprol the drug was complexed with two cyclodextrin molecules, hydroxypropyl-cyclodextrin (HPCD) and randomly methylated-cyclodextrin (RAMCD) to produce; mel/HPCD and mel/RAMCD. Cyclodextrins are cyclic oligosaccharides, widely used within the pharmaceutical industry to improve the solubility and oral bioavailability of poorly soluble lipophilic drugs. In this study, the trypanocidal activity of the melarsoprol cyclodextrin complexes was investigated in-vitro and in an in-vivo CNS stage model of T. b. brucei infection. The trypanocidal activity of melarsoprol is retained following its complexation with HPCD and RAMCD. The in-vitro trypanocidal activity of the melarsoprol cyclodextrin complexes against bloodstream T. b. brucei trypanosomes was comparable to that of contemporary melarsoprol. Furthermore, in an in-vivo murine model of CNS stage T. b. brucei the melarsoprol cyclodextrin complexes, mel/HPCD and mel/RAMCD produced 100% cure rates when administered orally at a dose of 0.05mmol/kg, daily, for seven consecutive days. Contemporary melarsoprol when administered by the same route and schedule only cured 33.3% of the animals. The cyclodextrins HPCD and RAMCD thus increase the oral bioavailability of melarsoprol whilst retaining the compounds trypanocidal activity. An oral administrable, water soluble formulation of melarsoprol instantly eliminates the problems associated with the intravenous administration of conventional melarsoprol. Furthermore, an orally available formulation would be of great benefit in the resource poor, isolated settings in which HAT occurs, as patients would not require hospitalisation during treatment thus alleviating the pressure on local hospitals. In the current investigation quantitative taqman PCR was utilised to investigate the rate of parasite clearance from the CNS during complexed melarsoprol treatment. Both mel/HPCD and mel/RAMCD were rapidly trypanocidal. Twenty-four hours after administration of one dose the number of trypanosomes within the brain was reduced by greater than 80% and all trypanosomes were eliminated from the brain by twenty-four hours after administration of four doses of mel/HPCD and five doses of mel/RAMCD. The elimination of all trypanosomes from the CNS following four doses of mel/HPCD and five doses of mel/RAMCD, indicates that it may be possible to reduce the dosage schedule from seven daily doses to four daily doses of mel/HPCD and five doses of mel/RAMCD. A short, simple, easily administrable treatment protocol is an essential requirement of any new trypanocide as if the treatment schedule is prolonged and complicated patients are unlikely to comply. CNS stage trypanosome infection is associated with a breakdown of the blood-brain barrier (BBB). Ideally following successful chemotherapy BBB function should be restored. In this investigation the effect of curative mel/HPCD treatment on the BBB was investigated in a murine model of CNS T. b. brucei infection using small bore MRI analysis. Mel/HPCD treatment results in a rapid restoration of BBB function as by twenty-four hours after the completion of mel/HPCD therapy the integrity of the BBB was fully restored. However, a very mild neuroinflammatory reaction persisted in the brain for up to fifteen days after completion of chemotherapy. This suggests that the BBB damage observed in trypanosome infection may be due to either the parasites directly or their secretory products and not as a result of the ongoing neuroinflammatory reaction. Despite melarsoprol being in use for over 60 years its pharmacokinetics are poorly understood and a sensitive assay by which to quantify the concentration of arsenic reaching tissues following administration of the compound is not available. In this study a gas chromatography mass spectrometry (GC-MS) technique was developed to quantify the concentration of arsenic reaching the plasma and brain following oral and intravenous administration of the melarsoprol cyclodextrin complexes, mel/HPCD and mel/RAMCD. The GC-MS assay had a limit of detection of 5ng/ml and a precision (expressed as the inter-day coefficient of variation) of 13.2%. The concentration of arsenic within the brain following the oral and intravenous administration of mel/HPCD was below the limit of quantification of the assay. The pharmacokinetics of mel/HPCD and mel/RAMCD could therefore not be determined in the present study. This study demonstrates that the melarsoprol cyclodextrin complexes mel/HPCD and mel/RAMCD are highly trypanocidal with no overt signs of toxicity and more importantly orally available. Following the oral administration of mel/HPCD or mel/RAMCD the melarsoprol is slowly released over a prolonged period of time from the cyclodextrin cavity. Patients are therefore not exposed to a ‘bolus’ of the drug as is the case in the intravenous administration of contemporary melarsoprol. The slow and sustained release of melarsoprol from the cyclodextrins should result in less adverse reactions and a decreased incidence of the PTRE. Furthermore, the complexed melarsoprol treatment protocol is shorter than the currently used 10 day concise melarsoprol treatment schedule therefore the total amount of melarsoprol administered to patients will be reduced. Patients should therefore experience fewer adverse reactions. In conclusion the results from this study demonstrate that the melarsoprol cyclodextrin complexes mel/HPCD and mel/RAMCD are promising oral candidates for the treatment of HAT.