The project had three aims: namely: - (i) to investigate the in vitro cell tropism of a range of field isolates from cats at different stages of disease and compare their phenotype with the well-characterised prototype viruses FIV-PET and FIV-GL8. (ii) to study the pathogenicity of these viruses in vivo in order to examine any correlation between virulence in vivo and tropism in vitro. (iii) to look at the role of the env gene in the pathogenicity of FIV. In vitro studies of cell tropism revealed that isolates from cats in the terminal stage of disease had a greater ability to utilise CXCR4 than isolates from cats displaying no clinical signs. In vivo, these symptomatic isolates, with greater CXCR4-tropism in vitro, displayed less virulence when compared with isolates from asymptomatic cats. Chimaeras were made by inserting the env genes of an isolate from the asymptomatic or terminal disease stages into a FIV-G8Mya backbone, allowing comparison of the cell tropism and receptor usage of these genes and the study of their phenotype with regard to virulence in vivo. The env genes from FIV-PET and the symptomatic isolate (F0827Hs) had a greater affinity to utilise CXCR4 for cell entry in vitro and this correlated with reduced virulence in vivo when compared to the asymptomatic isolate env and FIV-G8Mya. These studies highlight a trend where tropism in vitro can be correlated with virulence in vivo. Furthermore, the study indicated that viruses from asymptomatic cats (with a lesser ability to utilise CXCR4) have increased virulence. As these are the agents most likely to be transmitted in the field by the apparently healthy cat, vaccine development should focus on this population of viruses.

No description available.

The aims of this study were to assess community knowledge, awareness and practices on zoonoses, to gather baseline data on brucellosis in livestock and wildlife, to establish brucellosis seroprevalence in domestic ruminants and humans and risk factors associated with livestock seropositivity, to assess brucellosis dynamics and impact on livestock production and reproduction and to evaluate the performance of the Rose Bengal Plate Test (RBPT) in Tanzania. The results described in this study were carried out through Participatory Rural Appraisal (PRA), cross-sectional and longitudinal studies. In the PRA and cross-sectional studies, rabies, tuberculosis, anthrax and brucellosis were the zoonoses most frequently identified. Cattle were frequently identified as being associated with tuberculosis, anthrax and brucellosis, whereas dogs were frequently identified as being associated with rabies. Small ruminants, pigs, cats and poultry were either infrequently, or not identified as being associated with zoonoses. Recognition of clinical signs of zoonoses in humans was better than in animals. Ingestion of animal products was a route frequently identified as transmitting zoonoses to humans. During the baseline serosurvey, seroprevalences for brucellosis were 6.2% in cattle, 6.5% in small ruminants and 13% in wildlife, respectively. Seropositivity was significantly higher in the pastoral (13.2%), followed by agro-pastoral (5.3%), and lowest in the small holder dairy system (2.3%) (p<O.05). During the cross-sectional serosurvey, the seroprevalence was significantly higher in older animals and large herds (p<O.OOl). Variation in seropositivity between households was higher (1-30%) in the pastoral compared to agro-pastoral (1-14%) households. The model that best explained c-ELISA seropositivity included the feeding of dogs with foetuses and placentae, calving during the wet season, and the fanning system. In humans, 28% of families were seropositive for brucellosis with the highest levels in Ngorongoro district (46%), and lowest in Babati district (0%). Families with seropositive herds were 3.3 times more likely to be seropositive. However, 25% of families were seronegative when their herds were seropositive, and 48% of families were seropositive with seronegative herds. In the longitudinal study, the incidence was 73211,000 cases per animal-years at risk with an estimated survival probability of 0.836. Households with a high seroprevalence at the initial sampling had a high incidence of seroconversion in the subsequent visits. Occurrence of new seropositive cases was significantly higher in the wet season (p< 0.05). Calf serostatus was statistically associated with dam serostatus but no significant difference in growth rate was observed between calves suckled from seropositive and seronegative dams. Brucella melitensis type-1 was isolated from goats' milk following culture. Blood and placenta samples were negative on bacteriological culture. The RBPT was found to have low sensitivity in both field and laboratory settings. Brucellosis infection in livestock is widespread, but poses the greatest risk to human health and livestock production in pastoral systems in Tanzania.

The purpose of this study was to undertake detailed molecular and phenotypic characterisation of a MDR isolate of T. circumcincta (MTci5) with particular focus upon the mechanisms underlying benzimidazole (BZ) resistance.  MTci5 was isolated from a farm in Central Scotland, which employed a suppressive anthelmintic dosing regime and was closed in 2002 when control of the parasite population became unsustainable.  Underpinning all of the experiments in this study was an anthelmintic selection process whereby the MTci5 isolate was pressured individually with three broad-spectrum anthelmintics (benzimidazole, ivermectin and levamisole). There are three main areas of investigation in this study, the first being an investigation of the population genetic structure or a MDR isolate.  A central question was whether the MDR phenotype of MTci5 is conferred by the inheritance of genes present in a single interbreeding population or whether there is genetic sub-structuring, whereby discrete sub-populations of the isolate each show resistance to different anthelmintics.  Microsatellite analysis was employed to investigate the population genetic structure of the MTci5 isolate.  The results suggest that the MTci5 isolate is a single, freely interbreeding population with triple resistance, showing no evidence of genetic sub-structuring. The second area of investigation was the role of the F200Y isotype I ß-tubulin mutation in the determination of BZ resistance and the potential involvement of this mutation in resistance to ivermectin (IVM) and levamisole (LEV). There was no evidence of an effect of IVM or LEV selection upon the F200Y isotype I ß-tubulin mutation. The third area of investigation was the origin and diversity of BZ resistance alleles in the MTci5 isolate.  Single strand conformation polymorphism (SSCP) analysis of small region extending through exons 1 and 2 and intron 1 of the isotype I ß-tubulin gene was used to assess the genetic diversity of this locus in the MTci5 isolate and of five other UK T. circumcincta populations. Results are consistent with the theory of multiple independent, spontaneous mutations at the P200 locus of the isotype I ß-tubulin gene.

The studies reported in this thesis have been conducted in a high potential area of the country where no previous studies had been undertaken. The two major components of the research were an epidemiology study and an intervention study, both of which were conducted on farms in the peri-urban area of Kericho. An initial background study was conducted to collect data from the local Veterinary Investigation Laboratory, from extension staff in a participatory rapid appraisal (PRA) study and a cross-sectional socio-economic survey was undertaken with local farmers. These provided some understanding of the problems caused by helminths and the approaches taken to control them, together with socioeconomic data relating to the keeping of livestock in the area. Disease appeared to be a major constraint affecting ruminant productivity, with nematodoses appearing to be the most important helminth disease in the area particularly in small ruminants. In the cross-sectional survey, more than 60 % of farmers reported that they administered anthelmintics therapeutically and over 30 % gave routine treatments. On average, cattle were reported as being given up to 1.5 treatments per year, sheep just over 1.1 and goats 0.9 treatments per annum. In general, extension workers recommend that animals are wormed every 3 - 4 months, however this practice was not adopted by the farmers because of the expense involved. The epidemiology study conducted on 27 smallholder farms over a 22 month period obtained data on the prevalence of a) helminth ova in cattle, goat and sheep faeces b) helminth larvae on herbage and c) the different nematode species acquired by introduced Dorper tracer lambs which grazed on communal land. Grazing ruminants in Kericho appear to be exposed to infection with gastrointestinal nematodes throughout the year, there was no evidence of a marked seasonal influence on the availability of infective larvae on pasture. The two commonest genera Trichostrongylus and Haemonchus are both well adapted to the three ruminant species that are commonly grazed together.

This thesis describes a cell biological approach to studying lameness in the dairy cow. Lameness has been associated with altered keratinisation of the epidermis of the bovine hoof. Claw tissue was obtained by an in vivo claw biopsy technique. The biopsies were cultured in the presence of L-[35S]-methionine and [3H]-thymidine for measurement of protein synthesis and cell proliferation respectively. The influence of nutritional and environmental stressors on keratinisation in the claw was investigated. Protein synthesis was found to be significantly higher after challenge with these factors. It was not clear, however, if this was an effect of challenge or the biopsy procedure itself. Physiological and endocrinological changes associated with pregnancy and lactation also appeared to have an effect on claw keratinisation. In a long-term developmental study of first-calving heifers changes in cell proliferation and protein synthesis were related to reproductive state. A dramatic seasonal effect on claw cell biology was also demonstrated. The keratinocytes were actively proliferating and keratinising during the summer months but were quiescent during the winter. A final study investigated the cell biological changes which occur in the claw during the onset, development and recovery stages of weight-bearing challenge and the effects of concrete flooring. During challenge cell proliferation increased significantly in claw subjected to weight-bearing. The tissue may have been responding to challenge, however, the biopsy procedure itself could also have had an effect due to the short-time period between samplings. To conclude, altered keratinisation in the claw may be related to physiological and endocrinological changes associated with season and reproduction. However, changes in management may also be important and further investigation is required.

The principal objectives of the study presented in this thesis were to study the changes of milk proteomes, peptidomes and metabolomes during the course of bovine mastitis in comparison with normal milk samples and to discover new bovine mastitis biomarkers using various modern and up-to-date methodologies such as proteomics, peptidomics and metabolomics. Bovine mastitis caused by bacterial infection of the mammary gland of dairy cows is often associated with loss of milk production due to a reduction in milk composition and quality which in turns, lead to negative economic impact on dairy industry. Two important acute phase proteins (APPs) which serve as valuable biomarkers in bovine mastitis were investigated in every chapter using developed and validated enzyme linked immunosorbent assay (ELISA) for bovine milk haptoglobin and commercially available ELISA for bovine milk serum amyloid A3 (M-SAA3). These APPs were quantified alongside somatic cell counts (SCC) and California Mastitis Test (CMT) to confirm the disease status of each animal used in this study. Proteomic methodologies were applied including 1D gel electrophoresis, 2D gel electrophoresis, MALDI-TOF analysis and difference gel electrophoresis to investigate the changes of milk proteome in both subclinical and clinical mastitic milk samples in comparison with healthy milk samples. However these investigations did not reveal novel biomarkers for mastitis. Next, peptidomic methodologies were used to study the changes in milk peptidome and to detect the presence of any significant disease biomarkers in the presence of bovine mastitis by using CE-MS and LC-MS/MS. A total of 31 and 14 polypeptides can be used to discriminate control from infected groups and E. coli from S. aureus infected groups respectively. Lastly, metabolomic methodology was applied with an intention to study the changes in milk metabolome and ultimately to detect the presence of novel biomarkers in bovine mastitis. Di- and tri-peptides were found higher in S. aureus than in E. coli infected groups and based on metabolic pathways, arachidonic, arginine and galactose metabolites were seen increased in mastitic milk samples in comparison to healthy milk samples. Overall, the findings detailed in this thesis indicate that the use of advanced proteomic and metabolomic methodologies could deliver on their promise of the discovery of potential significant bovine mastitis biomarkers. Further studies are needed for validation of these proposed biomarkers and it was hoped that better prevention and treatment methods for bovine mastitis can be achieved in the future.

Salmonella is a Gram-negative bacterium that consists of two species; S. enterica and S. bongori. The species S. enterica can be further divided into 6 subspecies and subspecies I is predominantly associated with disease in warm blooded animals and contains over 2,500 antigenically distinct serovars. Each serovar is >90% identical at the DNA level but can infect a different range of hosts and cause different diseases. Poultry are an important reservoir of entry of Salmonella into the human food chain owing to the contamination of their eggs and meat. The molecular mechanisms underlying colonisation of food producing animals with Salmonella are unknown. Fimbrial genes encode proteinaceous surface exposed appendages which have been shown to mediate adhesion of bacterial cells but the precise role for fimbriae in the carriage and virulence of Salmonella is poorly defined. The purpose of this study was to annotate and characterise the fimbrial genes of the poultry-associated S. enterica serovars Enteritidis and Gallinarum and relate this role to host-specificity. The availability of the genome sequences of several strains of S. enterica allowed a comparison of the sequence, location and repertoire of fimbrial genes and although no unique fimbrial genes were identified all serovars possessed a unique repertoire. The host-specific serovars contain a higher number of pseudogenes within fimbrial operons than the ubiquitous serovars and the rate of attrition of fimbrial genes was 3-4 fold higher than the genomic mean. Such gene decay may partially explain the narrowing of host-range of the host-restricted and host-specific serovars. Polymorphisms that may alter transcription were identified along with targets that may be associated with phase variation of the fimbrial genes. Lambda red-mediated homologous recombination was used to construct a panel of S. Enteritidis P125109 and S. Gallinarum 287/91 strains lacking major fimbrial subunit genes which were examined in vitro and in vivo. Several fimbrial subunits played a role in the adherence to and invasion of different cell lines in different growth conditions and the role appeared to be serovar-specific. A mutation in the steA gene impaired interactions with different cell lines in vitro but this phenotype was found to be due to a polar effect on genes downstream of steA. The majority of fimbrial subunits played no significant role in the colonisation of the alimentary tract in an established chicken model. Mutation of the stcA gene resulted in the greatest degree of attenuation in vivo of all of the fimbrial mutants examined. This phenotype was trans-complemented and was not the result of a polar or second-site defect thereby fulfilling molecular Koch’s postulates. The stcA genes therefore play a significant role in the colonisation of the chicken caeca.

The internal carotid artery in the horse is of significant veterinary importance due to its intimate relationship with the guttural pouch, and mycotic disease thereof. The relevance of recognising and identifying variations involving the internal carotid artery lies in the fact that surgical occlusion of the artery is the treatment of choice for guttural pouch mycosis. However, occlusion could be hampered when there is doubt about the anatomy of this vessel and its variation. Conventional angiography and automatic rotational angiographic techniques were used to study the anatomy of the carotid trifurcation and the internal carotid artery on cadavers of three species included in the genus Equus; 50 horses, 26 donkeys and one zebra. Following angiography, arterial latex casting was performed on the horse and donkey specimens with subsequent dissection to harvest the hardened arterial casts. Rotational angiography with 3-dimensional image reconstruction represent a major advantage in the angiographic diagnosis of carotid and cerebral vascular variation compared to conventional angiography. This technique generated superior angiographic images of the carotid and cerebral vascular system of horses, donkey and zebra. In the horse, five variations of the internal carotid artery were identified as follows: [1] the internal carotid artery and occipital artery arising as a common trunk, [2] an aberrant branch of the internal carotid artery that unites with the basilar artery, [3] an aberrant branch of the internal carotid artery that does not unite with the basilar artery, [4] aberrant branch of the internal carotid artery that gives rise to several satellite branches, [5] aberrant branch of the internal carotid artery that has a satellite branch connected to the caudal branch of the ipsilateral occipital artery. Variations of the carotid arterial tree in donkeys were identified as follow: [1] the internal carotid and occipital arteries shared a common trunk, [2] the linguofacial trunk originated from the common carotid artery causing the common carotid artery to terminate as four branches, [3] a short external carotid artery before giving rise to the linguofacial trunk, mimicking the appearance of the common carotid artery terminating into four branches, [4] the internal carotid artery originating far more caudal from the common carotid artery termination. The carotid arterial anatomy of the one zebra studied here showed no discrepancy to the accepted common anatomical pattern of this structure. Aneurysm formation was not identified in any of the specimens.

The aim of this work was to develop a propofol (PPF) based Total Intravenous Anaesthesia (TIVA) technique to be used in dogs by veterinary surgeons in practice. As PPF is a poor analgesic agent, this work also looked into the development of a co-infusion scheme for the potent alpha2-adrenoceptor agonist medetomidine (MED) and its active enantiomer dexmedetomidine (DEX). The study was divided into 4 phases. In phase one, canine PPF pharmacokinetic (PK) parameters, derived from previous studies reported in the literature, were incorporated to a Target Controlled Infusion (TCI) system. This approach, comprising computer software incorporated into a syringe driver, which will deliver PPF to a predicted blood concentration, is widely used in humans. The performance of the system was investigated in 6 greyhounds and 10 mixed-breed dogs undergoing routine dental work by comparing predicted with measured PPF concentrations in venous blood samples obtained during and following TCI PPF anaesthesia. The optimal induction target was 3 mgml-1, and an adequate depth and a satisfactory quality of anaesthesia were achieved with mean maintenance targets of between 3.4 and 4.5 mgml-1 of PPF. The performance of the TCI system was considered clinically acceptable as the Median Prediction Error (MDPE%), a measure of bias, and the Median Absolute Performance Error (MADPE%), a measure of the accuracy, were -12.47 and 28.47 respectively, in the greyhounds and 1.56 and 24.79 respectively, in the mixed-breed dogs. The system was easy to use and the quality of anaesthesia was judged to be adequate for dental work. Phase 2 illustrated the inhibitory effect of MED and DEX on PPF metabolism at the level of the cytochrome P450 in rat and canine hepatic tissue and highlighted, therefore, the possible effect on the metabolism dependant performance of the TCI system. Before designing an infusion scheme for MED and studying its possible effect on PPF PK in vivo (phase 4), the purpose of phase 3 was to characterise cardiovascular and respiratory effects of MED administered IV to dogs anaesthetised with a TCI of PPF, and to assess its suitability for use in a TIVA regime. Eighty dogs, ASA 1 or 2, aged 0.5 to 8 years, were randomly allocated into 8 groups of 10 dogs according to the dose of MED administered (Groups 1-8: 0 (saline, 0.9%, 1 ml), 0.01, 0.03, 0.1, 0.3, 1, 3, 10 mcgkg-1 MED, respectively). Following premedication, anaesthesia was induced with a PPF target blood concentration of 3 mcgml-1 and maintained with a target concentration of 3.5 mcgml-1. Cardiovascular and respiratory parameters were recorded for 15 min post induction and before saline (group 1) or MED (groups 2-8) was injected slowly over 1 min. Medetomidine induced a dose-dependent reduction in heart rate (HR) and increase in systolic arterial blood pressure (ABP). At the time of maximum observed effect (2 min post MED injection), the ED50 for ABP and HR were 2.05 and 0.187 mcgkg-1 respectively, while the ED95 (doses of MED inducing 95% of the maximum effect) values were estimated to be 18.1 mcgkg-1 and 3.1 mcgkg-1, respectively. The no effect doses for MED were 0.01 mcgkg-1 for HR, and 0.1 mcgkg-1 for ABP. Minimal respiratory effects were observed in all groups except the group receiving 10 mcgkg-1 of MED where, by the end of the recording period (20 min post MED injection), 8 of 9 spontaneously breathing dogs became apnoeic after MED administration. Phase 4 was designed to develop and assess a stepped infusion scheme for MED and DEX in the TCI PPF anesthetised dog using MED PK parameters from O. Vainio (V1 = 470 mlkg-1, K12 = 0.0954, K21 = 0.0438, K10 = 0.0489); to observe the possible PK and PD (pharmacodynamic) interactions between PPF and the 2 alpha2-adrenoceptor agonists during co-infusion; to determine the minimum blood PPF infusion target (MIT) necessary to prevent purposeful movement during supramaximal noxious stimulation (tetanic twitch for 5 sec at the level of the 4th and 5th coccygeal vertebrae) with and without a co-infusion of MED or DEX and to confirm the DEX minimum analgesic blood concentration of 0.85 ngml-1. Six female beagle dogs, 7.3 (± 2.3) years old, were anaesthetised on 4 occasions, following a randomised cross over design: PPF TCI with either co-stepped infusion of saline (PS), MED (blood target of 1.7 ngml-1, PM), low DEX (blood target of 0.85 ngml-1, PLD) or high DEX (blood target of 1.7 ngml-1, PHD). The co-infusion was started 25 min after the start of anaesthesia (instrumentation period), while the MIT determination was conducted 15 min after the last step of the co-infusion. Venous blood samples were taken at specific times for determination of the PPF, MED and DEX plasma concentrations. The performance of the TCI system for PPF in the dog was only clinically acceptable in the PS and PLD treatments with MDPE% values of 18.85 and 25.94 respectively, and MDAPE% values of 18.85 and 35.80 respectively. In this study the use of DEX 0.85 ngml-1 had a similar PPF sparing effect to the equivalent MED blood concentration of 1.7 ngml-1, but with less effects on ABP, as well as on the performance of the TCI for PPF in the dog. Therefore, it could be concluded that DEX was more advantageous than MED given by infusion in PPF anaesthetised dogs. The study also confirmed the validity of the PK of MED from the previous study. The study redefined specific PK parameters for DEX, although the MED PK parameters could also be used. The study indicated that DEX blood concentrations as low as 0.85 ngml-1 decreased the measured PPF blood concentrations necessary to maintain anaesthesia during noxious stimulation by about 38%. However, although this study supported the suitability of the co-infusion of DEX during PPF anaesthesia in the dog, and the analgesic/sedative effects of DEX were present at the lowest blood concentrations with well maintained respiratory parameters, the CV effects were marked with a decrease in HR and CO and an increase in systolic and mean ABP. Further studies are therefore necessary to establish if a lower blood concentration of DEX will provide analgesia while preserving the CV system.