Veterinary Science - Theses
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ItemInvestigating the inflammatory pathways involved in placental detachment in the mare( 2018)The mechanism of placental detachment in the mare has not been investigated in detail and as such remains poorly understood. Research in other species provides strong evidence that parturition and fetal membrane detachment from the endometrium requires involvement of the immune response. Research in horses has also suggested that movement of leukocytes in the umbilical vessels, and their attraction to fetal membranes at parturition, contribute to placental detachment. The aims of this study were to 1) confirm whether a leukocyte “concentration gradient” exists between the equine neonatal umbilical artery and vein, and whether this gradient was associated with fetal membrane retention time 2) to investigate the expression of pro-inflammatory cytokines in the equine endometrium and fetal membranes at parturition using quantitative real time polymerase chain reaction (RT-qPCR) and 3) to localise pro-inflammatory cytokine proteins within the equine endometrial and fetal membrane tissues using immunohistochemistry to correlate with gene expression. Data was collected from 33 spontaneously foaling mares and their foals on a single Thoroughbred stud farm in New Zealand. Umbilical artery and vein blood, fetal membrane and endometrial biopsy tissue samples were obtained. The average fetal membrane retention time was 92 (+/-130) minutes. The median fatal membrane retention time was 47 minutes (IQR 30). There was a significantly higher number of total leukocytes, lymphocytes and neutrophils in the equine neonatal umbilical artery than the umbilical vein at parturition (p <0.001). Fetal membrane retention time tended to decrease with an increase in umbilical leukocyte concentration gradient but did not reach statistical significance (p = 0.098). The mRNA expression of IL-1, IL-6 and IL-8 was significantly greater in the endometrium of foaling mares compared to control mares (p < 0.001). The mRNA expression of IL-8 was significantly higher in the fetal membranes of foaling compared to control mares (p < 0.001) whereas mRNA expression of IL-6 in fetal membranes was significantly lower between the two groups (p< 0.001). Whilst mRNA expression for IL-1 was lower in the fetal membranes of foaling mares compared to controls, the difference was not significant (p = 0.16). This study has demonstrated the presence of an innate inflammatory immune response in the placenta of spontaneously foaling mares, suggesting the role of inflammation in the detachment of the fetal membranes from the endometrium. The results from this study have highlighted the need to further investigate the mechanism of placental detachment in the mare and also understand the pathophysiology behind abnormal fetal membrane retention.
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ItemBiomechanical testing of the calcified metacarpal articular surface and its association with subchondral bone microstructure in Thoroughbred racehorses( 2018)Subchondral bone adaptation and fatigue likely alter joint surface mechanical properties. The aim of this study was to evaluate the relationship of calcified articular surface biomechanical properties in the distal palmar metacarpal condyle of Thoroughbred racehorses with subchondral bone microstructure and training history. Subchondral bone microstructure was evaluated using both micro- and clinical-computed tomography. Biomechanical properties of the calcified articular surface were tested with reference point indentation at the micron level. Articular surface toughness increased with commencement of race training, and was lower in horses that died as a result of musculoskeletal fatigue injury, had evidence of palmar osteochondral disease, had higher subchondral bone connectivity with more concave surfaces, and higher subchondral bone volume fraction. Further investigation of bone microstructural properties is warranted based on bone connectivity and surface shape findings. At clinically applicable imaging resolution, evidence of subchondral bone modelling may be an important indicator of articular surface biomechanical properties.
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ItemInvestigation of the infectious causes of diarrhoea in Australian thoroughbred foals( 2017)Diarrhoea is a common disease in foals that is costly and labour intensive to manage. A large number of potential enteric pathogens have been detected in the faeces of foals, however, the role of these infectious agents in causing clinical disease is not clearly understood and their prevalence in Australia is unknown. In addition, timely methods for definitive diagnosis are not readily available for some of these agents. Therefore, this study aimed to develop rapid molecular detection assays to investigate the presence of equine rotaviruses, equine coronaviruses, Salmonella spp. and Clostridium difficile in Australian thoroughbred foals with and without diarrhoea. A prospective case control study was conducted on five thoroughbred breeding farms in the Hunter Valley (New South Wales, Australia) during the 2010 breeding season. Faecal samples were collected from age-matched foals with diarrhoea and without diarrhoea from the same farm (age-matched pair). In addition, faeces were collected from foals with diarrhoea from an equine veterinary hospital. All faecal samples were analysed for equine rotaviruses, equine coronaviruses, Salmonella spp. and Clostridium difficile by quantitative PCR (qPCR) assay. All faecal samples were also cultured for Salmonella spp. using selective growth media. Samples positive by qPCR and 15 randomly selected control foal samples negative by qPCR were cultured anaerobically for Clostridium difficile. Faecal samples were collected from 117 pairs of age-matched case control foals and 26 hospitalised foals with diarrhoea. In the age-matched case control foals, equine rotaviruses were the most frequently detected infectious agent (25% case foals, 5% control foals) and the only infectious agent significantly associated with the presence of diarrhoea. In hospitalised foals, Clostridium difficile (23%) was the most frequently detected infectious agent. In this investigation co-infections were detected in 4% of matched case foals and 4% of hospital foals, with equine rotaviruses and Salmonella spp. being the most frequent combination. Four different Clostridium difficile ribotypes were detected, including ribotype 012 and 078. Importantly, this is the first report of the detection of C. difficile ribotype 078 in Australian horses. As this ribotype has been associated with severe disease in humans, this finding may have public health implications. The availability of rapid molecular screening tests for infectious causes of foal diarrhoea enhances the veterinary practitioner’s ability to instigate appropriate therapy and control measures in foals with diarrhoea. However, the detection of pathogens in foals without diarrhoea highlights the need for more research into the role some of these pathogens play in clinical disease both individually and in combination.
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ItemRelationships between diet, obesity and insulin dysregulation in horses and ponies( 2016)Laminitis is a debilitating condition of equids that affects a significant proportion of domesticated horses and ponies (Equus caballus) worldwide. Prevention is the key to managing laminitis, as there are currently no effective treatments and crippling lameness can often necessitate the euthanasia of affected animals. The clinical clustering of obesity and insulin dysregulation as risk factors for laminitis has been referred to as equine metabolic syndrome. The studies reported in this thesis sought to further examine the relationships between diet, obesity and insulin dysregulation in horses and ponies. Differences in the innate glucose and insulin dynamics of different equine breeds were established by investigating the insulin responses of animals to oral and intravenous glucose challenges. Ponies and Andalusian horses were relatively insulin resistant and hyperinsulinaemic compared with Standardbred horses; a finding that occurred without the potentially confounding effects of obesity or modifying dietary factors. Studies of diet-induced weight gain were then undertaken, in which a high dietary glycaemic load was found to influence the development of insulin dysregulation more than the induction of obesity per se. Relatively low plasma levels of adiponectin were identified in animals with reduced insulin sensitivity, whilst evidence of significant systemic inflammation was not detected. Glucagon-like peptide-1 (GLP-1) levels were found to correlate with postprandial insulin responses in horses and ponies adapted to cereal-rich meals. These studies report for the first time the identification of innate differences in insulin metabolism between particular equine breeds. The induction of obesity was not associated with insulin dysregulation in horses and ponies fed a low glycaemic diet, suggesting that increased adiposity might be a consequence rather than a cause of insulin dysregulation in equids. Adiponectin may be a potentially useful biomarker for insulin dysregulation, although whether hypoadiponectinaemia is involved in the pathogenesis requires further investigation. Incretins such as GLP-1 could represent a potential therapeutic target for the control of equine hyperinsulinaemia. Understanding how genetic predispositions to insulin dysregulation can be aggravated by the environment is an essential first step in the development of countermeasures to reduce the incidence of laminitis in equine populations worldwide.
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ItemImmunological and virological investigations arising from the 2007 Australian equine influenza outbreak( 2016)During Australia’s equine influenza (EI) outbreak, horses were vaccinated in Victoria prophylactically using a recombinant canarypox- vectored vaccine. Humoral and cell–mediated immune responses were monitored following an accelerated primary course reduced to 14 days. To demonstrate proof of freedom from EI, nasal swabs were taken from diseased and normal horses. Quantitative PCR was performed on samples from 559 horses, all negative for EI. Shedding of equine herpesviruses -1,-2 and -4 was correlated with the horse’s clinical status.
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ItemEquine forelimb asymmetries and factors influencing hoof shape( 2014)This thesis provided detailed descriptions of bilateral asymmetries in equine distal forelimbs and explored factors influencing hoof shape. Measurement techniques described included the development of a photogrammetric method. This allowed for quick and highly accurate measurements to be taken from a 3-D model of the hoof, which was created from photographs taken at various locations surrounding the hoof. This is the first study to successfully recreate a hoof model from photographs, to measure proximal hoof circumference (PHC) using photogrammetry, and to demonstrate the feasibility of using it during an exercise study. Cadaver limbs were used to describe bilateral forelimb asymmetries. That study found significant differences in third metacarpal (MC3) bone weight, second phalanx (P2) specific gravity, and third phalanx (P3) angle. PHC also shared a linear relationship with several bone measurements; MC3 width, MC3 length, P3 angle, MC3 weight, and P3 weight. This was the first study to report bilateral differences in equine bone weight and specific gravity and to identify a relationship between PHC and internal bone measurements. There were also significant bilateral differences in ratios measured directly from yearling radiographs. Specifically A/B and G/H, and A/B shared a relationship with one performance variable. Other studies have reported radiographic asymmetries, but few have focused on young horses and performance. Factors that could influence hoof shape included increased load. Two pilot studies found that there were no changes in PHC with an increase in body weight or with the additional weight of a rider. The third acknowledged bilateral differences in hoof shape and loading using a pressure mat. It is one of the first studies to identify a difference in loading under the forelimbs, which warrants further investigation. Two preliminary studies found that there were no changes in PHC or hoof angle following a training period of exercise in a circle or immediately after exercise on a treadmill. This is one of the first studies to use horses exercising on a lunge-line. Previous research has found a change in PHC and hoof angle during race training, but this study did not show a difference after moderate intensity exercise. The farrier study indicated that the hoof changed shape as a result of trimming and growth. The farrier performing the trim affected some variables and hoof angle was more symmetrical post-trimming. This study supported previous work and contributed details on the magnitude of change. In the final experiment reported, shod horses exhibited a larger decrease in PHC and a decrease in hoof angle. This indicated that the shoes may limit deformation of the hoof which is supported by previous work. It is the first study to recognize PHC changes in shod horses. This thesis contributed to a greater understanding of equine asymmetries, their development, and factors influencing hoof shape. It aids in the design of beneficial hoof care practices.
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ItemAnalysis of physiological death in equine chondrocytes( 2007-07)Chondrocytes in growth cartilage undergo proliferation, hypertrophy, and then die by a mechanism that has not been characterised. The aims of the current study were to document the morphology of dying hypertrophic chondrocytes in equine growth cartilage and to establish a culture system in which the isolated chondrocytes can be induced to undergo the same modes of hypertrophy and physiological death seen in growth cartilage in vivo. Growth cartilage from foetal and growing postnatal horses was examined by electron microscopy. Ultrastructural studies of the tissue specimens suggested that the two types of hypertrophic chondrocytes that have previously been described as dark and light cells were dying by different non-apoptotic forms of cell death. Dying hypertrophic dark chondrocytes were characterised by a dark nucleus, and their cytoplasm appeared to undergo extrusion into the extracellular matrix, whereas light chondrocytes appeared to disintegrate within the cell membrane.
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ItemAnti-inflammatory effects of clenbuterol hydrochloride on leukocyte activation in the horse( 2013)The work reported in this thesis aimed to investigate the potential anti-inflammatory effects of clenbuterol hydrochloride in the treatment of equine endotoxaemia. Beta adrenergic agonists, such as clenbuterol hydrochloride reduce leukocyte activation and cytokine production through the up regulation of cyclic adenosine monophosphate. Investigations measured pro-inflammatory cytokine production (IL-1β and TNFα) by equine leukocytes, following stimulation with endotoxin, peptidoglycan and lipoteichoic acid and treatment with clenbuterol hydrochloride during in vitro and in vivo studies. The study also aimed to develop a model enabling correlation of pharmacokinetic and pharmacodynamic drug properties in order to predict an optimum dosing regimen for novel anti-inflammatory agents. The results of the study indicated that clenbuterol hydrochloride has significant anti-inflammatory effects on equine leukocytes challenged with Gram negative and Gram positive bacterial toxins in in vitro assays. Clenbuterol hydrochloride was most potent at inhibiting TNFα production in response to endotoxin, with the effects on peptidoglycan and lipoteichoic acid only being significantly inhibited at much higher clenbuterol hydrochloride concentrations (>10-6 M). Also a marked reduction in lipopolysaccharide stimulated IL-1β cytokine production following treatment with clenbuterol hydrochloride was recognised. In in vivo models of equine endotoxaemia, pre-treatment with oral clenbuterol hydrochloride caused a significant reduction in the peak rectal temperature and peak plasma TNFα concentrations. A model correlating clenbuterol hydrochloride pharmacodynamics and pharmacokinetics was developed. Clenbuterol may have a beneficial role alongside non-steroidal anti-inflammatory drugs and other previously investigated therapies in the treatment of inflammation associated with sepsis in horses. Further investigations in clinical cases of equine endotoxaemia are essential prior to the recommendation of clenbuterol hydrochloride as an adjuvant anti-inflammatory agent in the treatment of systemic inflammation.
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ItemDetection of subchondral bone pathology in the fetlock joint of Thoroughbred racehorses( 2013)REASONS FOR PERFORMING STUDY: Two common subchondral bone injuries occur within the fetlock joint; palmar osteochondral disease and condylar fracture of the distal metacarpus. Both result in lameness, whereas condylar fracture can potentially be life threatening. Pre-existing pathology is commonly identified in horses with condylar fractures, detection of which prior to fracture would have both welfare and economic benefits for the racing industry. However the close spatial association of these two conditions complicates imaging of the distal metacarpus/metatarsus. AIMS: To describe patterns of radiopharmaceutical uptake in fetlock joints of Thoroughbred racehorses and to evaluate racing performance post-scintigraphy and to assess the potential of high-resolution peripheral quantitative computed tomography to predict condylar fracture in Thoroughbred racehorses by imaging the subchondral bone of the distal palmar metacarpus and the cortical bone of the mid diaphysis. HYPOTHESES: Increased radiopharmaceutical uptake (IRU) in the metacarpal (MC) and metatarsal (MT) condyles is more common than IRU in the parasagittal grooves and is associated with poorer post-diagnosis performance than controls. Horses with lateral condylar fracture would have greater bone volume fraction, greater localised subchondral bone thickness at the fracture site and that articular surface fissures would be more prominent in the contralateral limb than controls. METHODS: Scintigraphic evaluation of the fetlock region - A retrospective, case control study was performed using horses undergoing scintigraphy for the investigation of lameness or poor performance. Location of IRU within the fetlock region was identified and graded in Thoroughbred (TB) racehorses presenting for scintigraphic examination. Performance variables were determined from race records for horses with moderate/marked MC/MT condylar IRU (cases), all other horses undergoing scintigraphy (scintigraphy controls), and age/sex matched controls from the last race in which a case participated (controls). Statistical analysis included quantile regression, Fisher’s exact test, Kaplan-Meier survival curves, log-rank test and Cox regression. High resolution peripheral quantitative computed tomography as a predictor of condylar fracture - A prospective, cross-sectional, case control study was performed using limbs from all TB racehorses that died or were euthanased on Victorian metropolitan racetracks between June 2007 and June 2009 (n=37). Groups were assigned as follows: horses with MC fractures with a condylar component (cases, n=13); horses with no limb fracture (controls, n=8); horses with fracture in other bones or disruption of suspensory apparatus (other fatal musculoskeletal injuries, n=16). The palmar distal aspect was removed and a 5cm length of midshaft of MC created and stored in 70% ethanol. The palmar condyles of MC and midshaft examined with high resolution peripheral quantitative computed tomography (HR-pQCT) and morphometric parameters computed. Statistical analysis included logistic regression and Spearman’s correlations. RESULTS: Scintigraphic evaluation of the fetlock region - Metacarpal/MT condylar IRU was identified in 103/220 horses with only 3/220 having parasagittal IRU. Moderate/marked IRU was identified in the forelimb MC and hindlimb MT condylar regions in 62 and 48 horses respectively with palmaromedial and plantarolateral IRU most common. Forelimb cases had fewer starts (P<0.001), reduced earnings (P<0.001), fewer wins (P=0.03) and fewer overall places (P<0.001) than controls. Hindlimb cases had fewer starts (P=0.003) and reduced earnings (P=0.01) than controls. Cases were more likely to return to racing later than controls following a rest period (P=0.003). Cases were older than scintigraphy controls (P<0.001). High resolution peripheral quantitative computed tomography as a predictor of condylar fracture - No significant differences were identified in HR-pQCT variables of distal MC and midshafts between cases and controls. Epiphyseal BV/TV was greater in injured limbs of horses with any fatal limb injury compared to controls (OR 1.20, 95% CI 1.01-1.42, P=0.034). An epiphyseal BV/TV >0.74 resulted in a sensitivity of 82.8% and specificity of 62.5% in identifying horses with fatal limb injury. In horses without condylar fracture, increased subchondral bone thickness was associated with POD lesions in the adjacent condyle (rs=0.65, P<0.001). CONCLUSIONS: The identification of condylar fractures prior to catastrophic injury remains challenging. Condylar fractures are rarely identified using bone phase scintigraphy and this may be because lameness is not observed by owners and trainers prior to fracture. Even with high resolution imaging of the distal metacarpus, early condylar fracture detection is challenging although increased BV/TV of the distal metacarpus may be useful for determining horses at risk of any fatal breakdown injury. HR-pQCT of the third metacarpal bone midshaft appears to be of no value for predicting fatal injury. Racehorses with moderate/marked condylar IRU have a shortened racing career, are less successful than age/sex matched controls and are older than other racehorses presented for scintigraphy. POTENTIAL RELEVANCE: Effective screening for condylar fractures prior to catastrophic breakdown would require imaging of horses that are not showing clinical signs of lameness or poor performance. Applying advanced imaging to all horses at regular intervals is not only cost prohibitive but the current evidence suggests it is unlikely to be highly effective. POD appears to be performance limiting in Thoroughbred racehorses.
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ItemAngiotensin-converting enzyme (ACE) in horses and its relationship to performance and fitness( 2010)This body of work concerns studies investigating aspects of ACE activity in the horse. Since very little information is available in the existing literature regarding this topic, basic research included assay validation, comparison of methods, and sample stability testing. Further investigations included the influence of environmental factors on circulating ACE activity, including diurnal and seasonal variations and the effects of training and acute exercise. This research sought to investigate whether there was an association between ACE phenotype and performance for different equestrian modalities, namely flat racing and endurance. Subsequently, aspects of the ACE genotype, ACE activity in tissues and whether there was an association between ACE activity and diseases of metabolic or pulmonary origins were investigated. Assay testing demonstrated that different methods are not comparable, therefore caution is required when interpreting data obtained with different protocols for ACE measurement. Also, sample stability testing provided evidence that equine ACE is more labile than its human counterpart, requiring strict handling and storage to achieve repeatable and consistent results. Data demonstrated that equine ACE has a seasonal variation, peaking during spring of the Southern Hemisphere, whereas no significant circadian rhythm of the enzyme was observed. Results demonstrate that greater ACE activity is associated with improved performance indices in maiden Thoroughbreds, and is also associated with distance successfully raced by Thoroughbred racehorses, both under flat racing conditions. ACE activity in equine plasma is responsive to both acute and chronic exercise, with increased levels proportional to workload. This has important implications with regard to selection of horses suitable for particular types of event, and also in the assessment of fitness. In relation to clinical conditions, although ACE activity was not associated with the systemic response to an experimental endotoxic challenge, horses admitted to a referral centre with colic had improved likelihood of being discharged if their ACE activity in plasma was higher at admission. ACE activity was not a good indicator of pulmonary disease diagnosed via endoscopy only, but there is an indication that ACE activity might be correlated to severity of chronic lung disease. ACE activity in equine lungs is higher than in the kidneys and the heart, and data suggests that acute lung injury may cause a reduction in both plasma and lung tissue ACE activity, whereas chronic pulmonary disease leads to an elevation in plasma ACE with reduced lung tissue ACE activity. ACE genotyping demonstrated that horses carrying a single copy of the H6 haplotype had lower levels of circulating ACE activity, but since only 9.5% of animals in the sample had the H6, the association was poor. ACE activity in equine plasma was demonstrated to be a better tool for assessing suitability for athletic events or training than ACE genotype, and to have potential as an accessory tool to monitor performance and as an indicator of disease course in some clinical situations.