Melbourne Veterinary School - Theses
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ItemThe role of osteopontin in muscle injury and regeneration(University of Melbourne, 2010)
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ItemPathogenesis of equine herpesvirus 2 (EHV2) infection and cellular processing of EHV2 glycoprotein B(University of Melbourne, 2010)
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ItemAssessing and addressing the welfare of extensively managed ewes( 2019)The sheep industry is currently facing increasing social demands for assurances of good animal welfare, which indicates that assessing and addressing the welfare of sheep is critical if the current ‘social license to farm sheep’ is to be maintained. Farmers are key players in improving sheep welfare because they are responsible for the care of their animals and make the management decisions on their farm. Hence, a better understanding of the main factors underpinning farmer behaviour is important. According to the Theory of Planned Behaviour, farmer attitudes towards sheep management are likely to influence their behaviour in terms of the implementation of improved management practices. Subsequently, farmer management behaviour may impact on the welfare of their sheep. The aim of this thesis was therefore to examine the relationships between farmer attitudes, management behaviour and sheep welfare outcomes. To examine these relationships, animal welfare assessments and questionnaire interviews with farmers were conducted. A total of 6200 ewes (aged 2-5 years) and 32 farmers were sampled across Victoria, Australia. Farms were visited at mid-pregnancy and weaning, and the ewes were examined using six animal-based measures: body condition score (BCS), fleece condition, skin lesions, tail length, dag score and lameness. In addition, the number of ewes that needed further inspection/care (such as sick or injured sheep) was recorded and reported to the farmers. The welfare of the ewe flocks, based on the six indicators measured, was good overall. However, individual welfare compromise was considered significant. There were 185 (3.0%) ewes needing further care and cases were identified in all farms. Main reasons for further inspection/care were lameness or foot-related issues, BCS ≤ 2 and active dermatophilosis or broken wool. Farmer attitudes to sheep and management were in general positive and welfare was considered an important aspect of farm productivity. In terms of management, it was found that farmers relied mostly on visual monitoring to assess the flocks and pasture quality and quantity. However, close inspection of the flock and more detailed practices such as body condition scoring of sheep, veterinary interventions or keeping accurate records were less common activities. Indeed, mortality rates were frequently underestimated. Positive farmer attitudes were associated with positive management behaviour (active management style), and positive management behaviour was associated with positive ewe welfare outcomes. Farmers with an active management style had fewer ewes in need of further inspection/care after both welfare assessments (mid-pregnancy and weaning). Main results indicated that farmers were more likely to perform an activity (management behaviour) if they perceived the activity was valuable (behavioural attitudes) and if they perceived the activity could be realised (perceived behavioural control). The results of this thesis demonstrate that there is an opportunity to create change in farmer management behaviour and potentially improve sheep welfare via education programs targeting attitudinal change. The results reported in this thesis provide what is believed to be the first comprehensive study investigating the on-farm welfare of extensively managed ewes and the farmer-sheep relationship in extensive systems. Controlled trials to assess if education programs or other interventions are able to improve farmer attitudes and management behaviour, as well as sheep welfare, would allow causality to be investigated and would be a valuable next research step following on from the current work.
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ItemPhylogeny and virulence factor genes of canine urinary Escherichia coli in relation to clinical disease and antimicrobial resistance( 2018)Traditionally, urinary tract infections (UTIs) have been categorised as either uncomplicated or complicated in veterinary medicine, with treatment differing for the two categories. In human medicine, there is an additional category: asymptomatic bacteriuria, which is the presence of bacteriuria without symptoms of infection. Escherichia coli is the most common bacterial species involved in UTIs in dogs. Clinical signs can be absent in dogs with complicated UTIs, and this has been likened to asymptomatic bacteriuria in people and has been termed subclinical bacteriuria (SBU). Treatment recommendations for SBU in dogs have been adapted from human recommendations. Many E. coli strains are resistant to multiple antibiotics and uropathogenic E. coli possess virulence factor genes that facilitate overcoming host defence mechanisms. These E. coli commonly belong to phylogenetic groups B2 and D. Some studies suggest that human E. coli isolated from asymptomatic bacteriuria differ from those causing clinical UTI. While the virulence factor genes and phylogeny of canine urinary E. coli isolated from UTIs are well characterised, little is known about virulence factor genes and phylogeny in E. coli isolated from SBU. Furthermore, these genomic characteristics have not been studied in detail in multi-drug resistant (MDR) canine urinary E. coli. Thus, the pathogenic potential of canine urinary E. coli is not well described and the benefit of antibiotic therapy in SBU and MDR infections is unknown. This study used whole genome sequencing to characterise 47 E. coli isolated from dogs with SBU and 67 E.coli from dogs with clinical UTI in terms of their phylogeny and virulence factor gene profile. From those strains, the 15 MDR strains were characterised. When the phylogeny and virulence factor gene profile of E. coli isolated from dogs with SBU were compared with clinical UTIs, results showed that most clinical UTI and SBU E. coli belonged to phylogenetic group B2. The virulence factor gene profile was similar between the two groups, and no association was found between them and the 83 virulence factor genes analysed. Many of the MDR E. coli belonged to phylogenetic group B1 and these isolates possessed fewer virulence factor genes than non-MDR E. coli. Based on the results of this study, it was concluded that phylogeny and the presence of virulence factor genes do not influence the manifestation of clinical disease. Host immunity and rather than presence and the expression of or mutations of virulence factor genes may have a role in the development of clinical disease. ii MDR E. coli have fewer virulence factor genes than non-MDR isolates, with MDR isolates commonly belonging to commensal phylogenetic groups. This suggests that treatment of MDR isolates is not always indicated because they tend to have commensal bacterial phylogeny, which can have implications for reducing the development of antimicrobial resistance. While whole genome sequencing is an accurate method for determining phylogeny and the presence of virulence factors, it is unable to easily differentiate the pathogenicity potential of urinary E. coli and therefore treatment recommendations cannot be made based on this technique.
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ItemChronic enteropathy in dogs: the role of macrophages and preliminary results in inflammatory cytokines( 2019)Chronic enteropathy (CE) is an umbrella term used in dogs to describe a group of diseases with different aetiologies, characterised by chronic gastrointestinal signs. These diseases are clinically classified according to treatment response as food-responsive (FRE), antibiotic-responsive (ARE), and immunosuppressant-responsive enteropathies (IRE). The first part of this PhD thesis prospectively describes the features of CE that are commonly seen in dogs presenting to a referral centre in Australia; information that has not been available previously. We found that similar to other countries, most dogs with CE are food-responsive, followed by antibiotic-responsive with a minority of immunosuppressant-responsive. Furthermore, our study raised concerns about prolonged antibiotic treatment for dogs with ARE. Firstly, most of these dogs do not respond to treatment completely for prolonged periods (as opposed to dogs with FRE that do), raising the question about the real benefit of antibiotic treatment. Secondly, half of the dogs with ARE required long-term or pulse treatment with antibiotics, which raises concerns about development of bacterial resistance. Our findings highlight the need to find alternative treatment for dogs with ARE in view of the poor long-term outcome. Although most dogs with FRE had long-term remission, adequate dietary trials were not performed until reaching the referral setting. This indicates that better education of general veterinary practitioners about the importance of performing adequate diet trial is needed to improve early disease remission in these dogs. The next focus of the research was to evaluate the role of macrophages in CE; this was achieved by using two macrophage markers: calprotectin and cluster of differentiation 163 (CD163) in immunohistochemical examination. Both immunohistochemical markers highlighted two different populations of macrophages in our intestinal biopsy specimens. Overall the number of CD163 positive cells was higher than calprotectin positive cells both in crypts and villi. Dogs with FRE and IRE had a decreased CD163:calprotectin ratio compared to healthy dogs with an increase in the ratio after treatment. Our results suggest that there is an imbalance in macrophage populations in dogs with FRE and IRE, with partial resolution following clinical response characterised by an increase in the ratio CD163:calprotectin. Interestingly, dogs with ARE not only have a poor long-term response, but also have different macrophage populations from dogs with FRE and IRE; and in fact, are very similar to healthy dogs without change in their macrophage populations with treatment response. These results suggest that macrophages play a role in the pathogenesis of FRE and IRE dogs with normalisation of macrophage populations with treatment response. The CD163 receptor is cleaved during macrophage activation and is released into the circulation as a soluble form. In view of the decreased number of CD163 cells in the intestine of dogs with FRE and IRE at diagnosis (and subsequent increase with clinical remission), we wanted to determine if soluble CD163 could be detected in dog serum, and therefore potentially serve as a biomarker. Two different ELISAs were tested and although one of them showed some signal, further testing of the antibodies used in the assay did not support that the signal was specific for CD163. With this experiment, we were not able to quantify soluble CD163 in dogs, but this molecule retains potential as a biomarker for diagnostic and monitoring purposes in CE as well as in other diseases characterised by macrophage activation. Biomarkers of systemic inflammation were also assessed in the same cohort of dogs and we showed that serum IL-6 decreased in dogs with CE after resolution of clinical signs. Similarly to soluble CD163, cytokines might play a role in further differentiating between the different causes of CE for prognostic and therapeutic purposes. Future studies are needed to assess these cytokines in a larger cohort of dogs to be able to study differences between FRE, ARE, and IRE, and further assess their role as biomarkers. Finally, we studied cytokine production by lymphocytes or monocytes in the peripheral blood of healthy dogs, and the effect of different immunosuppressive treatments on cytokine production. Differential activation of lymphocytes or monocytes can easily be achieved by using specific activators in whole blood. The advantage of this technique is that there is minimal handling of the cells with less risk of iatrogenic activation. Cytokine production was affected by cyclosporine and prednisolone, but not by mycophenolate, leflunomide, or azathioprine. Cyclosporine inhibited production of tumour necrosis factor (TNF), interferon gamma and IL-10 by lymphocytes whereas prednisolone inhibited TNF production by both lymphocytes and monocytes. Our findings suggest that this methodology can be used to monitor dogs treated with both drugs concurrently – although this needs to be further assessed with future studies. Future studies highlighted by our research suggest more in-depth assessment of serum cytokines as biomarkers for dogs with CE not only for monitoring purposes, but to determine if different patterns of cytokines can be useful to refine the classification of CE. Similarly, whole blood stimulation can be used to better assess underlying priming of the immune system and to monitor treatment response. Finally, our findings suggest that macrophages play a significant role in the pathophysiology of CE in dogs, particularly in FRE and IRE, but additional work is required to better understand their function in CE.
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ItemAssessment of a ropivacaine ultrasound-guided Transversus Abdominis Plane (TAP) block for peri-operative analgesia in the dog undergoing ovariohysterectomy surgery( 2019)Advances in veterinary medicine have led to the development of further techniques to provide analgesia for our patients. Ovariohysterectomy is a commonly performed surgery in female dogs which causes significant pain. Multimodal analgesia often combining opioids, non-steroidal anti-inflammatories and local anaesthetic techniques is implemented to treat this type of pain. The transversus abdominis plane (TAP) block is a local anaesthetic technique that provides analgesia to the afferent nerves supplying the parietal peritoneum, muscles and skin of the anterior abdominal wall. The TAP block is utilized in human anaesthesia and has been shown to reduce post-operative pain scores and opioid consumption. The TAP block has potential to provide local anaesthesia to the mid-caudal abdomen for dogs undergoing ovariohysterectomy. The initial pilot study was a cadaveric study carried out in 5 dogs with an ultrasound guided two-point TAP injection performed in each hemiabdomen. The study identified that a two-point TAP injection delivered consistent dye dispersion to adequately stain branches of thoracic nerve T13 and lumbar nerves L1, L2 and L3. It concluded that this technique should be assessed in vivo to evaluate the analgesic efficacy in mid to caudal abdominal surgeries. A subsequent randomized controlled trial assessed the two-point ultrasound-guided ropivacaine TAP block for dogs undergoing ovariohysterectomy, with the hypothesis that dogs receiving the ropivacaine TAP block would have a lower minimum alveolar concentration (MAC) isoflurane at skin incision and a lower opioid rescue analgesic requirement post-operatively. The results concluded that the estimated population MACiso for the treatment group was lower than the control group but that this was not significant. There was also no significant difference in pain scores between the groups as measured by the Glasgow composite pain scale short form and mechanical nociceptive threshold testing. The proportion of subjects receiving rescue analgesia in the treatment group was lower than the control group, but this was not significant. The study concluded that the two-point TAP block did not provide significant MAC reduction or additional intra-operative or post-operative analgesia compared with the control for dogs undergoing ovariohysterectomy. Multiple confounding factors such as the administration of morphine as premedication and the low concentration of local anaesthetic used, ropivacaine 0.2%, could have contributed to these findings. Although the cadaver study identified that a two-point TAP injection delivered consistent dye dispersion to thoracic nerve T13 and lumbar nerves L1, L2 and L3, the technique when performed with 0.2% ropivacaine in dogs undergoing ovariohysterectomy did not provide significant additional MACiso reduction or intra-operative or post-operative analgesia compared with the control for dogs. Further assessment of the toxic dose of ropivacaine in dogs and the minimum effective concentration of ropivacaine for TAP blocks in dogs needs to be evaluated in order to further develop and assess this technique.
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ItemEvaluation of the analgesic efficacy of methadone and buprenorphine for the treatment of acute postoperative pain in cats( 2018)Prior to the studies detailed within this thesis butorphanol was the only opioid registered for use in cats in Australia. There was a need for further clinical studies to be conducted in cats to evaluate the perioperative analgesic efficacy of other alternative opioids such as methadone and buprenorphine. While clinical studies had previously investigated the analgesic efficacy of methadone and buprenorphine, none had utilised a validated multidimensional pain assessment tool (PAT) designed to assess acute pain in cats, which called into question their findings. To address this need, the author conducted clinical studies in cats to evaluate the individual perioperative analgesic efficacy of each of methadone and buprenorphine compared to butorphanol. Surgical ovariohysterectomy (OVH) was used as a model of acute pain. Postoperative pain was assessed using a multidimensional PAT recently validated for assessment of acute pain in cats following OVH. Within the conditions of the studies documented within this thesis, methadone administered subcutaneously as a premedication at 0.6 mg/kg provided effective postoperative analgesia for at least 6 hours following OVH surgery in most cats. Buprenorphine administered intramuscularly as a premedication at 20 µg/kg with repeat administration of the same dose at the time of surgical wound closure, provided effective postoperative analgesia for at least 6 hours following OVH surgery. In contrast, intramuscular administration of a single premedication dose of 20 µg/kg IM buprenorphine prior to anaesthesia provided inadequate postoperative analgesia and additional analgesic drugs were required. All dosing regimens involving butorphanol resulted in unacceptable numbers of cats requiring rescue analgesia, suggesting that butorphanol is not a suitable choice of opioid for the provision of postoperative analgesia in the cat at these doses. An additional clinical study was conducted to examine the effect of atipamezole on analgesic requirement, and hence its potential confounding influence on the findings reported. Results from this follow up study suggested that the administration of atipamezole at a clinically recommended dose does not significantly affect the immediate postoperative pain scores in cats following OVH. Physiological data collected during the anesthetic period of these studies indicated that none of the opioids administered caused severe deleterious effects on vital functions and that all could be used safely for premedication prior to general anesthesia. In part dependent upon the findings from these studies, methadone and buprenorphine received registration approval with the Australian Pesticides and Veterinary Medicines Authority (APVMA) for use in cats within Australia. This will in part assist in addressing the undertreating of feline patients experiencing acute perioperative pain by providing clinicians with more efficacious registered drug options. In the course of evaluating the findings of the clinical studies reported in this thesis within the context of the wider peer-reviewed literature, it became apparent that inconsistencies existed in the way analgesic efficacy of opioids in cats is reported. Many of these inconsistencies arise due to variations in the quality and rigour with which a particular study is designed (including the use of non-validated PATs) and reported. To address these inconsistencies, a critical appraisal tool was developed for reviewing analgesia studies (CATRAS) involving animals and human patients who are incapable of self-reporting pain. Content validation was achieved using Delphi methodology via panel consensus. A panel of six experts reviewed the CATRAS in three rounds and quantitatively rated the relevance of the instrument and each of its quality items to their respective domains. The resulting 67-item critical appraisal tool will now provide a means of critically and quantitatively assessing the quality of analgesia trials involving subjects incapable of self-reporting pain for use in systematic reviews and meta-analysis studies.
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ItemInflammation and endothelial perturbation in canine abdominal surgery: the potential modulatory effect of lidocaine( 2019)Complication rates following emergency laparotomy surgery are high, with organ dysfunction being a commonly encountered post-operative complication. Given the endothelium acts as the interface between the systemic circulation and the organs, its function is vital to maintaining organ health. The endothelium is in a constant state of flux, impacted largely by the local environment of which it is a part. In the presence of wide-spread systemic inflammation, inflammatory mediators precipitate change to the structure of the endothelial glycocalyx. These changes result in shedding of the endothelial glycocalyx and alteration of the endothelial phenotype. The endothelium may, as a result, lose the capacity to regulate vasomotor tone, and shift toward a pro-inflammatory and pro-coagulant state. This predisposes to reduced tissue oxygen delivery, and organ dysfunction may ensue. This thesis aimed to answer two key questions: does surgical trauma induced in canine patients undergoing emergent abdominal surgery invoke a systemic inflammatory response and subsequent endothelial activation? And if so, does lidocaine, a proposed immunomodulatory drug, mitigate this effect when given in the post-operative period? Chapter two provides a detailed review of endothelial structure and function, and current literature pertaining to systemic inflammation and endothelial activation in the context of abdominal surgery. Chapter two also examines the literature regarding the proposed mechanisms through which lidocaine acts as an immunomodulatory drug, and reviews publications that investigate the use of lidocaine as an anti-inflammatory drug in human patients after abdominal surgery. Chapter three is a randomized, blinded clinical trial quantifying the effect of emergency abdominal surgery on the concentration of markers of systemic inflammation and endothelial perturbation in canine patients in the post-operative period. The trial also assessed the potential use of lidocaine as a post-operative immunomodulatory therapy in dogs having undergone laparotomy. Fifty canine patients undergoing abdominal surgery were enrolled in the study. Patients were randomized into two separate groups: a study group receiving lidocaine intravenously, and a control group receiving 0.9% NaCl intravenously for a twelve-hour period following abdominal surgery. Blood samples were gathered prior to surgery, followed by six and twelve hours post-operatively. Concentrations of markers of systemic inflammation (IL-6) and markers of endothelial perturbation (VEGF and HA) were quantified via means of ELISA at each time point. Results revealed a significant increase in the concentration of markers of systemic inflammation and endothelial perturbation in post-operative blood samples. No immunomodulatory or endothelial preserving effect of lidocaine was appreciated.
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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.