Veterinary Clinical Sciences - Theses
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Isocitrate dehydrogenase 1 R132H: epidermal growth factor receptor and Ki-67 expression in canine gliomas
The diagnosis, histological classification and grading of canine gliomas can be challenging, particularly in the antemortem. In humans, it is essential to accurately diagnose gliomas as the treatment and prognosis vary between tumour histologic type and grade. Numerous biomarkers have been investigated to aid in the diagnosis and classification of human gliomas, including epidermal growth factor receptor (EGFR), Ki-67 and a point mutation in the isocitrate dehydrogenase 1 gene at codon 132 (IDH1 R132H). Only limited studies looking at these biomarkers have been performed in dogs, from which clear conclusion cannot be drawn. The prognosis for canine glial tumours is considered guarded to poor, with the current treatment options having limited success. Consequently, there is a lack of gold standard treatment for canine glioma. In humans, gliomas are being investigated at a molecular level to identify therapeutic targets and to improve responsiveness to treatment. Numerous molecular targets have been identified including EGFR and IDH1 R132H. Canine gliomas share many morphologic, histologic and immunohistochemical characteristics with human gliomas, suggesting a translational approach to the classification, treatment and prognostication of canine gliomas may be possible. The objectives of this research were: (1) to describe the signalment, presenting clinical signs, diagnostic findings, treatment and survival time of a series of dogs with glial tumours, (2) to investigate the series of canine gliomas for the IDH1 R132H mutation, and for EGFR and Ki-67 expression, utilising immunohistochemistry and (3) to determine if immunoreactivity is associated with tumour histologic type and grade. The clinical data of thirty-one dogs with histologically confirmed glial tumours was reviewed retrospectively. The formalin-fixed paraffin-embedded tumour specimens were evaluated for IDH1 R132H, pan-IDH1 (IDH1 wild-type and mutated IDH1), EGFR and Ki-67 expression. IDH1 R132H and pan-IDH1 expression were recorded as positive or negative. EGFR immunoreactivity was evaluated using a semi-quantitative score, and Ki-67 expression was expressed as the Ki-67 labelling index (LI). Patient signalment, clinical presenting signs and diagnostic investigation findings were similar to previous reports. The IDH1 R132H point mutation was not identified in any (0%) of the canine glial tumours, while all 31 (100%) tumours were positive for pan-IDH1 expression. EGFR expression was identified in 16/31 (51.6%) tumours and expression was significantly greater in high grade gliomas when compared to low grade tumours (P = 0.04). Furthermore, EGFR expression was significantly greater in gliomatosis cerebri when compared to oligodendroglioma (P = 0.002), astrocytoma (P = 0.01), and oligoastrocytoma (P = 0.04). Ki-67 expression was identified in 28/31 (90.3%) gliomas, and the Ki-67 LI was significantly higher in high grade tumours (P = 0.02). A significant moderate correlation between the Ki-67 LI and EGFR immunoreactivity (r = 0.47, P = 0.007) was identified. The IDH1 R132H point mutation was not identified in this series of canine gliomas, and may not be an appropriate biomarker to aid in the classification and treatment of these tumours. EGFR, however, may be a suitable therapeutic target, particularly for gliomatosis cerebri. The Ki-67 LI may aid in the development of a grading scheme for canine gliomas.
Chronic enteropathy in dogs: the role of macrophages and preliminary results in inflammatory cytokines
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.
Associations between the radiographic appearance of vascular channels in proximal sesamoid bones, their microstructural characteristics and past racing performance in thoroughbreds
Reasons for performing the study: ‘Sesamoiditis’ is classically defined by equine practitioners as the presence of and abnormalities in vascular channel appearance within the proximal sesamoid bones (PSB). It is the most common finding in Thoroughbred yearling presale radiographs, as well as often being evaluated on radiographs of adult racehorses with lameness and poor performance. Despite this, the pathogenesis and clinical significance of changes in vascular channel morphology are poorly understood, and the association of ‘sesamoiditis’ with racing performance is inconsistently reported. Objectives: To determine the microstructural characteristics of the PSB associated with the radiographic appearance of vascular channels in Thoroughbred racehorses using micro-computed tomography (µCT), and to determine whether the presence, number and size of vascular channels has an association with past racing performance. Methods: Study design was cross-sectional. One pair of PSB were isolated from a randomly selected forelimb of 59 Thoroughbred racehorses presenting for post-mortem examination over the study period. Each PSB (n=118) was radiographed, assigned a vascular channel grade using previously published and novel radiographic grading systems, then imaged using µCT and similarly assessed. Racing history for each horse was collected. Uni- and multi-variable generalized linear models accounting for clustering at the horse level were generated to investigate associations between radiographic, µCT and performance variables. Results: All PSB had vascular channels observed on µCT originating from the abaxial border (mean 3.6, s.d 0.89), yet in only 63.6% (75/118) were channels observed radiographically. PSB with a higher bone volume fraction (OR 1.08; P=0.031) and wider channel diameter on µCT (OR 20.67; P=0.001) were more likely to have vascular channels identified on radiographs. Radiographic channel number (OR 0.96; P=0.043) and channel size (OR 0.96; P=0.049) were negatively associated with career placings. Main Limitations: Only the forelimb proximal sesamoid bones were collected, radiographs of isolated bones avoided normal superimposition of soft tissue encountered in the live horse, and a cross-sectional study design meant changes in sesamoid vasculature over time and work load could not be assessed. Conclusions: The ability to identify vascular channels radiographically indicates widening of channels and densification of the bone. Increased radiographic channel number and size is associated with poorer measures of past performance suggesting that these changes are not desirable.
Inflammation and endothelial perturbation in canine abdominal surgery: the potential modulatory effect of lidocaine
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.
Investigations on the pathophysiology of canine idiopathic epilepsy
The causes of canine epilepsy are poorly understood. The current aetiologic classification scheme for canine epilepsy includes the categories structural epilepsy, idiopathic epilepsy with genetic or suspected genetic cause, or idiopathic epilepsy of unknown cause. It is likely that dogs with idiopathic epilepsy of unknown cause have heterogeneous underlying pathologies, including subtle structural change that cannot be identified on conventional visual inspection of brain magnetic resonance images. This thesis examines the causes of epilepsy in a sample of Australian dogs presented to the University of Melbourne Veterinary Hospital. The general hypotheses explored by the studies described in this work are that some cases of canine idiopathic epilepsy have underlying structural brain pathologies similar to those identified in humans with epilepsy, and that canine idiopathic epilepsy is associated with increased cerebral levels of the excitatory neurotransmitter glutamate. The prevalence of epilepsy in dogs presented to the University of Melbourne Veterinary Hospital was 1.1%, and idiopathic epilepsy of unknown cause represented 75% of epilepsy diagnoses. Of the 25% of dogs with structural epilepsy, brain tumours were the most frequent cause (60%), followed by meningoencephalitis of unknown origin (11%). In dogs with idiopathic epilepsy, odds ratio analysis identified 21 breeds of dog with an increased risk of a diagnosis of epilepsy. The Hungarian viszla had a particularly strong association with this diagnosis, but in none of the dogs diagnosed with idiopathic epilepsy was a familial history of epilepsy reported. Based on retrospective, randomised and blinded, subjective review of brain magnetic resonance imaging scans of dogs with idiopathic epilepsy and controls, there was no convincing evidence of the magnetic resonance imaging findings of hippocampal sclerosis or focal cortical dysplasia. Brain MRIs were also evaluated using atlas-based segmentation and volumetry, based on a novel canine brain atlas developed for this research. Hippocampal atrophy was used as a biomarker for possible hippocampal sclerosis. Unilateral or bilateral hippocampal atrophy was identified in 15% of dogs with idiopathic epilepsy, based on identifying those dogs with a hippocampal formation volume below the lower 95% reference limit for hippocampal volume established in control dogs. Increased volume of the cerebral cortex was used as a biomarker for possible cortical dysplasia, and reduced volume of the cerebral cortex was a biomarker for cerebrocortical atrophy. Dogs with idiopathic epilepsy had statistically significant reductions in cerebrocortical volume in the left and right olfactory, temporal, occipital, and right parietal lobes. The second hypothesis was that dogs with idiopathic epilepsy had elevated cerebral glutamate, the major excitatory neurotransmitter in the brain. This was explored non-invasively in dogs with naturally occurring epilepsy, using proton magnetic resonance spectroscopy to measure in vivo brain glutamate. There was no significant difference in either glutamate or glutamate to creatine ratio between dogs with epilepsy and controls. Further work is required to establish whether hippocampal atrophy in dogs with epilepsy is due to hippocampal sclerosis. This work could use both volumetry, T2 relaxometry, and histopathologic evaluation of brain samples. Future investigation of the mechanisms of cerebrocortical atrophy in epilepsy may involve cortical thickness measures to allow targeted correlation of regions of localised cerebrocortical atrophy with brain histology, and fibre tracking to map epileptogenic networks, exploring the connections between EEG identified seizure focus and regions of cortical atrophy. The role of brain glutamate may be further investigated using multi-voxel magnetic resonance spectroscopy at 3 Tesla, and glutamate transporter proteins and receptors may be investigated through laboratory techniques.
Update on clinicopathological assessment of renal health in non-racing greyhounds
Background: Serum creatinine concentrations differ in greyhounds compared with non-sighthounds, but it is not known whether urine creatinine concentrations also differ and whether any difference would influence the interpretation of the urine protein to creatinine ratio (UPC). Additionally, there is some evidence for greyhounds having higher serum symmetric dimethylarginine (SDMA) than non-sighthounds, but this has yet to be confirmed in healthy non-racing greyhounds. Objectives: The objectives of this study were fourfold: (1) to compare the urine creatinine concentrations in healthy greyhounds and a control group of healthy non-sighthounds, (2) to determine the UPC reference interval in healthy greyhounds and to compare this with the UPC reference interval in a control group of healthy non-sighthounds, (3) to determine the serum SDMA concentration reference interval in healthy greyhounds and to compare this with the serum SDMA concentration reference interval in a control group of healthy non-sighthounds and with a previously established canine serum SDMA concentration reference interval, and (4) to establish whether lean body mass is correlated with serum creatinine and urine creatinine concentrations in greyhounds. Methods: The study used an observational cross-sectional design and included 98 clinically healthy non-racing greyhounds and 24 non-sighthound dogs with similar weight, age and sex distributions, as determined by t-test and chi-squared tests. SDMA, urine creatinine concentration and UPC values were measured from blood and urine samples. Linear regression was used to compare the greyhound and non-sighthound groups. Greyhound reference intervals were determined for SDMA and UPC using non-parametric methods. These were compared with the reference intervals for the non-sighthound group and with current International Renal Interest Society guidelines. In the greyhound sample, the association of urine creatinine with thigh circumference, height and weight was estimated using Pearson correlation. Statistical significance was set at P < 0.05 for all analyses. Results: Mean urine creatinine was approximately 22% higher in greyhounds than non-sighthounds after adjusting for urine concentration (P < 0.05). The upper limit of the greyhound UPC reference interval was 0.20 or 0.42, depending on whether strict or moderate exclusion criteria, respectively, were applied. The mean UPC was 29% lower in greyhounds than non-sighthounds, but this difference was not statistically significant (P = 0.1). The serum SDMA reference interval for greyhounds was 6.3–19.7 µg/dL (0.31–0.98 µmol/L). The upper end of this interval was higher than the upper limit of the published canine reference interval (6–13 µg/dL), and the mean concentration was statistically significantly higher in greyhounds (13.0 µg/dL) than non-sighthounds (10.2 µg/dL, P < 0.001). In greyhounds, there were weak correlations between the three morphometric measurements and both serum creatinine and urine creatinine after adjusting for urine concentration. Conclusions and clinical importance: These findings provide further evidence that greyhounds require several breed-specific reference intervals when evaluating renal function. Apart from having higher serum creatinine, greyhounds also have higher SDMA and higher urine creatinine when compared to non-sighthounds. Although UPC trended slightly lower in greyhounds, this finding was not significant, and therefore the threshold for non-proteinuria set by IRIS guidelines appears to be appropriate for greyhounds based on the calculated reference interval.
Improving disease surveillance in Australia’s sheep industries: investigations of syndromic surveillance, farmer behaviour and sheep trade networks
Designing and delivering effective, useful livestock health surveillance is a challenge for many countries. The observations of people in frequent contact with livestock, captured through passive surveillance, play an important role in many national surveillance systems. In Australia, the effectiveness of passive surveillance on sheep and beef farms has been limited by infrequent veterinary contact. Farm workers frequently observe signs of disease in livestock, but these observations are not captured by existing surveillance systems. This thesis therefore posed the question: can farmers’ observations be collected to generate useful surveillance information? Syndromic surveillance of farmers’ observations is one approach to increase data capture from extensive livestock farms. Chapter 3 describes the operation of a syndromic surveillance system collecting farmers’ observations of livestock health in Victoria, Australia, over its first two years of operation from 2014 to 2016. Survival analysis and classification and regression tree analysis were used to identify farm level factors associated with reliable participation, to inform future recruitment aimed at farmers who were willing and able to provide regular, timely reports. Farmers keeping only sheep were the most reliable and timely respondents, while farmers aged under 43 years or working full time on-farm had lower response rates than older farmers or part-time farmers. This chapter demonstrates that recording farmers’ observations of signs of disease using syndromes is a feasible and effective method to gather disease occurrence data. The utility of syndromic data is further investigated in Chapter 4, using the observations collected by the surveillance system to quantify ewe mortality on sheep farms in southern Australia. Ewe deaths were reported in 540 of 612 reports, describing 2106 individual deaths, with a median of 4 deaths per positive monthly report. Median mortality rates ranged between individual farms from 1 to 5 deaths/1000 ewes/month. The incidence rate ratio of mortality in the five months preceding and following lambing was 2.8 (95% CI 2.0 to 4.1) compared to the remaining seven months of the year. Overall ewe mortality could therefore be reduced through strategies targeted to improving peri-parturient ewe survival. In a subset of reports where veterinary contact was recorded, just 15% of reported deaths involved a veterinarian. Further investigation of how and why farmers respond to ewe deaths without veterinary support is needed, to determine the best farm management strategies to reduce mortality. Chapter 5 investigates Australian sheep farmers’ low rates of veterinary contact. The study aimed to understand why Australian sheep farmers chose not to contact veterinarians when their animals showed signs of disease, and what alternative approaches they took to managing unwell animals. Data were collected during three focus group discussions with sheep farmers in Victoria, Australia. Transcripts of those discussions were analysed using a modified grounded theory approach to develop a preliminary theory of Australian sheep farmers’ disease response behaviour. Critical steps in the decision-making process included the farmer recognising that action is needed, and then deciding what that action would be. The farmers reported having to decide whether they would act independently based on their previously experiences, or alternatively to seek advice. Veterinarians played a small but important role as potential advisors, alongside others including trusted farming friends and farmer discussion groups. Self-reliance and confidence in their knowledge and skills was highlighted as the main reason the farmers often chose not to seek veterinary advice. Rather than being seen as a barrier to effective passive surveillance, the actions that arise from farmers’ self-reliance when facing disease should be taken into account when designing novel surveillance approaches. A final consideration for observational disease surveillance is the selection of individuals to contribute data to the system. While characteristics associated with participation may guide recruitment as described in Chapter 3, it is also useful to target surveillance to farms that have increased risk of acquiring or disseminating disease. The movement of animals between farms contributes to infectious disease spread, and can be investigated through network analysis methods. Australia’s National Livestock Identification Scheme sheep movement records are suitable for such analyses, but are known to be a targeted subset of all sheep movement in the country. However, knowledge of the effect of sampling or incomplete network data on these studies is limited. In Chapter 6, a simulation algorithm is presented that provides an estimate of required sampling proportions based on predicted network size, density and degree value distribution. The algorithm may be applied a priori to ensure network analyses based on sampled or incomplete data provide population estimates of known precision. Results demonstrate that, for network degree metrics, sample size requirements vary with sampling method. Where simulated networks can be constructed to closely mimic the true network in a target population, this algorithm provides a straightforward approach to determining sample size under a given sampling procedure for a network metric of interest. Chapter 7 then presents analysis of National Livestock Identification Scheme sheep movement data for Victoria, Australia. The sheep movement network in Victoria shows typical livestock movement network characteristics including scale-free and small-world topology, small diameter and short average path lengths, supporting the assumption that disease could spread rapidly in the state through sheep movements if it were not detected rapidly. Victoria’s position as a net importer of sheep and sheep flow is confirmed, driven substantially by the activity of saleyards (livestock markets) and abattoirs. Little variation within or between years in overall movement patterns were detected. While most farms are connected to a very small number of properties in the network, small subsets of farms demonstrate high degree values (being directly connected to many other properties through incoming out outgoing animal movements) or high frequency of sheep purchases or sales. These farms may be useful targets for emerging surveillance methods that can be implemented on-farm. Together, these studies provide new information about the Australian sheep industry and the feasibility of new surveillance approaches to improve the effectiveness of surveillance. By describing farmer behaviour, livestock movements patterns and the feasibility of syndromic surveillance approaches to capture farmers’ observations of signs of disease, these studies justify further development and implementation of novel surveillance approaches in Australia and serve as an example for other countries facing similar surveillance challenges. While there is no ideal surveillance system, integrating new approaches into wider surveillance strategies can improve the quality of information generated by surveillance, to better describe true disease states in the population and drive appropriate response activities.
Viscoelastic coagulation changes in dogs with tiger snake envenomation
Snake venom induced consumption coagulopathy (SVICC) is an important yet poorly described clinical syndrome in the field of veterinary medicine. Publications referencing humans, on the other hand, are numerous in comparison, especially since the establishment of the Australian Snakebite Project. Since the introduction of Australian snake-specific antivenoms, SVICC has become the most common underlying reason for human fatality from snake envenomation in Australia.1 The annual canine snakebite caseload in Australia alone is vast and could be established as a model for the human condition. The overall objective of this study is to extend the scientific literature on SVICC in veterinary medicine, namely the role of thromboelastography (TEG) in tiger snake envenomed dogs. The study assessed the changing clot kinetics of canine whole blood after natural tiger snake envenomation using an established technique called thromboelastography (TEG). Specifically, we will be determining the TEG changes in dogs at several time points: T0 = time of presentation; T1 = 1 hour after antivenom administration; T18 = 18 hours after antivenom administration; and, finally, T24 = 24 hours after antivenom administration. Tiger snake venom caused alterations in TEG parameters, specifically the prolongation of R time (time to initiation of clot formation) during the first 24 hours of envenomation.2 No clinical benefit exists in using TEG over classical coagulation parameters in the identification of SVICC in tiger snake envenomed dogs. Hypercoagulability occurs for 24 hours after envenomation and VetSVICC appears to resolve 18–24 hours after antivenom, which is suggestive of a shorter period to apparent clinical recovery compared to humans, suggesting that VetSVICC may be a unique clinical syndrome. This project is a stepping stone to future research into both SVICC and tiger snake envenomation in veterinary medicine, including establishing an accessible and reliable diagnostic test for SVICC and further delineation of its importance regarding the severity of illness and outcome.
Investigation into the relationship between scrotal circumference, body weight, semen characteristics, daughter fertility and genomic breeding values, as well as monitoring behaviour in commercial pasture-based dairy breed natural-service sires
Use of natural-service sires in the pasture-based dairy industry of south-eastern Australia is common, however, research into their selection and management is sparse. This investigation included a cross-sectional study of the genetic merit of pasture-raised natural-service dairy breed sires and quantified the association between natural-service sire scrotal circumference and their daughter fertility breeding value. Additionally, investigations into the association within breeds between scrotal circumference (as an explanatory variable) and daughter fertility breeding value as the outcome variable in a linear regression model were conducted. And finally, this investigation aimed to monitor bull behaviour, including mounting and serving behaviour, as well as tracking distanced walked. Deoxyribose nucleic acid was submitted for genotyping from two groups of Tasmanian Holstein (n=124) and Jersey (n=85) bull calves to get a set of genomic breeding values. Scrotal circumference and body weight measurements and semen characteristics were recorded at 8 weekly intervals from the age of 6 months to 18 months of age. In addition, an observational study was conducted using collar mounted tri-axial accelerometers and global positioning systems on bulls (n=10) on a commercial pasture-based dairy farm in Tasmania, Australia. The genomic information from natural-service sires was compared to contemporary artificial insemination sires, however a clearly defined association between scrotal circumference and the daughter fertility breeding value could not be elucidated, despite the link in beef breeds. Linear regression quantified the relationship between scrotal circumference and body weight and classification and regression tree analyses were determined the predictive value of scrotal circumference and body weight on semen characteristics. Linear regression showed that 50 kg increase in a Holstein bull’s body weight was associated with a 2.9 (95% CI 2.8 to 3.0) centimetre change in scrotal circumference (P= <0.001). For Jerseys, 50 kg increase in body weight was associated with a 2.4 (95% CI 2.3 to 2.5) centimetre change in scrotal circumference (P= <0.001). Classification and regression tree analysis for Holsteins and Jerseys combined showed that 95% of animals that weighed greater had a scrotal circumference of at least 27 cm had a percent normal sperm score greater than 70%. Using learning algorithms bull behaviour was monitored including grazing (F = 0.86) and walking (F = 0.91) compared with ruminating (F = 0.19) and resting (F = 0.38). However, due to the short, yet explosive nature of the mounting and serving signatures, the algorithm was not able to detect them. Mean distance travelled per day ranged from 12.0km to 6.5km and maximum distance travelled per day of 28.0km to the least maximum distance of 14.6km. This study provides information about the genomic merit of natural-service sires and the relationship between scrotal circumference and daughter fertility, augments information on scrotal circumference and body weight relationships in pasture-raised Holstein and Jersey breed bulls and supports the use of the bull breeding soundness exam to manage the risk for sub-fertility in the dairy industry. Additionally, it was found that walking long distances may contribute to the lameness in dairy natural-service sires.
The effects of intravenous fluids on thromboelastrographic variables in dogs
Intravenous fluid resuscitation plays a fundamental role in treating dogs in haemorrhagic shock, as it can rapidly replenish lost intravascular volume and improve tissue perfusion. However, a consequence of intravenous fluid therapy is interference with haemostasis, which has a detrimental effect to trauma patients that are already haemostatically compromised. In people, intravenous fluid therapy can effect haemostasis and subsequently can increase haemorrhage, transfusion needs and mortality. The effects of intravenous fluids on haemostasis in dogs have not been widely established. More recently, viscoelastic devices like thromboelastography have been used to examine haemostasis. Compared to conventional coagulation testing, viscoelastic devices have the advantages to be able to assess the speed and kinetics of clot formation, clot strength and even the breakdown of the clot. The aim of our study was to determine the effects of intravenous fluids on coagulation in dogs with the use of thromboelastography. The objective of our study was to determine the effect of dilution of canine whole blood with clinically relevant doses of common intravenous fluids on thrombelastographic variables. Our hypothesis was that in vitro dilution of canine whole blood from healthy dogs with intravenous fluids will induce dose-dependent changes in thromboelastographic variables consistent with hypocoagulability. Further, we hypothesized that the characteristics of the fluids, such as its ionic strength and osmolality, will effect thromboelastographic variables in addition to those of dilution alone. The results of our study showed that in vitro dilution of canine whole blood with commonly used intravenous fluids lead to thromboelastographic changes consistent with hypocoagulability in a dose dependent manner. Besides dilution percentage, viscoelastic changes were influenced by fluid characteristics, specifically ionic strength, osmolality and colloidal properties. In our study, 7% hypertonic saline had the most severe effects on coagulation, followed by 20% Mannitol then 3.4% hypertonic saline. Hydroxyethyl starch 130/0.4 had minimal effects on coagulation besides a dilutional effect. The differential effect of fluid characteristics should be taken into consideration when resuscitating dogs with large fluid volumes, but clinical studies are still required to further delineate the importance of different resuscitation fluids and volumes on haemostasis in dogs.
Ensuring dairy cow welfare with increasing scale of production
Animal welfare is important to the general public and dairy consumers and the dairy industry is coming under increasing scrutiny. There is the potential for community concern arising from perceived intensification of the industry with an increasing number of large herds, and producers adopting a mix of grazing and confined feeding strategies. This thesis examines the particular welfare challenges associated with increased herd size in Australian pasture-based dairy herds including how they might be measured and managed. A survey of Australian dairy farmers was conducted to assess relationships between herd size and known or proposed risk factors for adverse animal welfare outcomes in Australian dairy herds in relation to increasing scale of production. Increasing herd size was associated with increases in stocking density, stock per labour unit and grain fed per day – all of which could reasonably be hypothesized to increase the risk of adverse welfare outcomes unless carefully managed. However, increasing herd size was also associated with an increased likelihood of staff with formal and industry-based training qualifications. Herd size was not associated with reported increases in mastitis or lameness. Large herds were more likely to use monitoring systems such as electronic identification in the dairy, computerised records, daily milk yield or cell count monitoring and pedometers or activity meters. Increasing herd size was related to increased herd milking time, increased time away from the paddock and increased distance walked. Animal welfare assessments were conducted on 50 Australian pasture-based dairy farms of varying herd sizes. Findings were generally consistent with the previous survey. Major challenges included heat stress, mastitis, lameness, and longer milking duration. All cows had access to water for more than 12 h in a 24 h period. More larger farms had water points on the farm tracks or at the dairy. Skin and joint lesion prevalence was not related to herd size and they were uncommon. All farms had some form of cooling strategy. Shade in all paddocks was more common on smaller farms than others while sprinklers were more common on large/very large farms. There was wide variation in the avoidance distance of humans but this was not related to farm size. Lameness scoring was conducted on 19154 cows from 50 farms as they left the dairy after being milked. We compared our Results with farmer estimates of lameness prevalence. Farmers detected only 24% of the cows observed/scored as lame. Whilst lameness scoring of the entire herd was necessary to detect all the lame cows, scoring just the last 200 cows milked could be used to estimate the prevalence of lame cows on a given day. In large Australian dairy herds, it is common for cows to be collected from the paddock as a group, to wait as a group in the dairy yard to be milked and to return individually to the paddock or feed pad immediately after milking. We demonstrated that even in these large herds there is consistency in the order cows are milked. This may have welfare implications for cows that are regularly milked last because they may spend several hours per day less in a paddock grazing. Lying is a high priority behaviour for cows. We used activity monitors to show that cows in large Australian dairy herds spend an average of 9.5 hours lying each day. Even in large herds, where milking can take up to 4 hours, the time taken to milk the cows did not affect their welfare by significantly impacting on their ability to lie down.
A pilot study of the seroprevalence of Q fever in cattle, sheep and goats in Victoria
Q fever is an important public health concern throughout the world and infection can result in debilitating and lifelong illness in some people. It is caused by the bacterium Coxiella burnetii and the most frequent source of infection for humans are domestic ruminants. Over a quarter of human Q fever cases in Victoria, 192 out of 659 (29%) analysed from 1994 to 2013 (Bond et al., 2018), are locally acquired indicating that it is endemic in the state. In addition, outbreaks of human Q fever in Victoria associated with local livestock is causing concern that the incidence of disease is increasing. However, information regarding the amount of infection present in the animal reservoir are lacking in Victoria. The aim of this pilot study was to survey cattle, sheep and goats in Victoria to estimate the individual animal-level, herd-prevalence and within-herd-prevalence of Coxiella burnetii infection. This survey was carried out from February 2015 to May 2017 in Victoria, Australia. Over 1500 blood samples were collected using a two-stage sampling process from farms and abattoirs. Sera were screened using a commercial Q fever ELISA kit for ruminants. True prevalence and intra-class correlation coefficient for each species was estimated using a Bayesian approach to account for known test imperfections. Post-hoc sample size estimates were calculated based on survey results to inform future study planning. Q fever is endemic in domestic ruminants in Victoria at low levels and may even be absent in some parts of the state. Of the herds and flocks that were sampled, 8% (95% confidence interval, 5 to 13%) had at least one animal seropositive for Q fever. Herd-level seropositivity rates were 0% (0, 4.7%) and 8.7% (2.4, 26.8%) for Goulburn Valley and Gippsland cattle respectively, 17.6% (9.6, 30.2%) for sheep and 8.0% (0.2, 25.0%) for goats. The overall pooled true animal-level prevalence was 1.3% (95% HPD, 0.9 to 2%). This was based on true animal-level prevalence of 0% (0, 0%) and 0.4% (0, 3.5%) for cattle in Goulburn Valley and Gippsland respectively, 2.1% (0, 3.7%) for sheep and 1.6% (0, 2.8%) for goats. Although the overall herd- and animal-level prevalence estimates were very low, the individual animal-level prevalence within infected herds (within-herd prevalence) was much higher, ranging from 12 to 19%, by species. Finding a small number of groups of animals with elevated levels of within-herd seroprevalence, whilst not detecting positive results in the rest of the sampled population, indicates that the prevalence of exposure to Coxiella burnetii was geographically uneven, being highly clustered within a small number of farms in Victoria. Post-hoc sample size estimates based on survey results indicate large numbers of farms and animals are required for further studies to confirm this very low rate or the absence of infection in some areas of Victoria. However, a greater priority and more cost-effective approach would be to establish why some farms are at higher risk, and this could be efficiently investigated using a case-control study. The findings of this pilot study present a step forward in understanding the epidemiology of Coxiella burnetii in this region of Australia and point to further areas of investigation and how to conduct such studies.