Veterinary Clinical Sciences - Theses
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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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ItemInvestigations on the pathophysiology of canine idiopathic epilepsy( 2018)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.
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ItemImproving disease surveillance in Australia’s sheep industries: investigations of syndromic surveillance, farmer behaviour and sheep trade networks( 2018)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.
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ItemEnsuring dairy cow welfare with increasing scale of production( 2018)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.
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ItemExploring oriental theileriosis in bovines using advanced molecular tools( 2018)Theileriosis is caused by tick-borne haemoprotozoan parasites of the genus Theileria spp. and is one of the most economically important diseases of bovines, particularly in tropical and sub-tropical regions of the world. Of the different forms of bovine theileriosis, oriental theileriosis, caused by one or more genotypes of T. orientalis, has become an important emerging tick-borne disease linked to substantial economic losses in cattle, particularly in the Asia-Pacific region. Based on the sequence analysis of the major piroplasm surface protein (MPSP) gene, at least 11 distinct genotypes of T. orientalis (types 1-8, and N1-N3) have been recognised globally. Of these, 11 currently known genotypes, types 1 (chitose) and 2 (ikeda) are usually associated with clinical oriental theileriosis in cattle in the Asia-Pacific region. The present thesis was chiefly aimed at exploring epidemiological aspects of oriental theileriosis in bovines from different countries and improving the diagnostic methods used for this disease. This thesis comprises of a literature review, seven result chapters and a general discussion. In Chapters 2 to 4, the applicability of a standard PCR and multiplexed tandem PCR (MT-PCR) followed by mutation scanning and phylogenetic analysis as well as different conventional diagnostic methods were used to assess the spread of T. orientalis infections associated with cattle movement within Australia and from Australia to Vietnam and to Pakistan. The results of these Chapters revealed the presence of genotypes buffeli, chitose, ikeda and type 5 of T. orientalis at high prevalence and intensity of infection, and genotypes chitose and ikeda were shown, for the first time, to be associated with oriental theileriosis outbreaks in South Australia (beef cattle) and Vietnam (imported dairy cattle). In addition, the MT-PCR assay was sensitive and specific for the rapid and reliable detection of four genotypes (buffeli, chitose, ikeda and type 5) of T. orientalis in cattle involved in oriental theileriosis in Australia and other countries. Cattle movement was shown to be a major risk factor for the spread of oriental theileriosis from endemic to non-endemic regions, and the recommendation was made that cattle should be tested before and after movement, in order to control the spread of the disease using advanced molecular tools such as MT-PCR. In Chapters 4 and 5, the established MT-PCR followed by MPSP-PCR phylogenetic analyses were applied in areas where the prevalence and genetic diversity of T. orientalis complex were previously unknown (e.g., in Ethiopia and Pakistan). Using these methods, four genotypes (buffeli, chitose, ikeda and type 5) of T. orientalis were detected in both countries with low prevalence and intensity of infection in bovines. The results indicated the broad applicability of the MT-PCR to T. orientalis infections in countries other than Australia and New Zealand. The impact of oriental theileriosis in bovines and local vectors are unclear in these countries, and further large-scale molecular epidemiological studies in different ecological and climatic zones are required. In Chapter 6, the cost-effectiveness of pooled cattle blood samples compared to the testing of large individual cattle blood samples by MT-PCR was assessed for the diagnosis of the two pathogenic genotypes chitose and ikeda of T. orientalis. The results revealed that the testing of pooled blood samples (pools of 5 or 10) was costeffective to screen herd-level prevalence and animal-level prevalence (in regions with low disease prevalence) for T. orientalis, and can significantly reduce the time and cost of testing large individual blood samples. This pooled blood sampling technique might also applicable to the detection of other infectious diseases of bovines. In Chapters 7 and 8, the established MT-PCR assay using multiple markers for the detection of four genotypes (buffeli, chitose, ikeda and type 5) of T. orientalis was evaluated and modified to use one marker i.e., MPSP gene to detect, differentiate and quantitate these four genotypes in different countries. The MPSP gene was found to be a reliable and suitable marker to investigate the T. orientalis complex by standard PCR and MT-PCR assays. The findings of the present thesis highlight the need for further large-scale international collaborations to understand the genetic diversity, epidemiology, transmissions, host-parasite interactions, role of reservoir hosts, vaccines and treatment of T. orientalis complex in different parts of the world. It also suggests the need to develop other alternative, advanced molecular tools to simultaneously detect all the currently known and novel genotypes of T. orientalis using a next-generation sequencing approach.