Veterinary Science Collected Works - Theses
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ItemNon-invasive measurements of stress, demeanour and body condition in horses and ponies, with and without pituitary pars intermedia dysfunction (PPID)( 2023-10)The population of aged horses and ponies is steadily growing as people keep their animals for longer and provide better care and nutrition. Pituitary pars intermedia dysfunction (PPID) is a common condition in older ponies and horses, affecting 1 in 5 animals aged over 15 years, and is associated with clinical signs such as hypertrichosis, muscle loss, polyuria/polydipsia, and laminitis. In addition, it has been suggested that PPID cases may exhibit a dull demeanour. Potentially, this could be caused by changes in pituitary-derived hormones or associated metabolic derangements; or due to stress or pain associated with subclinical laminitis or loss of muscle mass. If this does occur, it may contribute to loss of use and a general decline in wellbeing. Spontaneous blink rate (SBR) has recently been evaluated as a non-invasive measure of stress in horses. The aims of the current study were to determine whether a range of non-invasive measures (demeanour score, SBR, heart rate) could be used to assess demeanour and/or altered levels of alertness or stress in ponies and horses, and whether they would correlate with plasma cortisol and beta-endorphin concentrations. SBR was found to correlate well with demeanour score and also cortisol concentration. Teeth rasping was used as a mild ‘stressor’ to validate the methods, and the effects of a mild sedative drug were also assessed. The effect of short-term dopamine depletion in normal horses was also evaluated to assess the role of dopamine on demeanour, since PPID is caused by low dopamine concentrations in the brain. There were no changes in any of the parameters when dopamine concentrations were reduced using this protocol, although a change in SBR was observed when the mild sedative drug was administered. These methods were then used to determine whether PPID was associated with altered demeanour, alertness or stress compared with non-PPID animals of similar age. Out of a cohort of 32 ponies and horses greater than15 years old (that were likely candidates for PPID), 13 were diagnosed with PPID based on endocrine testing and the presence of clinical signs. There were no differences in SBR, demeanour score or heart rate between PPID cases (n is equal to13) and non-PPID controls (n equal to19). The hormonal changes that may accompany PPID, including potentially increased cortisol or Beta-endorphin, were also quantified to determine whether they may be associated with any observed changes in demeanour. There were no differences in total plasma cortisol; however, beta-endorphin concentrations were significantly higher in PPID cases. Although this hormone may be increased in exercise or stress, the increase in PPID cases appeared to be related to pituitary dysfunction and was not associated with any changes in demeanour or other parameters. PPID was associated with evidence of significant muscle atrophy, despite no difference in adiposity (measured by body condition score and cresty neck score) between PPID cases and non-PPID controls. The cause of muscle atrophy was not determined – there were no differences in plasma insulin-like growth factor 1 (IGF 1), or cortisol. Finally, the dopamine receptor agonist drug, pergolide, which is the standard treatment for PPID, was administered for 4 weeks in a randomised crossover study to examine the effect of treatment on demeanour and body condition. Demeanour scores were unchanged, but the drug did cause a significant decrease in beta-endorphin concentrations in PPID cases. There was also a negative effect on SBR, although this effects varied between individuals. Appetite (time taken to eat a standard meal) was also assessed, and there was no difference in appetite between PPID cases and controls; and also, no effect of treatment with pergolide. Furthermore, pergolide had no effect on the muscle atrophy score or body condition over the treatment period. In summary, this study has helped to further validate the use of SBR as a non-invasive index of demeanour and stress in ponies and horses; and has found no evidence for PPID cases exhibiting a dull demeanour or reduced appetite. Neither the dopamine agonist drug, pergolide, nor dopamine depletion caused any measurable changes in demeanour or appetite, and pergolide caused variable effects on blink rate, suggesting that dopamine does not appear to play a major role in these aspects. Therefore, the evidence indicates that ponies and horses with PPID should be able to enjoy a good quality of life without alterations in mental state such as a dull demeanour, provided that they are well managed and associated conditions such as chronic laminitis are avoided. Muscle atrophy is a significant issue with this condition, and so nutritional support is also crucial.
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ItemNo Preview AvailableVeterinary Forensic Radiology- Development of a cost effective and easy to perform Post-Mortem Computed Tomographic Angiography protocol( 2023-01)Post-mortem imaging (PMI) is a rapidly expanding field in human medicine. In veterinary medicine the literature published on PMI is limited to a few small case series and single case reports. Initially, we investigated the retrospective use of PMI at a university veterinary hospital over a ten-year period (January 2010 – March 2020). Documenting a five-fold increase in PMI between the first five years (2010-2014) and the last five years (2015-2020). The modality with the greatest increase in use was post-mortem computed tomography (PMCT) with a six-fold increase. PMI is an evolving field in veterinary radiology with rapid growth in use observed over the last 10 years. Veterinary post-mortem computed tomographic angiography (PMCTA) has the potential to act as an alternative or adjunctive to traditional necropsy. In human forensic radiology, the use of PMCTA is common practice and has clear benefits over plain PMCT. In veterinary medicine, there is currently no published PMCTA technique for dogs and cats that is repeatable and can be routinely performed in a referral veterinary hospital. This study shows PMCTA can be performed using iohexol mixed with a polyethylene glycol adjuvant and administered via a power pump injector and was successfully demonstrated in 5 dogs and 7 cats. We hypothesised that PMCTA will enhance the diagnostic capabilities of PMCT by increasing the visualisation of vasculature and soft tissue, as well as demonstrating contrast medium uptake characteristics of pathology. Our findings showed the cause of death determined from necropsy and PMCTA agreed in 83% of cases and 42%, the cause of death determined on PMCTA was aided by the administration of contrast medium. PMCTA outperformed necropsy in the detection of neurological and musculoskeletal pathology, detecting 3.3 times more pathologies. The addition of PMCTA to a post-mortem imaging examination was found to have increased diagnostic prowess for non-traumatic causes of death and enhanced characterisation of traumatic causes of death, compared to PMCT studies without contrast medium at our institution. This study represents the first of its kind as a case series describing the practical use of a veterinary PMCTA protocol.
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ItemInvestigation of immunology and neuropathology of influenza virus and arbovirus co-infection( 2024-02)Viral co-infections can substantially impact antiviral responses, reduce protection and enhance immunopathology. Limited data exist on whether co-infection with influenza and other non-respiratory viruses affect disease outcomes and/or immune responses towards sequential or concurrent co-infections. As influenza viruses are prevalent worldwide, their geographical distribution overlap with neurotropic arboviruses. Hence, it is of key importance to investigate influenza and encephalitic arbovirus co-infections. The aim of this PhD thesis is to investigate the immune responses and immunopathology during co-infection with a respiratory influenza virus and neurotropic Alphavirus. We established a novel mouse model to study co-infection with unrelated viruses, influenza A virus (IAV) and Semliki Forest virus (SFV), causing disease in different organ systems, respiratory and central nervous systems. In Chapter 3, we sought to define how sequential co-infection of Alphavirus and influenza virus affects host immunity towards influenza virus. Mice were given SFV infection eight days before IAV infection (SFV->IAV). We provided evidence that SFV->IAV co-infection resulted in compromised IAV immune responses, leading to inefficient clearing of pulmonary IAV, subsequently causing increased lung inflammation, measured by the elevated cytokine/chemokine levels and exacerbated lung pathology. This was associated with impaired lung IAV-specific CD8+ T cell responses, stemming from suboptimal CD8+ T cell activation and proliferation in draining lymph nodes, as well as dendritic cell paralysis. Furthermore, IAV CD8+ T cell were detected in the brain of SFV->IAV infected mice. Ex vivo IAV epitope-specific TCR analysis performed in the brain and lungs during single IAV and sequential SFV->IAV co-infection not only showed remodelled TCR repertoire in co-infected mice, but corroborates the presence of these IAV-specific CD8+ T cells in the brain during co-infection. Adoptive transfer experiments highlighted that recruitment of these IAV-specific CD8+ T cells were antigen-specific and not a of bystander event. In the long term, CD8+ T cell memory pool establishment and magnitude of recall CD8+ T cell responses were comparable between IAV-only and SFV->IAV infection. However, reversing the sequence of co-infection (IAV->SFV) significantly diminished long-term IAV-specific immune responses, and also abolished the immunodominance hierarchy of CD8+ T cell responses to DbNP366 and DbPA224 epitopes. In Chapter 4, we determined how sequential co-infection of influenza virus and Alphavirus affects host immunity towards Alphavirus. Here, mice were given IAV infection 8 days prior SFV infection (IAV->SFV). Our findings demonstrated that IAV->SFV co-infection led to milder disease compared to SFV-only infection. Contrary to previous findings in Chapter 3, IAV->SFV infection resulted in reduced overall brain SFV viral titre and hence, lower cytokine/chemokine levels in the CNS. While viremia in both IAV->SFV and SFV-only mice were comparable, the trend of increased levels of type I IFN in the CNS after influenza virus infection might have been sufficient to mediate protection towards SFV infection in the CNS. Further investigation revealed that the acute and long-term adaptive immune responses in the CNS of both SFV-only and IAV->SFV mice were comparable, suggesting that the early control of SFV replication in the CNS in IAV->SFV by IFN has led to an overall reduced adaptive response, given the lower number of innate immune cells and CD8+ T cells that were recruited to the brain in IAV->SFV infection. Taken together, Chapter 4 established that the order of sequential co-infection significantly impacts the immune response and thus disease aetiology. In Chapter 5, we investigated how simultaneous co-infection of influenza virus and Alphavirus affects host immunity towards both pathogens. Mice were given both SFV and IAV simultaneously at different site of infection and monitored across different time points (SFV+IAV). We demonstrated that SFV+IAV infection did not lead to exacerbated or attenuated disease compared to both the single infection groups. Our studies showed that the overall immune responses and inflammation in the brain in SFV+IAV co-infection were comparable to that of SFV-only. On the contrary, despite prolonged pulmonary IAV replication in SFV+IAV infection, we did not observe exacerbated lung pathology in SFV+IAV compared to IAV-only infection. However, the magnitude of IAV-specific CD8+ T cell responses in the lungs were diminished in SFV+IAV infection compared to that of IAV-only infection. Despite this, SFV+IAV infected mice had comparable brain and lung pathology to SFV- and IAV-only infection. The prolonged clearance of pulmonary IAV and reduced magnitude of IAV-specific responses in the lungs in SFV+IAV co-infection was parallel to our findings in Chapter 3, suggesting that SFV infection, whether given in combination or consecutively with IAV, hampers IAV immune responses. Thus, Chapter 5 highlighted the significance of timing in co-infection, influencing immune responses and disease trajectory. Overall, the knowledge from research performed in this PhD thesis provides key insights into our understanding of how infection with one virus can alter immunity towards the second virus, following sequential or concurrent viral co-infections. Furthermore, expanding our existing knowledge on immune responses towards viral co-infections can facilitate a rational design of successful vaccine regimens directed towards multiple viral diseases.
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ItemEffect of muscle fibre type and postmortem ageing on flavour of beef( 2023-12)Flavour is the most important sensory attribute for predicting consumer liking of beef. Flavour of beef is caused by the sensation of flavour-active compounds, including taste-active compounds and odour-active volatiles, during consumption. Research in this thesis was focused on the formation of flavour-active compounds and flavour development in beef. Flavour of beef is influenced by various intrinsic and extrinsic factors in the supply chain. It remains unclear in the literature how muscle fibre type influences formation of flavour-active compounds in beef. Postmortem ageing is known to improve flavour of beef, but the roles of flavour-active compounds in this process also remain unclear. Therefore, the overarching aim of this thesis was to investigate the effects muscle fibre type, postmortem ageing, and their interaction, on flavour development of beef. Dry ageing has been shown to result in higher flavour liking of beef compared to wet ageing. It was hypothesised that consumers’ preferences for dry aged beef could be explained by the volatile profile. To test this hypothesis, beef longissimus muscles were subjected to different ageing methods (dry vs. wet) and ageing time (35 days vs. 56 days). The volatile profile of cooked beef was analysed using gas chromatography-mass spectrometry (GC-MS), and important volatiles that influence aroma of beef were identified using gas chromatography-olfactometry (GC-O). Dry aged beef had higher concentrations of desirable volatiles, especially those derived from the Maillard reaction. 56-days ageing negatively impacted the volatile profile of both dry and wet aged beef compared to 35-days ageing. The high flavour liking of dry aged beef can be explained by the profile of volatiles. Results of this study demonstrated that the flavour development of beef during ageing was associated with the formation of odour-active volatiles. In addition to ageing, another aim of this thesis was to unravel the effect of muscle fibre type on formation of odour-active volatiles. Beef masseter and cutaneous trunci were used to prepare patties with predominantly oxidative or glycolytic fibres. The formation of volatiles in cooked patties was analysed using GC-MS, and the chemical composition of raw patties was also analysed. The results showed that predominantly oxidative and glycolytic patties had distinctive chemical characteristics such as pH, antioxidant capacity, total haem protein, and fatty acid composition. Results of volatile analysis indicate that oxidative patties had more desirable volatiles derived from the Maillard reaction but less volatiles derived from lipid oxidation. The higher formation of Maillard-derived volatiles in oxidative patties was associated with the higher pH. Whereas the lower formation of lipid-derived volatiles in oxidative patties can be explained by the high antioxidant capacity and haem protein content. This study revealed that oxidative muscles generate desirable odour-active volatiles during cooking due to their distinctive chemical characteristics. The role of haem protein in flavour development of beef was further investigated in a following experiment. Beef semitendinosus was spiked with different levels of haem proteins and free iron to study their effect on the formation of volatiles and aroma. Haem proteins could potentially reduce the livery odour of cooked patties by inhibiting the release of lipid-derived volatiles and enhancing the formation of Maillard-derived volatiles. Free iron negatively impacted aroma of beef by increasing lipid oxidation products that induce livery and oxidised fat odours. This study confirmed that haem protein contributes to the formation of odour-active volatiles and hence influences the flavour development of beef, especially for oxidative muscles. Water-soluble metabolites are also essential for flavour development of beef, as they contribute to taste of beef and are precursors of odour-active volatiles. A study was conducted to investigate the effect of predominant fibre types in a muscle (oxidative vs. glycolytic) and postmortem ageing (0 day vs. 14 days) on water-soluble metabolites in beef, using masseter (oxidative) and cutaneous trunci (glycolytic) muscles. Nucleotides, including inosine monophosphate, were initially higher and degraded slower during ageing in glycolytic muscles compared to in oxidative muscles, which could be explained by the profiles of nucleotide metabolism enzymes in these two types of muscles. Free amino acids were initially higher and increased faster during ageing in oxidative muscles compared to in glycolytic muscles, which may be explained by the abundance and activity of proteases. The distinctive metabolism of oxidative and glycolytic muscles leads to varied profiles of water-soluble metabolites, which could potentially influence the development of taste and aroma in beef. In conclusion, this thesis contributes to meat flavour chemistry knowledge via elucidating the effects of muscle fibre types and postmortem ageing on formation of flavour-active compounds in beef. Knowledge from this thesis is beneficial to the beef industry to manipulate and fine-tune supply chain factors to optimise beef flavour and hence maximise consumer preferences. This knowledge is also beneficial to the plant-based meat industry to formulate with innovative ingredients to recreate the unique flavour of beef.
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ItemEvaluation of in-house and commercial indirect elisas as diagnostic tools for Mycoplasma bovis surveillance in Australia and New Zealand( 2023-10)Mycoplasma bovis is an emerging pathogen in cattle worldwide and its ability to cause mastitis, pneumonia and arthritis in cattle results in production losses and has significant welfare implications. While the organism causes severe disease manifestations in clinically infected cattle, apparently healthy animals, or those subclinically infected, contribute most to its dissemination. Its capacity to develop resistance to most of the antimicrobials commonly used to treat it makes it hard to limit its spread in endemically infected population. There is a need for effective diagnostics to detect exposure to M. bovis at the animal and herd-levels and assist in the control of the spread of M. bovis and prevention of the disease it causes. The MilA ELISA and the ID Screen Mycoplasma bovis indirect ELISA are two diagnostic tests that have similar properties and are known to be the most reliable tests for detection of exposure to M. bovis, but our understanding of the application of these two diagnostic tests in detection of cattle infected with M. bovis in Australia and New Zealand is limited. The studies described in this thesis applied Bayesian latent class analysis (BLCA) to evaluate the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of the MilA ELISA and the ID Screen ELISA. In Chapter 2, an optimal cut-off, and the DSe and DSp at this cut-off, were estimated for use of the MilA ELISA for testing bulk tank milk (BTM) samples for antibodies against M. bovis in Australian dairy herds. The DSe of the MilA ELISA was found to vary with the stage of the milk production cycle, suggesting the importance of targeted application of BTM testing for antibodies against M. bovis in March in Australian dairy herds. In Chapter 3, the MilA ELISA was validated for use in testing serum for antibodies against M. bovis and its performance was compared to two commercially available ELISAs. The BLCA structure in this study accounted for the conditional dependence between these three tests. The commercially available ID Screen ELISA has been used extensively in New Zealand’s M. bovis eradication programme to test both BTM and serum samples. Chapter 4 evaluated the use of the ID Screen ELISA for testing BTM for antibodies against M. bovis in New Zealand dairy herds, using a BLCA structure to overcome the violation of the assumption of constancy of DSe or DSp. The model also accounted for repeated measures while including variables influencing the underlying true prevalence of detectable antibodies against M. bovis. The effects of stage of the milk production cycle and bulk milk tank volume on the DSe of the ID Screen ELISA were identified, highlighting the importance of targeted testing during mid-lactation, when there is a low bulk tank milk volume due to regularity of emptying collected milk from the bulk milk tank, to ensure the most sensitive use of the test. Chapter 5 investigated the performance of the ID Screen ELISA when used to test sera from dairy cattle and identified differences in the diagnostic performance of the ID Screen ELISA at different cut-points for the proportion of positive animals within a herd. The findings from both Chapter 4 and Chapter 5 have found application in the current surveillance for herds infected with M. bovis being undertaken to support the goal of achieving eradication in New Zealand. Overall, the studies described in this thesis evaluated the performance of the MilA ELISA and the ID Screen ELISA in detecting antibodies against M. bovis in cattle in New Zealand and Australia, applying complex BLCA structures to overcome assumptions that would have been violated in more standard models, enabling important breakthroughs in evaluation of these diagnostic tests that are proving crucial to the management of the disease in Australia, New Zealand and elsewhere.
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ItemReducing nitrogen loss and odour generation from broiler litter: the effect of lignite on litter physical, chemical and biological properties( 2024-02)In Australia, broiler production generates substantial quantities of organic waste, predominantly in the form of spent litter. This litter, composed of bird excreta, bedding material, spilt feed, and feathers, is a rich source of carbon (C) and nitrogen (N), which will ideally be recycled as an organic fertiliser. However, significant amounts of C and N are lost from litter before its removal from animal housing, resulting in the forfeiture of agronomically valuable nutrients and potentially leading to adverse environmental impacts. Additionally, odour from broiler farms can cause further issues, particularly regarding nuisance to surrounding communities. The encroachment of urban development into areas traditionally used for agriculture has reduced the spatial buffer between sources of odour and residents, often leading to conflict. These highlight the need for innovative and cost-effective technologies aimed at reducing C and N loss and abating odour emissions from broiler farms. The primary pathway of N loss from litter is the volatilisation of gaseous ammonia (NH3), a reactive form of N, which is produced as microbes hydrolyse uric acid in bird excreta. Likewise, the microbial decomposition of organic matter is the primary source of odour from litter. The rate at which these emissions occur is dictated by several in-house factors, including litter moisture, pH, temperature and ventilation. Lignite, which is a brown coal that is abundant in Victoria, has previously been shown to reduce NH3 emissions when applied to bedding in beef cattle feedlots and broiler litter in a laboratory incubation. However, its effectiveness in reducing NH3 emissions and odour from commercial broiler housing was unknown. This thesis investigated the capacity of lignite to reduce N loss and odour emissions from litter in commercial broiler housing and aims to improve the understanding of mechanisms by which any reductions occur. To investigate lignite's effect on N loss, it was applied to litter at a commercial broiler farm in Victoria, Australia, at a rate of 3.5 kg/m2 over three consecutive summer grow-out cycles. Lignite-treated litter retained more biomass, resulting in 32.1% more C and 45.8% more N per m2 than the untreated control. This was equivalent to an extra 70.1 g of C and 12.6 g of N per bird placed. Notably, lignite also reduced CO2 flux (microbial activity) in the litter by 46% at the end of the study. This reduction in microbial activity likely slowed litter decomposition, which contributed to the observed retention of C and N. Additionally, lignite consistently lowered litter moisture content by 3-7% and reduced litter water activity (Aw) to levels that were expected to inhibit microbial function. These results showed lignite's efficacy as a litter amendment, which enhanced litter nutrient value and reduced CO2 emissions. The main source of odour from broiler litter is the microbial decomposition of organic material. Thus, a reduction in microbial activity was expected to reduce odour emissions from broiler housing. A second experiment investigated the effect of lignite on odour emission rate (ou/s) using dynamic olfactometry over two consecutive summer grow-out cycles in Victoria, Australia, with lignite applied at the same rate as the previous experiment. The air quality dispersion model, AERMOD, was used to investigate how lignite’s effect on odour emission rate influenced the predicted odour dispersion distance and concentration in the context of the Australian regulatory framework. Additionally, the effect of lignite on bird production performance was also determined. This study showed that lignite reduced the odour emission rate from commercial broiler housing by an average of 55.5% over both grow-outs. This effect was observed for the duration of the trial, with maximal reductions of 54.9 and 71.3% per 1000 birds placed in week 7 for grow-out 1 and 2, respectively. Victorian EPA modelling requirements of a 3-minute averaging time and the 99.9th percentile provided a reduction in the total area under the 5 ou contour of 65.8%. There was no observed differences in feed conversion ratio, bird live weight or mortality throughout either grow-out. This study demonstrated the capacity of lignite to reduce odour emissions from commercial broiler housing without reducing bird production performance In a third experiment, custom tunnel ventilated chambers were used to simulate summer and winter conditions to assess the impact of lignite on water evaporation rates and the relationship between Aw and CO2 flux as a function of litter moisture content. The litter used was collected weekly over four weeks from two commercial broiler sheds, one treated with 3.5 kg/m2 lignite and the other left untreated. Under summer conditions (50% RH), lignite increased the evaporation rate by 0.06 L/m2/day for each 1% rise in moisture content (above 20% MC) at high ventilation (2.0 m/s), and by 0.03 L/m2/day at low ventilation. Under winter conditions (80% RH), the rates were 0.02 L/m2/day and 0.03 L/m2/day (above 15% MC) for high and low ventilation, respectively. Lignite-treated litter had a Aw of 0.90 at 25% moisture from week 4 of a grow-out compared to 0.95 in the control (while still being compared at 25% moisture content). This increased to 0.81 and 0.92 by week 7 for lignite and the control, respectively. Litter used in this study demonstrated a parabolic relationship with CO2 flux, with peak microbial activity consistently around 40% moisture content. However, from week 5 onwards, lignite-treated litter consistently reduced CO2 flux at all moisture levels, indicating lignite’s role in reducing microbial activity is independent of moisture content. Finally, an acid trap incubation experiment was conducted to assess the influence of lignite's physical and chemical properties on N transformations in litter, focusing on the effects of low Aw, humic acids, and lignite's surface area. Potassium chloride (KCl) was used to lower the Aw of litter (Aw 0.81) at a 40% moisture content where lignite had no effect on Aw (Aw 0.99). The KCl treatment reduced NH3 emissions by 78.1% compared to the control, whereas lignite resulted in an 83.4% reduction. In a second acid trap incubation, the effect of humic acids on N transformations were assessed. Similarly, at 40% moisture content, lignite reduced NH3 emissions by 61.8%, where the humic acid treatment reduced emissions by 89.2%, relative to the control. Lignite consistently increased ammonium-N (NH4-N) concentration, suggesting enhanced adsorption processes, in contrast to humic acid and K-Humate treatments increased urea levels, likely through inhibition of the urease enzyme. Conversely, the KCl treatments reduced microbial activity by lowering Aw, potentially restricting the hydrolysis of uric acid and thereby reducing N availability for further transformation. These findings indicate that lignite's capacity to mitigate N loss from broiler litter involves multiple mechanisms which contribute to its sustained efficacy. This thesis established that lignite effectively reduced N loss and odour from broiler litter under commercial conditions, primarily through the reduction of microbial activity. This reduction slowed down litter decomposition, which improves nutrient retention and reduces odour generation. Importantly, lignite's application did not impact bird production performance, including feed conversion ratio (FCR), live weight, or mortality. The effectiveness of lignite is linked to its physical and chemical properties, such as surface area, humic acid content, and the ability to lower litter Aw. These properties work in synergy to impact different forms of N, providing lignite with the sustained efficacy observed in this study (one month). This demonstrated lignite’s potential as an effective litter amendment for improved sustainability in commercial broiler housing.
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ItemRNA stability in alphavirus infected cells( 2022-04)Alphavirus is a genus of viruses in the family Togaviridae. Alphaviruses include notable human pathogens such as Chikungunya virus (CHIKV), which causes arthritis, and the equine encephalitis viruses, which cause encephalomyelitis. The only alphavirus vaccine approved for human use is the Ixchiq vaccine for CHIKV, approved in late 2023. Alphaviruses subvert many processes of host cells to facilitate their own replication and evade the immune response. By understanding these host-pathogen interactions, treatments can be potentially targeted towards these host processes on which the alphaviruses depend for replication. Preliminary data from the Fazakerley lab shows that the alphavirus Semliki Forest virus (SFV) replicates less efficiently in RNase L knockout mouse embryonic fibroblasts (MEFs) than in wild-type cells. This is surprising, as RNase L is known to be an antiviral protein, degrading RNA in response to viral infection. One explanation involves the interaction of alphaviruses with host RNA-binding proteins. Studies performed primarily with the alphavirus Sindbis virus (SINV) have shown the RNA binding protein HuR binds with high affinity to alphavirus RNA through sequence elements in the 3’ untranslated region (UTR), resulting in increased stability of viral RNA. Stability of some host transcripts is also reduced during infection. Tristetraprolin (TTP) is another RNA-binding protein that binds competitively with HuR and promotes RNA degradation. TTP has been found to recruit RNase L to cellular transcripts under mitogen stimulation, resulting in the degradation of these transcripts. We hypothesised that in SFV infected cells, viral RNA binds HuR and sequesters the protein from cellular transcripts. HuR protects viral RNA from being bound by TTP, preventing TTP from recruiting RNase L and degrading the viral RNA. Cellular transcripts with no HuR bound remain vulnerable to TTP binding and RNase L degradation, leading to reduction of cellular transcripts, and therefore reduced translation of cellular proteins. This reduces the competition for translation of viral proteins and therefore results in increased viral replication. Consistent with studies in SINV, we have shown using immunofluorescent microscopy that HuR relocalises from the nucleus to the cytoplasm during SFV infection. Preliminary results of siRNA knockdown experiments also suggest HuR increases SFV replication efficiency. However, we could not conclusively determine if TTP knockdown affected SFV replication. Through transcriptomic analysis of SFV-infected WT and RNase L KO MEFs we found that RNase L was not responsible for the majority of changes in cellular transcript levels or stability during SFV infection. Based on these results, we concluded that our initial hypothesis that RNase L and TTP increases SFV replication efficiency by degrading cellular transcripts is not correct. To find other ways RNase L may benefit alphavirus replication, we investigated the effect of RNase L on stress granules (SGs). Alphaviruses utilise the SG protein G3BP1 to enhance viral replication. To utilise this protein, alphaviruses induce the disassembly of SGs. As RNase L has also been shown to be involved in the disassembly of SGs, we hypothesised that RNase L may also be involved in the disassembly of SGs by alphaviruses. We used immunofluorescent microscopy to investigate whether SGs were present in SFV-infected WT and RNase L KO MEFs. We found that SGs were present in SFV-infected RNase L KO MEFs, but not in the WT. This suggests that RNase L is involved in alphavirus disassembly of SGs.
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ItemWhat are the triggers, challenges, attitudes and behaviours that contribute to poor welfare of livestock? Can these situations where livestock are at risk of poor welfare be predicted?( 2023-11)As incidents of poor welfare affecting non-dairy cattle, sheep and goats (key livestock) continue to occur, new ways to respond to them need to be considered. This thesis firstly aimed to understand livestock welfare non-compliance in Victoria and wider Australia and secondly develop an animal welfare risk assessment tool (AWRAT) that could be used to identify key livestock at risk of poor welfare, based on observable parameters on farm. If livestock at risk of poor welfare could be identified, it may facilitate early intervention and inform response planning and support to quickly resolve issues and prevent reoffending. This thesis began by analysing 10 years of substantiated animal welfare complaints (SWC) and 39 years of historical animal welfare investigation case (HAWC) notes from Victoria, Australia, to better understand the nature and extent of animal welfare non-compliance. There was a modest inverse correlation between rainfall and the number of SWC, but rainfall was not protective against all livestock welfare incidents. In total there were 2179 individuals or group of individuals that had an incident of poor welfare affecting livestock on at least one occasion, in the HAWC. Reoffending was significantly more likely when animals were found to be injured/unwell, recumbent, stuck in mud/yard/pen or in poor body condition, or when there was a failure to wean/cull, mark, dip/drench and draft. Using the learnings from the literature review, SWC and HAWC and a survey of industry (not including producers), 18 risk factors were selected to develop an AWRAT. The proposed AWRAT included factors relating to farm infrastructure, nutrition, treatment and mating times. Participants from across Australia trialled the AWRAT following farm visits for welfare and non-welfare related reasons. A novel algorithm was developed to generate an AWRAT-Risk Rating, based on the AWRAT assessment. Using linear regression, the relationship between the AWRAT-Risk Rating and a simple welfare rating was tested. The AWRAT was good at identifying livestock with poor welfare based on this preliminary testing. The adjusted R squared values varied from 0.791-0.803. The intra-observer reliability was strong, with 89% of the correlation coefficients (CC) for participants >0.8. Inter-observer reliability was good with 68% of participants with CC of >0.7. Participants in the survey also identified possible protective factors, that are more commonly observed on farms with good welfare. In the third section of the survey, industry participants (including producers) identified the management issues, contributing factors, challenges and possible solutions to issues of poor welfare affecting key livestock. The AWRAT developed in this thesis provided a structured framework to improve consistency in livestock welfare assessments. Initial testing has demonstrated a capacity to identify livestock with poor welfare, but further research is necessary to determine if the AWRAT can identify livestock at risk of poor welfare, by studying animal welfare incidents and reoffending over time. Livestock welfare non-compliance occurs when there is a failure of duty of care. The reason for this failure appears to be complicated. Developing new approaches to support both the farmer and the animals in instances of poor livestock welfare is likely to be beneficial.
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ItemEmbryo survival in the critically endangered Southern Corroboree frog (Pseudophryne corroboree) at Melbourne Zoo( 2023-10)The Southern Corroboree frog (Pseudophryne corroboree) is one of Australia’s most critically endangered amphibians and relies on captive assurance populations for survival. However, captive breeding at Melbourne Zoo is limited by high embryo mortality, previously found to be associated with hyphal fungal infections in abnormal embryos. To investigate the aetiology, I examined disease co-factors by characterising the epidemiology of embryo mortality as well as manipulating and assessing various husbandry conditions during the 2022 and 2023 Melbourne Zoo captive breeding seasons. I investigated 1) the epidemiology of embryo mortality, 2) the affect of three substrates on embryo survival, 3) the affect of physically separating eggs on embryo survival, and 4) fertility rates. Intense monitoring in 2022 revealed an overall 52.4% embryo survival rate with most loss occurring within two weeks of laying, and that mortality was higher for eggs laid later in the season. For males that fertilised multiple clutches, there was no decrease in survival in later clutches. Our experiments incubating embryos on three substrates (moss, live plants, gravel) and a trial of egg separation found no differences, indicating that altering these husbandry factors is unlikely to improve outcomes. Fertility did not appear to be a major issue in most of the clutches examined during the 2023 breeding season with a mean fertilisation rate of 77.2%. However, the high loss soon after laying suggests embryo quality rather than husbandry is an issue. A change in captive management in 2023 enabled more females to lay from the beginning of the breeding season rather than holding some for the second half, and embryo survival improved (63.5%). This suggests that egg quality reduces over time if laying is delayed, possibly related to over-maturation, but assessments in further years are needed to confirm that breeding females early in the season improves embryo survival rates. As most Southern Corroboree frog embryo loss occurs within the first two weeks after laying, further work is needed to determine the extent of infertility versus early embryonic death affecting this early period, and whether the fungi impacting unhealthy embryos is opportunistic or pathogenic. This may guide the development of other management options.
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ItemStudies on Intestinal Nematodes in Australian Thoroughbred Horses( 2023-10)Gastrointestinal nematodes (GINs) are the most important parasites of equines as they pose a significant threat to equine health and wellbeing, particularly in younger and geriatric horses. Strongylids (cyathostomins and strongylins) and ascarids (Parascaris spp.) are the significant GINs of horses, with the former constituting more than 75% of the total parasite fauna. Heavy burdens of these parasites such as Parascaris spp. in young horses, can cause impaction and rupture of the small intestine while cyathostomins can infect all age groups of horses, with variable severity of cyathostominosis, particularly when encysted larvae emerge synchronously from the intestinal wall. The literature review (Chapter 1) identified various research gaps related to the epidemiology, diagnosis, efficacy of commonly used anthelmintics and control of GINs in Australian horses. Over the last 50 years, only a few studies have investigated the epidemiology of GINs of equines in different states of Australia which were either restricted to some regions or involved only a small number of horses. To address some knowledge gaps on the GINs in Australian horses, this thesis aimed to (i) establish baseline epidemiological data on GINs in Australian Thoroughbred horses, (ii) develop and/or employ more sensitive and advanced molecular tools for the detection of GINs in horses in epidemiological and drug efficacy studies, (iii) ascertain the efficacy of commonly used anthelmintics against significant intestinal nematodes of horses, and (iv) assess worm control practices used by Thoroughbred farm managers and equine veterinarians. The longitudinal (Chapter 2) and cross-sectional (Chapter 3) epidemiological studies conducted using coprological methods showed high prevalence and egg-shedding patterns of GINs in various age groups of horses. Climatic zone and age had the highest impact on faecal egg shedding, particularly in the Mediterranean climate, the autumn season, and young horses (i.e., yearlings). The polymerase chain reaction-directed next-generation sequencing (PCR-NGS) method uncovered the diversity of strongylid nematodes as 31 species were detected in both epidemiological surveys and their occurrence varied across various climatic zones, seasons and age groups of horses. Traditionally, the faecal floatation method has been used to diagnose eggs of Strongyloides westeri – a free-living parasitic nematode of newborn foals. In Chapter 4, following the detection of S. westeri eggs in the faeces of foals using microscopy, a PCR-based diagnostic method was established for the first-time using DNA extracted from the parasite eggs. This method will help conduct future molecular epidemiological studies on S. westeri and assess the efficacy of commonly used anthelmintics in foals. Chapter 5 showed the extent of anthelmintic resistance (AR) in cyathostomins and Parascaris spp. prevalent in Australian Thoroughbred horses. An apparent failure of the efficacy of a combination of anthelmintic drugs (i.e., oxfendazole (OFZ) and pyrantel (PYR)) was observed for the first time against Triodontophorus brevicauda – a species of large strongyles. Cyathostomins were resistant to multiple anthelmintics, including abamectin (ABM), ivermectin (IVM), moxidectin (MOX) and OFZ, whether used individually or in combination with other classes of anthelmintics, i.e., OFZ+PYR. Furthermore, where anthelmintics were effective 2 weeks post-treatment, egg reappearance periods (ERPs) were reduced to four and/or five weeks. The major cyathostomin species identified 2 weeks post-treatment were from the two genera, Cylicocyclus and Cylicostephanus while those 5 weeks post-treatment with MLs were Cylicocyclus nassatus, Cylicostephanus longibursatus and Cylicocyclus ashworthi. Chapters 2 to 5 provided comprehensive information on the prevalence of GINs in Australian horses and resistance in ascarid and strongylid nematodes against commonly used anthelmintics. Subsequently, assessments of worm control practices surveys used by horse managers (Chapter 6) and veterinarians (Chapter 7) provided insights into their knowledge, aptitude and practices on GINs of horses, their diagnosis, treatment and control. Although both farm managers and veterinarians almost completely relied on anthelmintics to control GINs in horses, the latter seemed to use more targeted treatment strategies based on faecal egg count results. Multiple correspondence analyses used in Chapter 6 showed that the likelihood of suboptimal worm control practices on small farms (n = less than 50 horses) was greater than that of medium (n = 51-100) and large (n = above 100) farms. Furthermore, the findings highlighted a communication gap between veterinarians and horse managers. In conclusion, this thesis has contributed to addressing some key fundamental knowledge gaps on the GINs of Australian horses. Using conventional and advanced DNA-based diagnostic techniques, this thesis uncovered (i) the prevalence and egg-shedding patterns of GINs across various climatic zones during different seasons in various age groups of horses, (ii) the extent of AR against commonly used anthelmintics in ascarids and strongylid nematodes, and (iii) knowledge, aptitude and practices used by horse managers and veterinarians to control horse parasites. The novel findings of this thesis can pave the way for developing tailored guidelines for equine parasite control in Australia and globally.