Veterinary and Agricultural Sciences Collected Works - Theses
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ItemMonitoring Training Load in Thoroughbred Racehorses( 2019)Methods of monitoring training load are widely practiced amongst today’s elite, sub-elite and amateur athletes. Monitoring training load can range from simple, cost effective methods to complex, expensive methods. The aim of this research was to investigate different methods of monitoring training loads in young Thoroughbred racehorses in pre-training. Simple, cost effective measurements of proximal hoof circumference and hoof toe angle were investigated as a tool to monitor how the hoof responses to the loads experienced in the first two pre-training preparations in young Thoroughbred racehorses. Left and right proximal hoof circumference (n = 42) and hoof toe angle (n = 39) measurements were obtained weekly. During the first pre-training preparation, a significant reduction was recorded in both proximal hoof circumference and hoof toe angle. In contrast, proximal hoof circumference did not change significantly during the second pre-training preparation, whereas hoof toe angle showed a smaller significant decrease. The advancement of modern technology has allowed for improvements in the simplicity of radiographic measurements. However, the collection of radiographic images is still costly to the Thoroughbred racehorse owner. The response of the third metacarpal bone shape and size to training loads can be easily evaluated through the frequent collection of radiographic images. Firstly, weekly left and right mediolateral and lateromedial (respectively) radiographs of the third metacarpal bone were collected and radiographic measurements of bone shape and size were analysed with proximal hoof circumference. Secondly, measurements of supracondylar modelling were obtained the distal end of these third metacarpal bone radiographs. Fifty-four Thoroughbred racehorses were assessed during the first and second pre-training preparations for both sets of measurements. Forty-four Thoroughbred racehorses were assessed at follow-up supracondylar modelling measurement taken from radiographs at 1.5 or 2.5 years from the original measurements. The results showed a moderately strong positive correlation between supracondylar modelling and the number of days in training. Heart rate recovery (HRR) is a simple and effective method of analysing changing fitness levels in Thoroughbred racehorses. Predominantly, research to observe HRR values has been conducted in the laboratory rather than in the field. In the current study, HRR was assessed in thirty-six Thoroughbred racehorses during the first and last sessions of one pre-training preparation. Heart rate data were collected at peak heart rate, 60- and 120-seconds post peak heart rate, at the end of the training session, and 60- and 120-seconds post training. Heart rate recovery was calculated by subtracting the heart rate value at 60-seconds post peak heart rate from the peak heart rate. A significant difference was found between HRR at the first and last HRR assessments. Heart rate recovery assessments in the field may be more difficult to obtain than HRR assessments in the laboratory due to the separation of the trainer and the Thoroughbred racehorse. Therefore, the use heart rate monitors and GPS devices to collect and store HRR values may be more beneficial as it allows the trainer to analyse the data at the end of a mornings work, when there is sufficient time. After identifying and applying the above objective methods to monitor training load in the young Thoroughbred racehorse, a simple and efficient subjective method of monitoring training load was created. The Equine Perceived Exertion (EPE) scale was established from Borg’s Rate of Perceived Exertion scale and Borg’s CR 1 – 10 scale. Six subjective parameters were used to assess each of seventeen Thoroughbred racehorses immediately following their return from sixty-five separate pre-training session. These parameters were behavioural characteristics, respiratory rate, perspiration, capillary refill time, oral mucous membrane colour, and muscle engorgement. Each parameter was assigned a scale and the sum of all parameters corresponded to an algorithm score. The algorithm scores then corresponded to a specific EPE score. Training load was calculated by multiplying the EPE score by the duration of the training session in minutes. There were weak positive correlations between algorithm score or EPE or training load and heart rate recovery 60-seconds post peak heart rate. It was concluded that monitoring training load in Thoroughbred racehorses in a pre-training program can be accomplished and needs to be multifaceted. The trainer must not rely on one particular method of monitoring training load but use both internal and external methods simultaneously to successfully monitor training load. Overall, this work has identified six methods to monitor the training load of Thoroughbred racehorses in pre-training. Further investigation is required to determine if these methods can be used to monitor the training load of Thoroughbred racehorses in gallop training, undertaking different training programs.
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ItemPathogenesis of Bluetongue Disease( 2019)Bluetongue virus (BTV) is an important arthropod-borne virus that causes viral haemorrhagic disease in European adapted sheep. Scientific investigation of the pathogenic processes involved in bluetongue (BT) disease manifestation has been compromised by limitations in two key areas: (i) the detection of BTV antigens by immunohistochemistry (IHC) in formalin-fixed tissues has frequently proved to be problematic; and (ii) the choice between animal-derived virus or cell culture-passaged virus for use in experimental infection of animals remains contentious. In this study, BTV-specific antibodies NS1, NS2 and NS3/3a and VP7 were evaluated for use in IHC on formalin-fixed paraffin-embedded (FFPE) tissue specimens. It was shown that there was cross-reactivity between some of these antibodies with a range of BTV serotypes and other orbivirus species. BTV IHC methods developed as part of this evaluation were then employed to study BTV pathogenesis and pathogenicity using the embryonated chicken egg (ECE) as an alternative animal model. Using a pathogenic isolate of a reference BTV serotype 3 (BTV-3; isolated during the Cyprus 1943 outbreak) contained in the blood collected from an infected sheep, BTV-infected ECE demonstrated both endothelio- and neurotropic properties. Furthermore, the pathogenicity of BTV-3 derived from infectious sheep blood and cell-culture-passaged virus were compared in the ECE. BTV propagated in cell culture showed attenuated pathogenicity that was associated with a reduction in genetic diversity in BTV RNA genome Segment 7, encoding the viral structural protein VP7. A representative BT disease model in Australian Merino sheep was established through infection with the pathogenic BTV-3 derived from infectious sheep blood that was used in the ECE. In the sheep model, peak viraemia was observed at day 4 post-infection and this was associated with transient lymphopenia and onset of pyrexia. In addition, thrombocytopenia and prolongation of prothrombin time were detected during BTV infection, as well as the development of typical BT lesions at clinical end-point between 6 to 10 days post-infection. Immunohistochemical analysis demonstrated microvascular endotheliotropism of BTV in various organs, such as lung, tongue, lips, pulmonary artery and the coronary band; immunolabelling of mononuclear inflammatory cells were observed in lymphoid organs. Based on histopathological assessment, the findings of this study suggested that the development of vascular injury-related lesions, such as in the lung and tongue, could be associated with the recruitment of monocytes into alveolar septum and the submucosa of the tongue. To further understand the pathogenesis of BT disease in sheep, RNA-Sequencing was performed on peripheral blood mononuclear cells (PBMCs) and lungs derived from infected sheep collected at various time-points. Transient host responses were detected from the PBMCs and were associated with lymphopenia and peak viraemia. Upregulated differentially expressed genes in the PBMCs were involved in encoding chemokines and antiviral gene products. In the lungs, the host response increased progressively over the course of infection, which involved upregulation of genes associated with the monocyte-macrophage system, antiviral responses and vascular homeostasis. Importantly, upregulation of macrophage gene encoding CD163 in lung from BTV-infected sheep was confirmed by immunohistochemical analysis whereby the increased CD163+ pulmonary intravascular macrophages correlated with the development of pulmonary lesions. Similarly, activation of macrophages was associated with the pathological changes in the tongue. Based on the mechanistic analysis of BTV infection in sheep, BT disease is hypothesised to be a consequence of overt macrophage activation.
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ItemTowards unravelling the immunomodulatory mechanisms of S. mansoni & L. major parasites in a mouse model using transgenesis( 2019)Schistosoma mansoni and Leishmania major parasites use immunomodulatory strategies to evade the host’s immune response and little is known about the mechanisms by which these two parasites modulate host immune responses. As a result, these parasites develop different subversion mechanisms to escape host immunity and controlling such immunomodulatory pathogens is still not successful. In this project we hypothesized that an understanding of the ability of memory T cells to withstand pathogen manipulation is crucial for the development of effective vaccine strategies to control these pathogens. Therefore, in this study, OVA transgenic S. mansoni and OVA transgenic L. major were generated to use as a tool to measure the degree of immune memory resilience (defined as an ability of the immune memory to withstand pathogen manipulation) of T helper cells on the face of pathogen-mediated immune manipulation in vivo. The specific aims of this thesis are: i) Express recombinant OVA in both S. mansoni and L. major, ii) Demonstrate that OT-II TCR transgenic T cells are able to recognise the OVA expressed by the parasites, iii) Differentiate the OT-II cells into Th1, Th2 and Th17 cells in vitro and demonstrate their functional phenotype, and iv) Investigate whether the parasites expressing OVA are able to influence the cytokine expression profile of the Th1, Th2 and Th17 OT-II memory T cells. To achieve this, first OVA expression by S. mansoni and L. major was confirmed using RT-PCR and western blot. Subsequently, in vitro and in vivo analysis for proliferative responses of OT-II T cells revealed OVA was recognized by OT-II T cells. The proliferated cells also produced cytokine signatures following stimulation with the OVA expressing parasites both in vitro and in vivo. To measure the immune memory resilience of memory T cells against such pathogens, an OT-II mouse model was used as a source of naive OT-II T cells. Hence, Th1, Th2, and Th17 polarised memory cells were generated in vitro and these cells were adoptively transferred to recipient mice to investigate the immune memory resilience in the face of pathogen-mediated manipulation. After transferring memory cells, mice were challenged with OVA-transduced S. mansoni eggs and OVA transfected L. major parasites as well as wild-type controls. After recovery of Th memory cells, their proliferation rate and cytokine signature was analysed using flow cytometry. The in vitro differentiated Th1, Th2 and Th17 memory cells produced cytokines when challenged by OVA-expressing S. mansoni eggs and OVA expressing L. major. Indeed, Th1 memory cells produced significant level of IFN-g and TNF, while, Th2 memory cells produced high level of IL-2, IL-6 and IL-10, similarly, Th17 memory cells produced IL-17 when stimulated with OVA-expressing parasite eggs and OVA expressing L. major. However, Th1, Th2, and Th17 memory T cells didn’t proliferate in response to wild type S. mansoni eggs, and wild type L. major, and when they were left unstimulated. Therefore, the Th phenotypes of the memory T cells remains unaltered in the face of stimulation by two immune manipulative pathogens (S. mansoni and L. major). The ability of memory T cells to remain resilient to manipulation by these two pathogens has important implications for the prospect of developing vaccines against these parasites.
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ItemGenomics and pathogenesis of Australian avian coronaviruses( 2019)Studies of the molecular pathogenesis of coronaviruses are important as these viruses are major pathogens of animals and humans. The emergence during the 2000s of severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS) and more recently of SARS-2 as a result of interspecies transmission of coronaviruses from animals to humans has demonstrated the importance of understanding how these viruses evolve and what determines their virulence and tissue tropism. Chickens can be used as an animal model to study coronaviruses, as they are the natural host of the avian coronavirus infectious bronchitis virus (IBV). In the studies described in this thesis, different aspects of the molecular biology of infectious bronchitis virus were examined, including the patterns of recombination in the field and the relationship this may have with pathogenicity. IBVs have been constantly recombining and vaccines have played an important role in these recombination events. An attempt was made to generate a cloned genome of the VicS strain of IBV using Gibson Assembly, but was not successful, suggesting that an alternative approach is needed to generate this tool for further studies of the molecular pathogenesis of this virus. Finally, an \textit{in vivo} infection experiment was performed to study the pathogenicity of Australian strains of IBV. These studies demonstrated a tropism for a broader range of tissues than had previously been recognised for all but the most virulent viruses. Analysis of genomic and pathogenicity data suggested that nsp14 may be playing a significant role in the virulence of IBVs, and that further studies should be performed to examine the role of this protein in the replication of IBV.
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ItemTissue tropism and latency of infectious laryngotracheitis virus: A study in the natural host( 2019)Herpesviruses are evolutionarily successful pathogens that infect a large number of animal species. This success is partly attributed to their capacity to establish latency in the host. The avian alphaherpesvirus infectious laryngotracheitis virus (ILTV) causes an acute upper respiratory tract infection in chickens that has considerable impacts on world poultry economy and welfare. The disease caused by ILTV, infectious laryngotracheitis (ILT), is currently controlled by vaccination principally with live attenuated vaccines. Limitations associated with live attenuated vaccines, including the ability to establish latency, may provide avenues for the emergence of novel, more virulent, recombinant strains of ILTV, further complicating the epidemiology of ILTV. In this project, a sensitive nested polymerase chain reaction (NPCR) protocol was developed and the sensitivity of this assay was compared with that of other commonly used PCRs in ILTV research. A trigeminal ganglia (TG) co-culture system was established and optimised and a tracheal co-culture system was reproduced to study in vitro reactivation of latent ILTV. A genotyping system based on allelic variations in multiple genomic regions of ILTV to discriminate ILTV strains prevalent in Victoria, Australia, is also described. These methodologies revealed that a large proportion of the ILTV-vaccinated birds in a commercial layer flock, close to the end of their productive laying period, were shedding multiple vaccine strains of ILTV in the upper respiratory tract, presumably due to reactivation of latent infection. Further, co-culture systems showed in vitro reactivation of latent ILTV in TG and trachea of these birds. The capacity of four vaccine strains of ILTV (SA2, A20, Serva and a glycoprotein G deleted mutant vaccine candidate) to establish latency in specific pathogen free chicken (SPF) following eye-drop vaccination was investigated in vivo. This study revealed ILTV vaccines differed in their capacities to establish latency in TG, and also showed that nearly half of the population had detectable ILTV in their upper respiratory tract (URT), 21 days post vaccination, possibly due to reactivation of infection. A second in vivo experiment was performed to study latency characteristics and late systemic lymphocyte responses in SPF chickens following intratracheal inoculation with a vaccine ILTV (SA2) or a virulent field ILTV (class 9; CL9) strain. Results from this study indicated that latency characteristics did not significantly differ between these strains at 21 days post inoculation (dpi) or at 35 dpi, and suggested that the trachea may be a more significant site of latency and reactivation than the TG. Moreover, regardless of the ILTV strain inoculated, SPF birds showed lymphocytosis during the latent stage of infection. Additionally, tissue tropism of two newly emerged recombinant strains of ILTV (CL9 and class 10; CL10) was investigated using commercial broiler and SPF chickens. The possibility of using feathers as a diagnostic sample was explored. This study revealed that both CL9 and CL10 ILTV strains caused severe disease in both types of birds, distributed to visceral organs and persisted for up to 14 dpi in URT. The NPCR developed in this project detected ILTV DNA in feathers of infected broiler and SPF birds at 14 dpi. Taken together, these studies have shown that tissue tropism and latency is a complex area of research and to a large extent these properties are strain dependent. The studies reported in this thesis have enriched the literature on tissue tropism and latency of ILTV. The results will be valuable for future latency studies and for selection of vaccines to control ILTV.
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ItemChicken immune responses to infectious laryngotracheitis virus (ILTV) glycoproteins( 2019)Infectious laryngotracheitis virus (ILTV), an alphaherpesvirus, causes acute respiratory disease, primarily infecting the upper respiratory tract and conjunctiva. The disease results in significant economic losses to the poultry industry worldwide. Currently, the immune status of the vaccinated flocks or individuals is assessed by the presence of systemic antibodies using commercially available whole virus ELISAs. These ELISAs are good indicators of exposure to either field or vaccine virus, however, the assays are poor in predictors of the level of protective immunity. In this study, individual ILTV glycoproteins (gC, gD, gE, gG, gI and gJ) were assessed for their potential to predict the levels of protective antibody and/or cell-mediated immunity as well as for their capacity to induce neutralising antibodies. To examine whether antibody responses to individual ILTV glycoproteins are correlated with disease and protection, individual glycoproteins expressed in mammalian cells were used in indirect ELISAs to measure post-vaccination and/or post-challenge chicken serum antibodies. Serum optical density (OD) values detected by the whole virus, gC, gI and gJ were significantly higher in birds vaccinated with the Serva vaccine strain compared to the SA2 vaccine strain. However, the mean ODs detected by gD, gE and gG were not significantly different between the vaccine strains. Examination of post-vaccination pre-challenge antibodies to individual glycoproteins did not find a strong correlation between systemic antibodies to individual glycoproteins and protection. The possibility that the protective epitopes of glycoproteins may not be readily detectable in ELISA was investigated by evaluation of the antibodies to each glycoprotein in an in-vitro virus neutralisation assay. Monospecific polyclonal antibodies to individual ILTV glycoproteins gC, gD, gE, gI and gJ were generated in rats and examined for their capacity to neutralise the virus. Neutralising antibodies were detected to gC, gD and gJ individually or in combination. Polyclonal antibodies to gE and gI failed to neutralise the virus when tested individually or combined. In-vitro splenocyte stimulation assays using individual glycoprotein stimulating antigen were conducted to examine the transcription profiles of selected cytokines from nonvaccinated-nonchallenged (NVXNCh), vaccinated-challenged (VXCh) and nonvaccinated-challenged (NVXCh) groups of chickens. The transcription profiles of cytokines referencing Th1, Th2 and Th17 responses were then examined against tracheal pathology results to identify correlation between responses against viral glycoprotein(s) and protective immune response. Expression of IFN-gamma was significantly upregulated in VXCh and NVXCh groups of birds in response to stimulation with gD, with expression levels moderately correlated with protection. Stimulation with gE and gI resulted in significantly higher levels of IL-6 in VXCh birds and response to gE was moderately correlated with protection. Expression of IL-17a was significantly elevated in response to stimulation with gD in NVXCh birds and results were moderately linked to disease severity. The results of this study suggest that IFN-gamma and IL-6 cytokine responses to gD and gE, respectively, may be correlated with protective cell-mediated immunity, whereas IL-17a cytokine responses to gD may be linked to disease. Finally, whole-genome analysis of an unusually virulent “vaccine-like” isolate revealed a new class of ILTV, identified here as class 7b, emerged as a result of recombination probably between another recombinant strain and a vaccine strain. Further analysis of the genome detected recombination hotspots within the unique long (UL) region comprising of genes encoding gB, gC and gM and assembly proteins UL28 and ICP18.5. Boot-scanning analysis of concatenated glycoprotein sequence alignments detected recombination breakpoints within glycoproteins C and H, but not within glycoproteins located in the unique short (US) region. Further studies would be needed to explore the possible role of these glycoproteins in virulence. Thus, the studies reported in this thesis have contributed to understanding (1) the relationship between antibodies to ILTV glycoproteins and protective immunity, (2) virus neutralisation potential of specific antibodies to ILTV glycoproteins, (3) the potential role of ILTV glycoproteins to induce cell-mediated protective immunity, (4) and laid foundation to explore inter-strain genetic diversity within ILTV glycoproteins.
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ItemMolecular analysis of pathogenicity of Mycoplasma synoviae( 2019)Mycoplasma synoviae is a major pathogen of poultry globally, causing chronic respiratory disease and arthritis. Vaccination is an effective means for the control of the disease. The MS-H vaccine is a live attenuated temperature sensitive strain developed through chemical mutagenesis of an Australian field strain, 86079/7NS. Rarely, the temperature-sensitive MS-H strain may revert to a non-temperature-sensitive phenotype. Due to reversion to non-temperature-sensitive phenotype, vaccine isolates may be difficult to distinguish from field strains. Therefore, there is an urgent need for a reliable and rapid assay that can differentiate the MS-H vaccine from field isolates to evaluate the efficacy of the vaccination programs. However, developing such assays depends on a better understanding of molecular basis behind attenuation of MS-H. Comparison of the whole genome of MS-H with that of its parent strain 86079/7NS in previous studies has shown 32 mutations in the MS-H genome, one of which was a frameshift mutation early in an oligopeptide permease transporter gene oppF. In this study polyclonal antibodies raised against peptides upstream and downstream of the mutation in OppF revealed that only N-terminus of the OppF was expressed in MS-H while the full version was expressed in 86079/7NS. The potential of the recombinant full-length OppF, N- (OppF-N) and C termini (OppF-C) of OppF, upstream and downstream of the mutation site, was evaluated for the discrimination of antibody responses to MS-H versus field strains in indirect ELISA. Comparison of optical densities obtained from the ELISAs based on OppF, OppF-N and OppF-C revealed that the indirect ELISA based on OppF-C had the potential to differentiate between MS-H and field strain antibody responses. The impact of the oppF mutation on the MS-H phenotype was investigated in vitro using a mutant of MS-H complemented with a wild-type copy of oppF gene in the MS-H genome. The results revealed that truncation of oppF impacts on growth characteristics of the MS-Hand provide insight into the molecular pathogenesis of M. synoviae and perhaps of broader mycoplasma species. In addition, to characterise the function of OppF protein in MS-H, the metabolite profile of MS-H was compared with that of its field reisolate which only differed in its oppF gene. The liquid chromatography–mass spectrometry analysis revealed a significant decrease in the abundances of amino acids in MS-H compared to that in its field reisolate. This result was consistent with the role of OppF as a peptide transporter. Also, differences found in the level of other metabolites in MS-H reflected some compensation of the OppF function by pathways involving other metabolites. Moreover, in this study genomes of five M. synoviae isolates from MS-H vaccinated chicken flocks were determined and analysed to confirm their relationship with MS-H and also examine the stability of oppF mutation and other genetic markers of the MS-H vaccine. A total of 25 single nucleotide substitution/insertion/deletion including that of oppF existed among the isolates examined. Four of these mutations were those that had been reported between MS-H and its parent strain 86079/7NS, while the other 21 were found only in the MS-H reisolates tested in this study. Therefore, 28 out of 32 mutations previously detected between MS-H and 86079/7NS remained consistent in 5 MS-H reisolates. These results confirmed that M. synoviae isolates in this study were true MS-H reisolates and that majority of the mutations found in the MS-H vaccine are stable after passage in vivo under field condition. The studies described in this thesis have contributed to expansion of our understanding of putative virulence determinants in M. synoviae and laid foundations for the development of a DIVA (Differentiation of Infected and Vaccinated Animals) test to examine variants of the MS-H vaccine. Also, this thesis provided some information on the dynamics of MS-H population after passage in vivo.
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ItemDevelopment of an ‘equinised’ antibody to block the equine insulin-like growth factor-1 receptor (IGF-1R):A potential immunotherapy for endocrinopathic equine laminitis( 2019)Laminitis is a common and debilitating disease of horses’ feet that results in lameness, and in severe cases, may require euthanasia. Insulin toxicity appears to have a key role in the pathogenesis of the disease, and research has indicated that hyperinsulinemia may cause lamellar damage through excessive stimulation of insulin-like growth factor-1 receptors (IGF-1R). Inhibition of dysfunctional receptor activation has been the target of therapeutic (anti-cancer) monoclonal antibody (mAb) treatments for humans. Although common in human medicine, only a handful of therapeutic mAbs are under development for diseases in companion animals (dogs and cats), and none are available for horses. This approach demonstrated promise as a potential treatment of endocrinopathic laminitis. Therefore, the aim of this project was to convert a clinically-tested human IGF-1R-neutralizing mAb with high-affinity for the equine IGF-1R, into a horse-specific mAb while avoiding immuno-intolerance when administered in vivo. Development of the equine antibody was challenging due to the limited availability of immunoglobulin sequences (EquCab 2.0 database), in addition to the predominance of circulating light chain isotypes (Lambda), unlike the case in humans. Based on structural isotype characteristics, high binding affinity for the IGF-1R and minimal binding to insulin receptors, the parental antibody (IMC-A12) was chosen for chimerisation studies. Chimeric versions of this antibody were generated by combining the VH (Variable Heavy) and VL (Variable Light) domains of the human antibody to equine constant Fc regions. The mAbs were further engineered to abolish effector functions (activation of complement or the immune system) while retaining receptor binding functionality. Chimeric versions of IMC-A12 expressed at similar levels to the human parental mAb and bound to the equine receptor at similar affinities to human IMC-A12. The full ‘equinisation’ of chimeric mAbs was carried out using a novel speciation process known as “PETisation” and was adopted to generate thirteen candidate equine mAbs. Amongst three of the strongest equine mAb candidates (mAb8, mAb10, and mAb11), mAb11 displayed the highest expression yield purity (98 percent) and strong binding characteristics (KD of 0.2-0.3 nM and an EC50 of 127 pM, by BIAcore and ELISA, respectively). Cell proliferation assays demonstrated that mAb11 significantly inhibited the concentration-dependent proliferative effect of insulin in cultured lamellar cells (p<0.01). Further immunoassay assessment of lamellar growth factor signalling under hyperinsulinemic conditions demonstrated a significant reduction (p less than 0.05) in the phosphorylation of Ribosomal protein s6 (RPS6), Ras/extracellular signal-regulated kinase (ERK (1/2)) and Protein kinase B (AKT) after 24 h, in the presence of mAb11. As a novel approach, mAb therapies are an emerging option in the veterinary market. As far as I am aware, the fully-equine antibody mAb11, with its high-affinity for the target receptor (IGF-1R), is the first of its kind in the world and could open avenues for research on other equine indications and treatments. While promising results have been observed in vitro, further characterisation of mAb11 is required to establish its viability as a treatment for equine laminitis. A successful immunotherapy for equine laminitis would reduce the economic and welfare costs of this destructive disease while advancing our understanding of antibody therapeutics in the horse.
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ItemCharacterising the role of metabolic enzymes in the pathogenesis of Coxiella burnetii( 2019)Coxiella burnetii is a Gram-negative intracellular pathogen that replicates in a highly acidic and oxidative lysosome-derived vacuole known as the Coxiella-containing vacuole (CCV). The relatively recent identification of axenic medium that supports C. burnetii replication and the development of improved genetic tools has advanced knowledge of this pathogen and facilitated the identification of virulence factors. A previously conducted transposon mutant library screen using a human cell line identified a number of genes that appeared to be required for intracellular replication, including genes encoding metabolic enzymes such as nadB and sdrA. To investigate the role of nadB and sdrA in C. burnetii replication, and to link the metabolism of C. burnetii with pathogenesis, a number of approaches were used, such as advanced genetic and biochemical techniques, including targeted and untargeted metabolomics. Firstly, each transposon mutant was clonally isolated to avoid wild type contamination. Genetic complementation of both nadB and sdrA mutants and subsequent intracellular growth assays using both epithelial HeLa cells and macrophage-like THP-1 cells conclusively demonstrated that both nadB and sdrA are required for efficient C. burnetii replication and CCV formation. Analysis of the protein sequence revealed that NadB has a functionally conserved arginine residue at a position of 275 and this arginine was mutated to leucine using site-directed mutagenesis. Purification of both GST-NadB and R275L GST-NadB showed that GST-NadB had L-aspartate oxidase activity, an enzyme catalysing the first reaction of de novo NAD synthesis, whereas R275L NadB had lost enzymatic activity. Complementation of nadB mutant with a plasmid encoding this inactive R275L NadB was unable to rescue the intracellular replication defect, confirming the requirement of NAD de novo synthesis for intracellular replication of C. burnetii. Steady state metabolite analysis also showed key changes in the level of abundance of metabolites in the NAD biosynthetic pathway in the nadB mutant as compared to parent C. burnetii and the complemented nadB mutant, demonstrating the role of NadB in de novo NAD synthesis. This suggests that this pathway is an ideal target for development of therapeutics, and inhibition of this pathway using a compound non-toxic to mammalian cells reduced C. burnetii replication significantly. The role of SdrA in C. burnetii metabolism and pathogenesis was also further investigated in this thesis. SdrA is a putative short chain dehydrogenase containing a conserved glycine residue at position 12 that serves as an NADP binding site facilitating NADP(H) regeneration, a key process in resistance to oxidative stress in C. burnetii. This Gly residue was replaced by Ala using site directed mutagenesis and purified recombinant 6xHis-G12A was enzymatically inactive when compared to wild type 6xHis-SdrA that converted NADP+ to NADP(H) in vitro. Complementing the sdrA mutant with a plasmid encoding enzymatically inactive 3xFLAG-tagged G12A_SdrA failed to restore intracellular growth, confirming the link between NADP(H) regeneration and C. burnetii replication. The sdrA mutant showed greater susceptibility to oxidative stress in vitro induced by treatment with hydrogen peroxide, whereas both parent C. burnetii and the complemented sdrA mutant were less sensitive. Supplementation of a commonly available anti-oxidant, L-ascorbate, into the host cell growth medium partially restored the intracellular growth defect of the sdrA mutant. Metabolite profiling using GC-MS revealed significant changes in the level of abundance of metabolites of central carbon metabolism in the sdrA mutant as compared to parent C. burnetii NM II and the complemented mutant. Finally, stable isotope labelling studies using [13C] label glucose showed a change in flux through central carbon metabolic pathways in the sdrA mutant demonstrating the presence of oxidative stress. Overall, this thesis has demonstrated the crucial role of NadB and SdrA in C. burnetii metabolism and pathogenesis.
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ItemHuman-animal interaction in the modern zoo: Live animal encounter programs and associated effects on animal welfare.( 2019)Abstract Live animal encounter programs are an increasingly popular occurrence in the modern zoo. The welfare implications of animals participating in these programs has not been studied extensively to date. The aim of the current thesis was, therefore, to explore animal welfare effects associated with encounter programs in selected zoo-housed species, using a combined approach of survey data and empirical investigations. The survey assessment, which involved over 500 accredited zoos and aquariums in Australia and overseas, revealed that most surveyed institutions (85 %) engaged in an encounter program with one or more species, most of which were mammals. Behaviours indicative of a typical fight-or-flight response were the most commonly reported welfare concerns in relation to encounters. Behaviour indicative of positive welfare, as well as voluntary participation and interaction with visitors, were commonly mentioned in regard to positive welfare during encounters. Positive welfare experiences outweighed the number of reported concerns in birds and mammals, but the opposite was true for reptiles. The empirical studies involved zoo-housed servals, giraffes and shingleback lizards, and sought to identify a potential cause-effect relationship between behavioural and physiological welfare indices and short-term variations in encounter frequency. A similar methodology involving a repeated treatment design where the frequency of encounters was manipulated to reflect the regular frequency of encounters, a temporary withdrawal, and a temporary intensification of regime, was adopted across the three studies. Behavioural changes indicative of a positive welfare effect when participating in encounters were observed in the servals and giraffes. Servals exhibited a significant reduction in stereotypic pacing on weeks when participating in interactive presentations, or presentations and behind-the-scenes encounters combined. The giraffes engaged in amicable social interactions significantly more often when participating in visitor feeding encounters, at either regular or intensified frequency. By contrast, a potentially aversive effect of encounters was observed in the shinglebacks, who significantly increased their use of concealed locations within the enclosure when handled at either the regular or intensified frequency. Approach behaviour during encounters differed significantly between individual giraffes, and significant differences in coiling behaviour while handled was observed in the lizards. The findings of the empirical studies were in agreement with the survey data, which identified taxonomic as well as individual variation as important influences on the welfare of animals participating in encounters. The nature of the encounter the animals participate in was identified as another key factor, in which encounters that maximise choice and control, and where animals are rewarded for participation, were likely to contribute to a more positive welfare experience.