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.

Q fever is a zoonotic disease with a significant impact on public health worldwide. It is caused by the highly infectious bacterium Coxiella burnetii. In humans, Q fever typically manifests as a self-limiting febrile syndrome. C. burnetii infection can also lead to chronic disease presentations and be life-threatening for individuals with underlying risk factors. Australia ranks among the countries with the highest Q fever report rates. Livestock species are the main source of infection for humans and dairy goats have been linked to some of the largest Q fever outbreaks recorded in recent times. In Victoria, Australia, a Q fever outbreak linked to a large dairy goat enterprise occurred in 2012 – 2014. This was the largest farm-associated Q fever outbreak recorded in the country. In the context of a steadily growing dairy goat sector, an improved understating of Q fever dynamics and the efficacy of available control strategies in dairy goats is required. This thesis documents a series of studies designed to better understand factors contributing to the spread of Q fever in this herd and the potential efficacy of different control measures. As a first step, we developed a software prototype for reporting herd performance in intensively managed dairy goat herds. This allowed productivity, reproductive performance, health and mortality to be documented, and enabled the performance of Q fever positive and Q fever negative does to be compared. With this system in place, a panel study was conducted to document the prevalence of C. burnetii shedding at the time of kidding. A heterogeneous C. burnetii shedding pattern was found, characterised by the presence of small numbers of ‘super shedder’ does. Further, does identified as ‘super shedders’ had reduced total lactation milk yields compared with C. burnetii negative does. Demographic data gathered using the herd health software prototype and data on Q fever prevalence from our panel study were used to develop a within-herd transmission model of Q fever. The model was used to assess the expected time to eradication by means of vaccination. In addition, the efficacy of segregation of pregnant does and culling of C. burnetii shedders at the time of parturition or abortion was assessed. Vaccination consistently led to disease eradication, although it required this intervention to be sustained for at least 6.5 (95% CrI: 4.1 to 11.3) years. A combination of vaccination with segregation of pregnant does or culling of C. burnetii shedders at parturition or abortion shortened the median time to eradication. The model was adapted to account for heterogenous shedding patterns and used to test the potential efficacy of a test and remove intervention. We found the removal of super shedders does alone would lead to Q fever eradication. However, tests with a high sensitivity for early detection of super shedders are required for this strategy to be effective.

While previous studies of Leptospira Hardjo in Victoria demonstrated a relatively high prevalence of exposure in both cattle (40%; Milner et al. 1980) and humans (22%; Sutherland 1988) these estimates need to be revised since they were made more than 30 years ago and leptospirosis vaccination programmes are now commonplace on Victorian dairy farms. This was a cross-sectional study to estimate the prevalence of Leptospira borgpetersenii sv Hardjo and Leptospira interrogans sv Pomona in dairy herds in South-Western Victoria. Fifty-three herds were enrolled into the study. Herd managers were asked to present 15 late-lactation cows that had fertility issues (cows that had not conceived or had delayed calving to conception intervals). Furosemide 500 mg was injected into the tail vein of eligible cows and a mid-stream urine sample of the second voiding collected to increase the likelihood of sampling leptospira .At the time of each herd visit a questionnaire was administered to herd managers asking them to provide details of methods used for controlling leptospirosis, including vaccination. Urine samples were pooled at the herd level and tested for leptospira spp. using qPCR. Pooled samples were then tested individually and samples that were positive, were tested for Leptospira Hardjo and Leptospira Pomona using qPCR. Three of the 53 pooled urine samples returned a positive result. The leptospira positive pools returned a minimum of three positive individual cow urine samples. Testing of individual cow urine samples identified an additional positive herd (with one weak positive and one inconclusive result), giving an apparent prevalence of approximately 8 (95% CI 1 to 13) leptospira-positive herds per 100 herds at risk. Based on the 53 completed questionnaires, leptospirosis vaccination programs were non-compliant with label directions in 35 out of 52 vaccinated herds: 67 (95% CI 54 to 78) out of 100 herds that routinely vaccinate for leptospirosis were doing so incorrectly. Of the 53 herds that took part in this study, only one herd was completely unvaccinated. Based on the findings from this study, and assuming the herds that took part in this study were an unbiased sample of the dairy herd population at risk, we estimate that close to one out of 10 dairy farms in South-Western Victoria are leptospirosis positive. While most herds are vaccinating for leptospirosis, most are doing so incorrectly. We conclude that herd managers need to be better educated regarding leptospirosis vaccination programs.

Castration and disbudding are necessary husbandry practices commonly performed in farms which have the potential to induce stress and pain if not managed correctly. Depending on the severity of a stressor, potential stress responses can suppress immune function, lower resistance to disease-causing factors, contribute to, and result in, reduced animal welfare. Scientific literature is discordant on what recommendations should be transferred to farmers and practitioners regarding the preferred age to castrate and disbud calves. This lack of scientific support to guide recommendations is even more noticeable in suckler beef calves, since most of the disbudding studies have used dairy calves and most of castration studies have used dairy calves or calves older than 6-mo-old. In this thesis, a series of studies were conducted to investigate: a) the age-related differences in plasma cortisol, acute phase protein, metabolite concentrations, haematological variables, behaviour and performance of suckler beef calves in response to Burdizzo castration; b) the effect of sex, breed and age on horn bud size of dairy and beef calves at disbudding; c) the relationship between age and horn bud size of dairy and beef calves at disbudding; d) the horn bud growth in Holstein Friesian calves. Results indicate that Burdizzo castration caused significant stress in 2.5- and 5.0-mo-old calves, characterized by the increase in plasma cortisol concentrations, scrotal swelling, and abnormal behaviours observed after treatment. These responses were reduced by castrating calves at younger age. A secondary stress response was observed, which was greater in older castrated calves, suggesting that calves castrated at older ages have a prolonged overall stress response, compared to younger calves. There was no effect of castration on haematology profiles, haptoglobin, metabolites, body temperature and growth performance. Thus, if calves are to be castrated without analgesia or anaesthesia, then it would be preferable to do this at 2.5-mo-old rather than later, such as 5.0-mo-old, in order to minimize the physiological stress, scrotal swelling and pain-related behaviours associated with Burdizzo castration. Horn bud size at time of disbudding, irrespective of calf sex, was greater in Holstein-Friesian calves than Charolais, Simmental and Limousin calves. These results suggest that horn bud develop differently in beef calves compared with dairy breed calves. In addition, the relationship between age and horn bud size was very weak in all breeds studied with no difference due to calf sex. This means that the age of the calf is not a good predictor of the horn bud size. Therefore, the age of the calves is not a good parameter to guide recommendations on the best age to disbud calves and use of pain relief due to the great variation in horn bud size within and between breeds. The findings of this thesis will have implications for the welfare of cattle in relation to best recommendations regarding the age at castration of suckler beef calves and disbudding of dairy and suckler beef calves.

Snake venom-induced consumption coagulopathy (VICC) is an important syndrome resulting from snake envenomation. The prothrombin activator notecarin D is responsible for this coagulopathy in tiger snake (Notechis scutatus) envenomation. In dogs, the presence of this coagulopathy is used to assist with the diagnosis of snake envenomation and, in some cases, can produce clinical signs associated with a coagulopathic envenoming. There are subtle differences in this syndrome between dogs and people, particularly concerning the development of clinical signs. Chapter two reviews tiger snake envenomation and the consequent VICC in Australian dogs with comparisons to human literature. The VICC syndrome between the two species share many clinical similarities; the key difference lies in the significance of clinical haemorrhage in people when compared with dogs. A brief overview of coagulation and coagulometry is provided. Chapter three is a prospective observational cohort study characterising the coagulation factor activity patterns in VICC from naturally occurring tiger snake envenomation in dogs. The changes in coagulation factors in a study cohort of tiger snake envenomed dogs were compared to a healthy control cohort. Fibrinogen, factor V and factor VIII were identified as the most consumed factors and displayed the fastest recovery to control values. Tests of coagulation times (i.e., prothrombin time and activated partial thromboplastin time) were similarly affected. Additionally, higher serum tiger snake venom concentrations were associated with greater reductions in factors II, V and VIII, and greater prolongations in both coagulation times. The elucidation of the consumption and recovery pattern of coagulation factors in dogs, contrasted with that of people, provides insight into the underlying mechanisms of tiger snake VICC and its treatment. This information can be used for further diagnostic and therapeutic endeavours.

The objective of this thesis is to investigate the relationship between coxofemoral geometry and luxation of total hip replacements (THR) in dogs. This research question was explored in two parts. Examination of the relevant literature demonstrates that increased pre-operative soft tissue laxity is a risk factor for prosthesis luxation in dogs. However, there is little information pertaining to changes in this soft tissue tension in dogs after surgery and the impact this change may have on prosthesis stability. The first part of the thesis explores the association between radiographic measures of pre-operative soft tissue laxity, or offset, and the occurrence of luxation following total hip replacement. The offset measurements were validated in a pilot study, where ventrodorsal pelvic radiographs were assessed in a dog placed in varying degrees of pelvic rotation. Pre-operative and post-operative offset measurements, as well as measures of prosthesis orientation were made for dogs with confirmed dorsal luxation and compared with those of randomly assigned dogs with uneventful post-operative recovery using a case control study. Univariate generalised linear models were used to identify variables of interest and these were assessed in multivariate logistic models. Global offset of the hip was reduced after surgery in both groups, however, reduction was greater in the luxation group (17.7%) compared to the control group (7.4%) and was associated with an increasing odds of luxation. An open orientation of the acetabular cup was also positively associated with luxation. These results indicate that large changes in soft tissue tension after surgery may be a risk factor for luxation. In the second part of the study, a novel technique for surgical revision of luxated total hip replacements was explored. A triple pelvic osteotomy (TPO) was performed in 17 dogs with dorsal hip luxation and the efficacy of this procedure in preventing luxation recurrence, by altering the geometry of the acetabulum, was reported in a retrospective case series. Measurements of acetabular cup orientation were made from pre- and post-operative radiographs and compared using a parried t-test. The revision TPO was successful in preventing reluxation in fourteen cases and an excellent or good clinical outcome was reported in 12 cases. Ventral luxation occurred in three dogs due to over-rotation of the hemi-pelvis. The TPO significantly reduced the angle of lateral opening by 23.0o and increased the version angle of the acetabular cup by 9.0o. The results of this study show that the TPO is an effective procedure for managing THR luxation with retention of implants, however, careful patient selection is recommended to avoid subsequent ventral luxation. In the closing chapter, the challenges of managing surgical cases with significant pre-operative hip laxity are discussed and potential solutions are explored. Limitations of the studies presented in this thesis are also explored and future directions for research are proposed.

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.

Idiopathic pulmonary fibrosis is a lethal progressive respiratory disease with unknown aetiology that occurs predominantly in Western countries. The incidence and prevalence of this disease condition has been shown to be increasing over the years. IPF is localized to the lungs and diagnosed based on the radiologic and histopathological pattern of usual interstitial pneumonia (UIP). Two therapeutic drugs, pirfenidone and nintedanib, have been approved by the FDA in 2014 for treating IPF patients. While these drugs retard the progression of IPF, they do not cure it. Therefore, research is still required to better understand the underlying pathophysiology of IPF, and to develop therapies that halt and/or reverse the fibrosis in the lungs. Animal models are extensively used to elucidate the mechanisms that drive the fibrosis and to trial potential therapies for IPF. While bleomycin mouse models of pulmonary fibrosis are extensively used to investigate the human disease, these models fail to fully replicate the nature of the disease, and often fail in translating the gained knowledge to the clinic. Compared to the human lung, the small size of the murine lung and its dissimilar lung structure and function, can make it difficult to interpret data that is relevant to the human disease. Since many structural and functional aspects of the respiratory systems in large animals are closer to the human respiratory system, our laboratory developed a sheep model of pulmonary fibrosis, using bleomycin as the inducing agent. In this thesis, the sheep model of pulmonary fibrosis was used to study persistent fibrotic and functional changes in lung parenchyma at different stages of the disease progression after inducing fibrosis with bleomycin. A number of drugs were tested in the model, including the FDA approved drug pirfenidone. The effects of Mast cell activity on pulmonary fibrosis was also assessed in this Thesis. In a number of animal models, there are differences in the persistence of fibrosis after bleomycin insult. To determine the persistence of fibrosis in the sheep model, a study was conducted for sixteen weeks to characterise the time course and reversibility of fibrosis that was induced by bleomycin infusion into lung segments. Initially, a segmental approach was used to induce fibrosis with two infusions of bleomycin directed into the appropriate lung segments, two weeks apart. Saline was infused in the contralateral lung lobe to act as an internal healthy-control lung segment. A total of 10 sheep were used in this experiment. Changes in lung function throughout the experiment showed that the lung compliance was significantly poorer in bleomycin-induced lung segment up to eight weeks, and then improved to near normal levels at sixteen weeks after bleomycin. The improvement of lung function was confirmed with histopathological changes at the 16-week timepoint, where the lung parenchyma of bleomycin-infused lung segments exhibited normal tissue architecture. Importantly, results from this study demonstrate that bleomycin-induced fibrosis in sheep lungs resolves during the 8 to 16 week period after the last bleomycin dose. As the FDA approved anti-fibrotic drug, pirfenidone, is now recommended for treating IPF, an experiment was designed to test whether pirfenidone was also efficacious in the sheep model. The experiment was designed with 2 groups of ten sheep, both groups received bleomycin into a caudal lung segment, and saline into a contralateral control lung segment. One sheep group received 2 oral doses of pirfenidone/day for 5 weeks, starting two weeks after the final bleomycin infusion. The control group was treated identically, except that this group received 2 oral doses of vehicle/day. Pirfenidone was able to attenuate both histopathological and physiological readouts of established fibrosis in the sheep model of pulmonary fibrosis. Pirfenidone treatment improved bleomycin-induced fibrotic changes compared to vehicle control, with improved lung compliance, reduced fibrotic changes and collagen content, as well as a reduction in the density of TGFbeta positive cells and myofibroblasts. Overall, this study showed that pirfenidone can attenuate bleomycin-induced lung fibrosis in sheep. Therefore, in future studies with the sheep model, pirfenidone can be used as a benchmark drug to compare the efficacy of other novel therapeutics tested in this model. The role that mast cells play in pulmonary fibrosis is not well understood. Sheep are a good model for studying pulmonary mast cells because the phenotype and density of mast cells in the ovine lung are similar to that observed in the human lung. Two different drugs were used to investigate the role of mast cells in pulmonary fibrosis in sheep. Firstly, senicapoc, a KCa3.1 ion channel blocker which prevents mast cell activation, was compared with the current FDA approved drug, pirfenidone. Both drugs were given orally twice daily for 5 weeks, starting 2 weeks after the final bleomycin infusion. Without any drug treatments, mast cell density was significantly increased in the parenchyma of bleomycin-infused lung segments that were sampled at seven weeks after bleomycin injury. Mast cell density was significantly reduced in bleomycin-infused lung segments after a 5 week treatment with senicapoc. On the other hand, pirfenidone treatment did not attenuate mast cell density in bleomycin-infused sheep lung segments. This was interesting in that while both senicapoc and pirfenidone attenuated fibrosis and improved lung function in this model, only senicapoc retarded the increase in mast cell density that was attributed to bleomycin injury. Secondly, cromolyn sulphate, a mast cell stabilizing drug, was used in the sheep model to investigate whether it could attenuate the fibrosis resulting from bleomycin injury via its effects on mast cells. Cromolyn sulphate, when administered to bleomycin-infused lung segments, did not return either the mast cell density, or histopathological changes, or functional measurements to control levels that are normally observed in the healthy lung. Overall, results presented in this thesis show that the sheep model of pulmonary fibrosis can be used to study underlying pathophysiological mechanisms and treatments relating to IPF. It will be interesting to see whether the knowledge gained from this thesis can be translated to the clinic and contribute to better treatments outcomes.

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.

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.

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.

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.