Melbourne Veterinary School - Theses
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ItemAssessing and addressing the welfare of extensively managed ewes( 2019)The sheep industry is currently facing increasing social demands for assurances of good animal welfare, which indicates that assessing and addressing the welfare of sheep is critical if the current ‘social license to farm sheep’ is to be maintained. Farmers are key players in improving sheep welfare because they are responsible for the care of their animals and make the management decisions on their farm. Hence, a better understanding of the main factors underpinning farmer behaviour is important. According to the Theory of Planned Behaviour, farmer attitudes towards sheep management are likely to influence their behaviour in terms of the implementation of improved management practices. Subsequently, farmer management behaviour may impact on the welfare of their sheep. The aim of this thesis was therefore to examine the relationships between farmer attitudes, management behaviour and sheep welfare outcomes. To examine these relationships, animal welfare assessments and questionnaire interviews with farmers were conducted. A total of 6200 ewes (aged 2-5 years) and 32 farmers were sampled across Victoria, Australia. Farms were visited at mid-pregnancy and weaning, and the ewes were examined using six animal-based measures: body condition score (BCS), fleece condition, skin lesions, tail length, dag score and lameness. In addition, the number of ewes that needed further inspection/care (such as sick or injured sheep) was recorded and reported to the farmers. The welfare of the ewe flocks, based on the six indicators measured, was good overall. However, individual welfare compromise was considered significant. There were 185 (3.0%) ewes needing further care and cases were identified in all farms. Main reasons for further inspection/care were lameness or foot-related issues, BCS ≤ 2 and active dermatophilosis or broken wool. Farmer attitudes to sheep and management were in general positive and welfare was considered an important aspect of farm productivity. In terms of management, it was found that farmers relied mostly on visual monitoring to assess the flocks and pasture quality and quantity. However, close inspection of the flock and more detailed practices such as body condition scoring of sheep, veterinary interventions or keeping accurate records were less common activities. Indeed, mortality rates were frequently underestimated. Positive farmer attitudes were associated with positive management behaviour (active management style), and positive management behaviour was associated with positive ewe welfare outcomes. Farmers with an active management style had fewer ewes in need of further inspection/care after both welfare assessments (mid-pregnancy and weaning). Main results indicated that farmers were more likely to perform an activity (management behaviour) if they perceived the activity was valuable (behavioural attitudes) and if they perceived the activity could be realised (perceived behavioural control). The results of this thesis demonstrate that there is an opportunity to create change in farmer management behaviour and potentially improve sheep welfare via education programs targeting attitudinal change. The results reported in this thesis provide what is believed to be the first comprehensive study investigating the on-farm welfare of extensively managed ewes and the farmer-sheep relationship in extensive systems. Controlled trials to assess if education programs or other interventions are able to improve farmer attitudes and management behaviour, as well as sheep welfare, would allow causality to be investigated and would be a valuable next research step following on from the current work.
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ItemChronic enteropathy in dogs: the role of macrophages and preliminary results in inflammatory cytokines( 2019)Chronic enteropathy (CE) is an umbrella term used in dogs to describe a group of diseases with different aetiologies, characterised by chronic gastrointestinal signs. These diseases are clinically classified according to treatment response as food-responsive (FRE), antibiotic-responsive (ARE), and immunosuppressant-responsive enteropathies (IRE). The first part of this PhD thesis prospectively describes the features of CE that are commonly seen in dogs presenting to a referral centre in Australia; information that has not been available previously. We found that similar to other countries, most dogs with CE are food-responsive, followed by antibiotic-responsive with a minority of immunosuppressant-responsive. Furthermore, our study raised concerns about prolonged antibiotic treatment for dogs with ARE. Firstly, most of these dogs do not respond to treatment completely for prolonged periods (as opposed to dogs with FRE that do), raising the question about the real benefit of antibiotic treatment. Secondly, half of the dogs with ARE required long-term or pulse treatment with antibiotics, which raises concerns about development of bacterial resistance. Our findings highlight the need to find alternative treatment for dogs with ARE in view of the poor long-term outcome. Although most dogs with FRE had long-term remission, adequate dietary trials were not performed until reaching the referral setting. This indicates that better education of general veterinary practitioners about the importance of performing adequate diet trial is needed to improve early disease remission in these dogs. The next focus of the research was to evaluate the role of macrophages in CE; this was achieved by using two macrophage markers: calprotectin and cluster of differentiation 163 (CD163) in immunohistochemical examination. Both immunohistochemical markers highlighted two different populations of macrophages in our intestinal biopsy specimens. Overall the number of CD163 positive cells was higher than calprotectin positive cells both in crypts and villi. Dogs with FRE and IRE had a decreased CD163:calprotectin ratio compared to healthy dogs with an increase in the ratio after treatment. Our results suggest that there is an imbalance in macrophage populations in dogs with FRE and IRE, with partial resolution following clinical response characterised by an increase in the ratio CD163:calprotectin. Interestingly, dogs with ARE not only have a poor long-term response, but also have different macrophage populations from dogs with FRE and IRE; and in fact, are very similar to healthy dogs without change in their macrophage populations with treatment response. These results suggest that macrophages play a role in the pathogenesis of FRE and IRE dogs with normalisation of macrophage populations with treatment response. The CD163 receptor is cleaved during macrophage activation and is released into the circulation as a soluble form. In view of the decreased number of CD163 cells in the intestine of dogs with FRE and IRE at diagnosis (and subsequent increase with clinical remission), we wanted to determine if soluble CD163 could be detected in dog serum, and therefore potentially serve as a biomarker. Two different ELISAs were tested and although one of them showed some signal, further testing of the antibodies used in the assay did not support that the signal was specific for CD163. With this experiment, we were not able to quantify soluble CD163 in dogs, but this molecule retains potential as a biomarker for diagnostic and monitoring purposes in CE as well as in other diseases characterised by macrophage activation. Biomarkers of systemic inflammation were also assessed in the same cohort of dogs and we showed that serum IL-6 decreased in dogs with CE after resolution of clinical signs. Similarly to soluble CD163, cytokines might play a role in further differentiating between the different causes of CE for prognostic and therapeutic purposes. Future studies are needed to assess these cytokines in a larger cohort of dogs to be able to study differences between FRE, ARE, and IRE, and further assess their role as biomarkers. Finally, we studied cytokine production by lymphocytes or monocytes in the peripheral blood of healthy dogs, and the effect of different immunosuppressive treatments on cytokine production. Differential activation of lymphocytes or monocytes can easily be achieved by using specific activators in whole blood. The advantage of this technique is that there is minimal handling of the cells with less risk of iatrogenic activation. Cytokine production was affected by cyclosporine and prednisolone, but not by mycophenolate, leflunomide, or azathioprine. Cyclosporine inhibited production of tumour necrosis factor (TNF), interferon gamma and IL-10 by lymphocytes whereas prednisolone inhibited TNF production by both lymphocytes and monocytes. Our findings suggest that this methodology can be used to monitor dogs treated with both drugs concurrently – although this needs to be further assessed with future studies. Future studies highlighted by our research suggest more in-depth assessment of serum cytokines as biomarkers for dogs with CE not only for monitoring purposes, but to determine if different patterns of cytokines can be useful to refine the classification of CE. Similarly, whole blood stimulation can be used to better assess underlying priming of the immune system and to monitor treatment response. Finally, our findings suggest that macrophages play a significant role in the pathophysiology of CE in dogs, particularly in FRE and IRE, but additional work is required to better understand their function in CE.
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ItemEvaluation of the analgesic efficacy of methadone and buprenorphine for the treatment of acute postoperative pain in cats( 2018)Prior to the studies detailed within this thesis butorphanol was the only opioid registered for use in cats in Australia. There was a need for further clinical studies to be conducted in cats to evaluate the perioperative analgesic efficacy of other alternative opioids such as methadone and buprenorphine. While clinical studies had previously investigated the analgesic efficacy of methadone and buprenorphine, none had utilised a validated multidimensional pain assessment tool (PAT) designed to assess acute pain in cats, which called into question their findings. To address this need, the author conducted clinical studies in cats to evaluate the individual perioperative analgesic efficacy of each of methadone and buprenorphine compared to butorphanol. Surgical ovariohysterectomy (OVH) was used as a model of acute pain. Postoperative pain was assessed using a multidimensional PAT recently validated for assessment of acute pain in cats following OVH. Within the conditions of the studies documented within this thesis, methadone administered subcutaneously as a premedication at 0.6 mg/kg provided effective postoperative analgesia for at least 6 hours following OVH surgery in most cats. Buprenorphine administered intramuscularly as a premedication at 20 µg/kg with repeat administration of the same dose at the time of surgical wound closure, provided effective postoperative analgesia for at least 6 hours following OVH surgery. In contrast, intramuscular administration of a single premedication dose of 20 µg/kg IM buprenorphine prior to anaesthesia provided inadequate postoperative analgesia and additional analgesic drugs were required. All dosing regimens involving butorphanol resulted in unacceptable numbers of cats requiring rescue analgesia, suggesting that butorphanol is not a suitable choice of opioid for the provision of postoperative analgesia in the cat at these doses. An additional clinical study was conducted to examine the effect of atipamezole on analgesic requirement, and hence its potential confounding influence on the findings reported. Results from this follow up study suggested that the administration of atipamezole at a clinically recommended dose does not significantly affect the immediate postoperative pain scores in cats following OVH. Physiological data collected during the anesthetic period of these studies indicated that none of the opioids administered caused severe deleterious effects on vital functions and that all could be used safely for premedication prior to general anesthesia. In part dependent upon the findings from these studies, methadone and buprenorphine received registration approval with the Australian Pesticides and Veterinary Medicines Authority (APVMA) for use in cats within Australia. This will in part assist in addressing the undertreating of feline patients experiencing acute perioperative pain by providing clinicians with more efficacious registered drug options. In the course of evaluating the findings of the clinical studies reported in this thesis within the context of the wider peer-reviewed literature, it became apparent that inconsistencies existed in the way analgesic efficacy of opioids in cats is reported. Many of these inconsistencies arise due to variations in the quality and rigour with which a particular study is designed (including the use of non-validated PATs) and reported. To address these inconsistencies, a critical appraisal tool was developed for reviewing analgesia studies (CATRAS) involving animals and human patients who are incapable of self-reporting pain. Content validation was achieved using Delphi methodology via panel consensus. A panel of six experts reviewed the CATRAS in three rounds and quantitatively rated the relevance of the instrument and each of its quality items to their respective domains. The resulting 67-item critical appraisal tool will now provide a means of critically and quantitatively assessing the quality of analgesia trials involving subjects incapable of self-reporting pain for use in systematic reviews and meta-analysis studies.
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ItemImproving disease surveillance in Australia’s sheep industries: investigations of syndromic surveillance, farmer behaviour and sheep trade networks( 2018)Designing and delivering effective, useful livestock health surveillance is a challenge for many countries. The observations of people in frequent contact with livestock, captured through passive surveillance, play an important role in many national surveillance systems. In Australia, the effectiveness of passive surveillance on sheep and beef farms has been limited by infrequent veterinary contact. Farm workers frequently observe signs of disease in livestock, but these observations are not captured by existing surveillance systems. This thesis therefore posed the question: can farmers’ observations be collected to generate useful surveillance information? Syndromic surveillance of farmers’ observations is one approach to increase data capture from extensive livestock farms. Chapter 3 describes the operation of a syndromic surveillance system collecting farmers’ observations of livestock health in Victoria, Australia, over its first two years of operation from 2014 to 2016. Survival analysis and classification and regression tree analysis were used to identify farm level factors associated with reliable participation, to inform future recruitment aimed at farmers who were willing and able to provide regular, timely reports. Farmers keeping only sheep were the most reliable and timely respondents, while farmers aged under 43 years or working full time on-farm had lower response rates than older farmers or part-time farmers. This chapter demonstrates that recording farmers’ observations of signs of disease using syndromes is a feasible and effective method to gather disease occurrence data. The utility of syndromic data is further investigated in Chapter 4, using the observations collected by the surveillance system to quantify ewe mortality on sheep farms in southern Australia. Ewe deaths were reported in 540 of 612 reports, describing 2106 individual deaths, with a median of 4 deaths per positive monthly report. Median mortality rates ranged between individual farms from 1 to 5 deaths/1000 ewes/month. The incidence rate ratio of mortality in the five months preceding and following lambing was 2.8 (95% CI 2.0 to 4.1) compared to the remaining seven months of the year. Overall ewe mortality could therefore be reduced through strategies targeted to improving peri-parturient ewe survival. In a subset of reports where veterinary contact was recorded, just 15% of reported deaths involved a veterinarian. Further investigation of how and why farmers respond to ewe deaths without veterinary support is needed, to determine the best farm management strategies to reduce mortality. Chapter 5 investigates Australian sheep farmers’ low rates of veterinary contact. The study aimed to understand why Australian sheep farmers chose not to contact veterinarians when their animals showed signs of disease, and what alternative approaches they took to managing unwell animals. Data were collected during three focus group discussions with sheep farmers in Victoria, Australia. Transcripts of those discussions were analysed using a modified grounded theory approach to develop a preliminary theory of Australian sheep farmers’ disease response behaviour. Critical steps in the decision-making process included the farmer recognising that action is needed, and then deciding what that action would be. The farmers reported having to decide whether they would act independently based on their previously experiences, or alternatively to seek advice. Veterinarians played a small but important role as potential advisors, alongside others including trusted farming friends and farmer discussion groups. Self-reliance and confidence in their knowledge and skills was highlighted as the main reason the farmers often chose not to seek veterinary advice. Rather than being seen as a barrier to effective passive surveillance, the actions that arise from farmers’ self-reliance when facing disease should be taken into account when designing novel surveillance approaches. A final consideration for observational disease surveillance is the selection of individuals to contribute data to the system. While characteristics associated with participation may guide recruitment as described in Chapter 3, it is also useful to target surveillance to farms that have increased risk of acquiring or disseminating disease. The movement of animals between farms contributes to infectious disease spread, and can be investigated through network analysis methods. Australia’s National Livestock Identification Scheme sheep movement records are suitable for such analyses, but are known to be a targeted subset of all sheep movement in the country. However, knowledge of the effect of sampling or incomplete network data on these studies is limited. In Chapter 6, a simulation algorithm is presented that provides an estimate of required sampling proportions based on predicted network size, density and degree value distribution. The algorithm may be applied a priori to ensure network analyses based on sampled or incomplete data provide population estimates of known precision. Results demonstrate that, for network degree metrics, sample size requirements vary with sampling method. Where simulated networks can be constructed to closely mimic the true network in a target population, this algorithm provides a straightforward approach to determining sample size under a given sampling procedure for a network metric of interest. Chapter 7 then presents analysis of National Livestock Identification Scheme sheep movement data for Victoria, Australia. The sheep movement network in Victoria shows typical livestock movement network characteristics including scale-free and small-world topology, small diameter and short average path lengths, supporting the assumption that disease could spread rapidly in the state through sheep movements if it were not detected rapidly. Victoria’s position as a net importer of sheep and sheep flow is confirmed, driven substantially by the activity of saleyards (livestock markets) and abattoirs. Little variation within or between years in overall movement patterns were detected. While most farms are connected to a very small number of properties in the network, small subsets of farms demonstrate high degree values (being directly connected to many other properties through incoming out outgoing animal movements) or high frequency of sheep purchases or sales. These farms may be useful targets for emerging surveillance methods that can be implemented on-farm. Together, these studies provide new information about the Australian sheep industry and the feasibility of new surveillance approaches to improve the effectiveness of surveillance. By describing farmer behaviour, livestock movements patterns and the feasibility of syndromic surveillance approaches to capture farmers’ observations of signs of disease, these studies justify further development and implementation of novel surveillance approaches in Australia and serve as an example for other countries facing similar surveillance challenges. While there is no ideal surveillance system, integrating new approaches into wider surveillance strategies can improve the quality of information generated by surveillance, to better describe true disease states in the population and drive appropriate response activities.
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ItemViruses of the other mammals: genomics and epidemiology of marsupial herpesviruses( 2018)Improving wildlife population health requires an understanding of the infectious agents within those populations. Historical accounts of herpesviruses in marsupials indicate that they can have a significant impact on animal health. This evidence is strongest for the macropodid alphaherpesviruses, but with improvements in molecular diagnostics the discovery of novel viruses has outpaced our understanding of their impact and significance. This thesis aimed to expand our knowledge of marsupial herpesviruses by examining relationships between marsupial herpesviruses and other herpesviruses, and by describing the clinical significance of infection. This thesis also aimed to improve diagnostic tools for detecting herpesvirus infection in marsupials. The core genomes of three marsupial herpesviruses were determined; macropodid alphaherpesvirus 1 (MaHV1, infecting wallabies), phascolarctid gammaherpesvirus 1 (PhaHV1, infecting koalas) and vombatid gammaherpesvirus 1 (VoHV1, infecting wombats). MaHV1 had a similar genome arrangement to other simplexviruses, but contained gene clusters that may be unique to the macropodid simplexviruses. PhaHV1 and VoHV1 had a shared gene arrangement and were likely to have speciated from a common ancestor. Over 30 new ORFs were identified within the genomes. Functional enzymatic characterisation was performed on two viral NTPDase homologs encoded within the two gammaherpesviruses. NTPDase activity was confirmed for the PhaHV1 homolog but not the VoHV1 homolog. Koalas are host to two divergent gammaherpesvirus species, PhaHV1 and -2. To understand the clinical significance of each individual virus a large molecular epidemiological study of 810 koalas from 7 separate geographic regions was conducted. Samples were tested using a rapid and differential PCR-HRM assay. Available signalment and clinical observation data was analysed in comparison to infection status through univariable and multivariable logistic regression analysis. Additional factors considered were location, year, body condition, fecundity in females, as well as the presence of other infectious agents (Chlamydia pecorum and koala retrovirus). PhaHV1 and -2 were present in 17% and 22% of koalas tested (state-wide), although some variation from the state average was observed in particular populations. Neither virus was associated with a particular sex. PhaHV1 detection was uniquely associated with the presence of koala retrovirus as well as increasing age. PhaHV2 detection did not change with age, which may indicate differences in how these two viruses are acquired over the life of the animal. Both viruses were positively associated with genital tract abnormalities, lowered fertility in females, emaciated body condition, urinary tract infection (wet bottom) and detection of C. pecorum, although the strength of these associations varied by sex and herpesvirus species. To further the development of herpesviruses serological tools, this thesis examined the ability of four commercially-available immunoglobulin-binding reagents to bind serum antibodies from 17 species within the Marsupialia and Monotremata. Serum samples were assessed for binding using immunoblots and ELISAs to three microbially-derived proteins; staphylococcal protein A, streptococcal protein G and peptostreptococcal protein L, and to an anti-kangaroo antibody. The inter- and intra-familial binding patterns of the reagents to serum immunoglobulins varied and evolutionary distance between animal species was not an accurate predictor of the ability of reagents to bind immunoglobulins.
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ItemPharmacometrics of the bovine mammary gland( 2017)Intra-mammary antibiotics are widely used, and are an important contributor to the control of bovine mastitis. The concentrations of antibiotic drugs in milk after intra-mammary administration is highly important for food protection and treatment efficacy. In this project, novel mathematical and statistical models were developed to describe the pharmacokinetics of antibiotic drugs after intra-mammary administration. This approach combined physiologically-based pharmacokinetic models to capture the unique features of the mammary gland, and large-scale mixed-effects analysis to understand these effects at the level of the treated population. The developed models perform well across various drug types and production conditions, and it is proposed that these models are suitable as a generalized method for the description and analysis of drug concentration in milk. Further, the developed methods are applied to study the statistical performance of the various existing methods used to study milk residues in the regulatory context. A derived model is discussed, which is suitable for application to regulatory conditions for the determination of milk discard times.
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ItemEnsuring dairy cow welfare with increasing scale of production( 2018)Animal welfare is important to the general public and dairy consumers and the dairy industry is coming under increasing scrutiny. There is the potential for community concern arising from perceived intensification of the industry with an increasing number of large herds, and producers adopting a mix of grazing and confined feeding strategies. This thesis examines the particular welfare challenges associated with increased herd size in Australian pasture-based dairy herds including how they might be measured and managed. A survey of Australian dairy farmers was conducted to assess relationships between herd size and known or proposed risk factors for adverse animal welfare outcomes in Australian dairy herds in relation to increasing scale of production. Increasing herd size was associated with increases in stocking density, stock per labour unit and grain fed per day – all of which could reasonably be hypothesized to increase the risk of adverse welfare outcomes unless carefully managed. However, increasing herd size was also associated with an increased likelihood of staff with formal and industry-based training qualifications. Herd size was not associated with reported increases in mastitis or lameness. Large herds were more likely to use monitoring systems such as electronic identification in the dairy, computerised records, daily milk yield or cell count monitoring and pedometers or activity meters. Increasing herd size was related to increased herd milking time, increased time away from the paddock and increased distance walked. Animal welfare assessments were conducted on 50 Australian pasture-based dairy farms of varying herd sizes. Findings were generally consistent with the previous survey. Major challenges included heat stress, mastitis, lameness, and longer milking duration. All cows had access to water for more than 12 h in a 24 h period. More larger farms had water points on the farm tracks or at the dairy. Skin and joint lesion prevalence was not related to herd size and they were uncommon. All farms had some form of cooling strategy. Shade in all paddocks was more common on smaller farms than others while sprinklers were more common on large/very large farms. There was wide variation in the avoidance distance of humans but this was not related to farm size. Lameness scoring was conducted on 19154 cows from 50 farms as they left the dairy after being milked. We compared our Results with farmer estimates of lameness prevalence. Farmers detected only 24% of the cows observed/scored as lame. Whilst lameness scoring of the entire herd was necessary to detect all the lame cows, scoring just the last 200 cows milked could be used to estimate the prevalence of lame cows on a given day. In large Australian dairy herds, it is common for cows to be collected from the paddock as a group, to wait as a group in the dairy yard to be milked and to return individually to the paddock or feed pad immediately after milking. We demonstrated that even in these large herds there is consistency in the order cows are milked. This may have welfare implications for cows that are regularly milked last because they may spend several hours per day less in a paddock grazing. Lying is a high priority behaviour for cows. We used activity monitors to show that cows in large Australian dairy herds spend an average of 9.5 hours lying each day. Even in large herds, where milking can take up to 4 hours, the time taken to milk the cows did not affect their welfare by significantly impacting on their ability to lie down.
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ItemInitial development of an implantable drug delivery device for the treatment of epilepsy( 2018)Epilepsy is a chronic neurological condition, characterized by recurrent seizures. Treatment with conventional anti-epileptic drugs (AED) results in only 33% of patients having no seizure recurrence after a prolonged period. Some evidence suggests that the lack of effectiveness of AED penetration into the brain parenchyma could be one of the mechanisms for resistance to treatment. Furthermore, AED side effects often prevent large increases in their dose. It was for these reasons that a new alternative approach to therapy, intracranial implantation of polymer-based drug delivery systems aimed at improving the bioavailability of AED, was investigated in this PhD research project. Genetic Absent Epilepsy Rats from Strasbourg (GAERS) is a strain of rat that has 100% of the animals with recurrent generalised non-convulsive seizures. This animal model has become the gold standard to study the mechanisms underlying absence epilepsy. After validating an automated spike and wave discharges (SWDs) detection algorithm applied to GAERS’ electroencephalograms (EEG) and demonstrating some effects of enrofloxacin on the GAERS’ epileptic activity, in vitro characterisation and in vivo testing of the antiepileptic efficacy of Poly(D,L-lactide-co-glycolide) (PLGA) polymers loaded with anticonvulsants, either phenytoin or lacosamide, were performed. PLGA is a biodegradable copolymer that it is very well tolerated by the brain and can be used as a passive release substrate. In prospective randomized masked experiments, GAERS underwent surgery for implantation of skull electrodes, skull electrodes and blank polymers, or skull electrodes and AED loaded polymers. The polymers were implanted bilaterally and subdurally on the surface of the cortex. Electroencephalogram recordings were started at day 7 post-surgery and continued for eight weeks. The number of SWDs and mean duration of one SWD were compared week-by-week between the groups. Although temporary changes were seen, phenytoin loaded PLGA polymer sheets did not decrease seizure activity in GAERS. However lacosamide loaded PLGA polymer sheets affected seizure activity in GAERS by decreasing the mean duration of SWDs for a sustained period of up to 7 weeks. This provided proof of concept that intracranial implantation of polymer-based drug delivery systems could be used in the treatment of epilepsy. The last chapter of this thesis evaluated the in vivo biocompatibility of Polypyrrole (PPy) implanted subdurally and unilaterally on the surface of the motor cortex in GAERS. Polypyrrole offers the advantage of being electrically conductive which could allow a controlled release of the anti-epileptic medications. Due to the absence of evidence of toxic injury or immune mediated inflammation, it was concluded that PPy offers good histocompatibility with central nervous system cells. Furthermore, the comparison of immunohistochemical scores reveals that the amount of neuronal death and gliosis was significantly less in the PPy side than in the sham surgery side of the cortices (p values of 0.005 and 0.002 respectively) implying that the application of PPy could protect the CNS tissue after surgery. With improvements in polymer technologies and episodic release offering potentially much longer lasting release durations, intracranial polymer-based drug delivery systems may provide an effective therapeutic strategy for chronic epilepsy. While the development of such an antiepileptic device is worthwhile but still years away, the development of an implantable device combining the potential neuroprotective effect of PPy and an anti-inflammatory drug like dexamethasone would certainly be quicker to develop.
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ItemExploration of the basis of virulence attenuation and development of recombinant derivatives in a Mycoplasma synoviae live vaccine( 2017)Mycoplasma synoviae is an important avian pathogen that is responsible for significant economic losses in the poultry industry worldwide. It is able to infect chickens, turkeys and other avian species, causing subclinical respiratory tract infections, synovitis, and egg abnormalities. A live attenuated temperature-sentitive (ts+) vaccine strain, MS-H, is widely used in many countries to prevent infection with M. synoviae. Due to its ts+ phenotype, MS-H does not grow at the core body temperature of birds, but colonises the upper respiratory tract and establishes a solid protection against wild-type M. synoviae. In rare cases, the ts+ MS-H strain may revert to a non-temperature-sensitive (ts−) phenotype, but this is not associated with a significant increase in the pathogenicity in MS-H re-isolates, indicating that factors other than temperature sensitivity may be involved in the attenuation of the vaccine. To better characterise the molecular basis of the vaccine attenuation, MS-H and its parent 86079/7NS were subjected to whole genome sequencing. Comparative analysis of the genome organisations of the MS-H, 86079/7NS and other published M. synoviae strains revealed a number of features unique to MS-H that can be used as novel genetic markers in routine diagnostic tests. In M. synoviae strains MS-H and 86079/7NS, a 50-kb genomic inversion was observed for the first time amongst M. synoviae strains. Two newly identified horizontally transferred genes that encode a deoxyribose-phosphate aldolase and an ATP-dependent DNA helicase were noted for MS-H and 86079/7NS. Genomic comparison of MS-H and its parent strain 86079/7NS revealed 33 single nucleotide polymorphisms (SNPs) (excluding the highly variable vlhA gene), including 11 nonsynonymous SNPs that may havIe functional consequences. A frameshift mutation causing a premature termination of translation was found in oppF-1, a gene that encodes an ATP-binding protein that is a component of an oligopeptide transporter. This frameshift is likely to have inactivated the OppF protein function, suggesting that this mutation may play a significant role in the attenuation of MS-H. A nested PCR combined with high-resolution melting (HRM) curve analysis, based on the frameshift mutation within oppF-1 gene, was developed. This assay could reliably differentiate MS-H from all field strains tested in this study and was efficient for both cultures and swabs from clinical cases. In addition, this study investigated the potential for use of the MS-H vaccine as a delivery vector for foreign antigens using infectious bronchitis virus (IBV) as a model, aiming at the development of recombinant poultry vaccine candidates, especially for those pathogens affecting the upper respiratory system. To achieve this, expression vectors were constructed based on an oriC plasmid (pMAS-LoriC), which is capable of freely replicating in M. synoviae. Gibson assembly was used as a versatile strategy for constructing the vectors. The variable lipoprotein haemagglutinin gene (vlhA) promoter was used to drive expression of partial S1 and N IBV genes in MS-H. The expression of IBV N or S1 mRNA in MS-H recombinants was detected by quantitative real-time PCRs (qPCRs). Western blotting confirmed the expression of IBV partial S1 and N proteins using polyclonal antibodies specific for S1 and IBV respectively. This is the first report of successful expression of a non-native gene in M. synoviae. Thus the studies reported in this thesis have contributed to (1) development of our understanding of putative virulence factors in M. synoviae, (2) development of an assay to discriminate MS-H from field strains, and (3) laid the foundation for the development of novel recombinant vaccines to control other upper respiratory pathogens.
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ItemAntimicrobial stewardship in Australian veterinary practices( 2017)Antimicrobial use by the veterinary profession has been coming under increasing scrutiny by medical, public health and government officials as the threat of antimicrobial resistance becomes increasingly clear. The World Health Organisation has described antimicrobial resistance as one of the major public health challenges of our time. It is clear that at least some drug-resistant pathogens have evolved under selective pressure from antimicrobial use in agriculture and may be contributing significantly to resistance in clinical setting. Antimicrobial stewardship is the selection of the most appropriate antimicrobial for a given disease in a given animal, with the aim of reducing the risk of adverse effects in that animal, and reducing the likelihood of developing resistance on an individual level, on a farm level and on a national level. Currently none of the core elements of antimicrobial stewardship are widely available for veterinarians in Australia, and there is very sparse data available on which to base an antimicrobial stewardship program. This research project aims to address this paucity of data. A range of research methods were used to assess detailed antimicrobial use by veterinarians in Australia and the enablers and barriers to antimicrobial stewardship. These included quantitative methods such as surveys and analysis of pet insurance data, and qualitative methods such as interviews and focus groups. While antimicrobials with low importance rating were predominately used in all species, under-dosing and inappropriate timing of antimicrobial therapy were common particularly in horses and cattle. Few veterinary practices in Australia had antimicrobial stewardship policies in place, or were using antimicrobial use guidelines. The key barriers to implementing antimicrobial stewardship programs were a lack of antimicrobial stewardship governance structures, client expectations and competition between practices, the cost of microbiological testing, and a lack of access to education, training and antimicrobial stewardship resources. The enablers were, firstly, concern for the role of veterinary antimicrobial use in development of antimicrobial resistance in humans, secondly , a sense of pride in the service provided, and thirdly , preparedness to change prescribing practices. This research culminated in the development of a proposed antimicrobial stewardship policy and procedure documents, to enable veterinarians to institute antimicrobial stewardship programs that suit their individual practice requirements. However, it is likely that governance changes will be necessary to compel veterinary practice owners to implement antimicrobial stewardship on a large scale.