Veterinary Science Collected Works - Theses
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ItemRNA stability in alphavirus infected cells( 2022-04)Alphavirus is a genus of viruses in the family Togaviridae. Alphaviruses include notable human pathogens such as Chikungunya virus (CHIKV), which causes arthritis, and the equine encephalitis viruses, which cause encephalomyelitis. The only alphavirus vaccine approved for human use is the Ixchiq vaccine for CHIKV, approved in late 2023. Alphaviruses subvert many processes of host cells to facilitate their own replication and evade the immune response. By understanding these host-pathogen interactions, treatments can be potentially targeted towards these host processes on which the alphaviruses depend for replication. Preliminary data from the Fazakerley lab shows that the alphavirus Semliki Forest virus (SFV) replicates less efficiently in RNase L knockout mouse embryonic fibroblasts (MEFs) than in wild-type cells. This is surprising, as RNase L is known to be an antiviral protein, degrading RNA in response to viral infection. One explanation involves the interaction of alphaviruses with host RNA-binding proteins. Studies performed primarily with the alphavirus Sindbis virus (SINV) have shown the RNA binding protein HuR binds with high affinity to alphavirus RNA through sequence elements in the 3’ untranslated region (UTR), resulting in increased stability of viral RNA. Stability of some host transcripts is also reduced during infection. Tristetraprolin (TTP) is another RNA-binding protein that binds competitively with HuR and promotes RNA degradation. TTP has been found to recruit RNase L to cellular transcripts under mitogen stimulation, resulting in the degradation of these transcripts. We hypothesised that in SFV infected cells, viral RNA binds HuR and sequesters the protein from cellular transcripts. HuR protects viral RNA from being bound by TTP, preventing TTP from recruiting RNase L and degrading the viral RNA. Cellular transcripts with no HuR bound remain vulnerable to TTP binding and RNase L degradation, leading to reduction of cellular transcripts, and therefore reduced translation of cellular proteins. This reduces the competition for translation of viral proteins and therefore results in increased viral replication. Consistent with studies in SINV, we have shown using immunofluorescent microscopy that HuR relocalises from the nucleus to the cytoplasm during SFV infection. Preliminary results of siRNA knockdown experiments also suggest HuR increases SFV replication efficiency. However, we could not conclusively determine if TTP knockdown affected SFV replication. Through transcriptomic analysis of SFV-infected WT and RNase L KO MEFs we found that RNase L was not responsible for the majority of changes in cellular transcript levels or stability during SFV infection. Based on these results, we concluded that our initial hypothesis that RNase L and TTP increases SFV replication efficiency by degrading cellular transcripts is not correct. To find other ways RNase L may benefit alphavirus replication, we investigated the effect of RNase L on stress granules (SGs). Alphaviruses utilise the SG protein G3BP1 to enhance viral replication. To utilise this protein, alphaviruses induce the disassembly of SGs. As RNase L has also been shown to be involved in the disassembly of SGs, we hypothesised that RNase L may also be involved in the disassembly of SGs by alphaviruses. We used immunofluorescent microscopy to investigate whether SGs were present in SFV-infected WT and RNase L KO MEFs. We found that SGs were present in SFV-infected RNase L KO MEFs, but not in the WT. This suggests that RNase L is involved in alphavirus disassembly of SGs.
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ItemWhat are the triggers, challenges, attitudes and behaviours that contribute to poor welfare of livestock? Can these situations where livestock are at risk of poor welfare be predicted?( 2023-11)As incidents of poor welfare affecting non-dairy cattle, sheep and goats (key livestock) continue to occur, new ways to respond to them need to be considered. This thesis firstly aimed to understand livestock welfare non-compliance in Victoria and wider Australia and secondly develop an animal welfare risk assessment tool (AWRAT) that could be used to identify key livestock at risk of poor welfare, based on observable parameters on farm. If livestock at risk of poor welfare could be identified, it may facilitate early intervention and inform response planning and support to quickly resolve issues and prevent reoffending. This thesis began by analysing 10 years of substantiated animal welfare complaints (SWC) and 39 years of historical animal welfare investigation case (HAWC) notes from Victoria, Australia, to better understand the nature and extent of animal welfare non-compliance. There was a modest inverse correlation between rainfall and the number of SWC, but rainfall was not protective against all livestock welfare incidents. In total there were 2179 individuals or group of individuals that had an incident of poor welfare affecting livestock on at least one occasion, in the HAWC. The majority of livestock welfare incidents were associated with neglect (99%) and 27% of cases reoffended. Good predictors of increasing welfare severity were identified using ordinal regression and included: failure to draft (z=-17.3), dip/drench (z=-13.1), mark (z=-11), provide proper and sufficient nutrition (z=-16.8), unsuitable use of males (z=-11.1) and overstocking (z=-17.2). Using the learnings from the literature review, SWC and HAWC and a survey of industry (not including producers), 18 risk factors were selected to develop an AWRAT. The proposed AWRAT included factors relating to farm infrastructure, nutrition, treatment and mating times. Participants from across Australia trialled the AWRAT following farm visits for welfare and non-welfare related reasons. A novel algorithm was developed to generate an AWRAT-Risk Rating, based on the AWRAT assessment. Using linear regression, the relationship between the AWRAT-Risk Rating and a simple welfare rating was tested. The AWRAT was good at identifying livestock with poor welfare based on this preliminary testing. The adjusted R squared values varied from 0.791-0.803. The intra-observer reliability was strong, with 89% of the correlation coefficients (CC) for participants >0.8. Inter-observer reliability was good with 68% of participants with CC of >0.7. Participants in the survey also identified possible protective factors, that are more commonly observed on farms with good welfare. In the third section of the survey, industry participants (including producers) identified the management issues, contributing factors, challenges and possible solutions to issues of poor welfare affecting key livestock. The AWRAT developed in this thesis provided a structured framework to improve consistency in livestock welfare assessments. Initial testing has demonstrated a capacity to identify livestock with poor welfare, but further research is necessary to determine if the AWRAT can identify livestock at risk of poor welfare, by studying animal welfare incidents and reoffending over time. Livestock welfare non-compliance occurs when there is a failure of duty of care. The reason for this failure appears to be complicated. Developing new approaches to support both the farmer and the animals in instances of poor livestock welfare is likely to be beneficial.
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ItemStudies on Intestinal Nematodes in Australian Thoroughbred Horses( 2023-10)Gastrointestinal nematodes (GINs) are the most important parasites of equines as they pose a significant threat to equine health and wellbeing, particularly in younger and geriatric horses. Strongylids (cyathostomins and strongylins) and ascarids (Parascaris spp.) are the significant GINs of horses, with the former constituting more than 75% of the total parasite fauna. Heavy burdens of these parasites such as Parascaris spp. in young horses, can cause impaction and rupture of the small intestine while cyathostomins can infect all age groups of horses, with variable severity of cyathostominosis, particularly when encysted larvae emerge synchronously from the intestinal wall. The literature review (Chapter 1) identified various research gaps related to the epidemiology, diagnosis, efficacy of commonly used anthelmintics and control of GINs in Australian horses. Over the last 50 years, only a few studies have investigated the epidemiology of GINs of equines in different states of Australia which were either restricted to some regions or involved only a small number of horses. To address some knowledge gaps on the GINs in Australian horses, this thesis aimed to (i) establish baseline epidemiological data on GINs in Australian Thoroughbred horses, (ii) develop and/or employ more sensitive and advanced molecular tools for the detection of GINs in horses in epidemiological and drug efficacy studies, (iii) ascertain the efficacy of commonly used anthelmintics against significant intestinal nematodes of horses, and (iv) assess worm control practices used by Thoroughbred farm managers and equine veterinarians. The longitudinal (Chapter 2) and cross-sectional (Chapter 3) epidemiological studies conducted using coprological methods showed high prevalence and egg-shedding patterns of GINs in various age groups of horses. Climatic zone and age had the highest impact on faecal egg shedding, particularly in the Mediterranean climate, the autumn season, and young horses (i.e., yearlings). The polymerase chain reaction-directed next-generation sequencing (PCR-NGS) method uncovered the diversity of strongylid nematodes as 31 species were detected in both epidemiological surveys and their occurrence varied across various climatic zones, seasons and age groups of horses. Traditionally, the faecal floatation method has been used to diagnose eggs of Strongyloides westeri – a free-living parasitic nematode of newborn foals. In Chapter 4, following the detection of S. westeri eggs in the faeces of foals using microscopy, a PCR-based diagnostic method was established for the first-time using DNA extracted from the parasite eggs. This method will help conduct future molecular epidemiological studies on S. westeri and assess the efficacy of commonly used anthelmintics in foals. Chapter 5 showed the extent of anthelmintic resistance (AR) in cyathostomins and Parascaris spp. prevalent in Australian Thoroughbred horses. An apparent failure of the efficacy of a combination of anthelmintic drugs (i.e., oxfendazole (OFZ) and pyrantel (PYR)) was observed for the first time against Triodontophorus brevicauda – a species of large strongyles. Cyathostomins were resistant to multiple anthelmintics, including abamectin (ABM), ivermectin (IVM), moxidectin (MOX) and OFZ, whether used individually or in combination with other classes of anthelmintics, i.e., OFZ+PYR. Furthermore, where anthelmintics were effective 2 weeks post-treatment, egg reappearance periods (ERPs) were reduced to four and/or five weeks. The major cyathostomin species identified 2 weeks post-treatment were from the two genera, Cylicocyclus and Cylicostephanus while those 5 weeks post-treatment with MLs were Cylicocyclus nassatus, Cylicostephanus longibursatus and Cylicocyclus ashworthi. Chapters 2 to 5 provided comprehensive information on the prevalence of GINs in Australian horses and resistance in ascarid and strongylid nematodes against commonly used anthelmintics. Subsequently, assessments of worm control practices surveys used by horse managers (Chapter 6) and veterinarians (Chapter 7) provided insights into their knowledge, aptitude and practices on GINs of horses, their diagnosis, treatment and control. Although both farm managers and veterinarians almost completely relied on anthelmintics to control GINs in horses, the latter seemed to use more targeted treatment strategies based on faecal egg count results. Multiple correspondence analyses used in Chapter 6 showed that the likelihood of suboptimal worm control practices on small farms (n = less than 50 horses) was greater than that of medium (n = 51-100) and large (n = above 100) farms. Furthermore, the findings highlighted a communication gap between veterinarians and horse managers. In conclusion, this thesis has contributed to addressing some key fundamental knowledge gaps on the GINs of Australian horses. Using conventional and advanced DNA-based diagnostic techniques, this thesis uncovered (i) the prevalence and egg-shedding patterns of GINs across various climatic zones during different seasons in various age groups of horses, (ii) the extent of AR against commonly used anthelmintics in ascarids and strongylid nematodes, and (iii) knowledge, aptitude and practices used by horse managers and veterinarians to control horse parasites. The novel findings of this thesis can pave the way for developing tailored guidelines for equine parasite control in Australia and globally.
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ItemDeveloping live attenuated vaccines against Avian Colibacillosis( 2023-04)Avian colibacillosis, caused by avian pathogenic Escherichia coli (APEC), is a disease of significant economic importance for the global poultry industry. Controlling APEC infections through vaccination is an effective strategy that can also contribute to reducing antibiotic use, an important goal in an era of ever-growing antibiotic resistance. An aroA mutant of E. coli serotype O78 has previously been commercialised as a live attenuated vaccine against avian colibacillosis. This vaccine provides O-antigen-specific protection against a homologous challenge but is ineffective against a heterologous challenge. This thesis aimed to develop a vaccine that can provide a broader protection against APECs. Four live attenuated vaccine candidates were constructed by deleting the aroA gene or the tonB and oppD genes in genetically distinct APEC strains. The presence of tonB is essential for the activity of outer membrane iron siderophore receptors/transporters that are common in all APEC strains. The deletion of tonB gene is expected to cause a substantial depletion of the pool of intra-bacterial iron, which in turn will suppress the inhibition of iron uptake by the Fur protein. The de-repression of the Fur regulon would increase the expression of siderophore receptors on the surface of the APEC cell. Therefore, it is hypothesized that the immune response against overexpressed iron siderophore receptors could induce serotype-independent, cross-protective immune responses following vaccination. The deletion of the oligopeptide transporter gene oppD was introduced in the tonB mutants as a second virulence attenuation step, to reduce the probability of a reversion to a wildtype phenotype. In parallel to these studies, the aromatic amino acid pathway aroA gene was deleted in APEC strains of the same genetic background, to compare the protective efficacy of aroA against tonB/oppD deletion mutants. The vaccine candidate strains APEC 102026 (O78) and APEC 10-578 (O127) were selected from a collection of isolates from cases of avian colibacillosis in our laboratories. The selection criteria included (i) genetic distance between strains to take into account the phylogenetic diversity of APECs, (ii) absence of antibiotic resistance genes to avoid a potential barrier to the commercialisation of a future vaccine product and (iii) virulence of the parent APEC strains to ascertain and characterize the effectiveness of the attenuation strategies. The parent APEC strains were also entirely sequenced to provide baseline information on the genome, which may be used to trace back potential genetic variations that may occur in the live vaccine over time under field conditions. The markerless single gene deletion mutants were constructed using chloramphenicol resistance cassette flanked by loxP or FRT sites to target tonB and aroA genes respectively, and kanamycin resistance cassette flanked by FRT sites to target oppD gene, followed by removal of these antibiotic resistance cassettes using the FLP-recombinase system. For the tonB/oppD double gene deletion mutants, the antibiotic resistance gene was removed using the FLP/FRT system for oppD deletion while the CRE/Lox system for tonB deletion. Confirmation of successful gene deletions was done using conventional PCR and Sanger sequencing, followed by biochemical characterization of mutants confirming the loss of function of the aroA, tonB and oppD genes products. To evaluate the colonization ability and safety of vaccine candidates, this study also developed an aerosol infection model, compatible with the strict containment requirements of handling genetically modified organisms (GMO). Nebulizer-based and atomizer-based aerosol infection methods were compared, by exposing young chicks to the virulent APEC strain E956 directly within negative pressure housing isolators. Birds exposed twice (days 1 and 4) to aerosols produced by the nebulizer developed a rapidly progressing disease mimicking field cases of avian colibacillosis. By contrast, birds exposed to aerosols produced by an atomizer did not develop colibacillosis even after three exposures on days 1, 4 and 7. The nebulizer infection model was then used to analyse the safety and colonization ability of aroA and tonB/oppD mutants constructed in APEC strains 102026 and 10-578. Birds exposed to aroA and tonB/oppD deletion mutants from both lineages were able to colonize in the upper respiratory tract (trachea) of birds. The aroA and tonB/oppD mutants from APEC 10-578, but not APEC 102026, also colonized the lower respiratory tract (air sacs) of birds. Birds exposed to aroA and tonB/oppD mutants also had significantly lower air sac lesion severity scores than birds exposed to the wildtype parent strains, indicating virulence attenuation in both APEC lineages following the deletion of either aroA or tonB/oppD. These findings reflected safety of both lineages and set foundation for future investigation onto the efficacy of the candidate vaccines against virulent APEC strains. In conclusion, this thesis described the construction of new vaccine candidates to overcome the limitations of an existing commercial vaccine and also provided their detailed biochemical and genetic characterization. The confirmation of the safety and colonization ability of these mutants using an improved aerosol infection model was validated during this thesis and can be used in future studies on the pathogenesis and vaccine development for avian colibacillosis.
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ItemInvestigation on Wound Healing Properties of Novel Selenium Compounds and Honey Bioactives( 2022-12)When the functional or structural integrity of the skin is compromised by various physical, chemical or microbial injuries, an automatic and organised multi-step process of wound healing occurs to repair the damaged tissues. However, various factors, such as age or underlying conditions, including cardiovascular disease, ischaemia, diabetes and infection, may contribute to impaired healing and result in non-healing chronic wounds. The chronicity of wounds has become a major global challenge impacting on a patient’s quality of life by increasing the morbidity rate and leading to amputations, imposing a high economic burden on health systems. Knowledge about the physiology of the wound healing process and its underlying mechanisms has been advanced over the past few decades using animal models. Various treatments have been proposed, which have focused directly on their effects on the immunohistopathological appearance of wounds or indirectly on infection, a major complication that may occur during wound healing. Despite the advances that have been made, the findings from animal studies conducted in rodents may not translate well into clinical trials because of the significant anatomical differences between the skin of humans and rodents. In addition, the mechanisms underlying wound healing in rodents differ considerably from those involved in wound healing in humans. Therefore, using large animal models, such as pigs, which share more similar structural features and wound healing mechanisms with humans may result in better translation into clinical trials. Therefore, the potential of 1,4-dideoxy-4-seleno-D-talitol (SeTal), a selenosugar, was tested in a pig model of wound healing in the studies described in this thesis using 2 different drug carriers, phosphate buffered saline (PBS) and 80% glycerol. Several macroscopic and histopathological aspects of different phases of wound healing were examined in three animal trials. The increasing prevalence of MDR (multi-drug resistance) in bacteria over the past few decades has given rise to concerns about the potential future lack of efficacy of most commercially available antimicrobials. To overcome this global health issue, development of new therapies based on using natural antimicrobials, drug repurposing and drug combination, have gained increasing interest. Drug repurposing or drug repositioning aims to find new applications for existing compounds with established pharmacological and toxicological profiles that are currently used for other clinical purposes. In the studies described in this thesis, two main bioactives of honey, bee glucose oxidase and defensin-1, were expressed in vitro and were examined for their functionality. The defensin-1 and glucose oxidase genes from Apis mellifera were successfully cloned in the pFASTBAC1 vector and were introduced into recombinant baculoviruses using a Bac-to-Bac system in an insect cell line. Both proteins were successfully cloned in a bacmid. Glucose oxidase was successfully expressed, and the activity of the expressed protein was confirmed in vitro using an enzymatic assay. Furthermore, in recent decades manuka honey and its therapeutic properties has resulted in numerous scientific investigations and various clinical trials have been conducted. Manuka honey has attracted particular attention because of its notably greater antimicrobial effects, particularly when compared with other varieties of honey. Antimicrobial activities of seleno-compounds, including SeTal, sodium selenite, seleno-L-methionine and ebselen, in combination with the main bioactive of manuka honey, methylglyoxal (MGO), were examined against some of the most common wound pathogens, S. aureus, E. coli and P. aeruginosa, using in vitro disc diffusion and checkerboard assays. Analyses for synergy were performed using fractional inhibitory concentration index (FICI) and fractional bactericidal concentration index (FBCI) determination. Overall, the pig model used in the studies described in this thesis will potentially contribute to enhanced translation of studies in animal models into successful clinical trials. While the studies suggested that repurposing of seleno compounds and combinational therapy may have potential, there is a need for in vivo studies in animal models and subsequently clinical trials.
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ItemCoxiellosis on Commercial Dairy Goat Farms in Australia: Prevalence, Risks Factors, Risk Assessments, and Surveillance( 2022-12)Coxiella burnetii is the causative agent of coxiellosis in animals and Q fever in humans. Multiple animal species can acquire coxiellosis. Being highly infectious and resilient, C. burnetii is a threat to both animals and humans. Clinical signs of coxiellosis rarely occur in animals except for reproductive disorders such as abortion, stillbirths, weak offspring, reduced milk yields and mastitis. Infected domestic small ruminants can excrete C. burnetii from their milk, urine, faeces and birthing products, therefore being a crucial source of human infections. Once excreted outside the host animal, C. burnetii takes its small cell variant (SCV) form, which can withstand high temperatures and disinfectants, and travel long distances as airborne particles. In 2012, the largest Australian farm-related Q fever outbreak was reported in an intensive dairy goat farm in Victoria. This thesis aims to improve the understanding of C. burnetii status among commercial dairy goat farms in Australia and attempt to establish a framework of a program to minimise the possibility of C. burnetii infection among commercial dairy goat farms. This aim was achieved by a series of studies on the prevalence of C. burnetii among commercial dairy goat farms, risk perceptions among commercial dairy goat farmers and an evaluation of different surveillance methods. A cross-sectional study (Chapter 2) was conducted to quantify the prevalence of C. burnetii infections among commercial dairy goat farmers in Australia and identify risk factors associated with farm positivity. The apparent herd prevalence was 10% (95% CI: 4, 22) and the true herd prevalence estimated to be 3% (95% CI: 0, 18). Samples from herds with >900 milking goats were 6.75 (95% CI: 1.65, 27.7) times more likely to return a C. burnetii positive result compared with farms with no less than 900 milking does. Farms with an increased dairy goat density had higher odds of BTM sample positivity, increasing by a factor of 2.53 (95% CI: 1.51, 4.22) for each order of magnitude increase in the number of goats per acre. In the following chapter (Chapter 3), a study was conducted to identify the pattern of C. burnetii Com1 PCR results in bulk tank milk (BTM) samples as well as production factors that may affect testing results. This longitudinal BTM test study found that Com1 PCR tests fluctuate in positivity. C. burnetii DNA concentration in BTM was associated with the season, farm and fat concentration of the BTM sample. These findings are important for informed decisions when making BTM surveillance plans for C. burnetii infection in dairy goat herds. Based on the findings from Chapter 3, risk perceptions of farmers from test-negative farms for C. burnetii introduction into their herds were investigated and comprehensive risk assessments were undertaken (Chapter 4). Participants perceived Q fever as an important risk but their self-efficacy level was ambiguous. Medium overall risk of C. burnetii introduction was reported by four out of seven participating farms. The introduction of infected goats was perceived to be the most important introduction route, followed by transmission on fomites, introduction from neighbouring domestic animals and spillovers from wildlife. An evaluation of different surveillance methods for detecting C. burnetii infections for herds with different starting probabilities of freedom was conducted in Chapter 5. Seven surveillance strategies were constructed from three candidate surveillance system components, and their performance was evaluated quantitatively. The results show that the most efficient combination of surveillance system components depends on a good understanding of initial herd C. burnetii status and the probability of introduction of infection. Collectively, the findings of this thesis identify, at the time of writing, a relatively low C. burnetii prevalence among commercial dairy goat farms in Australia. However, the risk factors for detecting C. burnetii infection on a farm were related to farm size/intensity and the industry is undergoing change in this regard. Overall, this thesis presents many elements of a framework for developing a market assurance program to achieve confidence in C. burnetii freedom and maintain such status
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ItemMolecular approaches to limit herpesvirus recombination in ILTV( 2023-02)Gallid alphaherpesvirus1 (infectious laryngotracheitis, ILTV) is an alphaherpesvirus that causes respiratory infection in chickens. It causes economic loss in poultry industries. Recombination between field strains of ILTV, and between live attenuated vaccines, has created more virulent strains of virus that have caused severe disease. This highlights the necessity for developing safer ILT vaccines. In this project, we manipulated the ILTV genome with the aim of creating attenuated viruses with a reduced capacity to recombine with other viruses. Specifically, the virus genome was manipulated with the aim of decreasing virus co-infection (a requirement for viral recombination) and with the aim of disrupting the recombination molecular machinery. The resultant viruses were characterised in vitro in order to identify any promising candidate vaccines for further testing and development.
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ItemImproving dairy fertility through genetic selection( 2022-09)Fertility has declined in dairy cattle populations all over the world as an unintended consequence of genetic selection for high milk yield. Fertility is an essential component of profitable dairy systems for several reasons: firstly, cows must calve in order to lactate; secondly, cows must re-calve to achieve optimal production efficiency in early lactation; thirdly, replacement heifers are required to maintain or expand herd sizes; and finally, good fertility allows herd managers to time the energetic requirements of lactating dairy cows to match feed availability in pasture-based systems. By maximizing the intake of low-cost feed, herd managers maintain profitable margins and optimize production efficiency. Unfortunately, fertility is difficult to improve, being complex from both a physiological and a management perspective. The global development of fertility Estimated Breeding Values (EBV) has been an essential strategy to improve fertility. These are easy to use – with most herd managers already selecting sires for artificial insemination – and are capable of producing permanent and cumulative change. However, new technologies must be carefully implemented to maximize herd manager uptake. The traditional 'top down' adoption model posits agricultural research, development, and extension (RD&E) as a unidirectional pipeline. This assumes that industry bodies and scientists are best placed to choose the practices that herd managers should adopt, which risks devaluing herd manager knowledge, skills, and adaptive abilities. Conversely, a 'bottom up' participatory approach integrates herd manager input at all stages of the RD&E process, producing solutions better tailored for real-world requirements. For theoretical models to be relevant, they must be applied to real world problems. In my thesis, I apply a participatory approach to the fertility EBV by using the Theory of Planned Behavior framework. Through qualitative interviews, I identify salient beliefs regarding the selection of high fertility EBV sires in 35 herd managers from northern Victoria. Perceived barriers to selection include: a lack of trust in the fertility EBV and/or Australian EBV system, low bull reliability, low confidence in the impact of genetic selection for fertility, and the feeling that fertility is not important. This is significant as it suggests that we can improve uptake of the fertility EBV by addressing these beliefs; the remainder of my thesis pursues this aim. Firstly, I address the belief that 'fertility is not a problem' using 438,578 mating and pregnancy diagnosis records for 86,974 cows collected from 38 herd managers in northern Victoria. With these data, I confirm that herd reproductive performance has significantly declined over fifty years, with a concomitant shift towards split calving. This is an often-involuntary system adaptation to poor fertility which can lead to undesirable changes in labor management, feed inputs, and business risk. Excellent herd reproductive performance allows herd managers to operate the management system that best meets their physical environment and farming goals, re-affirming the importance of genetic selection for fertility. The second barrier I address is low confidence in the fertility EBV. Using multilevel Cox proportional hazard and logistic regression models, I demonstrate that commercial dairy cattle with higher fertility EBVs are more likely to be inseminated earlier, conceive faster, and calve earlier than their low fertility EBV counterparts. With an effect size comparable to other management and environmental factors – such as age, milk production, and heat stress – my results show that herd managers can be confident that continued selection for high fertility is likely to result in improved phenotypic performance in Australian dairy herds. Finally, I address the perception that the fertility EBV is unreliable. It is important to iterate on the fertility EBV, especially with new developments in our understanding of the genetic architecture of complex traits, in the automated capture of fertility phenotypes, and genomic techniques. Using the latter, I develop a novel method for understanding the physiological mechanisms underlying correlated traits, specifically finding that alternative splicing may play a role in the antagonistic relationship between fertility and milk yield. This method also identifies clusters of variants that may allow geneticists to fine-tune selection, enabling herd managers to breed dairy cattle that are both fertile and highly productive into the future. Together, the work in this thesis shows that the fertility EBV is a relevant and effective tool for improving herd reproductive performance. It also describes methods which can be used to refine the next generation of EBVs. More broadly, my results demonstrate how herd manager input can be incorporated into setting research priorities, increasing herd manager adoption, and improving the technological outputs of agricultural research.
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ItemEnvironmental Distribution and Risks Associated with Canine Soil-transmitted Helminths and Canine Lungworms Contaminating Parks in Australia( 2022)In this thesis, 1581 canine faecal samples from 190 parks across Australia were collected and subjected to faecal floatation as well as newly developed qPCR assays to detect a range of canine soil transmitted helminths (cSTHs) and lungworms. In addition, questionnaires were administered to Australian dog owners and veterinarians to establish their knowledge, practices and perceptions with regards to canine endoparasites. In the first research chapter of my PhD project, I set out to develop and optimise Taq-Man based multiplex real-time PCRs (qPCRs) for the detection and characterisation of the species of canine hookworms from faeces. In order to validate these newly developed qPCRs we sourced canine faecal from Vietnam and Queensland (Australia) previously confirmed positive for A. caninum, A. ceylanicum and U. stenocephala by PCR- Restriction Fragment Length Polymorphism (PCR-RFLP). These qPCRs demonstrated a higher diagnostic sensitivity compared to the PCR-RFLP and allowed the detection of additional single and mixed hookworm species infections missed by the PCR-RFLP. In order to validate these tools for A. braziliense (Chapter 3), we sourced hookworm microscopy-positive canine faecal samples from a cross-sectional survey of gastrointestinal parasites of dogs in Nigeria and subjected these to the newly developed qPCRs. The qPCRs were confirmed as a highly sensitive tool for the detection of A. braziliense from field samples and revealed for the first time, the occurrence of this parasites in the country. Further, I updated the occurrence and distribution of hookworm species affecting dogs in Nigeria and highlighted the suitability of the newly developed multiplex qPCR assays as high-throughput tools for the surveillance of zoonotic hookworms, globally. In Chapter 4 extensive fieldwork and sampling of canine faecal samples contaminating parks across seven of the eight states of Australia was performed. Faecal samples collected were subjected to faecal floatation and a set of qPCRs to detect a range of canine soil-transmitted helminths (cSTHs). In total, 44.2% of the parks sampled were contaminated with at least one species of cSTHs, with hookworms being the most prevalent parasites (10.2%) followed by Trichuris spp. (1.3%) and Strongyloides spp. (1.2%). This study revealed a high rate of contamination with cSTHs in dog parks in urban Australia, most of which having proven zoonotic potential. Preventive measures, including awareness-raising educational programs promoting responsible pet ownership, immediate disposal of pet faeces in public areas were encouraged to minimise the health risks associated with cSTHs to both dogs and humans. In Chapter 5, to investigate the faecal prevalence of canine lungworms in Australia, we developed, optimised and validated a multiplex qPCR for the detection of Angiostrongylus vasorum, Crenosoma vulpis, and respiratory species of Eucoleus spp. and subjected canine faecal samples collected in Chapter 4 to the newly developed qPCR. The multiplex qPCR was validated on canine faecal samples from Switzerland and Cambodia previously subjected to Baermann examination and faecal floatation to screen for canine lungworms. The multiplex qPCR was able to detect all the species of canine lungworms targeted with higher diagnostic sensitivity and specificity compared to microscopy-based techniques. This qPCR allows for the simultaneous detection of the main species of canine lungworms from faeces and provides a relatively rapid diagnostic compared to time-consuming traditional techniques such as Baerman, faecal floatation and conventional PCR. Furthermore, qPCR does not require fresh faecal material opposite to Baermann technique hence representing an ideal diagnostic tool for large epidemiological studies. Based on sample size and the diagnostic parameters of the assay, and failure of the multiplex qPCR to detect any samples positive for lungworm DNA, lead to the assumption that the maximum prevalence of canine lungworms in faecal samples contaminating urban parks across Australia is less is than 0.19%. Lastly, Chapter 7 and 8 investigated pet owners and veterinarians’ behaviour, knowledge, and attitudes towards the control of canine intestinal and cardiopulmonary parasites in Australia. Overall, this PhD project found that the majority of dog owners and veterinarians in Australia are not complying with guidelines on the control of canine endoparasites. For instance, although the majority of dog owners dewormed their dogs (89.6%), only the 27.8% followed best practice guidelines, i.e., administered a monthly prophylactic treatment all-year round. A large proportion of respondent dog owners administered prophylactic treatment at an inappropriate frequency (47.8%) or did not treat for canine gastro-intestinal (GI) parasites at all (24.4%). Just over a half of veterinarians (55.9%) recommended prophylactic treatment of nursing or pregnant bitches for canine GI parasites, and recommended puppies to be first dewormed before three weeks of age (55.2%). Furthermore, many veterinarians were also unaware of the most common or significant species of GI parasites occurring in their practice area. This study demonstrates that dog owners and veterinarians in Australia are not complying with best practice regarding the control of canine GI parasites and are exposing themselves and their dogs to the risk of infections. As veterinarians constitute the main source of knowledge to dog owners it is imperative that Australian veterinarians focus on improving their knowledge on companion animal parasitology by accessing up-to-date, reputable online resources and attending continuing professional educational seminars. Furthermore, veterinarians are called to implement dog owner’s education, raise their awareness on the threats canine parasitic diseases pose to both dogs and humans and finally, encourage them to follow a monthly prophylactic treatment for canine GI parasites all year round.
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ItemCoxiella burnetii environmental contamination from a large multi-site goat farm and its spatial risk profile( 2022)Coxiella burnetii is a zoonotic bacterial pathogen that can infect multiple animal species. Animals rarely develop clinical disease from infection with C. burnetii. When infection occurs in animals, C. burnetii can be found in relatively high concentrations in the reproductive tract and are released into the environment during parturition. Intensively managed small ruminant farms can play an important role in the epidemiology of C. burnetii due to the potential for abundant release of the bacteria into the environment when large numbers of animals give birth during synchronized kidding/lambing events. Outside the host, C. burnetii can attach itself to dust particles and travel by the wind to places distant from the site of disposition. Human infection, Q fever, manifests itself as clinical disease in about 40% of cases and has the potential to be fatal if not treated. Q fever is endemic in Australia, with 2 cases per 100,000 population notified annually and Q fever seroprevalence in Australia is (at the time of writing) the second highest in the world. However, the knowledge of its spatial transmission from infected sources and validated methods to detect C. burnetii in the environment are limited. This thesis assesses the level of C. burnetii environmental contamination in and around a known infected intensively managed multi-site dairy goat farm in Victoria, Australia. The overarching aim of this work was to improve understanding of the environmental epidemiology of C. burnetii using as a case example the geographic distribution of contamination around a known C. burnetii-positive source. As a first step in addressing this aim, a systematic review (Chapter 3) was conducted to identify the main environmental substrate types, sampling, and testing methods available. Critical appraisal of the available evidence showed that a variety of factors play a role in the ability to detect the organism during field sampling and laboratory testing. Chapter 3 concludes with a framework that can be used by future researchers as a guide for environmental field sampling to detect C. burnetii. Given that the primary mode of transmission of C. burnetii is inhalation, determining the level of bacteria circulating in air is important when considering environmental contamination. In Chapter 4, three air sampling devices were compared and validated in a laboratory-based experiment to determine their ability to detect known concentrations of C. burnetii. This chapter showed that the air samplers performed similar at detecting aerosolized C. burnetii and provided detection and quantitation limits for each sampler with the PCR protocol validated in the study. Chapters 5 and 6 were field sampling studies centred around the dairy goat farm in which coxiellosis was endemic. In Chapter 5, an understanding of the level of environmental contamination in and around the kidding sheds was obtained while standardizing laboratory testing methods for different environmental substrates. Chapter 5 served as an assessment of the feasibility and assisted in the design of the larger-scale geospatial field study presented in Chapter 6. The field study found that C. burnetii soil positivity was higher closer to rivers and creeks. The detected association could be due to either contamination of the environment arising from wildlife preferentially aggregating around waterways or runoff of deposited material on topsoil accumulating in and around waterways. Considering the findings of this thesis and previous work in this field, it is evident that C. burnetii environmental contamination is context specific, depending on many factors including but not limited to the source of bacterial release, surrounding terrain and weather conditions. Overall, the work presented in this dissertation serves as a guiding model for research on C. burnetii geospatial contamination elsewhere.