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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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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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.
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ItemDevelopment of a live-attenuated vaccine against canine pyometra( 2022)Pyometra is a disease affecting intact bitches and is fatal if left untreated. It is caused by the bacterial invasion of the uterus under the influence of progesterone resulting in the accumulation of pus. Uropathogenic E. coli (UPEC) are one of the most predominant pathogens isolated from the uteri of affected animals. UPEC carry uropathogenic virulence factor (UVF) genes which influence their virulence in the urogenital tract. The urogenital tract is a low iron and nutrient-deficient environment making the UPEC’s iron acquisition system and the oligopeptide uptake system critical for their virulence and survival in the urogenital tract. To date, there are no medical alternatives to ovariohysterectomy for the prophylaxis of pyometra. The main objective of the studies described in this thesis was to develop a bacterial vaccine targeting the TonB-mediated iron uptake system, the Fur regulon and the oligopeptide uptake pathway. The hypothesis was that targeting the iron uptake, iron homeostasis and oligopeptide uptake systems at once would allow the vaccine to provide protection despite the genetic diversity between UPEC strains and the redundancy of many of their metabolic systems. The first step was to assemble and characterise the complete genome of canine UPEC isolates using Illumina Miseq and Oxford Nanopore MinION sequencing technologies. Whole genome comparative genomics was carried out between canine isolates and other pathogenic and non-pathogenic Escherichia coli (E. coli) strains from various sources with a special focus on human UPEC isolates. The genomes of the canine UPEC isolates P4 and YP3 contained a single circular chromosome of 5.09 mega basepairs (Mbp) and 5.08 Mbp, respectively. The genomes were annotated using the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline (PGAP) and 18 pathogenicity-associated islands carrying virulence-associated genes were predicted in both genomes. Both canine uterine isolates belonged to the phylogroup B2, multilocus sequence type ST12 and serotype O4. The evolutionary relatedness between the canine UPEC isolates and other E. coli based on the cgMLST scheme and core single nucleotide phylogeny (SNP) showed a close phylogenetic relationship between the canine pyometra strains and human UPEC strains UTI89 and NU14. The virulence gene profiles of P4 and YP3 were nearly identical to human cystitis causing UPEC subtypes. The canine faecal isolate YF8 was identified as Escherichia marmotae and contained a single chromosome of 4.55 Mbp and an extra-chromosomal plasmid pYF8. The assembled genomes of the canine UPEC isolates will improve the understanding of the disease processes involved in pyometra and help explain similarities to urinary tract infections. They also facilitated genetic manipulation of the isolates for developing vaccine candidates in this thesis. The strain P4 was selected as the parent strain for vaccine development described in this thesis. The strain P4 was subjected to mutagenesis. The genes tonB, fur and oppD were insertional inactivated by lambda red recombination to generate single (P4-ΔtonB; P4-ΔoppD; P4-Δfur), double (P4-ΔtonB::ΔoppD) and triple (P4-ΔtonB::Δfur::ΔoppD) knockout mutant strains. In vitro growth assays of the mutants were carried out in iron limiting (2,2’ dipyridyl) and iron excess (ferric chloride) conditions and in the presence of the iron-dependent antibiotic Streptonigrin, to confirm their phenotypes. Dipyridyl sensitivity assays (200 µM) showed that the tonB mutants (P4-ΔtonB, P4-ΔtonB::ΔoppD, P4-ΔtonB::Δfur::ΔoppD) did not grow as efficiently as the field strain (P<0.05). Even in iron excess conditions, these strains grew less when compared to the field strain P4 (P<0.05), indicating the mutants’ inability to utilise environmental iron. Streptonigrin sensitivity assays (5 µg/mL) showed that the tonB mutants were more resistant to the antibiotic compared to the parent strain (P<0.05). This further confirmed the mutants’ impaired iron uptake ability. Together, these in vitro growth studies demonstrated the central role of tonB in iron starvation. Following on from this, the expression of the iron regulated outer membrane proteins (OMPs) was analysed in the vaccine candidates in comparison to the parent strain P4 under iron limiting and iron excess conditions. The outer membrane fraction was isolated by sonication and high-speed centrifugation using N-lauroylsarcosine and separated by Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). To identify and evaluate the antigenicity of the OMPs, immunoblotting was carried out using monospecific polyclonal rabbit anti-IroN antibodies. The results of this study showed that OMPs were expressed in iron limiting conditions in all the vaccine strains and in the parent strain. However, when the Fur regulon was inactivated, OMPs were expressed even in iron excess conditions. This is suggestive of successful deregulation of the Fur and that fur mutant strains are capable of expressing the OMPs irrespective of the iron levels in the media. The results of Western blotting showed that anti-IroN sera bound to a 79 kDa protein, indicating that the surface-exposed iron-regulated OMPs are strong antigenic proteins. In the final study, the safety and efficacy of the vaccine candidates were evaluated in an in vivo study. To do this, a murine model was developed in C57BL/6 mice to evaluate the pathogenicity of the canine UPEC strain P4 in the urinary tract and the reproductive tract by transurethral and intravaginal inoculation, respectively. P4 was mildly to moderately pathogenic in the urogenital tract of mice and produced mild to moderate inflammatory changes in the urinary bladder and uterus of C57BL/6 mice. This model was therefore chosen to evaluate the safety and efficacy of the vaccine candidates P4-ΔtonB, P4-ΔtonB::ΔoppD and P4-ΔtonB::Δfur::ΔoppD in the reproductive tract of female mice. Intravaginal inoculation of the vaccine candidates was found to be safe with no adverse effects recorded in the vaccinated mice. While up to 60% of mice vaccinated with the single P4-ΔtonB or the double knockout vaccine candidate P4-ΔtonB::ΔoppD were protected from challenge with the parent strain P4, the triple knockout strain did not confer any protection. The humoral immune response was poor in the mice inoculated intravaginally with any of the vaccine candidates. The total serum IgG levels did not vary between vaccinated and unvaccinated mice. Subcutaneous inoculation of the single knockout strain P4-ΔtonB produced severe systemic illness in the vaccinated mice. The double P4-ΔtonB::ΔoppD and triple knockout P4-ΔtonB::Δfur::ΔoppD strains caused severe inflammatory reaction at the injection site resulting in the premature abortion of this part of the experiment. The initial humoral immune response to P4-ΔtonB::Δfur::ΔoppD inoculated subcutaneously was promising. In summary, the studies described in this thesis characterise the whole genome of canine UPEC isolates and assess their virulence in a murine model. The live attenuated vaccine candidates developed by inactivating TonB, Fur and OppD expressed multiple antigenic OMPs and showed reduced growth potential in vitro. The preliminary outcomes of the in vivo studies are promising but more work is necessary to further evaluate their potential as vaccines.
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ItemInvestigation of equine hoof biomechanics using experiment, finite element and machine learning methods( 2022)This thesis investigates the biomechanics of the horse hoof, emphasising on its shape, deformation during movement, and influencing variables. Previous in vivo studies evaluated hoof shape parameters, such as proximal hoof circumference, to examine hoof conformation. In the majority of cases, a traditional measuring method, such as a measuring tape, was used, and, in a few instances, advanced measuring technology was utilised. To replace conventional measurement tools with modern technologies, it is necessary to assess their accuracy, reliability, and practicality. Therefore, the current study conducted a technical comparison between the measuring tape, 3D scanning and photogrammetry for measuring proximal hoof circumference. Considering the 3D scanner to be the most accurate method, the measuring tape was more accurate than photogrammetry. In addition, the inter- and intra-rater reliabilities of tape measurements were reported to be excellent. In the next step, a finite element analysis of the horse hoof deformation was performed. An artificial neural network was trained to obtain the whole deformation response of the hoof for varied tissue hydration levels, and trotting and standing locomotion modes. Results showed that increasing environmental hydration significantly increased strains on the hoof wall. The study also demonstrated how adopting advanced material models, such as hyperelastic compared to linear elastic, improved the accuracy of finite element analysis. Dynamic finite element analysis of the hoof was then conducted over a full trot stride and in vivo measurements were obtained to examine the impact of a toe-in conformation on the hoof's biomechanical response. The study hypothesised that different deformation patterns and hoof kinematics in toe-in hooves are caused by a different path of the centre of pressure under the hoof. Consequently, the model was allocated two distinct centres of pressure paths associated with normal and toe-in conformations. The finite element model generated similar strain patterns to those seen in vivo on the hoof walls and successfully demonstrated the distinct kinematics of toe-in and normal hooves. Lastly, the study examined the effect of toe-in conformation on laminitis. A total of 100 trotting loading cycles were applied to the model. The laminar junction injury was simulated by decreasing the tissue's elastic modulus in the presence of excessive maximum principal stresses. In both the normal and toe-in models, the injury began in the quarters. In the normal hoof, however, laminar junction tissue degeneration was distributed symmetrically at the quarters, whereas the toe-in model had a lateral concentration of degeneration. The models replicated clinical observations, indicating the third phalanx dorsal rotation, a symmetric distal displacement of the third phalanx dorsal in the normal model, and an asymmetric distal displacement of the third phalanx dorsal in the toe-in model.
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ItemInvestigating bone health in dairy cows during pregnancy and lactation( 2022)Selection of dairy cattle for high milk yields creates a significant metabolic demand for all nutrients including calcium, which is met by changes in bone homeostasis. The effects of selective breeding to increase milk yield on bone loss and skeletal health in dairy cows have not been fully studied and recent reports of fractures in dairy cattle suggest that there may be significant welfare implications related to poor bone health in these animals. The current study undertook to investigate the bone health of cows during pregnancy and lactation. It was hypothesised that biochemical markers of bone turnover, the structure of cortical bone and systemic levels of cytokines are related to stages of production in dairy cattle. Investigation of biochemical markers of bone turnover showed that the plasma CTX-I levels were highest during early lactation and its levels declined with decrease in milk yield while plasma osteocalcin levels were highest during mid lactation and constant at other stages of lactation. To further investigate the changes in bone turnover at tissue level, rib biopsies were collected for micro-CT and dynamic histomorphometry analysis. The micro-CT and dynamic histomorphometry data demonstrated loss in bone mass during early lactation while increase in bone mass and decrease in porosity with decreased milk yield during late lactation. However, the newly formed bone was found to be less mineralised during periods of rapid bone formation. Investigation of the systemic levels of osteoclastogenic and anti-osteoclastogenic cytokines demonstrated high levels of IL-6 at late pregnancy and IFN-gamma and IL-4 at late lactation. This study provides evidence that dairy cows adapt to high demands of calcium during lactation and pregnancy by changes in bone turnover. The high levels of bone resorption during early lactation corresponded with high circulating levels of the osteoclastogenic cytokine IL-6 and increased porosity of the cortical bone of cattle. Decreased milk yield in late lactation was accompanied by increased the bone formation and higher circulating levels of the anti-osteoclastic cytokines IFN-gamma and IL-4 and reduced porosity of the cortical bone. Together these results suggest that systemic cytokines may in part regulate bone resorption and formation associated with stages of production and that in late lactation bone formation increases to recover the calcium reserves lost during peak lactation. Further work is required to determine if bone loss is fully recovered by the increased bone formation observed during late lactation or if over multiple production cycles, the cortical bone mass decreases significantly or cortical bone becomes increasingly less mineralised.
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ItemBoosting amphibian resilience to the pandemic fungal disease chytridiomycosis using vaccines and artificial environmental refugia( 2022)Amphibian biodiversity is under threat from an infectious disease called chytridiomycosis (caused by Batrachochytrium dendrobatidis-Bd). Due to a lack of solutions for mitigating the disease in the wild, many species are maintained in captivity to prevent extinction. This, however, is not sustainable and the ecological function of the species is not restored in the wild. My PhD research approaches this challenge by providing disease mitigation approaches that can be applied in nature. In laboratory studies, I found that low virulence strains of Bd can be used as a transmissible inoculum that can spread between frogs and provide protection against more virulent strains. Transmissible, resistance-boosting strains of Bd (similar to a transmissible vaccine) would be uniquely suited for controlling chytridiomycosis in nature, because only a fraction of populations would need to be directly inoculated. I also found that I could similarly boost amphibian resistance to Bd by infecting frogs with Bd and then treating them with heat or antifungals (exposure-clearance). In a surprising experiment, I found that treating frogs with the antifungal itraconazole alone can increase amphibian resistance to chytridiomycosis. This approach and the exposure-clearance approach would be useful for captive breeding for release programs because frogs could be resistance-boosted prior to release. In a separate set of experiments, I exploited differences in host and pathogen physiology to develop thermal refugia that can protect frogs from Bd. The Bd pathogen is a psychrophile, preferring cool, moist environments. Under warm temperatures (> 28 C) the pathogen will stop growing and may die. Using an optimized artificial refuge, I found that thermophilic frogs that were able to access high temperatures in these refugia had drastically lower infections. These structures could serve as self-propagating resistance engines, allowing infected frogs to cure their infections and become resistant to future infections through acquired immunity.