Veterinary and Agricultural Sciences Collected Works - Theses

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    In-water antimicrobial dosing of pigs
    Little, Stephen Bennett ( 2021)
    Antimicrobials are an essential tool for safeguarding the health, welfare and productivity of pigs. On many farms, groups of growing pigs are mass-medicated with antimicrobials for short periods through their building’s piped water distribution system (WDS) at pre-planned intervals for metaphylaxis, and, when necessary, to treat clinical disease caused by bacterial pathogens. In-water antimicrobial dosing offers several advantages over continuous antimicrobial administration in feed: the ability to respond rapidly if disease is detected, to target administration of antimicrobials to specific groups of pigs, and to adjust readily the dose administered and the dosing commencement time and duration. The process of in-water antimicrobial dosing a group of pigs in a weaner or grower/finisher building requires sequential steps that determine the antimicrobial concentration over time at the infection site in each pig. The process is complex and dynamic and influences the clinical efficacy of treatment and suppression of emergence of antimicrobial resistance. The between-animal variability in systemic exposure to an antimicrobial is substantial, resulting in under-dosing or over-dosing of many pigs. There are three sources of between-animal variability in exposure: 1) variability in the dose supplied, i.e., the concentration of antimicrobial in water available to pigs at drinking appliances (drinkers) in each pen over time; 2) variability in the dose consumed, i.e., the pattern of consumption of medicated water by pigs in each pen over time; and 3) variability in pharmacokinetics, i.e., oral bioavailability, volume of distribution and clearance between pigs and within pigs over time. The pig industry has been practising in-water antimicrobial dosing since the 1980s. However, the factors operating in each farm building that influence the range of systemic exposures of pigs in a group to an antimicrobial when administered in-water have remained poorly understood. Furthermore, evidence-based strategies have not been developed to increase the probability that most pigs in a group medicated in-water attain the systemic exposure to the antimicrobial required for high clinical efficacy and suppression of antimicrobial resistance. The studies described in this thesis aimed to address these deficiencies using a multi-disciplinary approach, generating data in commercial pig production environments. The studies focused on the first two sources of between-animal variability in systemic exposure to antimicrobials described above. The dosing practices of the farm manager, the characteristics of the building’s WDS and the daily water demand and water use pattern within each day of each group of pigs affect the flow of water and antimicrobial through the WDS during and after an in-water dosing event, and therefore affect the antimicrobial dose supplied at drinkers, consumed by pigs over time and passing to the site of infection. The studies described here found that all of these factors varied widely across pig farms. The WDSs within many conventional buildings and some eco-shelters were ‘over-sized’, comprising large -diameter main pipelines of substantial length with high holding volumes. This resulted in low velocity water flows through sections of a WDS’s main pipeline that increased variability in the dose supplied and dose consumed. Many buildings had insufficient drinkers per pen and WDS sanitisation was not practised on many farms. The water use patterns of cohorts of pigs within each day varied; some were unimodal, while others were bimodal. It was therefore concluded that the water use pattern of a cohort of pigs could not be used reliably to predict the patterns of other cohorts of pigs, even if reared in the same building. When a particular group of pigs in a building was dosed, the antimicrobial concentration in water delivered to pigs at drinkers in each pen over time was profoundly influenced by the characteristics of the WDS (looped or branched configuration, length and diameter of its main pipe sections, and head pressure), the daily water demand and use pattern of the pigs within each day, and the dosing event’s commencement time and duration. Differences in the antimicrobial concentration at drinkers over time during a dosing event due to the natural, demand-driven hydraulic behaviour of a looped WDS could be eliminated by using a circulator pump to establish and maintain a high, steady, uni-directional water flow; however, there were several factors that should be considered before opting for this approach. Alignment of a dosing regimen with the water use pattern of a group of pigs within each day had a substantial impact on the antimicrobial dose consumed by pigs drawing water from drinkers at different points along the length of the WDS over the first few hours that the antimicrobial was available to them, and therefore on pigs’ systemic exposure to the antimicrobial over time. Water wastage can be a confounding factor when in-water dosing. A study described here showed that it is feasible to quantify the water consumption and wastage behaviour of groups of pigs in farm buildings using a water metering system. This system could support more accurate dosing calculations and regimen design, while helping to reduce the quantities of antimicrobials used and disseminated into the environment. The studies described in this thesis contributed to development of a process to evaluate alternative in-water antimicrobial dosing regimens, based on different WDS characteristics, water use patterns and dosing practices, for a specific group of pigs in a building, and determine the optimal regimen. Several strategies were developed that should increase the proportion of a group of pigs that attain the systemic exposure to an antimicrobial required for high clinical efficacy and suppression of antimicrobial resistance. The studies culminated in development of a concept for an ‘intelligent’, adaptive dosing system that would be a significant advance on in-water dosing as currently practised on pig farms. Much of the new knowledge acquired in the studies described here is applicable to other additives administered to pigs through their building’s WDS for which the degree of efficacy is dependent on the dose administered. These additives include vaccines, parasiticides, organic acids, electrolytes, minerals, vitamins, amino acids, sweeteners, direct-fed microbials, essential oils and potential new therapeutic products, such as bacteriophages.
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    Sensitivity of canine haematological cancers to BH3 mimetics
    Jegatheeson, Selvi ( 2022)
    Background: Haematological cancers such non-Hodgkin lymphoma (NHL) and acute and chronic leukaemias are common in both humans and dogs. Whilst these cancers can be treated with cytotoxic chemotherapy (and immunotherapy in people), development of treatment resistance is common. A frequently identified mechanism associated with resistance to chemotherapy-induced cell death is overexpression of the antiapoptotic B cell lymphoma 2 (BCL2) protein. Highly specific small molecule inhibitors of antiapoptotic BCL2 proteins, known as B cell lymphoma Homology 3 (BH3) mimetics, result in rapid induction of apoptosis in vitro and in vivo in human haematological cancer cells. This has led to the approval of the BCL2-specific inhibitor, venetoclax (VEN), for the treatment of chronic lymphocytic leukaemia (CLL) and acute myeloid leukaemia (AML) in Australia, North America and Europe. Expression of BCL2 has been reported in canine nodal lymphoma, however sensitivity of primary canine cells to BH3 mimetics has not been evaluated. Objectives: This study aimed to assess the in vitro sensitivity of non-neoplastic lymphocytes and primary haematological cancer cells from dogs to VEN or the dual BCL2/BCLxL inhibitor, navitoclax (NAV). The second aim was to evaluate the association between BCL2 protein expression and sensitivity to VEN. Methods: Nine dogs without cancer and 30 dogs with haematological cancers were recruited. Lymphocytes were isolated from peripheral blood, lymph node and/or bone marrow and incubated with VEN or NAV for 24 hours. Viable cells were enumerated using flow cytometry and the half maximal effective concentration (EC50) was calculated; BCL2 protein from whole cell lysates was assessed via immunoblotting. Results: Non-neoplastic lymph node-derived B and T canine lymphocytes were more sensitive to VEN than circulating lymphocytes (P = 0.02). Eighteen dogs with haematological cancers were included in the final analysis, including six cases of non-indolent multicentric B cell lymphoma, four cases of acute leukaemia, three cases of non-indolent multicentric T cell lymphoma, two cases each of indolent T-zone lymphoma and T-cell CLL, and one case of multiple myeloma. Neoplastic T lymphocytes (7/7) showed marked sensitivity to BH3 mimetics, with an EC50 <100nM, whilst 6/7 samples of non-indolent B cell cancers were resistant to VEN, with an EC50 >1000nM. All samples of acute leukaemia showed sensitivity to NAV, however sensitivity to VEN varied. Canine BCL2 protein was detected in all samples sensitive to VEN and was variably detected in resistant samples. All samples that lacked BCL2 were resistant to VEN. Conclusion and Clinical Importance: Neoplastic canine T lymphocytes are sensitive to VEN at concentrations achievable in vivo, thus VEN may be a novel therapeutic agent for treatment of canine T cell cancers. Detection of BCL2 protein is insufficient to predict in vitro sensitivity to VEN.
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    Studies of Exercise induced pulmonary haemorrhage in Thoroughbred and Standardbred Racehorses
    Sullivan, Stacey Louise ( 2022)
    Exercise induced pulmonary haemorrhage (EIPH) or ‘bleeding’ is an important disease of exercising horses. Though it is a disease commonly identified by equine veterinary practitioners, there is a lack of information regarding the impact of an episode of EIPH to career after examination or whether occurrence of EIPH is predictive of a horse’s lifetime athletic potential. There also exists a lack of consensus as whether the commonly used treatment, frusemide (a loop diuretic) is an effective medication to reduce or prevent EIPH. This research project addresses these two questions, with the aim of producing high quality information which can be used to underpin evidence based clinical recommendations for EIPH in horses.
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    Construction of feline alphaherpesvirus 1 derived immunocontraceptives for the population management of feral and domestic cats in Australia
    Cottingham, Ellen Peterson ( 2022)
    Virally vectored immunocontraception (VVIC) has been identified as a potential population control method for invasive vertebrate species. The technique relies on stimulation of the host immune system to suppress either the occurrence or continuation of a pregnancy. Properly designed, immunocontraceptives have the potential to be more humane and effective, while requiring less human input with regards to delivery, than current population suppression methods. Since their introduction, the impact of feral cats on Australian native wildlife has been devastating. Feral cats are responsible for killing approximately 75 million native animals every day. Controlling feral cat populations in Australia has proven to be exceptionally difficult, but it’s possible that VVIC could be used for this purpose. This thesis reviews the features of feline alphaherpesvirus 1 (FHV-1) that make it attractive for use as an immunocontraceptive vector. The thesis then describes the development of three immunocontraceptive candidate vaccines derived from FHV-1. Each candidate vaccine was engineered to contain genes essential for reproductive-related processes, specifically the feline zona pellucida subunit 3 (ZP3) and gonadotropin releasing hormone (GnRH) genes. It is predicted that viral expression of these genes will induce an immune-directed disruption of the natural activity of both genes, rendering the cat partially or fully sterile. The immunocontraceptive candidates were engineered to differ in their level of attenuation. This was achieved by codon usage bias deoptimization of the non-essential viral virulence gene thymidine kinase (TK). The TK-disrupted candidate vaccines were developed as an alternative contraceptive option to traditional surgical sterilisation, particularly for owned cats where horizontal transmission of the vaccine is not a sought-after feature. A candidate vaccine with wildtype TK was also constructed. This vaccine would be expected to retain the ability to spread horizontally and thus could be suitable for use as a self-disseminating vaccine in feral cat populations. Feral cats in Australia have long been identified as a target species that may benefit from VVIC control due to the absence of any native feline species in Australia that could be impacted by the vaccines acting in off-target species. This thesis further investigated the species specificity of FHV-1 and FHV-1 derived immunocontraceptives in vivo in a murine model and in vitro in a variety of non-feline cell lines. In the in vivo murine model, FHV-1 and the derived immunocontraceptives were unable to replicate, induce an adaptive immune response towards the reproductive antigens, or induce changes in reproductive tissues due to immune disruption of reproductive pathways. The in vitro studies in non-feline cell lines demonstrated that an FHV-1 derived immunocontraceptive was unable to infect or replicate in any of the non-feline cell lines. These results support previous observations of restriction of FHV-1 infection to the Felidae family. Finally, this thesis investigated the seroprevalence of FHV-1 in Victorian feral and domestic (owned) cats. The results indicate an overall low exposure to FHV-1 in feral cats, consistent with similar studies conducted 40 years prior. This may indicate a naIve population of feral cats where a self-disseminating VVIC could be capable of spreading efficiently due to the absence of a protective response. The VVIC candidates developed in this thesis provide preliminary insights into a novel contraceptive tool for feral and domestic owned cats in Australia. This is an important start to exploring FHV-1 based immunocontraceptives. Future in vivo studies in cats will be crucial for further development of these vaccine candidates.
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    Functional and computational approaches to understand post transcriptional regulation in parasitic protist Giardia duodenalis
    Balan, Balu ( 2022)
    Understanding how complex systems like "central dogma" (DNA encodes RNA encodes protein) developed is one of biology's fundamental concerns. Central dogma includes layers of regulatory networks — pre-transcriptional, transcriptional, post-transcriptional (PTR), translational, and post-translational (PTM) – that guide a cell's genotypic to phenotypic transition. Pre- and post-transcriptional regulators (chromatin-modifying enzymes and transcription factors), as well as readers, writers, and erasers involved within PTM, attained complexity in higher metazoans. Critical components of PTR are unexpectedly retained and appear to be less evolvable, implying that it was one of the earliest arms of "central dogma" to arise. PTR is a multiphasic control of RNA, mechanistically regulated by ribonucleoprotein complexes containing RNA binding proteins (RBPs). Even though RBPs are mostly conserved across the tree of life, there are substantial transition phases, notably during eukaryogenesis. As eukaryotic lineages developed, they built on bacterial and archaeal RNA Binding domains (RBDs) and created "new" RBD-containing RBPs. The discovery of these "eukaryotic RBPs" was a crucial breakthrough that underpins cell fate decisions that control eukaryotic stage transition, cellular development, and embryogenesis. Surprisingly, RBP biology and functionality among single-celled eukaryotes are mostly studied in yeasts which falls in the same phylum as metazoans (phylum Opisthokonta). And most RBP functionalities remained conserved from yeast to metazoans. This raises concerns regarding when eukaryotic RBP regulation developed, what functionality these early RBP systems have, and what the early eukaryotic RBPome resembled. Such basal eukaryotic systems are required to research RBP biology and regulation features in their most basic form. Given that the Last Eukaryotic Common Ancestor (LECA) is a theoretical notion based on the most recent tree of life, protists, a polyphyletic collection of unicellular eukaryotes reflects the eukaryotic tree's basal lineages. Giardia duodenalis is one such earliest diverging eukaryotic lineages and is believed to be one of the oldest lineages in which innovative eukaryotic RBPs, including Pumillio proteins, is known to be encoded. In this context, this thesis hypothesises that highly complex eukaryotic RBP systems emerged within basal eukaryotes and can be studied in protist G.duodenalis as a model. To this end, the objective of this thesis was to research RBP biology within G.duodenalis and gain a deeper insight into the emergence of innovative eukaryotic RBPs within basal eukaryotes. In this endeavour, this thesis encompasses a literature review, three original research chapters and a general discussion. Elaborate curation of scientific literature (Chapter 1) suggests a distinct lack of system-wide understanding of PTR, particularly RBP biology in protists, notably G.duodenalis. Therefore, the aims of this thesis were: A) To gain insight into RBP-mediated PTR, primarily translational repression in G.duodenalis through transcriptome-proteome correlation studies, by establishing a high-resolution quantitative proteome atlas for G.duodenalis during differentiation (encystation), B) Mapping G.duodenalis genome encoded RBPs by computational strategies, complemented by experimental in vitro RNA interactome capture (RIC) method followed by Liquid chromatography-mass spectrometry (LC-MS/MS) across encystation, C) Functional delineation of specific RBPs identified by RIC, through CRISPRi mediated RBP knockdown, followed by quantifying the RBP knockouts through quantitative proteomics. D) To adapt the established computational RBD mapping strategy and employ it to curate the RBPs within eight representative protists Trichomonas vaginalis, Giardia duodenalis, Entamoeba histolytica, Trypanosoma brucei, Toxoplasma gondii, Cryptosporidium parvum, Plasmodium falciparum and Plasmodium vivax. Then, to explore the behaviour of these RBPs over each parasite’s life cycle and for major ecological transitions. Initially (Chapter 2), by developing the highest-resolution proteome draft of G.duodenalis encystation to date, I performed the first transcriptome-proteome correlation analysis in Giardia (Chapter 2); then mapped the differential protein expression of known Giardia RBPs, pointing to the possibility of RBP-mediated PTR within Giardia. Furthermore, by mapping the RNA binding domain (RBDs) across the tree of life (Chapter 3) revealed Giardia RBPome is shaped by bacterial and archaeal RBDs and “eukaryotic innovative” RBDs and RBP families. Moreover, computational, and experimental RNA interactome capture (RIC) profiling identified both canonical RBPs and “enigm/moonlighting RBPs” in Giardia, with a high RBP orthologues sharing with humans, suggesting a highly conserved eukaryotic RBPome. Further, CRISPRi-based knockout of the earliest known Pumilio homologs (PUF, PUM) and two significant helicases, DDX3X and EIF4A in RIC data, hints to active regulatory role for these RBPs. Next, well-curated map of genome-encoded RBPs in seven protists (Chapter 4), revealed protists encoding an elaborate cell fate RBP suite, and further transcriptomic studies of these cell fate RBPs over each parasite life cycle and significant ecological transitions hinting significant developmental regulatory role for these RBPs within protists. In Chapter 5 (General discussion), I briefly contextualise the significant findings and broader implication of this work, and the pivotal role protists, notably G.duodenalis play in giving insights into early eukaryotic RBP networks. And the relevance of this information in understanding and tracing the complexity of eukaryotic cell fate decisions. In conclusion, this thesis addressed a significant knowledge gap in eukaryotic RBP biology, by presenting the most comprehensive RBP resource within protists, particularly in Giardia. Our study strongly indicates that Giardia RBP-mediated PTR is possibly complex and sophisticated as those of higher eukaryotes, thus hinting that complex RBP regulation emerged at the base of the eukaryotic trees. We propose protists, notably Giardia as an attractive model for understanding “cell fate decisions” in its most rudimentary form at the base of eukaryogenesis, and evolution of “cell fate” across higher scales of evolution.
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    Novel platforms for full genomic characterisation of avian pathogens directly from clinical samples
    Asif, Kinza ( 2021)
    Identification of the microbial strains involved in infectious disease is imperative for epidemiological investigation and to implement control strategies using vaccination. The conventional techniques used for strain identification including microneutralisation assays and polymerase chain reaction (PCR)-based assays such as PCR-restriction fragment length polymorphism (PCR-RFLP), and PCR-High resolution melt (PCR-HRM) curve analysis are not only cumbersome but also their results are difficult to interpret. Analysis of the whole genomes of the pathogens is considered the gold standard for the characterisation of strains. However, whole-genome sequencing (WGS) requires the in vitro isolation of the pathogens which can potentially introduce variations in the genome. In addition, some pathogens are not cultivable in vitro. Therefore, WGS directly from clinical tissues would be the most suitable option. In this study, four viral pathogens namely Fowl adenovirus (FAdV), avian hepatitis E virus (aHEV), fowlpox virus (FPV), and infectious laryngotracheitis virus (ITLV) affecting the hepatic, cutaneous, and respiratory tissues were targeted in four independent studies to assess the suitability of WGS directly from clinical tissues using Illumina and Nanopore sequencing platforms. To extract sequencing grade viral DNA/RNA directly from hepatic tissues, the liver homogenate was treated with 5% kaolin hydrated aluminium silicate to remove excess lipid tissue present in the liver before proceeding to the phenol-chloroform extraction. FAdV DNA extracted from treated tissue, resulted in the complete genome assembly of FAdV using both Illumina and Nanopore sequencing platforms. A similar extraction technique was used to extract aHEV RNA directly from liver tissues followed by long range RT-PCR and sequencing. . Analysis of the resultant WGS from clinical samples revealed that Australian aHEV isolates had emerged as a result of recombination between the US and European strains possibly following the importation of poultry into Australia and dissemination through vertical transmission. To evaluate the suitability of WGS directly from cutaneous tissues affected with FPV, homogenate of the affected comb tissue was subjected to DNA extraction and sequencing. The sequencing results were compared with the same FPV isolate grown in chorioallantoic membranes (CAMs). Complete genome sequence of FPV was obtained directly from affected comb tissue using a map to reference approach. FPV sequence from cutaneous tissue was highly similar, but not identical to that of the virus grown in CAMs with a nucleotide identity of 99.8%. Detailed polymorphism analysis revealed the presence of a highly comparable number of SNPs in the two sequences when compared to the reference genome, essentially classifying the two sequences as the same strain, but also highlighting the impact of in vitro passage on WGS of viral pathogens. Sequencing the whole genome of ILTV was attempted directly from tracheal scrapings of experimentally infected birds to circumvent in vitro culturing. Despite the high number of quality reads obtained from sequencing, assembling the genome was not possible due to poor overlapping sequences and the presence of multiple gaps. A concatenated sequence covering 91% of the ILTV genome was obtained after excluding the regions with low coverage. Further analysis performed on the concatenated genome classified the ILTV isolate as the same class used for infection of the birds (class 9) but revealed the presence of 50 single nucleotide polymorphisms (SNPs) between the two. The results of this study suggest that despite the failure to assemble ILTV genome directly from clinical tissues, the technique has a potential to replace the current PCR-HRM technique used for ILTV typing since it provides far more detailed information about the genome of the ILTV. Thus, the studies reported in this thesis have served as a proof of concept and have contributed to the evaluation of the suitability of WGS as a tool for accurate strain identification directly from clinical specimens of these pathogens. Also, this thesis provided insights into the origin of aHEV isolates currently circulating in Australia.
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    Phylogenetics and taxonomy of the strongyloid nematodes parasitic in macropodid and vombatid marsupials
    Sukee, Tanapan ( 2021)
    Nematodes of the superfamily Strongyloidea that inhabit the gastrointestinal tracts of Australasian macropodid and vombatid marsupials are one of the most speciose groups of mammal parasites. These nematodes are prevalent and occur in high abundance in almost all species of macropodid and vombatid marsupials. A review of the literature (Chapter 1) revealed that most studies of strongyloid nematodes of marsupials are species descriptions and taxonomic revisions, based on morphological features. Currently, there are over 300 morphospecies, most of which occur in the stomachs of macropods and are placed in the subfamily Cloacininae, and the remaining species, found mostly in the intestines of macropods and wombats, represent the subfamily Phascolostrongylinae. From the early 1990s onwards, the use of molecular approaches in taxonomic studies accelerated the discovery of marked population variation and cryptic species, allowed relationships of taxa to be resolved and provided improved insight into speciation processes. However, studies combining molecular and morphological approaches have been conducted mainly on the subfamily Cloacininae, whereas the subfamily Phascolostrongylinae has been neglected, resulting in very limited knowledge of the systematic status of this subfamily. Additionally, a monophyletic hypothesis has been proposed for the subfamilies Cloacininae and Phascolostrongylinae; however, the relationship between these subfamilies based on morphological features has not been established beyond speculation. To address these gaps, the research aims of this thesis were to assess the genetic variability within the Phascolostrongylinae, elucidate any species complexes and determine their phylogenetic relationships using sequence data sets derived from the internal transcribed spacers (ITS) of nuclear ribosomal DNA and/or the inferred mitochondrial proteome. This thesis also aimed to determine the relationship between the Phascolostrongylinae and Cloacininae to test the hypothesis of their monophyly. Initially, a molecular characterisation of Macropostrongyloides baylisi, a species complex within the Phascolostrongylinae uncovered four genetically distinct groups that were mostly host-specific (Chapter 2). Comparative morphological studies of these genotypes using light and scanning electron microscopy (SEM) found that each of them represented different species (Chapter 3). Using amino acid sequences inferred from mitochondrial genomes, the phylogenetic relationship of members within the genus Macropostrongyloides were resolved, and speciation within this genus was found to be influenced by host colonisation and phylogeography (Chapter 4). Subsequently, phylogenetic analyses of the entire subfamily Phascolostrongylinae using ITS sequence data suggested that the species Macropostrongyloides dissimilis and Paramacropostrongylus toraliformis were distinct from their respective congeners (Chapter 5). An assessment of their morphology, including SEM, revealed novel features, warranting their separation at the generic level (Chapter 6). Molecular data also indicated that, in phascolostrongyline species from macropods, the predilection sites within the host species were linked to their phylogenetic relationships. For instance, stomach-inhabiting species including Wallabicola (formerly Macropostrongyloides) dissimilis and Paramacropostrongylus were more closely related to each other than to the species occurring in the intestines of their hosts (Chapters 5 and 7). To address the last aim of the thesis, a species of Macroponema was studied as a representative of the Cloacininae. To clarify the status of species within this genus, the cryptic status of Macroponema cf comani was investigated. Two new species of Macroponema were identified by molecular and morphological means (Chapter 8). Subsequently, phylogenetic analysis of representatives of the subfamilies Cloacininae and Phascolostrongylinea inferred from mitochondrial amino acid sequence data supported their monophyly. However, the subfamily Phascolostrongylinae was found to be paraphyletic due to the separation of wombat-specific genera from those occurring in macropods. The sister relationship between the genera in vombatid hosts and those in macropods supports an alternative hypothesis that the strongyloid nematodes in marsupials may have evolved initially in the Vombatiformes (Chapter 9). In conclusion, this thesis has contributed to addressing some key fundamental knowledge gaps for members of the order Strongyloidea of macropodid and marsupials, particularly the systematics of the subfamily Phascolostrongylinae. Through integrating molecular markers and microscopical techniques, this thesis has uncovered genetic and morphological diversity within the subfamilies Phascolostrongylinae and Cloacininae. The outcome of this work indicates that future studies are required to gain deeper insights into the evolution of the cloacinine and phascolostrongyline nematodes and that these studies will necessitate comprehensive sampling of strongyloid nematodes from non-Australian hosts, not just from marsupials but also from extinct marsupial hosts if accessible via museum collections (Chapter 10).
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    Mechanism of immunity to Mycoplasma synoviae infection in chickens
    Omotainse, Oluwadamilola Samuel ( 2021)
    Mycoplasma synoviae is regarded as the second economically most important poultry Mycoplasma, causing a large economic loss to the poultry industry worldwide. Clinical signs in infected chickens often include sub-clinical to clinical respiratory signs, lameness, decreased egg production, and/or eggshell abnormalities. The consequences of M. synoviae infection include increased production of second-class eggs, reduced growth, and increased feed conversion ratio, and downgraded carcasses at slaughter. A combination of biosecurity measures and vaccination has proven to be practically effective in controlling M. synoviae infection in most countries. MS-H is a live attenuated temperature-sensitive (ts+) vaccine used in the prevention and control of M. synoviae infection by several countries including Australia. However, the mechanism of protective immunity after vaccination has not been fully investigated. There is also no study differentiating between immune responses induced by vaccine versus those by the field strain. This information would be very critical in defining protective and non-protective immune responses. The studies performed during this project aimed at addressing these knowledge gaps. To examine local and systemic cellular and humoral immune responses, specific-pathogen-free chickens were inoculated with MS-H, its parent strain 86079/7NS (7NS) and/or the virulent M. synoviae strain 94011/v-18d (v-18d). Local cellular responses were investigated through the examination of lymphocytes phenotypes infiltrating the tracheal mucosa and associated cytokines by immunofluorescence and RT-qPCR respectively. MS-H- and 7NS-inoculated chickens showed an early T-helper 2 (Th-2) response closely followed by a Th-1 type response. Vaccinated-challenged (+V+C) chickens had Th-1 cytotoxic mediated responses while unvaccinated-challenged (-V+C) chickens had Th-2, T regulatory and Th-17 type responses. Cellular infiltrates consisted of CD4+, CD8+, CD4+CD25+ T-cells, B-cells and macrophages. To examine whether local antibody responses to M. synoviae correlated with vaccination or challenge responses, upper respiratory mucosal immunoglobulins (Ig) A and IgY in the nasal washing were measured using an indirect MSPB ELISA. Anti-M. synoviae MSPB IgA and IgY antibody were detected in nasal washings of all groups of chicken exposed to vaccine and/or field strain. Anti-MSPB IgA and IgY antibodies were found to be strain-dependent and correlations between these antibodies and tracheal mucosal thickness suggested them as poor indicators of local immunity against M. synoviae infections. Examination of systemic cellular responses was evaluated through ex-vivo splenocytes stimulation assays and flow cytometry. Vaccinated-challenge (+V+C) chickens had a Th-1 skewed immune response while -V+C chickens had Th-2, Th-17 and T regulatory skewed immune responses. T-helper to cytotoxic T-cells ratio (CD4+CD8- : CD4-CD8+) of +V+C chickens was significantly (P = 0.009) higher than -V+C chickens suggesting increased immunocompetence in +V+C chickens. Host systemic antibody responses were examined through an indirect ELISA using purified recombinant M. synoviae surface proteins, MSPB and variants of MSPA (MSPA B1, MSPA B2, MSPA B3, MSPA B4, MSPA C1, MSPA C2, MSPA C4 and MSPA C5’). Anti-MSPA B1, MSPA C1, MSPA C2 and MSPA C4 antibodies appeared early after inoculation and/or challenge with MS-H, 7NS and v-18d. Antibody production against MSPA B3 and MSPA B4 appeared to be different between MS-H-inoculated and 7NS-inculated chickens. Anti-MSPA B1, MSPA C1, MSPA C2 and MSPA C4 antibodies appeared early v-18d challenge. Anti-MSPA C5’ antibody also appeared to be different between +V+C and -V+C chickens. This suggests that the host antibody responses against these MSPA variants are time- and strain-dependent. Thus, the studies reported in this thesis have contributed to the understanding of the role of local and systemic antibody and cell-mediated responses in immunity against M. synoviae and laid foundations for the development of novel diagnostic assays and vaccines that could potentially lead to better control of M. synoviae infection.
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    Heterogeneity of Horse Fibrinogen
    Russell, Elise Bernadette ( 2021)
    Fibrinogen has long been used diagnostically as a positive acute phase protein in horses. Fibrinogen heterogeneity has been observed in humans and can influence fibrinogen measurements when using the modified Clauss assay. To date, no studies have evaluated fibrinogen heterogeneity in other species. In this thesis we investigate the existence of fibrinogen heterogeneity in horses, and evaluate the precision of a method for fibrinogen fraction purification and relative quantification. Lastly, we evaluate the stability of fibrinogen fractions with freezing.
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    Production effects, diagnosis and control of small lungworms in sheep in southeastern Australia
    Hanks, Jenny Elizabeth ( 2021)
    The effects of small lungworm (Muellerius capillaris and Protostrongylus rufescens) on sheep production in southeastern Australia are not clear. However, there is evidence that small lungworm can be a significant problem in other parts of the world, which suggests that direct and indirect losses may be incurred from these infections in Australia. Recent monitoring in abattoirs and anecdotal evidence from producers and veterinary advisors suggest a very high prevalence of small lungworm infection of adult sheep and lambs, particularly from farms in the southeast area of South Australia (SA). Limitations of current diagnostic tests mean that these high estimates may be even greater. As a result, producers in this region are concerned about potential economic loss from such high prevalences of small lungworm. Furthermore, there are no recommendations on how to control small lungworm if management is indicated due to production loss. Consequently, this thesis sought to improve understanding of the production effects, diagnosis, and control of small lungworm in southeastern Australia through a series of studies over three years (2018-2020). Three field and abattoir studies together describe the seasonal pattern of small lungworm infections under different grazing management, assess the production effects, and evaluate a method of control of small lungworm. This is followed by a laboratory study seeking to improve the diagnosis of small lungworm. Firstly, chapter 2 outlines a field study describing the prevalence of small lungworm in sheep mobs on a heavily infected farm, from August 2018 to March 2019, including farm management factors which had the potential to influence the prevalence. Liveweight, lungworm and gastrointestinal nematode infection, as well as pasture type grazed and snail density, were measured at five farm visits. Rather than a distinct seasonal pattern of infection, this study found that small lungworm can occur throughout the year, with its prevalence most influenced by pasture production system (irrigated vs. dryland), grazing management and the population density of the intermediate host snails. Importantly, this chapter suggests that small lungworm infection did not reduce lamb liveweights. In chapter 3 the severity and prevalence of small lungworm infections, and their association with sheep carcass characteristics, were assessed from 1332 lambs and adult sheep bred on three farms in southeast SA. Liveweight and measures of lungworm infection were measured on farm, then lung lesions and carcass characteristics were assessed at slaughter. The overall prevalence of small lungworm lesions at slaughter was 79%, with a prevalence of 87% in lambs, and 69% in adults. Small lungworm infected lambs and adults had a similar hot standard carcass weight and dressing percentage compared to non-infected animals, both overall and within their respective cohort. This study confirmed a very high prevalence of small lungworm infection in sheep bred on farms in this region of SA, but their hot standard carcass weights were not reduced by this infection. The limitations of the currently available diagnostic tests for small lungworm were also demonstrated, indicating a need for the development of more sensitive tests to assess lungworm infections, both in live sheep on the farm and at abattoirs. The effect of pasture molluscicide treatment on the prevalence and severity of small lungworm infections, and the productivity of lambs grazing improved pastures was evaluated in chapter 4. A randomised control field trial of 260 Merino-cross lambs was conducted on a commercially managed farm in SA with a history of high small lungworm prevalence. Separate groups of lambs rotationally grazed irrigated lucerne paddocks that were either treated with iron chelate molluscicide or remained untreated control paddocks. There was a higher population of adult Prietocella barbara snails (a known intermediate host of small lungworms) in the Control paddocks compared to the Treatment paddocks after molluscicide had been applied and prior to grazing commencing, although the overall mollusc density was similar between Control and Treatment. A similar proportion of Treatment and Control lambs had evidence of small lungworm infection lesions at slaughter (both 68%). Control lambs grew slightly faster than Treatment lambs, with an average daily gain of 202 g/head/day for Control and 190 for Treatment during the 112-day trial. Thus, iron chelate molluscicide treatment prior to lambs grazing the pasture had no demonstrable effect on the prevalence and severity of small lungworm infections, nor the productivity of lambs grazing these pastures. This study indicates that for a commercial sheep farm, additional molluscicide treatments after pastures are established, to reduce the population of intermediate hosts and decrease the prevalence of small lungworm infection, may not be warranted. Diagnosis is limited by the current tests available, namely the isolation of first stage larvae from faeces using the Baermann test, or the post-mortem detection of lung lesions at an abattoir. The final experimental chapter (5) presents the development of a novel multiplex real-time PCR (qPCR) for the detection of Muellerius capillaris and Protostrongylus rufescens, the two small lungworm species infecting sheep in Australia. Diagnostic sensitivity and specificity of the qPCR assay was confirmed using field samples. DNA extracted from whole faeces and isolated larvae (qPCR-Baermann) was compared with that of the Baermann test and post-mortem diagnosis of lung lesions. There were 50% more samples which were detected positive by qPCR-Baermann (21%, 23/112) compared to the Baermann test (13%, 15/112). In 70% (16/23) of cases, species-level identification of the small lungworms by qPCR-Baermann was in agreement with the diagnosis of lesions at post-mortem, in contrast to none by the Baermann test. Whilst further validation is required, this novel qPCR has the potential to improve the detection and surveillance of small lungworm infections. Together, these complementary studies indicate that small lungworms are unlikely to be causing significant production and therefore economic loss to sheep producers in southeastern Australia. They reassure producers that, despite the high prevalences of small lungworm infections detected, there is no requirement for additional anthelmintic treatments, nor the control of intermediate host molluscs for indirect management of small lungworm in this region. Rather, this series of studies reaffirms that well established determinants of production should be managed first, such as optimising pasture quality through correct grazing management and effective control of gastrointestinal nematode infections.