Veterinary Science Collected Works - Theses
Permanent URI for this collection
Search Results
1 - 10 of 14
-
ItemIn-water antimicrobial dosing of pigs( 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.
-
ItemNovel platforms for full genomic characterisation of avian pathogens directly from clinical samples( 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.
-
ItemPhylogenetics and taxonomy of the strongyloid nematodes parasitic in macropodid and vombatid marsupials( 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).
-
ItemMechanism of immunity to Mycoplasma synoviae infection in chickens( 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.
-
ItemProduction effects, diagnosis and control of small lungworms in sheep in southeastern Australia( 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.
-
ItemThe Likely Effect of Varroa destructor on Australian Honey Bee Populations( 2021)Varroa destructor, an invasive ectoparasitic mite of honey bees, is believed to be responsible for a large proportion of reported honey bee deaths globally. Australia remains the only Varroa-free country in the world with a beekeeping industry and horticulture sector reliant on Apis mellifera, the European honey bee. This thesis addresses issues essential to prevent the introduction of Varroa into Australia and better manage Varroa if an incursion (and then establishment) were to occur. The managed honey bee population in Australia is unusual, compared with other livestock industries, because a large sector of the industry (hobby beekeepers) are concentrated in locations where incursions of exotic diseases are likely to occur — urban areas adjacent to sea and international airports. For this reason, hobby beekeepers play an important role in early detection of honey bee diseases and limiting the spread of these diseases once an incursion has occurred. Hobby beekeeper networks need to be documented and appropriate communication and biosecurity resources developed for this sector of the industry to ensure roles and responsibilities are clearly defined (Chapter 3). A quantitative assessment of the Sugar Shake Team surveillance program for Varroa surveillance using scenario tree methods was made in Chapter 4 showing that if one of the 23,300 feral and managed apiariesin the Melbourne Metropolitan area were infested with Varroa there was only a 0.40% chance that it would be detected using the Sugar Shake Team surveillance program during one of the three or four surveillance events held each year. Reasons for low surveillance system sensitivity include poor sensitivity of the sugar shake method as a diagnostic test for the presence of Varroa in a colony and the relatively low frequency of testing of high-risk colonies in the greater Melbourne area. If the Sugar Shake Team program is to be used as a credible means for Varroa incursion detection the frequency of testing and the number of participants in the program needs to be increased. Research effort should be directed towards identifying more sensitive diagnostic tests to detect the presence of Varroa in honey bee colonies. If Varroa were to be introduced into Australia and there was a need to control the parasite using miticides, the cost per hive per year, if recommended controls were applied, would be in the order of AUD 51 (Q1 40; Q3 65) per hive per year (Chapter 5). In 2020 this represents a 17% reduction in net hive profitability. Assuming colony annual mortality rates attributable to Varroa ranging from 30% to 50% the percentage of apiary-year simulations where the cost of recommended Varroa controls was less than a suboptimal Varroa control strategy ranged from 32% to 43% (assuming a 30% Varroa attributable mortality rate) and 40% to 57% (assuming a 50% Varroa attributable mortality rate). While the use of suboptimal controls may be a pragmatic and rational economic choice for a beekeeper this represents a classic ‘tragedy of the commons’ situation whereby individual users, acting independently according to their own self interest, behave contrary to the common good of the industry. On the basis of these analyses, thought should be given to what changes can be made to the industry to make application of recommended Varroa controls cost effective for beekeepers. The first is to consider ways by which Varroa inspection and treatment times might be reduced. The second is to ensure that the cost of colony replacement is kept relatively high. A model was developed to simulate the introduction and spread of resistant Varroa Sensitive Hygienic (VSH) genetics into the managed honey bee population as a means for enhancing resistance of the feral population (Chapter 6). Introduction of VSH queens into the managed honeybee population had relatively little effect on the development of resistance in the feral population because the spread of Varroa resistance from the managed population to the feral population is slow compared with the rapid collapse of the feral population following Varroa the introduction of the mite. The only exception to this is that if the size of the resistant, managed population is large relative to the size of the feral population. This being the case there is scope for managed honey bee colonies in high risk areas to be seeded with Varroa-resistant queens to form a ‘barrier’ to limit the spread of the mite away from its point of entry into the country. If VSH genetics are to be used in this way as a means for either promoting Varroa resistance in the feral colony population or for iideveloping a Varroa resistance barrier around likely incursion sites (i.e., ports and international airports) it is essential that programs are established well in advance of a Varroa introduction.
-
ItemThe Epidemiology and Chemoprevention of Canine Vector-Borne Pathogens in the Asia-Pacific Region Using Novel Molecular Diagnostics( 2021)Canine vector-borne pathogens (VBPs) are a diverse range of arthropod-transmitted organisms that encompass viruses, bacteria, protozoa and multicellular metazoan parasites. Together they can generate a spectrum of disease in dogs from relatively benign infections to ones that can be fatal, whilst some are also zoonotic and therefore represent a concurrent risk to humans. Although canine VBPs have been extensively studied in Europe and North America there is substantially less relevant information on these pathogens in the tropics, particularly across the Asia-Pacific. The potential for novel and emerging VBPs to surface from such regions is great, whilst international travel and the movement of pets and livestock may facilitate a global spread of such novel diseases, as has been aptly demonstrated by the ongoing COVID-19 pandemic. Taking this into consideration, my PhD project set out to firstly optimise a novel next-generation sequencing (NGS) based diagnostic for the holistic characterisation of canine bacterial, apicomplexan and kinetoplastid VBPs. Following validation of my method it would be employed to unbiasedly characterise pathogen communities from stray dogs across Cambodia - a country suspected of being hyperendemic for such diseases based on limited prior data. Building upon these findings I then aimed to use our broad-scope NGS data to design a new high-throughput and economical qPCR diagnostic for detection of the common canine VBPs in the region. Such a diagnostic would then be utilisable within the context of a field efficacy trial, to compare the VBP-blocking efficacy of a contemporary ectoparasiticide collar product against a topical ectoparasiticide typically used in the region. Initial design, testing and optimisation of my NGS metabarcoding method identified it as being more sensitive for the detection of canine VBP than conventional PCR (cPCR) and better able to detect rarer/unexpected pathogens (Chapters 2 and 3). With respect to the detection of bacterial VBPs my method was further augmented in Chapter 4 by the development of a canine DNA blocking primer that reduced the amount of extraneous amplification and sequencing of host DNA and increased the amount of bacterial pathogen DNA detected, thereby increasing my method’s sensitivity. Next, extensive fieldwork and sampling of stray and semi-domesticated dogs from across five Cambodian study sites, followed by use of my novel NGS method identified a diverse range of hyperendemic canine VBPs (Chapter 5). Across all sites 62% of dogs were infected with at least one confirmed VBP with Anaplasma platys, the causative agent of infectious cyclical thrombocytopenia, the most common at 32%, followed by Ehrlichia canis the agent of the often lethal canine monocytic ehrlichiosis in 20% of dogs. Other key pathogens identified included Hepatozoon canis (18%), Babesia vogeli (14%), Mycoplasma haemocanis (13%), the zoonotic pathogen Bartonella clarridgeiae (3%), Candidatus Mycoplasma haematoparvum (0.2%) and Trypanosoma evansi (0.2%) Critical metadata was also collected and analysed which identified the presence of ectoparasites, abnormal mucous membrane condition, anaemia and elevated total protein as significant risk indicators for canine VBP exposure; information that will be key for veterinarians working in the region that may have limited resources to molecularly diagnose dogs. Subsequently, information gleaned through my NGS-based survey and prior studies permitted the identification of the most prevalent canine VBPs in the Asia-Pacific. Using such data we designed and validated a novel Taq-Man based multiplex qPCR to rapidly and economically test for such pathogens from canine blood samples, in Chapter 6. Finally, to explore measures for reducing canine VBP transmission within the Asia-Pacific a 12-month Cambodian field efficacy trial was commenced to compare the efficacy of a long-acting ectoparasiticide collar (Seresto, Bayer) against a monthly application of topical fipronil, a product commonly used throughout the region (Chapter 7). Despite this trial not reaching completion within the duration of my PhD, initial 6-month data suggests that both products are highly effective at blocking the transmission of canine VBPs. Moreover, information accrued at baseline suggests that topical ectoparasiticide products are better than systemic ones, such as the isoxazolines, at preventing VBP infection in dogs; a critical piece of information for veterinarians in the tropics. Overall, my thesis has garnered data and employed techniques in contexts never previously conducted. For example, I was the first to use NGS metabarcoding to detect pathogens from canine blood and my research has been the first to molecularly identify the canine VBPs A. platys, T. evansi and B. clarridgeiae from Cambodian dogs. Moreover, to the best of my knowledge there has not been such a country-wide epidemiological survey of bacterial and protozoan VBPs using metabarcoding for any host animal to date. I envision that the data and methods presented within this thesis will provide researchers with a critical baseline understanding of common and rare canine VBPs within the Asia-Pacific, whilst also generating valuable tools for future epidemiological surveys that can be employed in a wide range of contexts, not solely restricted to diagnosis in canine populations. Such tools will assist in unpicking the complex interplay between VBPs infecting companion animals, wildlife and humans, allowing researchers to better comprehend transmission cycles, emerging zoonotic threats and the importance of a One Health approach to managing our environment and future public health risks.
-
ItemInvestigation into the Contribution of Fascia to the Functional Anatomy of the Hindlimbs of Domestic Dogs( 2021)The hindlimb has a crucial role in the propulsion of the body through the interaction of interdependent anatomical structures. The objective of this study was to investigate how the fascia contributes to the functional anatomy of the canine hindlimb. The importance of employing a simple and reliable tool to determine the range of motion of joints, and which could be evaluated for normal and abnormal joints, motivated this work to use plastic goniometry (Jaegger, G., Marcellin-Little, D. J., & Levine, D., 2002). The joint angles of the stifle and tarsus of forty-four dogs (26 greyhounds and 18 other breeds) were measured before and after each step of a 12-step dissection processes. Significant changes to the angles of stifle and tarsus were observed following removing the skin, cutting of the biceps femoris insertion into the fascia lata and crural fascia, and after transverse incision of the deep fascia proximal and distal to the tarsal joint. This suggested that these structures play a significant role in stifle and tarsal function and may assist in stabilizing joints during periods of loading by restricting hyper-flexion and hyper-extension. Research then focused on the wider field of fascial anatomy to provide the foundations for understanding the distribution of loading and tension across the entire hindlimb. Twenty hind limbs were obtained from greyhound dogs, and their fascial anatomy was examined by dissection. The fascial layers and their connection to musculoskeletal, and collagen fibre alignment were described to deduce the fascial functional significance. Histological analyses of deep fascia and its attachment to muscles showed the fascia as a sheath of connective tissue made of irregular collagen and a few elastic fibres. This deep fascia was connected to the underlying muscle and superficial fascia by loose connective tissue. In addition, deep fascia had several myofascial expansions and various types of mechanoreceptors, as well as variable thickness according to the region of the limb. Hence, it can participate in joint coordination and load distribution during movement. The superficial fascia appeared to have the same structure as the deep fascia. It provided adhesion to the deep fascia and skin over bony prominences to create several fascial bursae, which suggested its role in reducing friction and assisting motion.
-
ItemSuperinfection exclusion of alphaviruses in Aedes mosquitoes( 2021)Chikungunya virus (CHIKV), Semliki Forest virus (SFV) and Ross River virus (RRV) are arthropod-borne alphaviruses transmitted by Aedes mosquitoes. Among alphaviruses, CHIKV has especially become a health concern over recent years. With no vaccine or treatment currently available, mosquito control remains the best strategy to limit arbovirus disease transmission. Despite the current use of vector control strategies, the continuing emergence of arbovirus disease outbreaks around the world, warrants the development of new strategies to reduce transmission. Superinfection exclusion, a phenomenon whereby a primary virus infection prevents the replication of a second closely related virus, has the potential to control arbovirus disease emergence and outbreaks. This phenomenon has been previously observed in plants, insects, and mammalian cells. In this study, I investigated superinfection exclusion of alphaviruses in Aedes mosquito cells and mosquitoes to identify and characterise the mechanism behind this phenomenon. In this study I established superinfection exclusion with a panel of alphaviruses in both Ae. aegypti and albopictus cells and Ae. aegypti mosquitoes. I demonstrated that superinfection exclusion of CHIKV could vary based on the primary infecting virus, the CHIKV strain, and the mosquito specie. Further experiments allowed us to establish that superinfection exclusion can be triggered at early stage of infection and that non-structural and structural proteins might not be individually involved in superinfection exclusion. Results allowed us to identify which viral replication stage is involved in superinfection exclusion. Furthermore, results found in this study also allowed us to assess the impact of other infection factors, like mosquito specificity or the immune system, on superinfection exclusion. This knowledge will not only improve our understanding of arbovirus-mosquito interactions but also set the basis for mosquito control strategies and reduction of burden of mosquito-transmitted diseases in humans and livestock worldwide.
-
ItemWelfare assessment of Australian bobby calves using blood parameters( 2021)Non-replacement dairy calves in Australia, known as bobby calves, are typically fasted and transported to abattoirs for slaughter at around 5-10 days of age. Bobby calves are particularly vulnerable to poor welfare outcomes due to their low body fat reserves, immature immune systems, and their lack of herding behaviour, which makes them difficult to handle. The main aim of this research was to assess the welfare of bobby calves after transport, fasting, handling and lairage, and to identify risk factors for poor calf welfare. An additional aim was to evaluate the welfare of cows and calves in two prolonged cow-calf contact systems. Avoiding early separation of cows and calves has potential health, welfare, and social licence benefits. To start with, a method for collecting blood samples at exsanguination was validated for blood parameters relevant to calf welfare, including packed cell volume (PCV), plasma glucose, and serum total protein, beta-hydroxybutyrate (BHB) and urea. Haematology and biochemistry reference intervals for dairy calves aged 5-12 days old were then established. A method for estimating calf age using total protein, immunoglobulin G and gamma-glutamyl transferase was tested, but estimates of calf age were not reliable enough for further use. Next, blood samples were collected at exsanguination from 4484 bobby calves at three Australian abattoirs. Blood samples were analysed for indicators of energy status (glucose and BHB), hydration (urea, PCV, and total protein), muscle damage/fatigue (creatine kinase (CK)) and colostral immunity (total protein). Transport distance was estimated based on scanned ear tag data. Transport distance had clinically small, though statistically significant, effects on most blood parameters measured. With increasing transport distance, glucose was more likely to be low, and PCV and total protein high. Unexpectedly, CK was more likely to be low with longer transport distances. It was theorised that this could be due to loading being a high risk time for muscle damage. A number of bobby calves had blood measurements outside of the reference intervals. Twelve percent of calves had PCV values consistent with anaemia, 11% of calves had urea values consistent with dehydration, and 35% of calves had total protein values indicative of failure of passive transfer of immunity. These results show that a large proportion of calves were at risk of illness due to failure of passive transfer, while a smaller proportion of calves may have been compromised due to pathology or fasting. Two systems for managing cows and calves together on dairy farms were also compared. It was found that cows separated from their calves for half days were more restless in the dairy, and, when reunited with their calves, avoided nursing more and performed more agonistic interactions, compared with cows separated from calves for milking only. Half day separation may, therefore, have some negative welfare impacts on cows, compared to separation for milking only. However, further research is needed to compare the half day separation to early cow-calf separation, as there may be relative benefits to half day contact. The overall conclusions of this body of work were that transport distance had relatively minor effects on the blood parameters measured, but very long distances appeared to affect calves more per km travelled. Additionally, around a third of bobby calves had failure of passive transfer, which puts them at risk of infectious disease. Minimising transport distances and improving colostral immunity are thus areas where the risk of poor bobby calf welfare can be reduced in the current system. A smaller proportion of calves showed evidence of dehydration and anaemia, which could indicate sub-optimal health and welfare. Further research is warranted to investigate the aetiology of these conditions. Finally, the research presented here on two cow-calf contact systems provides evidence of a negative welfare impact on cows and calves separated for half days, compared with those separated for milking only. Some of these impacts could potentially be managed with the provision of milk to calves during the separation period, however this would require further research to confirm. The results of this study have helped to identify areas within the current system that can be targeted to improve bobby calf welfare. However, a whole system change, including the abandonment of both early life slaughter and early cow-calf separation, will likely be needed in order to align with public values and ensure the dairy industry has a social licence to operate.