Veterinary and Agricultural Sciences Collected Works - Theses
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Tissue tropism and latency of infectious laryngotracheitis virus: A study in the natural host
Herpesviruses are evolutionarily successful pathogens that infect a large number of animal species. This success is partly attributed to their capacity to establish latency in the host. The avian alphaherpesvirus infectious laryngotracheitis virus (ILTV) causes an acute upper respiratory tract infection in chickens that has considerable impacts on world poultry economy and welfare. The disease caused by ILTV, infectious laryngotracheitis (ILT), is currently controlled by vaccination principally with live attenuated vaccines. Limitations associated with live attenuated vaccines, including the ability to establish latency, may provide avenues for the emergence of novel, more virulent, recombinant strains of ILTV, further complicating the epidemiology of ILTV. In this project, a sensitive nested polymerase chain reaction (NPCR) protocol was developed and the sensitivity of this assay was compared with that of other commonly used PCRs in ILTV research. A trigeminal ganglia (TG) co-culture system was established and optimised and a tracheal co-culture system was reproduced to study in vitro reactivation of latent ILTV. A genotyping system based on allelic variations in multiple genomic regions of ILTV to discriminate ILTV strains prevalent in Victoria, Australia, is also described. These methodologies revealed that a large proportion of the ILTV-vaccinated birds in a commercial layer flock, close to the end of their productive laying period, were shedding multiple vaccine strains of ILTV in the upper respiratory tract, presumably due to reactivation of latent infection. Further, co-culture systems showed in vitro reactivation of latent ILTV in TG and trachea of these birds. The capacity of four vaccine strains of ILTV (SA2, A20, Serva and a glycoprotein G deleted mutant vaccine candidate) to establish latency in specific pathogen free chicken (SPF) following eye-drop vaccination was investigated in vivo. This study revealed ILTV vaccines differed in their capacities to establish latency in TG, and also showed that nearly half of the population had detectable ILTV in their upper respiratory tract (URT), 21 days post vaccination, possibly due to reactivation of infection. A second in vivo experiment was performed to study latency characteristics and late systemic lymphocyte responses in SPF chickens following intratracheal inoculation with a vaccine ILTV (SA2) or a virulent field ILTV (class 9; CL9) strain. Results from this study indicated that latency characteristics did not significantly differ between these strains at 21 days post inoculation (dpi) or at 35 dpi, and suggested that the trachea may be a more significant site of latency and reactivation than the TG. Moreover, regardless of the ILTV strain inoculated, SPF birds showed lymphocytosis during the latent stage of infection. Additionally, tissue tropism of two newly emerged recombinant strains of ILTV (CL9 and class 10; CL10) was investigated using commercial broiler and SPF chickens. The possibility of using feathers as a diagnostic sample was explored. This study revealed that both CL9 and CL10 ILTV strains caused severe disease in both types of birds, distributed to visceral organs and persisted for up to 14 dpi in URT. The NPCR developed in this project detected ILTV DNA in feathers of infected broiler and SPF birds at 14 dpi. Taken together, these studies have shown that tissue tropism and latency is a complex area of research and to a large extent these properties are strain dependent. The studies reported in this thesis have enriched the literature on tissue tropism and latency of ILTV. The results will be valuable for future latency studies and for selection of vaccines to control ILTV.
Chicken immune responses to infectious laryngotracheitis virus (ILTV) glycoproteins
Infectious laryngotracheitis virus (ILTV), an alphaherpesvirus, causes acute respiratory disease, primarily infecting the upper respiratory tract and conjunctiva. The disease results in significant economic losses to the poultry industry worldwide. Currently, the immune status of the vaccinated flocks or individuals is assessed by the presence of systemic antibodies using commercially available whole virus ELISAs. These ELISAs are good indicators of exposure to either field or vaccine virus, however, the assays are poor in predictors of the level of protective immunity. In this study, individual ILTV glycoproteins (gC, gD, gE, gG, gI and gJ) were assessed for their potential to predict the levels of protective antibody and/or cell-mediated immunity as well as for their capacity to induce neutralising antibodies. To examine whether antibody responses to individual ILTV glycoproteins are correlated with disease and protection, individual glycoproteins expressed in mammalian cells were used in indirect ELISAs to measure post-vaccination and/or post-challenge chicken serum antibodies. Serum optical density (OD) values detected by the whole virus, gC, gI and gJ were significantly higher in birds vaccinated with the Serva vaccine strain compared to the SA2 vaccine strain. However, the mean ODs detected by gD, gE and gG were not significantly different between the vaccine strains. Examination of post-vaccination pre-challenge antibodies to individual glycoproteins did not find a strong correlation between systemic antibodies to individual glycoproteins and protection. The possibility that the protective epitopes of glycoproteins may not be readily detectable in ELISA was investigated by evaluation of the antibodies to each glycoprotein in an in-vitro virus neutralisation assay. Monospecific polyclonal antibodies to individual ILTV glycoproteins gC, gD, gE, gI and gJ were generated in rats and examined for their capacity to neutralise the virus. Neutralising antibodies were detected to gC, gD and gJ individually or in combination. Polyclonal antibodies to gE and gI failed to neutralise the virus when tested individually or combined. In-vitro splenocyte stimulation assays using individual glycoprotein stimulating antigen were conducted to examine the transcription profiles of selected cytokines from nonvaccinated-nonchallenged (NVXNCh), vaccinated-challenged (VXCh) and nonvaccinated-challenged (NVXCh) groups of chickens. The transcription profiles of cytokines referencing Th1, Th2 and Th17 responses were then examined against tracheal pathology results to identify correlation between responses against viral glycoprotein(s) and protective immune response. Expression of IFN-gamma was significantly upregulated in VXCh and NVXCh groups of birds in response to stimulation with gD, with expression levels moderately correlated with protection. Stimulation with gE and gI resulted in significantly higher levels of IL-6 in VXCh birds and response to gE was moderately correlated with protection. Expression of IL-17a was significantly elevated in response to stimulation with gD in NVXCh birds and results were moderately linked to disease severity. The results of this study suggest that IFN-gamma and IL-6 cytokine responses to gD and gE, respectively, may be correlated with protective cell-mediated immunity, whereas IL-17a cytokine responses to gD may be linked to disease. Finally, whole-genome analysis of an unusually virulent “vaccine-like” isolate revealed a new class of ILTV, identified here as class 7b, emerged as a result of recombination probably between another recombinant strain and a vaccine strain. Further analysis of the genome detected recombination hotspots within the unique long (UL) region comprising of genes encoding gB, gC and gM and assembly proteins UL28 and ICP18.5. Boot-scanning analysis of concatenated glycoprotein sequence alignments detected recombination breakpoints within glycoproteins C and H, but not within glycoproteins located in the unique short (US) region. Further studies would be needed to explore the possible role of these glycoproteins in virulence. Thus, the studies reported in this thesis have contributed to understanding (1) the relationship between antibodies to ILTV glycoproteins and protective immunity, (2) virus neutralisation potential of specific antibodies to ILTV glycoproteins, (3) the potential role of ILTV glycoproteins to induce cell-mediated protective immunity, (4) and laid foundation to explore inter-strain genetic diversity within ILTV glycoproteins.
Molecular analysis of pathogenicity of Mycoplasma synoviae
Mycoplasma synoviae is a major pathogen of poultry globally, causing chronic respiratory disease and arthritis. Vaccination is an effective means for the control of the disease. The MS-H vaccine is a live attenuated temperature sensitive strain developed through chemical mutagenesis of an Australian field strain, 86079/7NS. Rarely, the temperature-sensitive MS-H strain may revert to a non-temperature-sensitive phenotype. Due to reversion to non-temperature-sensitive phenotype, vaccine isolates may be difficult to distinguish from field strains. Therefore, there is an urgent need for a reliable and rapid assay that can differentiate the MS-H vaccine from field isolates to evaluate the efficacy of the vaccination programs. However, developing such assays depends on a better understanding of molecular basis behind attenuation of MS-H. Comparison of the whole genome of MS-H with that of its parent strain 86079/7NS in previous studies has shown 32 mutations in the MS-H genome, one of which was a frameshift mutation early in an oligopeptide permease transporter gene oppF. In this study polyclonal antibodies raised against peptides upstream and downstream of the mutation in OppF revealed that only N-terminus of the OppF was expressed in MS-H while the full version was expressed in 86079/7NS. The potential of the recombinant full-length OppF, N- (OppF-N) and C termini (OppF-C) of OppF, upstream and downstream of the mutation site, was evaluated for the discrimination of antibody responses to MS-H versus field strains in indirect ELISA. Comparison of optical densities obtained from the ELISAs based on OppF, OppF-N and OppF-C revealed that the indirect ELISA based on OppF-C had the potential to differentiate between MS-H and field strain antibody responses. The impact of the oppF mutation on the MS-H phenotype was investigated in vitro using a mutant of MS-H complemented with a wild-type copy of oppF gene in the MS-H genome. The results revealed that truncation of oppF impacts on growth characteristics of the MS-Hand provide insight into the molecular pathogenesis of M. synoviae and perhaps of broader mycoplasma species. In addition, to characterise the function of OppF protein in MS-H, the metabolite profile of MS-H was compared with that of its field reisolate which only differed in its oppF gene. The liquid chromatography–mass spectrometry analysis revealed a significant decrease in the abundances of amino acids in MS-H compared to that in its field reisolate. This result was consistent with the role of OppF as a peptide transporter. Also, differences found in the level of other metabolites in MS-H reflected some compensation of the OppF function by pathways involving other metabolites. Moreover, in this study genomes of five M. synoviae isolates from MS-H vaccinated chicken flocks were determined and analysed to confirm their relationship with MS-H and also examine the stability of oppF mutation and other genetic markers of the MS-H vaccine. A total of 25 single nucleotide substitution/insertion/deletion including that of oppF existed among the isolates examined. Four of these mutations were those that had been reported between MS-H and its parent strain 86079/7NS, while the other 21 were found only in the MS-H reisolates tested in this study. Therefore, 28 out of 32 mutations previously detected between MS-H and 86079/7NS remained consistent in 5 MS-H reisolates. These results confirmed that M. synoviae isolates in this study were true MS-H reisolates and that majority of the mutations found in the MS-H vaccine are stable after passage in vivo under field condition. The studies described in this thesis have contributed to expansion of our understanding of putative virulence determinants in M. synoviae and laid foundations for the development of a DIVA (Differentiation of Infected and Vaccinated Animals) test to examine variants of the MS-H vaccine. Also, this thesis provided some information on the dynamics of MS-H population after passage in vivo.
Development of an ‘equinised’ antibody to block the equine insulin-like growth factor-1 receptor (IGF-1R):A potential immunotherapy for endocrinopathic equine laminitis
Laminitis is a common and debilitating disease of horses’ feet that results in lameness, and in severe cases, may require euthanasia. Insulin toxicity appears to have a key role in the pathogenesis of the disease, and research has indicated that hyperinsulinemia may cause lamellar damage through excessive stimulation of insulin-like growth factor-1 receptors (IGF-1R). Inhibition of dysfunctional receptor activation has been the target of therapeutic (anti-cancer) monoclonal antibody (mAb) treatments for humans. Although common in human medicine, only a handful of therapeutic mAbs are under development for diseases in companion animals (dogs and cats), and none are available for horses. This approach demonstrated promise as a potential treatment of endocrinopathic laminitis. Therefore, the aim of this project was to convert a clinically-tested human IGF-1R-neutralizing mAb with high-affinity for the equine IGF-1R, into a horse-specific mAb while avoiding immuno-intolerance when administered in vivo. Development of the equine antibody was challenging due to the limited availability of immunoglobulin sequences (EquCab 2.0 database), in addition to the predominance of circulating light chain isotypes (Lambda), unlike the case in humans. Based on structural isotype characteristics, high binding affinity for the IGF-1R and minimal binding to insulin receptors, the parental antibody (IMC-A12) was chosen for chimerisation studies. Chimeric versions of this antibody were generated by combining the VH (Variable Heavy) and VL (Variable Light) domains of the human antibody to equine constant Fc regions. The mAbs were further engineered to abolish effector functions (activation of complement or the immune system) while retaining receptor binding functionality. Chimeric versions of IMC-A12 expressed at similar levels to the human parental mAb and bound to the equine receptor at similar affinities to human IMC-A12. The full ‘equinisation’ of chimeric mAbs was carried out using a novel speciation process known as “PETisation” and was adopted to generate thirteen candidate equine mAbs. Amongst three of the strongest equine mAb candidates (mAb8, mAb10, and mAb11), mAb11 displayed the highest expression yield purity (98 percent) and strong binding characteristics (KD of 0.2-0.3 nM and an EC50 of 127 pM, by BIAcore and ELISA, respectively). Cell proliferation assays demonstrated that mAb11 significantly inhibited the concentration-dependent proliferative effect of insulin in cultured lamellar cells (p<0.01). Further immunoassay assessment of lamellar growth factor signalling under hyperinsulinemic conditions demonstrated a significant reduction (p less than 0.05) in the phosphorylation of Ribosomal protein s6 (RPS6), Ras/extracellular signal-regulated kinase (ERK (1/2)) and Protein kinase B (AKT) after 24 h, in the presence of mAb11. As a novel approach, mAb therapies are an emerging option in the veterinary market. As far as I am aware, the fully-equine antibody mAb11, with its high-affinity for the target receptor (IGF-1R), is the first of its kind in the world and could open avenues for research on other equine indications and treatments. While promising results have been observed in vitro, further characterisation of mAb11 is required to establish its viability as a treatment for equine laminitis. A successful immunotherapy for equine laminitis would reduce the economic and welfare costs of this destructive disease while advancing our understanding of antibody therapeutics in the horse.
Characterising the role of metabolic enzymes in the pathogenesis of Coxiella burnetii
Coxiella burnetii is a Gram-negative intracellular pathogen that replicates in a highly acidic and oxidative lysosome-derived vacuole known as the Coxiella-containing vacuole (CCV). The relatively recent identification of axenic medium that supports C. burnetii replication and the development of improved genetic tools has advanced knowledge of this pathogen and facilitated the identification of virulence factors. A previously conducted transposon mutant library screen using a human cell line identified a number of genes that appeared to be required for intracellular replication, including genes encoding metabolic enzymes such as nadB and sdrA. To investigate the role of nadB and sdrA in C. burnetii replication, and to link the metabolism of C. burnetii with pathogenesis, a number of approaches were used, such as advanced genetic and biochemical techniques, including targeted and untargeted metabolomics. Firstly, each transposon mutant was clonally isolated to avoid wild type contamination. Genetic complementation of both nadB and sdrA mutants and subsequent intracellular growth assays using both epithelial HeLa cells and macrophage-like THP-1 cells conclusively demonstrated that both nadB and sdrA are required for efficient C. burnetii replication and CCV formation. Analysis of the protein sequence revealed that NadB has a functionally conserved arginine residue at a position of 275 and this arginine was mutated to leucine using site-directed mutagenesis. Purification of both GST-NadB and R275L GST-NadB showed that GST-NadB had L-aspartate oxidase activity, an enzyme catalysing the first reaction of de novo NAD synthesis, whereas R275L NadB had lost enzymatic activity. Complementation of nadB mutant with a plasmid encoding this inactive R275L NadB was unable to rescue the intracellular replication defect, confirming the requirement of NAD de novo synthesis for intracellular replication of C. burnetii. Steady state metabolite analysis also showed key changes in the level of abundance of metabolites in the NAD biosynthetic pathway in the nadB mutant as compared to parent C. burnetii and the complemented nadB mutant, demonstrating the role of NadB in de novo NAD synthesis. This suggests that this pathway is an ideal target for development of therapeutics, and inhibition of this pathway using a compound non-toxic to mammalian cells reduced C. burnetii replication significantly. The role of SdrA in C. burnetii metabolism and pathogenesis was also further investigated in this thesis. SdrA is a putative short chain dehydrogenase containing a conserved glycine residue at position 12 that serves as an NADP binding site facilitating NADP(H) regeneration, a key process in resistance to oxidative stress in C. burnetii. This Gly residue was replaced by Ala using site directed mutagenesis and purified recombinant 6xHis-G12A was enzymatically inactive when compared to wild type 6xHis-SdrA that converted NADP+ to NADP(H) in vitro. Complementing the sdrA mutant with a plasmid encoding enzymatically inactive 3xFLAG-tagged G12A_SdrA failed to restore intracellular growth, confirming the link between NADP(H) regeneration and C. burnetii replication. The sdrA mutant showed greater susceptibility to oxidative stress in vitro induced by treatment with hydrogen peroxide, whereas both parent C. burnetii and the complemented sdrA mutant were less sensitive. Supplementation of a commonly available anti-oxidant, L-ascorbate, into the host cell growth medium partially restored the intracellular growth defect of the sdrA mutant. Metabolite profiling using GC-MS revealed significant changes in the level of abundance of metabolites of central carbon metabolism in the sdrA mutant as compared to parent C. burnetii NM II and the complemented mutant. Finally, stable isotope labelling studies using [13C] label glucose showed a change in flux through central carbon metabolic pathways in the sdrA mutant demonstrating the presence of oxidative stress. Overall, this thesis has demonstrated the crucial role of NadB and SdrA in C. burnetii metabolism and pathogenesis.
Human-animal interaction in the modern zoo: Live animal encounter programs and associated effects on animal welfare.
Abstract Live animal encounter programs are an increasingly popular occurrence in the modern zoo. The welfare implications of animals participating in these programs has not been studied extensively to date. The aim of the current thesis was, therefore, to explore animal welfare effects associated with encounter programs in selected zoo-housed species, using a combined approach of survey data and empirical investigations. The survey assessment, which involved over 500 accredited zoos and aquariums in Australia and overseas, revealed that most surveyed institutions (85 %) engaged in an encounter program with one or more species, most of which were mammals. Behaviours indicative of a typical fight-or-flight response were the most commonly reported welfare concerns in relation to encounters. Behaviour indicative of positive welfare, as well as voluntary participation and interaction with visitors, were commonly mentioned in regard to positive welfare during encounters. Positive welfare experiences outweighed the number of reported concerns in birds and mammals, but the opposite was true for reptiles. The empirical studies involved zoo-housed servals, giraffes and shingleback lizards, and sought to identify a potential cause-effect relationship between behavioural and physiological welfare indices and short-term variations in encounter frequency. A similar methodology involving a repeated treatment design where the frequency of encounters was manipulated to reflect the regular frequency of encounters, a temporary withdrawal, and a temporary intensification of regime, was adopted across the three studies. Behavioural changes indicative of a positive welfare effect when participating in encounters were observed in the servals and giraffes. Servals exhibited a significant reduction in stereotypic pacing on weeks when participating in interactive presentations, or presentations and behind-the-scenes encounters combined. The giraffes engaged in amicable social interactions significantly more often when participating in visitor feeding encounters, at either regular or intensified frequency. By contrast, a potentially aversive effect of encounters was observed in the shinglebacks, who significantly increased their use of concealed locations within the enclosure when handled at either the regular or intensified frequency. Approach behaviour during encounters differed significantly between individual giraffes, and significant differences in coiling behaviour while handled was observed in the lizards. The findings of the empirical studies were in agreement with the survey data, which identified taxonomic as well as individual variation as important influences on the welfare of animals participating in encounters. The nature of the encounter the animals participate in was identified as another key factor, in which encounters that maximise choice and control, and where animals are rewarded for participation, were likely to contribute to a more positive welfare experience.
Discovery of natural product scaffolds with anthelmintic activity against Haemonchus contortus
The present thesis focused on discovering new candidate compounds in natural products against economically important parasitic worms of livestock to circumvent current problems associated with resistance to commercially-available drugs (anthelmintics) (cf. Chapter 1). One of the most pathogenic parasites of small ruminants, Haemonchus contortus (barber’s pole worm), was used as the screening tool. This whole-organism bioassay was used to identify active compounds/extracts that reduced motility and/or development or altered the morphology (phenotype) of H. contortus larvae in vitro. Selected compounds were purified from active extracts using a bioassay-guided fractionation approach. The screening of two compound libraries containing a total of 1,000 natural products, natural product-inspired or synthetic compounds identified 34 compounds with notable activity against H. contortus (Chapters 2 and 3). Most of the ‘hits’ (n=32) identified from the first library were analogues of arylpyrrole (a natural product scaffold), while deguelin and rotenone (two plant rotenoids) were identified from the second library. Encouraged by these findings, a library of 7,500 extracts from different plant species was screened (Chapter 4). This screen identified three active extracts from the leaves and roots of Cryptocarya novoguineensis and the roots of Piper methysticum. Bioassay-guided fractionation of active extracts yielded four known alpha-pyrones, namely goniothalamin from C. novoguineensis, and dihydrokavain, desmethoxyyangonin and yangonin (= kavalactones) from P. methysticum. Three kavalactones induced a lethal ‘evisceration’ phenotype in treated larvae of H. contortus, and had limited toxicity on mammalian epithelial (MCF10A) cells. This is the first report on the activity of such natural compounds on a parasitic nematode of animals. The work was extended to explore compounds from Australian marine species. A library of 2,000 extracts from marine invertebrates was screened (Chapter 5) and identified three active extracts from marine sponges - one from Monanchora unguiculata and two extracts from Haliclona sp. The compounds with moderate activity against H. contortus were fomiamycalin from Monanchora unguiculata and halaminol A from Haliclona sp., although the activity of these compounds was not entirely selective. Subsequently, a structure-activity relationship (SAR) investigation of one of the identified natural product scaffolds, alpha-pyrone was conducted (Chapter 6). This work identified three analogues (designated W-408, W-415 and W-417) with anthelmintic activity similar to, or greater than the synthetic parent kavalactones (desmethoxyyangonin or yangonin), with the most potent analogue W-408 achieving a marked increase in potency (7-fold) against and selectivity (> 21) for H. contortus. Taken together, this drug discovery effort identified or purified 40 natural products representing distinct chemical classes (Chapters 2-5), and the SAR investigation of the alpha-pyrone scaffold identified three key analogues with enhanced potency and/or selectivity (Chapter 6). Collectively, the findings of this thesis indicate that some of the identified natural product scaffolds have the potential to be developed as ‘lead’ candidates. Developing such anthelmintic candidates via future chemical optimisation, efficacy and safety assessments, broad spectrum activity assessments, and target identification represents an exciting prospect (cf. Chapter 7) and, if successful, could pave the way to subsequent pre-clinical and clinical evaluations.
Compressive fatigue life and micromorphology of equine metacarpal subchondral bone
Bone fatigue injuries are an important cause of lameness in racehorses and as such are an animal welfare issue and a cause of losses to the industry. The metacarpal condyles are commonly affected by stress fractures and palmar osteochondral disease, a fatigue injury of subchondral bone. Bone fatigue injuries should be preventable through training adjustments. Knowledge about the behaviour of equine bone, and particularly subchondral bone, under cyclic loading is important to inform training recommendations. Computational models have been developed to investigate the effects of training changes on stress fracture development but require equine subchondral bone material properties as an input to meaningfully model metacarpal condylar fatigue injuries. A method for compressive fatigue testing of subchondral bone was developed and the fatigue life of equine subchondral bone determined. The fatigue life curve followed a power law, similar to cortical and cancellous bone. Subchondral bone stiffness increased during fatigue testing over hundreds to thousands of cycles, which was initially attributed to a method artefact. Initial specimen stiffness was positively associated with actual density but not with horse and training related factors. Micro-CT was used to investigate the micromorphology of the palmar metacarpal subchondral bone. Micromorphology parameters were analysed with principal component analysis and mixed-effects linear regression models. The largest differences in micromorphology were observed in untrained horses between the age of 16 and 20 months. In racehorses in training, age and duration of a training period had no influence on micromorphology. Horses with palmar osteochondral disease had more pores in cross-section than those without. Tissue mineral density increased, and bone volume fraction decreased with increasing distance from the articular surface. Several alterations to the fatigue testing method were explored to accommodate the curved articular surface and eliminate the stiffness increase during compression-compression cyclic loading. The stiffness increase during the first 50 cycles was demonstrated to be reversible and no microdamage or trabecular compaction was detected in specimens loaded up to 200 cycles. Stiffness increase during compression-compression fatigue testing at a physiological frequency is thus likely the result of normal viscous material properties of subchondral bone. Finally, compressive fatigue life was positively associated with bone volume fraction in the deeper layers of subchondral bone. Initial stiffness was positively associated with tissue mineral density in the deeper layers and bone volume fraction in the superficial layer. Both maximum stiffness and cycles to maximum stiffness were positively associated with fatigue life. Cycles to 10% reduction of maximal stiffness correlated strongly with cycles to gross fracture. The results showed that subchondral bone’s viscous material properties led to a prolonged stiffness increase during cyclic compressive loading at physiological frequencies. Further research is required to investigate the physiological implications of this finding. Subchondral bone sclerosis measured as bone volume fraction is positively associated with compressive fatigue life and thus has a protective effect on subchondral bone. Further research is required to reconcile this finding with the common colocation of fatigue damage in sclerotic subchondral bone of racehorses.
A study into the genotypes of Escherichia coli from pyometra affected dogs
Pyometra is one of the most common reproductive diseases in female intact dogs. It affects nearly 25% of all intact female dogs before they reach 10 years of age. This disease not only affects the animal’s health but also its breeding value. The predominant pathogen that has been isolated from dogs with pyometra is Escherichia coli (E. coli). The research described in this thesis focused on characterising the particular strains found in dogs affected by pyometra and comparing them with those found in the rectum of control dogs. The objectives were to identify differences regarding the genotype, the presence of genes transcribing for uropathogenic virulence factors (UVF) and the bacteria’s abilities to form biofilms. The initial aim of investigating differences between bacteria isolated from young and old pyometra patients was hindered by the limited availability of E. coli strains from young dogs. To achieve these aims uterine, vaginal and rectal samples were collected from 32 pyometra patients and the patients’ age was recorded. Rectal samples were collected from 45 clinically healthy dogs to serve as controls. Samples were analysed by polymerase chain reactions for genotyping by Clermont’s scheme and evaluation of UVF gene presence. A subset of samples was further investigated to analyse their biofilm forming potentials using crystal violet assays. Finally, the ability of biofilm producing bacteria to withstand antimicrobial treatment was evaluated using assays to determine the minimum inhibitory concentration and time-kill curve studies. These investigations demonstrated that there is a significantly different UVF gene profile in E. coli isolated from pyometra patients when compared to control dogs. In contrast, there were only few differences between the uterine, vaginal and rectal samples taken from the same dogs. Similarly, little difference was detected between phylogenetic groups with the majority of all samples being classified as B2. Isolates with strong biofilm forming potential were only identified from those collected from pyometra patients but not control dogs and they were subsequently shown to be more resistant to antimicrobials. These studies have shown that there are major differences between the E. coli strains present during pyometra and those present as part of the normal microflora in the intestinal tract. It is also the first time that the biofilm forming potential was evaluated for bacteria involved in pyometra. It becomes apparent that care must be taken in the medical treatment of pyometra patients to consider the impact of biofilms on their efficacy.
Investigation into the Morphology of the Canine Sacrum and its Relationships with Selected Structures of the Vertebral Column and the Hind Limb
In dogs, the sacrum consists of three fused vertebrae (Evans & De Lahunta, 2013) and recently the population of greyhounds in Victoria, Australia have shown a remarkably high incidence of sacrocaudal fusion in which the sacrum consists of four fused vertebra (Oheida, Philip, Yen, & Davies, 2016). Scientific data and the available literature regarding how the occurrence of sacrocaudal fusion might influence the morphology of other parts of the locomotory system in dogs is not clear enough to explain why sacrocaudal fusion appears to be so prevalent in the Victorian greyhound population. This study set out to explore the hypothesis that there are measurable differences in the morphology of the sacrum (S. Weight, S. Length, and S. Width) between greyhounds with standard (3 fused vertebrae) and those with different types of fused sacra (4 fused vertebrae, Type B, C, and D). Also, it was hypothesised that there is an association between the sex and body mass of the greyhounds and morphology of the sacrum (S. Weight, S. length, and S. Width). In addition, this study included an exploration into the influence of sacrocaudal fusion on the morphology of related anatomical structures within the spine such as the L.7 vertebra, and selected bones in hind limbs such as the calcaneus, talus, and patella. The sacra were collected from 171 greyhounds and classified using two systems. For the first system, sacra were classified based on the number of fused vertebrae and the type of fusion into four types: Type A (standard sacra), Type B (complete fusion between the transverse processes and between the bodies of the S3 and Ca1 vertebrae), Type C (fusion only between the transverse processes of the S3 and Ca1 vertebrae), and Type D (fusion only between the bodies of the S3 and Ca1 vertebrae). For the second classification system, sacra were classified based on the presence, reduction, or absence of the median sacral crest between the spinous processes of the S1 and S2 vertebrae into the following three types: Type F (a complete fusion or crest), Type R (reduction in the height of the crest between successive spinous processes), and Type N (absence of the crest). The length, width, and weight of sacra, calcanei, tali and patellae were recorded and compared between the greyhounds with standard and fused sacra. Also, the length of the L.7 vertebra and the angle of the lumbosacral junction and the angle of the spinous process of the 1st sacral vertebra were compared between the greyhounds with standard and fused sacra. There were variable numbers of the population in various chapters because some bones were too difficult to measure or because of breakage of bones during processing. The exact sample numbers are indicated in each table illustrating the results. The results of the studies showed that there was an increase in the convexity of the curvature of the pelvic surface of the sacrum and an increase in the roughness of the articular surface of the sacral wing in association with the occurrence of sacrocaudal fusion. Sacrocaudal fusion was found in 71 out of 171 greyhounds (41%). Overall, the mean weight, length, and width of the fused sacra were found to be significantly greater (P < 0.01) than standard sacra. Among the fused sacra, 13.5% (23/71) were Type B, 21.1% (36/71) Type C, and 6.4% (11/71) Type D. Type B sacra (complete fusion) were the heaviest (P <0.001), broadest (P <0.001), and longest (P <0.001) sacra compared to Type A, C or D sacra. Regarding standard and different types of fused sacra, there was no association between the sex, body mass and the occurrence of sacrocaudal fusion. In addition, different scenarios were suggested for the evolution of sacrocaudal fusion in the greyhound. The first scenario was termed “Continuous Dependent Sacrocaudal Fusion”, in which it was proposed that fusion occurred first between the S3 and Ca1 vertebral bodies, followed by the occurrence of fusion (Type C), with a final result of complete fusion (Type B). The second scenario was termed “Continuous Independent Sacrocaudal Fusion”, in which the sacrum might change from Type A to either Type C or D, and then further evolve into Type B (complete fusion). The third proposed scenario was termed “Non-continuous Independent Sacrocaudal Fusion”, in which a sacrum may evolve directly from Type A (standard) to be Type B, C or D without going through the possible sequences assumed in the first or second scenarios. The reduction (R) or absence (N) of the median sacral crest between the spinous processes of the S1 and S2 vertebrae, which has been observed in association with the occurrence of sacrocaudal fusion, allowed us to suggest this new classification of sacra in greyhounds. This classification divided sacra into three different types (F, R, and N) and the percentage of each was found to be significantly different between standard and fused sacra (P <0.001) with the fused sacra more commonly showing the N type, and not associated with sex or body mass. In addition, the angle of the spinous process of the 1st sacral vertebrae of sacra with median crest type N was statistically significantly less (more upright) than those in sacra with median crest type F (P <0.042). Investigating the morphology of the L.7 vertebra as an anatomical structure articulating directly with the sacrum and lumbosacral junction, showed that the L.7 length was significantly (P ≤ .008) longer and the angle of the lumbosacral junction in greyhounds with fused sacra was significantly increased (P <0.028) in dogs with sacrocaudal fusion compared with those with standard sacra and this variation was not associated with sex (P <0.765) or body mass (P <0.802). The results showed that the right and left calcanei, tali, and patellae in greyhounds with standard and fused sacra were anatomically similar. Among all greyhounds, asymmetry between some of measurements was found, including in the width of the calcaneus (P <0.009) and the talus (P <0.025) and the length of the calcaneus (P <0.001). Measurements were higher in those bones from left hind limbs. Studying specifically the greyhounds with standard sacra also showed asymmetry in the length of the calcaneus (P <0.008) and again those calcanei from left hind limbs (L.C.L) were longer than those from right hind limbs (R.C.L). For greyhounds with fused sacra, asymmetry was found in the width of the talus (P <0.024) and again those of left hind limbs were wider than those from right hind limbs. Regarding the influence of sacrocaudal fusion on bones, there were no significant differences in the mean of measurements in bones between greyhounds with standard and those with fused sacra except for the mass of the right (M.R.C, P <0.003) and left (M.L.C, P <0.006) calcaneus. Also, the measurements of these limb bones from greyhounds with fused sacra were heavier than those with standard sacra. This thesis provides quantitative data about the occurrence of sacrocaudal fusion and is the first study to investigate the morphological anatomy of the two types of sacrum: standard or fused, as well as selected bones in the vertebral column and hind limbs of greyhounds, such as the L.7 vertebra, calcaneus, talus, and patella. In summary, sacrocaudal fusion in greyhounds was associated with a change in the morphology of the sacrum, which included an increased angle of the spinous process in the 1st sacral vertebra, a reduction or absence of the median sacral crest between the spinous processes of the S1 and S2 vertebrae, and changes in related structures, such as an increase in the length of the L.7 vertebra and angle of the lumbosacral junction. Furthermore, it seems that sacrocaudal fusion has a type of influence on certain hind limbs bones such as the mass of the right and left calcanei. These variations in the anatomical features of the sacrum between dogs with or without sacrocaudal fusion may influence the effect of forces applied to the sacral region and also the incidence of injuries. In conclusion understanding the morphological variations of different types of sacra, in addition to the data provided by this thesis, is establishing a solid base for further research, which might help to explain why sacrocaudal fusion is so common in the Victorian greyhound racing population. Overall, this work provides an anatomical foundation for understanding the function of the sacrum in greyhounds and highlights how investigation into the sacral anatomy and related structures of the greyhound body may further the current understanding of dog locomotion.
Comparative analysis of the faecal virome of dogs with various inflammatory intestinal diseases
Little is known about the population of viruses present in the intestine of dogs, the virome, despite advances in molecular biology techniques. The viral flora in the intestine of dogs has been mostly studied in disease settings, targeted to known pathogenic viruses. There is currently a gap in our knowledge of the faecal canine virome present in healthy conditions. This study aimed to identify and characterise the virome present in faeces of healthy dogs compared to the virome present in dogs with acute diarrhoea and dogs with chronic enteropathy. Faecal samples were collected from 8 healthy dogs, 8 dogs with acute diarrhoea and 8 dogs with chronic enteropathy. A viral enrichment protocol, using a mixture of endonucleases and bacterial filtration was used to enrich viral DNA and RNA. The total DNA and RNA isolated from the stool samples were amplified using a sequence-independent single-primer amplification (SISPA) protocol and subsequently sequenced by next-generation sequencing using the Illumina MiSeq platform at the Australian Genome Research Facility. Two bioinformatic pipelines were used to analyse the viral population present in each sample. After selecting high-quality reads (HQRs) and removing dog and bacterial sequences, sequence information was compared against CAMERA viral reference database in one pipeline and against the NCBI non-redundant nucleotide database in the other. Faecal samples from all groups showed a large occurrence of bacteriophages, from different families mainly under the order Caudovirales. Eukaryotic viruses were also identified. Consistent results, obtained from both bioinformatic analysis pipelines, were found for only 8 viral eukaryotic families. Interestingly, sequences with high similarity to viruses within the Astroviridae and Caliciviridae families were identified only in samples from the dogs with acute diarrhoea. Sequences similar to those of Picornaviridae were identified in one dog with acute diarrhoea and in one dog with chronic enteropathy. Only one group of sequences similar to a known virus family with known pathogenic members, Reoviridae, was identified in all groups (healthy and diarrhoeic samples). In conclusion, the largest proportion of viruses identified belonged to bacteriophages. The eukaryotic virome detected in this study contained viral sequences from a range of different virus families, including some with known enteric pathogens. These results were complemented with the complete genome characterisation of a canine kobuvirus, a canine astrovirus and a canine rotavirus. Furthermore, a prevalence analysis of these viruses, in a wider population, was performed. This project gives the first description of the canine faecal virome of healthy dogs and dogs with chronic enteropathy, thus contributing new knowledge that complements what was already known about the faecal virome of dogs with acute diarrhoea. Results from this study indicate that metagenomic analyses are useful for the investigation of viral populations in the faeces of dogs with various inflammatory intestinal diseases. Studies to further elucidate the epidemiological and biological relevance of these findings are warranted.
Studies on gastrointestinal nematodes of alpacas in Australia
The Australian alpaca industry has grown remarkably since the re-introduced of alpaca into the country in the 1980s. This relatively new and small industry faces various challenges that affect the overall production (fibre, meat etc.) of animals. For example, production losses and deaths associated with gastrointestinal nematodes (GINs) have become one of the major concerns for alpaca producers in Australia and elsewhere as there is a scarcity of reliable information about the epidemiology of GINs in alpacas, worm control practices used by alpaca farmers and the efficacy status of widely used unregistered anthelmintics. The present thesis aimed to (i) assess worm control practices used by Australian alpaca farmers, (ii) determine the epidemiology GINs and (iii) the efficacy of commonly used anthelmintics against GINs of alpacas in Australia. A comprehensive literature review along with seven results chapters and a general discussion chapter are included in this thesis. In Chapter 2, an online questionnaire survey was conducted to assess worm control practices used by Australian alpaca farmers. Results showed that more than half of the respondents perceived that GINs was an important health problem. Macrocyclic lactones (MLs) were the most commonly used anthelmintics in alpacas. Almost half of the respondents (47%) used anthelmintics at the dose rate recommended for sheep. The majority of alpaca farmers were unaware of pasture spelling and one-third of respondents had shared paddocks with ruminants. This study led to subsequent studies to understand the epidemiology of GINs of Australian alpacas using sensitive diagnostic methods and assess the efficacy of commonly used anthelmintics against GINs of Australian alpacas. In chapters 3 and 4, sensitive diagnostic methods were developed to determine the faecal eggs counts and identify nematode genus/species in the faeces of alpacas. In chapter 3, a newly developed diagnostic method, the FECPAKG2, was compared to a routinely used method, the McMaster technique, for the counting of GIN eggs in the faeces of alpacas. Data revealed moderate to good agreement between the two methods. This was the first study to assess the agreement of measurements between two methods for estimating nematode eggs in the faeces of alpacas. In Chapter 4, a molecular diagnostic tool based on multiplexed-tandem PCR (MT-PCR) was developed. This tool is faster and is capable of identifying common nematode genera/species of alpacas, including Camelostrongylus mentulatus which was not possible using traditional larval culture method. The epidemiology of GINs of alpacas in Australia was assessed using cross-sectional (Chapter 5) and longitudinal studies (Chapter 6). A range of GINs are prevalent in Australian alpacas with variable worm burdens in different climatic zones and seasons. The results of both studies were comparable. Both studies showed an overall prevalence of GINs in SACs from 61 – 66%. Weaners had the highest prevalence in both studies ranging from 73 - 80%. However, the pattern of prevalence was not same across the climatic zones. In cross-sectional study, the highest prevalence of GINs (77%) were observed in the summer rainfall zone, whereas in longitudinal study the winter rainfall zone had the highest prevalence (68%). In addition, a mixed-effects zero-inflated negative binomial (ZINB) regression model has been used to design parasite control interventions. To assess worm burden and the spectrum of GINs infecting Australian alpacas, 100 gastrointestinal tracts of alpacas were examined (Chapter 7). Results revealed a mean burden of 1,300 worms, with the highest burden of 29,000 worms. Nineteen different species of GINs were identified from Australian alpacas, including three camelid specific nematodes: Graphinema auchenia, Camelostrongylus mentulatus and Trichuris tenuis. Haemonchus contortus was the most prevalent nematode followed by C. mentulatus and Trichostrongylus spp. In Chapter 8, the efficacy of commonly used anthelmintics against GINs of alpacas was assessed. The faecal egg count reduction tests were conducted on 20 alpaca farms across the country. The results showed that a commercially available combination of levamisole, closantel, albendazole and abamectin was the most effective dewormer followed by single anthelmintic such as, monepantel, moxidectin, closantel, fenbendazole and ivermectin. Haemonchus spp. were the most commonly resistant nematodes. This was the first comprehensive study to investigate the efficacy of commonly used anthelmintics against GINs of alpacas. This study provides significant information on GINs of Australian alpacas. Results of this study advance our knowledge on the epidemiology and control of GINs and efficacy status of most commonly used anthelmintics which could be used to develop control strategies against GINs of alpacas in Australia.