Veterinary Science - Theses
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ItemThe role of the intestinal microbiota in the pathogenesis of chronic enteropathies and their interplay with the immune system( 2018)The intestinal microbiota and its associated genome is collectively called the gastrointestinal (GI) microbiome, and is composed of crucial components that help not only to determine host biology but also to maintain host physiology. Dysregulation of the gastrointestinal microbiome has been associated with a range of diseases in people such as inflammatory bowel disease (IBD), diabetes and obesity. Previous studies have found dysbiosis and a reduced bacterial diversity in dogs with chronic enteropathies (CE). However, the precise nature of the intestinal microbiota dysfunction and whether the microbiota has a causative role or is secondarily affected remain to be elucidated. The first step in understanding the relationship between the gut microbiota and disease is the characterisation of the normal gut microbiota, how it is established and how stable it is during different periods of life. In this work, we assessed the dynamics and stability of faecal microbiota over time in healthy dogs of different age groups, and the development of the microbiota from birth in puppies, and the association with the maternal microbiome. Next, we characterised highly immunoglobulin A and G coated bacteria in faecal samples from dogs with chronic enteropathies using flow cytometry and 16S rRNA sequencing and assessed their correlation with disease stage and resolution of the clinical signs. Finally, we characterised the expression of thymic stromal lymphopoetin (TSLP), a cytokine that is produced in response to bacterial contact, in the intestine of healthy dogs and its correlation with disease activity in dogs with chronic enteropathies. The results reported here, help to understand the assembly of the gut microbiota, its interaction with the immune system and emphasise on the importance of longitudinal studies and personalised approach in order to understand the pathogenesis and the role of the microbiota in intestinal diseases in dogs.
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ItemRadiographic assessment of bone morphometry, alignment and loading stability of the equine carpal joint in racehorses( 2018)The carpus is the most complex joint of the equine forelimb and lameness secondary to carpal injuries accounts for up to 41% of forelimb lameness in racehorses. However, despite suggestions that carpal conformation is a contributory factor to the orthopedic health, performance and forelimb lameness, few attempts have been made to objectively measure radiographic variations of carpal bone morphometry and alignment in horses due to insufficient measurable carpal parameters. Furthermore, non-physiologic loading of carpal bones is believed to result in osteochondral fractures, ligament ruptures and axial instability of the equine forelimb; however, the mechanism of carpal damage due to non-physiologic loading of the carpus is largely unknown. The aims of this thesis were: 1. To investigate the radiographic anatomy of the equine carpus and develop reliable measurable parameters that can be used to consistently and objectively measure carpal conformation in horses 2. To provide baseline data for the developed parameters from radiographs of a group of racing thoroughbred horses. 3. To use some of the developed parameters to assess the stability of the equine carpal joint under incremental load. 4. To observe the roles of the equine carpal ligaments to the load redistribution within the carpus and stability of the carpal joint during axial compressional loads. A pilot study on 6 cadaveric equine forelimbs from 3 adult horses (5.67±2.08 years), was used to investigate the radiographic anatomy of the carpus in “Zero Lateromedial” (ZLM) and “Zero Dorsopalmar” (ZDP) views and 17 measurable parameters with validated anatomical landmarks were developed. Six parameters were developed from the ZLM view and 11 parameters from the ZDP views consisting of angles, ratios and linear measurements. Subsequent studies established: i.) baseline data of these parameters from carpal radiographs of a group of 20 two-year old thoroughbred racehorses in training; ii.) effects of limb postural changes, vertical rotations of radiographic plate, vertical and horizontal rotations of projection angles of primary X-ray beam on the quality of radiographs and carpal measurements; iii.) changes in positions of carpal bones during flexion, extension and incremental load and iv.) a 3-D finite element model of the bony components of the equine carpus. The proximo-distal gliding movement within the carpus enabled transverse movement of the proximal carpal row which in turn allowed the proximal and distal articular surfaces of the radial (Cr), intermediate (Ci) and ulna (Cu) carpal bones to slide into and out of congruity with the distal articular surfaces of the radius and the proximal articular surfaces of the distal carpal row during extension (loading) and flexion. Increased load on the carpus produced carpal hyperextension with measurable radiographic changes in the position and alignment of the carpal bones. A relaxed intercarpal ligament between Cr and Ci (Cr-Ci ICL) during loading, as indicated by decrease in the width of the groove diameter of Cr-Ci ICL (GD.Cr-Ci ICL), signifies minimal or no stretch (strain) on Cr-Ci ICL. This would facilitate absorption and redistribution of concussion forces within the carpal joint during loading, thereby providing a useful mechanism to minimize carpal damage. In conclusion, the carpal bone geomorphometric and loading data, along with information generated on the ligaments of the proximal carpal row in this study, will allow reliable quantitative assessment of carpal conformation and eliminate judgmental errors or variation between observers using subjective visual assessment for the carpus. This data will improve our understanding of carpal biomechanics and pathogenesis of injury. The measurement protocols will require further investigation on large groups of different breeds of horses for wider acceptance, adaptability and validation.
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ItemThe epidemiology of Salmonella transmission in chicken meat( 2018)A longitudinal study was conducted between January 2013 and September 2014 in a vertically integrated chicken meat enterprise under commercial farming conditions. Using methods routinely used for Salmonella surveillance in poultry production systems, environmental sampling was conducted in two generations (parent and broiler) at multiple locations within the production system. Data was collected on all product movements during the study period and social network analysis was used to describe product movements and identify locations for the potential introduction and dissemination of Salmonella. The results showed that the structure of a vertically integrated enterprise enhanced the transmission of Salmonella between poultry generations and locations, even at a very low prevalence, and that the hatchery was a critical point of amplification. The use of phenotyping (phage typing) and genotyping (MLVA) tools were not sufficient in the absence of good sampling (methodology or intensity) or epidemiological evidence to determine the source of introduction or dissemination within this complex environment. Whole genome sequencing allowed the genetic relatedness of the S. Typhimurium isolates to be elucidated and confirmed that transmission was occurring between generations within the enterprise with little to no change. Diversity and cluster analysis findings suggest that these salmonellae were not a significant source of infection to the human population during the study period.
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ItemEnhancing control of virulent recombinant strains of laryngotracheitis virus using vaccination( 2018)Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes acute upper respiratory tract disease in chickens. Attenuated live ILTV vaccines are often used to help control the disease, but these vaccines have well-documented limitations including natural recombination between different vaccine strains. Recently, two novel ILTV field strains (class 8 and 9 ILTV viruses) emerged in Australia due to natural recombination involving two distinct commercial ILTV vaccines. These recombinant field strains became dominant in important poultry producing areas and caused severe disease in commercial poultry flocks, showing that more options are needed to enable control of ILTV. The work described in this thesis investigated tools to better control disease due to ILTV. Firstly, different commercial ILTV vaccines and a developmental candidate vaccine, glycoprotein G-deficient ILTV (ΔgG ILTV, registered as Vaxsafe ILT, Bioproperties Pty Ltd) were investigated for their ability to protect commercial broiler chickens against challenge with the virulent recombinant class 9 ILTV after drinking water vaccine delivery. All vaccines induced partial protection by direct (drinking-water) and indirect (contact) exposure when birds were subsequently challenged with the virulent class 9 challenge strain. A vaccination and challenge study was then performed to determine the minimum effective dose of ∆gG ILTV that, when delivered by eye-drop to layer birds, would protect the birds from a robust challenge with class 9 ILTV. A dose of 103.8 plaque forming units per bird was the lowest dose capable of providing a high level of protection against challenge, as measured by clinical signs of disease, tracheal pathology and viral replication after challenge. Finally, attempts were made to develop suitable tools to measure the level of immunity induced by ILTV vaccination. To this end, an ELISA that measures the amount of chicken interferon gamma (IFN-γ) was developed and used to quantitate IFN-γ production from splenocytes stimulated with control mitogens, or with ILTV antigen. The assay could detect IFN-γ released from chicken splenocytes after stimulation by concanavalin A. However, when splenocytes were incubated with semi-purified ILTV antigens in vitro, there was no increase in the level of ILTV specific IFN-γ production by splenocytes from ILTV infected birds, compared to uninfected birds. A number of potential avenues for further development of this assay were identified. The work described in this thesis demonstrates that currently available vaccines and the new Vaxsafe ILT vaccine can be used to help control class 9 ILTV when delivered by drinking water. When delivered by eye-drop the Vaxsafe ILT vaccine candidate can induce a high level of protection against class 9 ILTV at a commercially feasible dose. Taken together, the results from this work lay the foundations on which a commercial vaccine may be developed, thereby offering the potential to provide producers with another important tool to help control ILTV. Future development of a tool to measure protective immunity after vaccination is needed and would be a valuable addition to disease control programmes
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ItemEpidemiological investigations into the ecology and transmission of environmental mycobacteria.( 2018)Part 1: Investigations of Mycobacterium ulcerans. Real-time PCR investigations have detected M. ulcerans DNA in a variety of Australian environmental samples, including the faeces of native possums with and without clinical evidence of infection. Characterisation of the disease in possums and attempts try to ascertain what role, if any, possums may have as reservoir hosts for this organism were undertaken. It was found that there is a significant disease burden in Pseudocheirus peregrinus (especially males) in some areas of Victoria and that these animals may become systemically, and potentially fatally affected. Some mildly affected Trichosurus vulpecula and Trichosurus cunninghami can spontaneously overcome the infection. Subclinical gut carriage of M. ulcerans DNA in possums is common and in some T. vulpecula and T. cunninghami this is transient. Culture of M. ulcerans from the gut contents of clinically affected possums was successful on two occasions. Localised infection caused by M. ulcerans in seven dogs and two alpacas is also described. Part 2: Investigations of fastidious mycobacteria causing cutaneous nodular disease in cats (feline leprosy). A detailed and comparative molecular and clinical epidemiological description of feline leprosy disease in 145 cats referable to Candidatus ‘Mycobacterium tarwinense’, Mycobacterium lepraemurium and Candidatus ‘Mycobacterium lepraefelis’ is presented.
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ItemInvestigation of the infectious causes of diarrhoea in Australian thoroughbred foals( 2017)Diarrhoea is a common disease in foals that is costly and labour intensive to manage. A large number of potential enteric pathogens have been detected in the faeces of foals, however, the role of these infectious agents in causing clinical disease is not clearly understood and their prevalence in Australia is unknown. In addition, timely methods for definitive diagnosis are not readily available for some of these agents. Therefore, this study aimed to develop rapid molecular detection assays to investigate the presence of equine rotaviruses, equine coronaviruses, Salmonella spp. and Clostridium difficile in Australian thoroughbred foals with and without diarrhoea. A prospective case control study was conducted on five thoroughbred breeding farms in the Hunter Valley (New South Wales, Australia) during the 2010 breeding season. Faecal samples were collected from age-matched foals with diarrhoea and without diarrhoea from the same farm (age-matched pair). In addition, faeces were collected from foals with diarrhoea from an equine veterinary hospital. All faecal samples were analysed for equine rotaviruses, equine coronaviruses, Salmonella spp. and Clostridium difficile by quantitative PCR (qPCR) assay. All faecal samples were also cultured for Salmonella spp. using selective growth media. Samples positive by qPCR and 15 randomly selected control foal samples negative by qPCR were cultured anaerobically for Clostridium difficile. Faecal samples were collected from 117 pairs of age-matched case control foals and 26 hospitalised foals with diarrhoea. In the age-matched case control foals, equine rotaviruses were the most frequently detected infectious agent (25% case foals, 5% control foals) and the only infectious agent significantly associated with the presence of diarrhoea. In hospitalised foals, Clostridium difficile (23%) was the most frequently detected infectious agent. In this investigation co-infections were detected in 4% of matched case foals and 4% of hospital foals, with equine rotaviruses and Salmonella spp. being the most frequent combination. Four different Clostridium difficile ribotypes were detected, including ribotype 012 and 078. Importantly, this is the first report of the detection of C. difficile ribotype 078 in Australian horses. As this ribotype has been associated with severe disease in humans, this finding may have public health implications. The availability of rapid molecular screening tests for infectious causes of foal diarrhoea enhances the veterinary practitioner’s ability to instigate appropriate therapy and control measures in foals with diarrhoea. However, the detection of pathogens in foals without diarrhoea highlights the need for more research into the role some of these pathogens play in clinical disease both individually and in combination.
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ItemMonophasic Salmonella Typhimurium in Australian pigs( 2017)Salmonella enterica enterica 1,4,[5],12:i:- colonization in Australian pig herds was investigated. The research considered: the distribution of S. 1,4,[5],12:i:- in the Australian pig industry; dynamics of colonization in herds; diversity in the Australian porcine population; comparison of study strains with related domestic serovars and strains reported internationally; antimicrobial resistance characteristics and determinants; and implications for optimal typing and surveillance. In total, 773 faecal samples were collected from Australian pig herds in cross-sectional (16 herds) and longitudinal (five herds) observational epidemiological studies. Samples were cultured and where Salmonella was confirmed multiple colonies were collected, 2326 isolates in total. Representative isolates were characterized by serotyping, phage typing, antimicrobial susceptibility testing and multiple-locus variable-number tandem-repeat analysis (MLVA). In addition, the genomes of a sample of the study collection isolates were sequenced. The results indicated that S. 1,4,[5],12:i:- has spread rapidly through the Australian pig industry. Persistent S. 1,4,[5],12:i:- shedding and considerable escalation among weaners was observed in the sampled herds. High levels of shedding were also observed among finisher pigs, indicating a possible pathway into the human food chain. Low S. 1,4,[5],12:i:- phenotypic and MLVA profile diversity was observed, suggesting the Australian porcine S. 1,4,[5],12:i:- population is closely related. Comparative genomic studies demonstrated that the S. 1,4,[5],12:i:- had undergone clonal expansion, consistent with the population having emerged from a single event. The characteristics of the study S. 1,4,[5],12:i:- strains closely resembled those of the European clone strains, supporting the hypothesis that S. 1,4,[5],12:i:- was recently introduced to Australia from overseas. In spite of the close relatedness of the study strains, phylogenetic analyses readily differentiated S. 1,4,[5],12:i:- strains on the basis of source. Little resistance to critical antimicrobials for the treatment of human salmonellosis was observed. Salmonella resistance types varied considerably between herds and were serovar associated within herds. The majority of S. 1,4,[5],12:i:- were multidrug resistant, whereas the majority of non-S. 1,4,[5],12:i:- serovars were pansusceptible. The variation in resistance types between contemporary serovars within herds indicated that antimicrobial use on farm was not driving selection for Salmonella resistance types. However, selection pressure for resistance types appeared to vary between herds. In some herds resistance diminished over time due and gene loss was identified. In other herds, there were indications of horizontal resistance gene acquisition among some of the more resistant strains. The most common resistance genes identified among the study S. 1,4,[5],12:i:- isolates also matched reports from overseas. Phage typing proved to be of limited value in differentiating Australian porcine S. 1,4,[5],12:i:- strains but MLVA showed promise for surveillance and broader epidemiological purposes. However, these studies further illustrated the value of comparative genomics for surveillance, source attribution and broader epidemiological purposes. This research has generated original insights into the epidemiology of S. 1,4,[5],12:i:- in pig herds. The findings have implications for pig industry and public health risk mitigation and risk management.
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ItemProtease-activated receptor-2 in bone morphogenesis and pathology: Is par2 essential in osteoblastic bone homeostasis?( 2017)Protease activated receptor-2 (PAR2) is a member of the small family of protease-activated G protein-coupled receptors. PAR2 is widely expressed in most tissues including bone and has been shown to regulate cell proliferation and survival, cytokine production and release as well as nociception. Recent unpublished data suggest that PAR2 plays a role in bone homeostasis by regulating the osteogenic and adipogenic differentiation of mesenchymal stem cells (MSCs). Due to the potential impact of such regulation on age-related bone loss and osteoporosis, the current study undertook to investigate the hypothesis that PAR2 activation regulates the determination of mesenchymal cell fate such that osteoblastogenesis is preferred over adipogenesis both in vivo and in vitro and that such regulation impacts the potential outcome of ageing in bone. Moreover, as PAR2 is a pro-inflammatory receptor, it was also hypothesised that PAR2 is a mediator of inflammation induced osteopoenia and as such its deletion will improve the resulting bone phenotype. Comparison of the bone phenotype in ageing global PAR2 null mice with the wildtype controls showed that the global lack of PAR2 in vivo was associated with a low bone mass profile and bone tissue mineral density which was compounded by a relatively rapid structural deterioration. These observations were more pronounced in the male knockout mice. In vitro gene silencing experiments with bipotential Kusa 4b10 mesenchymal cells suggested a pro-osteogenic and anti-adipogenic role for the receptor in the differentiation of MSCs. Gene expression studies revealed a number of novel genes downstream of PAR2 that were not previously associated with these processes. These included Cnr1, Il6, Ramp3 and Enpep which were more highly expressed following PAR2 knockdown and C1qtnf3, Snorc and Gpr35, which were suppressed. Investigation of IL-6 concentrations in the medium by ELISA as well as use of anti-IL-6 neutralising antibody suggested that PAR2 promoted osteogenesis and inhibited adipogenesis partly by suppressing expression of IL-6. To further examine the role of the receptor in osteoblasts in vivo, osteoblast-specific deletion of PAR2 was achieved through the expression of Cre-recombinase that was driven by the Osterix (Osx) promoter. Surprisingly, osteoblast-specific ablation of PAR2 in growing mice not only resulted in an increased bone formation rate in 7 week old females, but also culminated in a higher bone mass profile at 13 weeks whilst males remained unaffected. The paradoxical finding may be explained by the presence of PAR2 during the critical earlier stages of MSC differentiation and its ablation only after cells have become committed Osx-expressing osteoprogenitor cells. These results suggest that PAR2 plays a role in the determination of osteoblasts prior to the stage at which gene deletion occurs in the osteoblast-specific PAR2 null mice. To investigate the role of the receptor in the pathogenesis of inflammation-induced osteopoenia, the bone phenotype of PAR2 null and wildtype dystrophin deficient mice (mdx mice) was compared. Results revealed that PAR2 deficiency protected the mdx mice from the impact of muscular dystrophy on bone structural and material properties. This study provides evidence that PAR2 slows ageing-related bone loss, possibly by promotion of osteoblast differentiation and suppression of adipogenesis in MSCs. In contrast, evidence is provided that PAR2 contributes to inflammation-induced bone loss.
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ItemCoxiella BurnetII infections in intensively-managed dairy goats: elucidation of transmission dynamics and vaccination strategies( 2017)Coxiella burnetii causes Q fever, a zoonotic disease commonly associated with exposure to infected animals, particularly ruminants. In this study, the dynamics of C. burnetii infections were elucidated by detecting serological responses to C. burnetii and DNA of the organism in samples from adult and kid goats on a large dairy enterprise in Australia on which 24 cases of Q fever had occurred since 2013. An indirect immunofluorescence assay (IFA), initially developed for human serology, was adapted and standardised to detect IgG and IgM antibodies to phase 1 and to phase 2 antigens of C. burnetii. A cross-sectional study was then undertaken in 164 pregnant goats of different parities (86 primiparous and 78 multiparous) to determine the changes in seroprevalence over the kidding period. Following kidding, the seroprevalence in adult goats increased by 17.7%; the increase was higher (26.7%) in primiparous than in multiparous goats (7.7%) as 47.4% of the multiparous goats were seropositive pre-partum compared to 4.7% of the primiparous goats. However, the risk of seroconversion in multiparous and primiparous goats that were seronegative pre-partum was comparable; 24.4% and 26.8%, respectively, suggesting a constant risk of infection with C. burnetii to all susceptible goats across the kidding period, irrespective of parity status. A longitudinal study in 95 kids from two successive kidding seasons was performed to elucidate the infection dynamics of C. burnetii. Maternally-derived antibody was detected after feeding pooled goat colostrum but this subsided within 9 weeks and new infections (shown by a rise in phase 2 IgM) occurred soon after. As kid goats were infected very early in life and well before they were mated, it was concluded that there is a need to vaccinate young goats, before they are 9 weeks of age, in addition to the conventional strategy of vaccinating adult goats shortly before breeding. Since a vaccine registered for use in goats is not currently available in Australia, an inactivated autogenous vaccine, prepared from an isolate from the goat farm, was trialled in 8-week-old goats. Two injections, four weeks apart elicited specific IgM and IgG responses in all vaccinated goats (n = 11), while no antibodies were detected in two control groups (n = 22). Swelling at the site of inoculation was observed in all of the vaccinated and in 91% of the control group but receded after 3 weeks. The data indicated that this autogenous vaccine could be suitable for immunising young goats (< 3 months of age) to break the cycle of infection with C. burnetii on this property although further trials to determine level of protection against field challenge are required.
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ItemAn investigation of two significant infectious diseases in populations of Victorian koalas (Phascolarctos cinereus)( 2017)The koala (Phascolarctos cinereus), an iconic Australian marsupial, is considered a vulnerable species in parts of Australia due to recent rapid population declines. The role of infectious diseases in population declines in northern koalas (New South Wales (NSW) and Queensland populations) has been highly studied. Chlamydia pecorum and koala retrovirus (KoRV) have both been given considerable attention. C. pecorum in koalas is associated with infertility and blindness through infection of the urogenital tract and conjunctiva, respectively. The prevalence of C. pecorum in northern koalas is as high as 87%. Different genotypes of KoRV have been identified in northern koalas. KoRV-A has been identified in all northern koalas tested to date. KoRV-B appears to be less prevalent but has been implicated as a cause of neoplasia. Molecular testing of C. pecorum and KoRV prevalence in Victoria, a southern population of koalas, has been limited. No genotyping studies have been undertaken on either organism in Victorian koalas. This thesis conducted an extensive survey of Victorian koalas across seven separate regions to establish a prevalence estimate for both C. pecorum and KoRV. A genotyping study for each pathogen was also completed. The estimated prevalence of C. pecorum in Victorian koalas was 15.2% (125/820, 95% confidence interval (CI) 12.9, 17.9%). Molecular evidence of C. pecorum infection in French Island koalas was detected for the first time. Only a single ocular C. pecorum infection was identified in Victorian koalas (1/459). A total of six C. pecorum genotypes were detected, the majority of which were genotype B, which has only been detected in southern koalas. Three of the genotypes were novel, each of which were found in distinct populations. Male koalas were more likely to be infected than females. C. pecorum infection was associated with ‘wet bottom’ (a sign of urinary incontinence and inflammation) in male koalas and reproductive tract disease in female koalas. Not all koalas with ‘wet bottom’ had detectable C. pecorum, suggesting another potential cause. Analysis of the genetic diversity of the bacteria present in urogenital tract samples from ten koalas, of which only five displayed wet bottom, identified 13 operational taxonomic units that occurred at a higher abundance in wet bottom-affected koalas. These bacterial families are of interest for future studies. The genomes of 57 C. pecorum samples from koalas across Australia were sequenced and assembled. The results showed that C. pecorum genomes from southern koalas were distinct from those of northern koalas. KoRV prevalence in Victorian koalas was 24.7% (160/648, 95% CI 21.5, 28.2%). Only KoRV-A was detected. Koalas with ‘wet bottom’ were almost twice as likely to have KoRV detected. There was no association between KoRV and C. pecorum detection. This research highlights that Victorian koalas are experiencing a reduced burden of infection compared to northern koalas, and this may be a factor in southern populations outgrowing the available habitat resources in Victoria, compared to northern populations. Victorian koalas free from C. pecorum and KoRV infection could be sourced from over-abundant populations to assist re-establishment of populations where koalas have become locally extinct. Overall, this research provides valuable information for both future research and koala population management.