Veterinary Science - Theses
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ItemClinical use of plasma lactate concentration and evaluation of the analytical performance of two benchtop analyzers for the measurement of L-lactate in canine plasma( 2018)Lactate has been used in small animal veterinary medicine since the 1990’s. Ongoing reviews of the literature had revealed that an up to date, comprehensive review of the use of lactate in small animal clinical practice was overdue. Consequently, this became the first objective of this thesis (Chapters 1 and 2). Through this review process, a surprising gap was identified in the veterinary literature. Despite widespread integration into clinical practice and validation of hand-held lactate analyzers against benchtop methods, there was no actual published evidence validating these benchtop methods for use in dogs (Chapter 3). Accordingly, this became the objective of the experimental component of this thesis (Chapter 4). A partial method validation study was designed to satisfy requirements outlined by the American Society of Veterinary Clinical Pathologists (ASVCP). The objective was to evaluate the analytical performance and equivalency of two commercially available benchtop lactate analyzers: a high-end point-of-care analyzer, the Radiometer ABL 800 (ABL800), and a clinical laboratory analyzer, the Cobas Integra 400 (Cobas400). As a gold-standard, definitive method for lactate measurement is poorly defined, we elected to use canine plasma manually spiked with reference standard sodium lactate as our test medium. After a preliminary assessment of linearity, intra-assay precision, and inter-assay precision using manufacturer controls, we used the spiked plasma to assess linearity, intra-assay precision, accuracy, range and equivalency of the two analyzers. Both analyzers demonstrated excellent precision. The Cobas400 was accurate over a wider range than the ABL800, however the ABL800 was more accurate within the most clinically relevant range. Method comparison was performed using the Cobas400 as the nominated reference method. Passing-Bablok linear regression and Bland-Altman analysis revealed that although the methods were in agreement, with ≈ 95% of measured differences falling within ± 1.96 standard deviations of the mean difference, they cannot be considered equivalent due to the presence of a small but clinically significant amount of constant and proportional bias. In conclusion, both analyzers demonstrated adequate analytical performance for use in clinical practice and future validation studies. However, they cannot be considered equivalent due to the presence of both constant and proportional bias and should not be used interchangeably. Consequently, analyzer specific reference intervals should be applied, and the same analyzer should be used when serially trending lactate concentrations in an individual patient.
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ItemSelect bacterial and viral pathogens of potential zoonotic or biosecurity importance in Australian brushtail (Trichosurus spp.) and ringtail (Pseudocheirus peregrinus) possums( 2018)Possums and gliders represent a diverse group of marsupial mammals native to Australia, including multiple vulnerable and endangered species. Common brushtail (Trichosurus vulpecula) and common ringtail (Pseudocheirus peregrinus) possums are two urban adapted species frequently found in gardens and parks throughout eastern Australia. Their presence in urban areas provides opportunities for transfer of zoonotic pathogens through both direct and indirect contact with humans. Diseases affecting possums are relatively poorly understood, as is the role of possums in the maintenance and transmission of zoonotic pathogens. This study aimed to investigate the significance of these possums as host species for a select range of pathogens, particularly in Victoria. Opportunistically collected serum samples from common ringtail, common brushtail and mountain brushtail possums (Trichosurus cunninghami) from Victoria and South Australia were tested for antibodies against Ross River virus (RRV) and flaviviruses. Cloacal swabs from common brushtail and ringtail possums from Victoria and New South Wales were screened for the presence of Mycobacterium ulcerans (the cause of Buruli ulcer), Campylobacter spp. and Salmonella spp. Spleen samples were screened for Francisella tularensis (the cause of tularaemia) and Coxiella burnetii (the cause of Q fever) by PCR. Antibodies to RRV were detected in 6.2% (16/259) of all possum samples, which were sampled during a concurrent epidemic of RRV in humans. Geographical areas with a moderate to high human case rate were associated with a higher seroprevalence rate in sampled possums. There was a significantly lower rate of detection in possums from urban environments compared to rural and mountainous environments. Seroconversion to an unspecified flavivirus was also present in 5.2% (5/97) of common ringtail possums and 6.5% (2/31) of mountain brushtail possums, but not in common brushtail possums (0/137). Two of the seropositive animals displayed clinical signs of encephalitis. Salmonella spp. was detected in 3.1% (7/229) of possums, while Campylobacter spp. Was found in 27.7% (41/229), with a significant predilection for common brushtail possums (38/106). The exact Campylobacter species was not identified, however, the main human pathogens C. jejuni and C. coli were excluded. No samples were positive for F. tularensis (n = 187), C. burnetii (n = 184) or M. ulcerans (n = 229). These findings indicate that, while urban-adapted possums may potentially carry zoonotic pathogens, they are unlikely to represent a high risk to public health, at least in regards to the infectious agents that were the focus of this study in the areas that were investigated. Finally, serum samples from all three possum species were screened for antibodies to Wobbly Possum Disease virus (WPDV), a recently identified virulent nidovirus in common brushtail possums in New Zealand. Although thought to be absent from Australia, antibodies to WPDV, or an antigenically similar nidovirus, were detected in all three species of possum, originating from both Victoria and South Australia. Overall, 16% (30/188) of samples were positive and 11.7% (22/188) were equivocal, with the two brushtail species most commonly affected. This is the first published report of serological evidence of WPDV, or an antigenically similar nidovirus, in Australian possums. However, attempts to detect viral RNA in spleen samples by PCR were unsuccessful.
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ItemInvestigating the inflammatory pathways involved in placental detachment in the mare( 2018)The mechanism of placental detachment in the mare has not been investigated in detail and as such remains poorly understood. Research in other species provides strong evidence that parturition and fetal membrane detachment from the endometrium requires involvement of the immune response. Research in horses has also suggested that movement of leukocytes in the umbilical vessels, and their attraction to fetal membranes at parturition, contribute to placental detachment. The aims of this study were to 1) confirm whether a leukocyte “concentration gradient” exists between the equine neonatal umbilical artery and vein, and whether this gradient was associated with fetal membrane retention time 2) to investigate the expression of pro-inflammatory cytokines in the equine endometrium and fetal membranes at parturition using quantitative real time polymerase chain reaction (RT-qPCR) and 3) to localise pro-inflammatory cytokine proteins within the equine endometrial and fetal membrane tissues using immunohistochemistry to correlate with gene expression. Data was collected from 33 spontaneously foaling mares and their foals on a single Thoroughbred stud farm in New Zealand. Umbilical artery and vein blood, fetal membrane and endometrial biopsy tissue samples were obtained. The average fetal membrane retention time was 92 (+/-130) minutes. The median fatal membrane retention time was 47 minutes (IQR 30). There was a significantly higher number of total leukocytes, lymphocytes and neutrophils in the equine neonatal umbilical artery than the umbilical vein at parturition (p <0.001). Fetal membrane retention time tended to decrease with an increase in umbilical leukocyte concentration gradient but did not reach statistical significance (p = 0.098). The mRNA expression of IL-1, IL-6 and IL-8 was significantly greater in the endometrium of foaling mares compared to control mares (p < 0.001). The mRNA expression of IL-8 was significantly higher in the fetal membranes of foaling compared to control mares (p < 0.001) whereas mRNA expression of IL-6 in fetal membranes was significantly lower between the two groups (p< 0.001). Whilst mRNA expression for IL-1 was lower in the fetal membranes of foaling mares compared to controls, the difference was not significant (p = 0.16). This study has demonstrated the presence of an innate inflammatory immune response in the placenta of spontaneously foaling mares, suggesting the role of inflammation in the detachment of the fetal membranes from the endometrium. The results from this study have highlighted the need to further investigate the mechanism of placental detachment in the mare and also understand the pathophysiology behind abnormal fetal membrane retention.
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ItemEndothelial activation in dogs with severe sepsis( 2018)Two components of endothelial biology – endothelial glycocalyx, endothelial activation – have been explored this two-part, in-depth research project. An extensive literature review discusses the endothelial glycocalyx in health and critical illness. A prospective observational clinical research study then measures the concentration of soluble biomarkers of endothelial activation in severely septic dogs. The study found a significant difference in the concentration of biomarkers between the septic dogs and controls, a result consistent with the presence of endothelial activation in dogs with severe sepsis.
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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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ItemBiomechanical testing of the calcified metacarpal articular surface and its association with subchondral bone microstructure in Thoroughbred racehorses( 2018)Subchondral bone adaptation and fatigue likely alter joint surface mechanical properties. The aim of this study was to evaluate the relationship of calcified articular surface biomechanical properties in the distal palmar metacarpal condyle of Thoroughbred racehorses with subchondral bone microstructure and training history. Subchondral bone microstructure was evaluated using both micro- and clinical-computed tomography. Biomechanical properties of the calcified articular surface were tested with reference point indentation at the micron level. Articular surface toughness increased with commencement of race training, and was lower in horses that died as a result of musculoskeletal fatigue injury, had evidence of palmar osteochondral disease, had higher subchondral bone connectivity with more concave surfaces, and higher subchondral bone volume fraction. Further investigation of bone microstructural properties is warranted based on bone connectivity and surface shape findings. At clinically applicable imaging resolution, evidence of subchondral bone modelling may be an important indicator of articular surface biomechanical properties.
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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.