Veterinary Science - Theses
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ItemGenomic resources and genetic studies of parasitic flukes, with an emphasis on Clonorchis sinensis( 2019)Clonorchiasis is a complex hepatobiliary disease caused by the foodborne parasite, Clonorchis sinensis (family Opisthorchiidae). This disease can induce cholangiocarcinoma (CCA), a malignant cancer of the bile ducts, and has a major socioeconomic impact on ~ 35 million people predominantly in East Asia. Currently, no vaccine is available to prevent clonorchiasis, and repeated use of the only recommended drug, praziquantel (PZQ) increases the risk of developing drug resistance. Further understanding of the disease epidemiology relies on the knowledge of genetic variation of C. sinensis in endemic areas. Moreover, evidence of karyotypic variation within C. sinensis highlights the importance of comparing the genomes of geographically distinct isolates of this parasite. The two predominant research aims of this thesis were to decode the mitochondrial (mt) and nuclear genomes of a Korean isolate of C. sinensis and assess genetic variation, using high-throughput sequencing technologies and advanced bioinformatic workflows. The mt and nuclear genomes for a C. sinensis isolate from Korea (Cs-k2) were sequenced, assembled, characterised and compared with one or more isolates. In addition, a refined bioinformatic workflow was designed to infer high quality syntenic blocks between the nuclear genome and a previously published draft genome of another isolate from China. The results not only reveal a high level of nucleotide similarity within the syntenic regions, but also pinpoint variable genes that might be central to infection and/or adaptive process. The mt and nuclear genomes and the syntenic blocks now serve as a solid foundation for a future genetic analysis of C. sinensis. The mt genome, on one hand, confirmed the specific identity of the specimen, on the other hand, highlighted potential challenges with using mtDNA markers for genetic analyses. In contrast to the mt genome, the syntenic blocks of the nuclear genome exhibit major potential for future genetic studies due to a vast extent of nucleotide differences in coding regions. These blocks also contain a substantial number of genetic loci that might enhance knowledge of host- parasite relationships in an evolutionary context. Compared with coding regions, the genetic variation in the intronic regions showed an improved phylogenetic signal at both the whole genome and individual gene levels. In future, improved quality of the assembly and annotation of nuclear genomes should be achieved using long read data, allowing a broader range of genetic and structural variation to be identified using whole genomic data of representative individuals. Furthermore, a systematic bioinformatic framework is required to discover individual variants, infer population structure and identify adaptive selection, with the consideration of parasitic life cycle and demographic history. Although the present thesis focused predominantly on C. sinensis, the work extended logically to another trematode. A third research aim was addressed to explore the population genetic structure of a related trematode parasite, Schistosoma japonicum in China and to identify genes under positive selection in particular geographical locations. Clearly, the findings of this thesis and the approaches established should have important and broad implications for studies of a range of flatworm parasites.
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ItemChronic enteropathy in dogs: the role of macrophages and preliminary results in inflammatory cytokines( 2019)Chronic enteropathy (CE) is an umbrella term used in dogs to describe a group of diseases with different aetiologies, characterised by chronic gastrointestinal signs. These diseases are clinically classified according to treatment response as food-responsive (FRE), antibiotic-responsive (ARE), and immunosuppressant-responsive enteropathies (IRE). The first part of this PhD thesis prospectively describes the features of CE that are commonly seen in dogs presenting to a referral centre in Australia; information that has not been available previously. We found that similar to other countries, most dogs with CE are food-responsive, followed by antibiotic-responsive with a minority of immunosuppressant-responsive. Furthermore, our study raised concerns about prolonged antibiotic treatment for dogs with ARE. Firstly, most of these dogs do not respond to treatment completely for prolonged periods (as opposed to dogs with FRE that do), raising the question about the real benefit of antibiotic treatment. Secondly, half of the dogs with ARE required long-term or pulse treatment with antibiotics, which raises concerns about development of bacterial resistance. Our findings highlight the need to find alternative treatment for dogs with ARE in view of the poor long-term outcome. Although most dogs with FRE had long-term remission, adequate dietary trials were not performed until reaching the referral setting. This indicates that better education of general veterinary practitioners about the importance of performing adequate diet trial is needed to improve early disease remission in these dogs. The next focus of the research was to evaluate the role of macrophages in CE; this was achieved by using two macrophage markers: calprotectin and cluster of differentiation 163 (CD163) in immunohistochemical examination. Both immunohistochemical markers highlighted two different populations of macrophages in our intestinal biopsy specimens. Overall the number of CD163 positive cells was higher than calprotectin positive cells both in crypts and villi. Dogs with FRE and IRE had a decreased CD163:calprotectin ratio compared to healthy dogs with an increase in the ratio after treatment. Our results suggest that there is an imbalance in macrophage populations in dogs with FRE and IRE, with partial resolution following clinical response characterised by an increase in the ratio CD163:calprotectin. Interestingly, dogs with ARE not only have a poor long-term response, but also have different macrophage populations from dogs with FRE and IRE; and in fact, are very similar to healthy dogs without change in their macrophage populations with treatment response. These results suggest that macrophages play a role in the pathogenesis of FRE and IRE dogs with normalisation of macrophage populations with treatment response. The CD163 receptor is cleaved during macrophage activation and is released into the circulation as a soluble form. In view of the decreased number of CD163 cells in the intestine of dogs with FRE and IRE at diagnosis (and subsequent increase with clinical remission), we wanted to determine if soluble CD163 could be detected in dog serum, and therefore potentially serve as a biomarker. Two different ELISAs were tested and although one of them showed some signal, further testing of the antibodies used in the assay did not support that the signal was specific for CD163. With this experiment, we were not able to quantify soluble CD163 in dogs, but this molecule retains potential as a biomarker for diagnostic and monitoring purposes in CE as well as in other diseases characterised by macrophage activation. Biomarkers of systemic inflammation were also assessed in the same cohort of dogs and we showed that serum IL-6 decreased in dogs with CE after resolution of clinical signs. Similarly to soluble CD163, cytokines might play a role in further differentiating between the different causes of CE for prognostic and therapeutic purposes. Future studies are needed to assess these cytokines in a larger cohort of dogs to be able to study differences between FRE, ARE, and IRE, and further assess their role as biomarkers. Finally, we studied cytokine production by lymphocytes or monocytes in the peripheral blood of healthy dogs, and the effect of different immunosuppressive treatments on cytokine production. Differential activation of lymphocytes or monocytes can easily be achieved by using specific activators in whole blood. The advantage of this technique is that there is minimal handling of the cells with less risk of iatrogenic activation. Cytokine production was affected by cyclosporine and prednisolone, but not by mycophenolate, leflunomide, or azathioprine. Cyclosporine inhibited production of tumour necrosis factor (TNF), interferon gamma and IL-10 by lymphocytes whereas prednisolone inhibited TNF production by both lymphocytes and monocytes. Our findings suggest that this methodology can be used to monitor dogs treated with both drugs concurrently – although this needs to be further assessed with future studies. Future studies highlighted by our research suggest more in-depth assessment of serum cytokines as biomarkers for dogs with CE not only for monitoring purposes, but to determine if different patterns of cytokines can be useful to refine the classification of CE. Similarly, whole blood stimulation can be used to better assess underlying priming of the immune system and to monitor treatment response. Finally, our findings suggest that macrophages play a significant role in the pathophysiology of CE in dogs, particularly in FRE and IRE, but additional work is required to better understand their function in CE.
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ItemRecombination of infectious laryngotracheitis virus (ILTV) and the role of vaccination( 2019)Infectious laryngotracheitis virus (ILTV; Gallid alphaherpesvirus-1) is an alphaherpesvirus that causes respiratory disease in chickens, resulting in significant production losses in poultry industries worldwide. Recombination between different ILTV strains has recently been identified. Recombination in alphaherpesviruses was first described more than sixty years ago and since then, different techniques have been used to detect recombination in both natural (field) and experimental settings. In the past, natural recombination events between ILTV strains has resulted in the emergence of virulent recombinant viruses that have caused severe disease outbreaks in Australia. In this work, a single nucleotide polymorphism (SNP) genotyping assay was developed to study ILTV recombination in vivo. This assay was used to study recombination in a large number of viruses retrieved from chickens co-inoculated with two Australian ILTV field strains. Further application of this SNP genotyping assay helped unveil other aspects of ILTV recombination such as viral diversity over time and dominant recombination patterns in the recombinant progeny. Whole genome sequencing (WGS) of dominant viruses was performed in order to analyse their recombination breakpoint locations. This latter analysis revealed the presence of recombination hot-spots. The location of these hotspots were consistent with those found after the analysis of publicly available whole genome sequences of ILTV from different geographical regions, such as Australia and the United States (US). Additionally, the recombination output was determined in chickens after vaccination with three commercially available Australian ILTV vaccines (SA2, A20 and Serva), or two vaccines from the US (CEO-Tachivax and HVT-LT). For this later analysis a second SNP genotyping assay was develop to detect recombination between the USA field strains of ILTV used in that study. Results from these analyses indicated that vaccination can limit the number and diversity of recombinant progeny viruses and introduced new research questions about the role of the immune system in limiting recombination. The studies reported in this thesis have provided new insights into recombination in alphaherpesviruses that will be useful for future studies regarding vaccine development and use in both Australia and elsewhere.
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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.