Veterinary Science - Theses
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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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ItemHealth and fitness effects of Anaplasma species infection in African buffalo (Syncerus caffer)( 2017)Anaplasma marginale and A. centrale are intra-erythrocytic bacteria of domestic and wild ruminants and are mainly transmitted by ixodid ticks. Most of the work on anaplasmosis has been done on A. marginale infections in cattle, as it can cause disease with varying levels of severity, from icterus and anaemia, to abortions and death. However, wildlife, such as African buffalo (Syncerus caffer), appear to be only subclinically infected with A. marginale and A. centrale. This thesis aimed to characterise A. marginale and A. centrale in African buffalo from Kruger National Park (KNP), South Africa, and investigate the effects of the burden of Anaplasma species on the health and fitness of their host. Firstly, the major surface protein 1α (msp1α) and heat-shock protein (groEL) genes were used to characterise A. marginale and A. centrale, respectively, from African buffalo. Sequence variation and phylogenetic analyses revealed that sequences of Anaplasma spp. from African buffalo were unique and that they grouped separately when compared with previously published sequences of both species. Sequencing the same species in cattle from the same area in the future will allow for more conclusive evidence as to whether African buffalo are a reservoir for anaplasmosis, thereby providing insights into the interface of domestic and wild ruminants. Secondly, the burdens of A. marginale and A. centrale in blood samples from African buffalo were determined, using an established quantitative PCR, and then various statistical models were run to investigate associations between Anaplasma burden, co-infection dynamics and health outcomes for African buffalo. There appeared to be a time-lag between infection and host response, or co-infection response, for some of the parameters examined, showing the importance of considering such delays in studies of disease. Despite finding a positive association between the concurrent burdens of infection with the two Anaplasma species examined, once the time-lag was accounted for, there was a negative association between the species, possibly indicating resource competition or the development of cross-immunity. African buffalo did not have an anaemic response to infection with either A. marginale or A. centrale; in contrast, for animals infected with A. marginale, there was an increase in haematocrit levels in response to infection. On the other hand, there were higher serum total protein levels associated with increased burdens of A. centrale, which may be due to the development of an immune response. Host responses to infection were also affected by external factors, including season and resource availability, and host factors such as gender. Younger buffalo appeared to be infected with higher burdens of A. marginale and A. centrale. In calves, infection with A. marginale appeared to occur before infection with A. centrale, and more frequently, which could be the result of different invasion and evasion techniques of the two species in this host. This study provides an insight into the effects of a subclinical infection on a wildlife host, caused by a pathogen which may cause severe clinical disease in domesticated animals.
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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.
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ItemMucin 1 regulation of the NLRP3 inflammasome( 2017)Mucin 1 (MUC1) is a cell membrane associated, heavily glycosylated protein, widely expressed on mucosal epithelial cells lining the respiratory, gastrointestinal and urogenital tracts of the human body and on immune cells. MUC1 performs multiple functions on epithelial surfaces that confer protection against bacterial pathogens. It can limit the access of pathogens to the host epithelium both by steric hindrance and by acting as a releasable decoy. Additionally, MUC1 functions as a negative regulator of Toll-like receptor signalling pathways, suppressing the downstream production of pro-inflammatory cytokines, thereby reducing inflammation induced by pathogens and their products. While these functions of MUC1 on the epithelial surface have been fairly well investigated, its role on immune cells, and specifically on macrophages, is comparatively unexplored. Infection with the gastric pathogen, Helicobacter pylori is the underlying cause of many pathologies in humans including, atrophic gastritis, gastric and duodenal ulcers, gastric mucosa-associated lymphoma and gastric cancer. Previous studies demonstrated that it is MUC1 on haematopoietic cells that plays a critical protective role during H. pylori-induced gastritis. MUC1 dampened the production of the pro-inflammatory cytokine IL-1β by suppressing activation of the NLRP3 inflammasome in immune cells, thereby protecting mice from severe H. pylori-associated disease. The current thesis was aimed at extending these findings by identifying the immune cell subset(s) in which MUC1 modulates the activation of the NLRP3 inflammasome in mice to protect against H. pylori pathogenesis and to evaluate the importance of this finding in a pulmonary bacterial infection model and in the function of human immune cells. Using an in vivo mice model of H. pylori infection, it was shown that MUC1 on monocytes and macrophages of the gastric mucosa negatively modulates NLRP3 inflammasome activation induced by H. pylori infection in mice. During investigations of the role of MUC1 during infection with the NLRP3-activating respiratory bacterial pathogen Streptococcus pneumoniae, a completely novel function of MUC1 on macrophages was discovered. Specifically, MUC1 was found to facilitate pneumococcal phagocytosis by murine macrophages, assisting in protection against pneumococcal pneumonia and bacteraemia. Thus, using two different pathogens at two different site, the studies presented in this thesis have identified that MUC1 has at least two quite distinct functions on macrophages which contributes to host defence against important mucosal bacterial pathogens. Another salient finding of this thesis is that MUC1 length, and not just the cytoplasmic domain, can affect the production of IL-1β in response to NLRP3 inflammasome activation. The length of MUC1 was not previously considered to have any direct effect on cytoplasmic immune signalling; this thesis now provides evidence to suggest the opposite, opening up a new area of future research. An interesting discovery made by studies in this thesis is the apparent function of MUC1 on eosinophils. Eosinophils are present in the immune cell infiltrate in the gastric mucosa of humans and mice, however their functional significance in H. pylori-associated disease is unknown. In the current thesis, H. pylori infection was found to induce caspase-1 activation in eosinophils, suggesting that these cells may be the source of IL-1β during H. pylori infection. MUC1 on these cells was found to function as a suppressor of caspase-1 activation, demonstrating a novel function of this molecule in eosinophils. In conclusion, studies presented in this thesis have clearly demonstrated that MUC1 specifically on macrophages and eosinophils plays a protective role during innate defences against bacterial pathogens. Thus, this study has added to the previously known functions of MUC1 and laid foundation for future studies to investigate the impact of MUC1 regulation of the aforementioned macrophage functions on infectious and non-infectious inflammatory diseases. Additionally, the possibility of an immunomodulatory role of MUC1 on eosinophils has opened up a new exciting area of research.
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ItemKinomes of selected parasitic helminths - fundamental and applied implications( 2017)Worms (helminths) are a large, paraphyletic group of organisms including free-living and parasitic representatives. Among the latter, many species of roundworms (phylum Nematoda) and flatworms (phylum Platyhelminthes) are of major socioeconomic importance worldwide, causing debilitating diseases in humans and livestock. Recent advances in molecular technologies have allowed for the analysis of genomic and transcriptomic data for a range of helminth species. In this context, studying molecular signalling pathways in these species is of particular interest and should help to gain a deeper understanding of the evolution and fundamental biology of parasitism among these species. To this end, the objective of the present thesis was to characterise and curate the protein kinase complements (kinomes) of parasitic worms based on available transcriptomic data and draft genome sequences using a bioinformatic workflow in order to increase our understanding of how kinase signalling regulates fundamental biology and also to gain new insights into the evolution of protein kinases in parasitic worms. In addition, this work also aimed to investigate protein kinases with regard to their potential as useful targets for the development of novel anthelmintic small-molecule agents. This thesis consists of a literature review, four chapters describing original research findings and a general discussion. A detailed assessment of the literature (Chapter 1) revealed that, despite a recent increase in the availability of transcriptomic and genomic data sets for parasitic worms, very little is known about the protein kinases encoded in these genomes and their associated functions. In addition, the bioinformatic tools currently used for kinase identification and classification do not permit the accurate characterisation of kinase complements of parasitic worms as they do not take into account the draft state of genomic data sets and the substantial diversity in kinases of species that are only distantly related to well-curated model organisms. Therefore, the aims of this thesis were: (i) to comprehensively identifiy, classify, curate and functionally annotate the full complements of protein kinases in the genomes of parasitic worms, by (ii) establishing an advanced bioinformatic workflow system to carry out this task; (iii) to explore fundamental aspects of kinase signalling in worms based on developmental transcriptomes and cross-species comparisons; and (iv), from an applied perspective, to identify protein kinases with potential as anthelmintic targets. An integrated bioinformatic workflow relying on a pairwise-comparative approach was established and used to define the complete kinomes of the blood flukes Schistosoma haematobium and S. mansoni (phylum Platyhelminthes; class Trematoda; Chapter 2). For such flatworms, scant kinome data and vast phylogenetic distance from any well-curated model organism represented a challenge for kinase identification and classification. Therefore, trematode-specific stochastic models were inferred to identify and classify kinases prior to pairwise curation, which proved superior to the generalised models used in kinase identification tools available at the time. The transcription profiles of the curated kinase genes were then investigated and, employing this and other functional information, kinases were assessed in detail for their potential as drug targets. Subsequently, using an in silico approach, small-molecule effectors were inferred. Next, using the well-curated kinome of the best-characterised metazoan organism, Caenorhabditis elegans (a free-living nematode) as a reference, the complete kinome of Haemonchus contortus, a parasitic nematode of ruminants, was defined (Chapter 3). Based on this curated data set, the transcriptional regulation of kinase genes across parasite development was investigated, and these data were then integrated into an improved, ranking-based drug target prediction pipeline. This study showed that, using the kinome of the free-living nematode as the reference, the curation of the H. contortus kinome was readily possible. However, this was not the case for species that were distantly related to C. elegans, such that a distinct approach had to be taken. Therefore, for the identification, characterisation and curation of kinase sequences of distant taxa, such as those in in the class Enoplea, pairwise analyses were undertaken between closely related species within each of the genera Trichinella and Trichuris (Chapters 4 and 5). Kinomes of four species of enoplean, with unique biology and evolution, were investigated and compared. These analyses showed that enopleans have remarkably compact kinomes compared with other worms, and complemented with advanced three-dimensional modelling, revealed a novel enoplean-specific protein kinase. In conclusion, the present thesis has contributed significantly to gaining a deep understanding of the protein kinomes in socioeconomically important parasitic worms (Nematoda and Trematoda), and has provided a bioinformatic framework for the exploration of kinomes of the plethora of parasitic worms (Chapter 6). Although established for worm kinomes, the workflow system developed will have broad applicability to almost any group of eukaryotic organisms. Importantly, the findings presented in this thesis provide a practical resource for future functional investigations of signalling pathways in parasitic worms, which has considerable fundamental implications for studying worm biology, physiology and evolution. Given that protein kinases are recognised as attractive targets for small molecule drugs, the results should also have significant applied implications for future anti-parasitic drug discovery, repurposing and development.
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ItemEquine adenovirus: molecular and host cell receptor characterisation( 2017)Equine adenoviruses (EAdV) are members of the family Adenoviridae. While EAdV-1 causes respiratory and ocular infections, EAdV-2 has been associated with gastrointestinal infections. Little is currently known about the way these viruses interact with the host cell, and how the genetic composition of EAdV-2 compares to other adenoviruses. There is also a need for more sensitive detection methods to understand the role of EAdV-2 in foals with diarrhea. The aims of this research project were to characterise the full genome of EAdV2-385/75.4, to develop a quantitative PCR (qPCR) for detection of this virus in faecal samples, and to characterise the putative EAdV receptor molecules on the surface of equine foetal kidney (EFK) cells. By applying next generation sequencing, the size of the EAdV-2 genome was determined to be 33,010 bp in length, with a GC content of 48%. Comparative genome analysis showed EAdV-2 to be a member of the Mastadenovirus genus but has a distinct arrangement and genetic content in the genome termini, which placed the virus in a separate cluster within the genus Mastadenovirus. A high throughput qPCR assay using primers targeting a 109 bp region of the hexon gene and capable of differentiating EAdV-2 from EAdV-1 were also designed and optimised. The assay was specific and sensitive for the detection of EAdV-2. The detection limit of the assay was 27 genome copies per reaction. The performance of the qPCR assay was assessed using archived faecal samples and was shown to exclusively amplify the targeted EAdV-2 hexon fragment. To test the possible role of the fibre knob domain in the infectious cycle of both EAdV-1 and EAdV-2, a recombinant baculovirus was constructed for the expression of the EAdV fibre knob proteins from both serotypes. A soluble EAdV-2 fibre knob protein was successfully expressed and was immunogenic when analyzed by Western blot. Polyclonal antibodies against purified EAdV-1 and EAdV-2 virion were also produced. Sensitive neutralisation assay has shown heterologous neutralisation of both virus serotypes, suggesting the presence of some cross neutralisation antibodies in these sera. An immune-fluorescent infectivity assay was developed to characterise potential host cell surface receptors for infection of EAdV-1 and EAdV-2 in EFK cells. EAdV-2 infection was significantly inhibited by NaIO4, neuraminidase, wheat germ agglutinin and Sambucus nigra agglutinin treatments, suggesting a sialic acid molecule with an α(2,6) linkage is involved in the attachment and infection of EAdV-2 to the EFKs. Integrin blocking experiment results using vascular cell adhesion molecule 1(VCAM-1) also showed EAdV-2 utilised α4β1 integrins for subsequent internalisation. Similarly, heparan sulfate (HS), heparinase I and III, NaIO4 and neuraminidase significantly inhibited EAdV-1 infection, suggesting HS plays crucial role in promoting EAdV-1 infection and the possible involvement of sialic acid as a secondary receptor. Future studies using cells which express little or no sialic acid containing molecules would be helpful to unravel whether the sialic acids and integrins are used by EAdV-1 and EAdV-2, respectively, as secondary receptors. This study complements new knowledge on the full genome structure of EAdV-2 and has elucidated some of the mechanisms by which EAdVs interact with their receptor, advancing our knowledge of the EAdV-host interactions.
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ItemA bacteriological disease investigation in a captive breeding colony of Lord Howe Island Stick insects (Dryococelus australis)( 2017)The Lord Howe Island stick insect (Dryococelus australis) is critically endangered in the wild. Once driven to near-extinction, the species now exists within just a few captive breeding programs that have been established to conserve the species, to increase population numbers through breeding, and to support the eventual reintroduction of the species into the wild. In the current study, the bacteria Serratia marcescens was investigated as a possible cause of mortality in a captive breeding colony of D. australis housed at Melbourne Zoo. S. marcescens was isolated from both dead insects and from the environment. The prevalence of the bacteria in dead insects was estimated, and the gross pathological and histopathological changes in infected insects were characterised. Strain types of S. marcescens were characterised using pulsed-field gel electrophoresis, and the strain type A1 was implicated as the most likely outbreak strain associated with insect deaths. Epidemiological risk factors for mortality were also investigated, and a novel PCR test was developed to facilitate rapid detection and identification of S. marcescens. The overall findings of this research suggest that S. marcescens is a potential opportunistic pathogen of D. australis, and is associated with haemocoelomitis and death. However, high virulence was not demonstrated in infected insects under experimental conditions. Recommendations for the future management of D. australis and the future reintroduction of the species to the wild are discussed in light of the findings from this research.