Dissertationen zum Thema „Marsupial diseases“

Four main types of viruses capable of causing systemic and gastrointestinal infections, namely Coronavirus, Rotavirus, Parvovirus or Morbillivirus (Tennant et al, 1991) have been investigated in marsupials. A pilot study to develop and optimise the methodology was undertaken using Canine Coronavirus and the study was then extended to marsupials and other target viruses.In the marsupial portion of the study, a fragment of the correct size for the amplification of pol gene, 409 bp, was obtained from two different faecal samples from tammar wallaby (from Macquarie Fauna Park) and one western grey kangaroo (from Taronga Zoo). The results from tests are studied and compared. It is suggested that future further investigation should be directed at: 1/. Applying the protocols in this body of work to the testing of faecal samples from animals that have been diagnosed as positive with other diagnostic protocols. 2/. Examining more faecal samples from animals that present with active diarrhoea
Master of Science (Hons)

Four main types of viruses capable of causing systemic and gastrointestinal infections, namely Coronavirus, Rotavirus, Parvovirus or Morbillivirus (Tennant et al, 1991) have been investigated in marsupials. A pilot study to develop and optimise the methodology was undertaken using Canine Coronavirus and the study was then extended to marsupials and other target viruses.In the marsupial portion of the study, a fragment of the correct size for the amplification of pol gene, 409 bp, was obtained from two different faecal samples from tammar wallaby (from Macquarie Fauna Park) and one western grey kangaroo (from Taronga Zoo). The results from tests are studied and compared. It is suggested that future further investigation should be directed at: 1/. Applying the protocols in this body of work to the testing of faecal samples from animals that have been diagnosed as positive with other diagnostic protocols. 2/. Examining more faecal samples from animals that present with active diarrhoea

Chlamydia pneumoniae is a wide spread human and animal pathogen, associated with a number of acute and chronic disease states. In this thesis, next-generation sequencing technologies were used to identify genetic markers involved in the diverse presentation of Chlamydia pneumoniae infections. The findings presented in this study describe a phylogenetically distinct human Chlamydia pneumoniae line, as well as a number of minor DNA sequence changes that may account for the biological fitness of particular strains to different diseases and animal hosts. The outcomes of this thesis have considerably expanded our knowledge of the genetic and evolutionary diversity of this common pathogen.

This thesis examined the hypotheses that the mucus lining of the gastrointestinal tract (GIT) of Australian marsupials is colonised with large populations of spiral and fusiform shaped bacteria, many of which belong to the genus Helicobacter and that these Helicobacter species are likely be unique. The presence of spiral and fusiform shaped bacteria in the GIT of 8 Australian marsupial species (32 animals in total) was examined using microscopy, culture and Helicobacter genus specific PCR. The marsupials studied included the brushtail possum, ringtail possum, koala, wombat, Eastern grey kangaroo, Tasmanian devil, Eastern quoll and long nosed bandicoot. The spiral and fusiform shaped isolates were characterised and identified using morphological appearance, Helicobacter genus specific PCR and 16S rRNA gene sequence comparisons. The spatial distribution of Helicobacter species in the GIT sections was examined microscopically in silver stained sections of the GIT and using Fluorescent in situ hybridisation (FISH) with a Helicobacter genus specific probe. Spiral and/or fusiform shaped bacteria were detected and/or isolated from all marsupials studied. The prevalence and bacterial load of these organisms was found to differ in each marsupial species. These bacteria were found to belong to 3 different genera (Helicobacter, Campylobacter and Desulfovibrio). Each marsupial species appeared to be colonised with one or more unique Helicobacter species. Comparison of the detection of Helicobacter species in different groups of marsupials (herbivores, omnivores and carnivores) suggests that diet as well as the function and structure of the GIT may have a significant impact on their colonisation. Phylogenetic analysis of the new possum Helicobacters showed that they shared a common ancestor. Comparison of Helicobacter species isolated from different species of marsupial and placental mammals, as well as birds, showed that differences in environmental location i.e. gastric vs lower bowel had a major impact on the position of the Helicobacters on the phylogenetic tree.

Between 1901 and 1931, there were at least six anecdotal records of disease outbreaks in mainland quokkas (Setonix brachyurus) that were associated with mass mortalities. This time period pre-dates the arrival of the red fox (Vulpes vulpes). Despite these outbreaks, little or no research has been carried out to establish health and disease baseline data of the fragmented and scattered, extant populations. Epidemiological data was determined for a range of potential pathogens, and established physiological reference intervals of apparently healthy, wild quokkas on Rottnest Island and mainland locations. There were significant differences between Rottnest Island and mainland quokkas. Rottnest Island animals had haemograms with mark evidence of oxidative injury and bone marrow response consistent with a regenerative normocytic hypochromic anaemia. Except alkaline phosphatase (ALP), all blood chemistry analytes where higher in mainland animals, with particular emphasis on creatine kinase (CK), alanine amino transferase (ALT), aspartate amino transferase (AST) and vitamin E. Some other key findings include a widespread presence of a novel herpesvirus (MaHV-6), the recovery of Cryptococcus neoformans var. grubii from quokkas in highly altered ecosystems on Rottnest Island, and new Salmonella spp. serovars in Rottnest Island quokkas. Atypical lymphocytes resembling those in proliferative disorders of the lymphoid and haematopoietic tissues in other species were observed in blood smears of animals on Rottnest Island but not on the mainland. The presence of potentially-pathogenic organisms is likely to increase synergistic effects of ongoing and future threats (e.g. habitat clearing, climate change), and could increase quokka extinction risk. Disease surveillance would make a valuable contribution to Recovery Plans for the quokka, enabling preparedness for a rapid response if clinical disease is to happen, and to manage populations in a more integrated way.

The Gilbert’s potoroo (Potorous gilbertii) is a small marsupial endemic to the Two Peoples Bay Nature Reserve in the south-west of Western Australia. The Gilbert’s potoroo is classified as Australia’s most critically endangered mammal (IUCN 2006) with an estimated population of only 35 individuals. This thesis examines the health and disease status of the Gilbert’s potoroo, presenting a strong case for the relatively new concept of disease as a potential threatening factor and modifier of population decline. Specific diseases, including Cryptococcus, ectoparasitism, endoparasitism, haemoparasitism, Toxoplasma and a novel Treponema organism are extensively studied. An assessment of the clinical significance of these diseases is made, and management strategies are recommended to minimise the impact of these diseases on both the wild and captive population. The novel Treponema organism which clinically presents with tenacious, green discharge and an associated balanoposthitis in males is molecularly characterized. Epidemiological studies show the effects of this agent on reproductive function and a penicillin-based treatment regime is trialled in the analogous long-nosed potoroo (Potorous tridactylus) with a recommendation to then trial this treatment regime in the critically endangered Gilbert’s potoroo. Standard haematological and urinalysis findings are tabulated to form reference ranges for this species. A treatment regime for Cryptococcus in the analogous long-nosed potoroo is reported and parasitological findings, including the identification of a novel tick species are discussed. This thesis addresses key health issues, which have subsequently been incorporated into the Recovery Plan of the Gilbert’s potoroo. A document encompassing multiple disciplines and expertise to support the recovery of this critically endangered marsupial in its current environment. In addition, this thesis outlines a recommended health monitoring and treatment protocol for future translocation procedures and provides a working example of the emerging importance of health monitoring in threatened species recovery programs.

Thesis by publication -- 8 co-authored articles.
Thesis (PhD)--Macquarie University, Division of Environmental and Life Sciences, Department of Biological Sciences.
Includes bibliographical references.
Preface -- Management issues of urban common brushtail possums (Trichosurus vulpecula): a loved or hated neighbour -- Effects of deslorelin implants on reproduction in the common brushtail possum (Trichosurus vulpecula) -- Brushtail possums (Trichosurus vulpecula) in metropolotan Sydney: population biology and response to contraceptive implants -- Strategic survey for Toxoplasma gondii and Neospora caninum in the common brushtail possum (Trichosurus vulpecula) from urban Sydney, Australia -- Leptospirosis serology in the common brushtail possum (Trichosurus vulpecula) from urban Sydney, Australia -- Conclusions.
The common brushtail possum (Trichosurus vulpecula) is indeed a common inhabitant of many Australian citites, and one of the few marsupials that has adapted well to the urban environment. Their close proximity to people provides a great opportunity to experience native wildlife in the backyard, however, their utilization of house roofs, bold behaviour and appetite for garden plants often leads to conflict with householders. Population numbers are sufficiently high to require ongoing management to minimise negative impacts for humans and brushtail possums alike in a socially acceptable manner. The aim of this thesis was to identify current management issues and address the need for improved and novel management strategies. The potential of slow-release implants, containing the GnRH agonist deslorelin, as a contraceptive agent for brushtail possums was tested on a captive population. Males appeared resistant to treatment, but deslorelin was found to inhibit reproduction in female brushtail possums for at least one breeding season, making it a promising tool to control fertility in some wild populations. A further aim was to trial deslorelin implants on a wild urban population, to collect more information about the urban biology of this species and to point out issues which have previously not been addressed. Close proximity and interaction of urban brushtail possums with humans and their domestic animals can increase the risk of disease exposure and transmission and influence the health of wild populations. Serosurveys showed that animals were readily exposed to Leptospira spp. and Toxoplasma gondii. This thesis also provides the first data on brushtail possum dispersal in urban areas, knowledge which is highly relevant to the development of management strategies such as fertility control. The findings from this research broaden our knowledge about urban brushtail possums and should assist wildlife authorities in developing alternative or improved management procedures.
Mode of access: World Wide Web.
xxv, 287 p. ill., maps

The reasons for the population decline of the marsupial the Eastern Barred Bandicoot, Perameles gunnii, in Tasmania, and the probable role of their susceptibility to protozoan diseases such as Toxoplasma gondii remain elusive. The pathological hallmarks of the disease have been extensively investigated in other animal species and although some species are more vulnerable to toxoplasmosis than others, the reason for marsupial vulnerability remains to be fully defined. This thesis has sought to address these and other factors that might be associated with the relationship between the marsupial, its microbial and parasitic diseases, ecological surroundings and immune system. Secondly, it was essential to examine the current disease status of P. gunnii in relation to its zoonotic potential in the Tasmanian setting. Finally, it was necessary to attempt to investigate the immunological status of this animal. The major conclusions from these investigations were that P. gunnii is extremely susceptible to even low doses of T. gondii oocysts. The development of clinical disease is rapid, with high morbidity and mortality. The species appears to be susceptible to other Apicomplexan species including Hepatozoon sp., and the flagellate Giardia. The close association of P. gunnii with humans and the infringement of humans into their habitat makes this marsupial vulnerable to trauma, predation, shelter and food deprivation, and exacerbation of current parasitic and microbial diseases. In summary, this study further clarifies the relationship between humans, P. gunnii and parasite and the possible zoonotic potential that exists within Tasmania.

"The T cell signalling cascade is an important biochemical event in the adaptive immune system. In this study, various molecules involved in the T cell biology of marsupials were characterized. Investigation of possible differences in the molecular sequence of these genes was undertaken to explain why marsupials appear to respond differently to other mammals in their reaction to challenge by pathogens. Prior to this study it has been generally accepted that the adaptive immune system in marsupials may not function in the same manner as in other mammals"--Abstract.

This thesis describes a comprehensive study of the cellular responses of a number of endangered marsupial species with a principal focus on the tammar wallaby (Macropus eugenii) as a model macropod species. The development of in vitro experimental assays for the assessment of immune responses in this model species are described, which provided a set of benchmarks for comparisons with other members of the Macropodidae and with eutherian mammals. Once this data was collected and protocols were established, the study was extended to include investigations of the immune responses in opportunistic samples obtained from the Rufous Hare-wallaby (Lagorchestes hirsutus), the Long-footed potoroo ( Potorous longipes) and the more common, but nonetheless still vulnerable, Long-nosed potoroo (Potorous tridactylus) with a view to investigating their apparent susceptibility to infection with intracellular pathogens, particularly mycobacterial species. The findings from the application of these assays suggest that the cellular immune responses of these species are relatively complex and involve a level of sophistication that rivals their eutherian counterparts. Specifically peripheral blood and tissue leukocytes were morphologically similar to those of other mammals, with the exception of tammar wallaby monocytes that appeared to contain few lysosomal granules, and the basophils of the Rufous Hare-wallaby that contained very large atypical granules. The overall findings of this study suggest that the immune systems of macropod species possess most of the sophistication associated with that of eutherian mammals. Whilst some differences were apparent in cells and their products in the test species, no single factor common to all macropods was identified as a cause for immune dysfunction. It appears likely that as yet undefined factors related to their confinement rather than an inherent defect in their immunocapacity is responsible for the apparent disease susceptibility of these animals.
Doctor of Philosophy (PhD)

This thesis describes a comprehensive study of the cellular responses of a number of endangered marsupial species with a principal focus on the tammar wallaby (Macropus eugenii) as a model macropod species. The development of in vitro experimental assays for the assessment of immune responses in this model species are described, which provided a set of benchmarks for comparisons with other members of the Macropodidae and with eutherian mammals. Once this data was collected and protocols were established, the study was extended to include investigations of the immune responses in opportunistic samples obtained from the Rufous Hare-wallaby (Lagorchestes hirsutus), the Long-footed potoroo ( Potorous longipes) and the more common, but nonetheless still vulnerable, Long-nosed potoroo (Potorous tridactylus) with a view to investigating their apparent susceptibility to infection with intracellular pathogens, particularly mycobacterial species. The findings from the application of these assays suggest that the cellular immune responses of these species are relatively complex and involve a level of sophistication that rivals their eutherian counterparts. Specifically peripheral blood and tissue leukocytes were morphologically similar to those of other mammals, with the exception of tammar wallaby monocytes that appeared to contain few lysosomal granules, and the basophils of the Rufous Hare-wallaby that contained very large atypical granules. The overall findings of this study suggest that the immune systems of macropod species possess most of the sophistication associated with that of eutherian mammals. Whilst some differences were apparent in cells and their products in the test species, no single factor common to all macropods was identified as a cause for immune dysfunction. It appears likely that as yet undefined factors related to their confinement rather than an inherent defect in their immunocapacity is responsible for the apparent disease susceptibility of these animals.

Orientação : Pedro Faísca ; co-orientação : Vanessa Almagro
A urolitíase é uma doença, bem documentada, do sistema urogenital em animais domésticos e humanos, tendo vindo a ser relatada cada vez mais em espécies selvagens, tanto em cativeiro como em estado selvagem. Contudo o seu relato em marsupiais, nomeadamente em espécies de Macropus, é raro. O presente estudo retrospetivo, relata 6 casos de urolitíase, como achados de necrópsia, com ausência de sintomatologia clínica, em duas espécies do género Macropus (Macropus rufus e Macropus rufogriseus), parte da coleção do Jardim Zoológico de Barcelona, relatados entre 2010 e 2015. Os animais afetados eram todos fêmeas, com idades entre os 5 e os 17 anos. Quatro dos seis casos são da espécie Macropus rufogriseus e os restantes dois casos da espécie Macropus rufus. Os urólitos analisados, eram compostos por carbonato de cálcio puro. Em três dos seis casos, os urólitos apresentavam-se com localização na pélvis renal, bilateral; em dois casos apresentavam-se com localização na pélvis renal do rim esquerdo; num caso, encontravam-se na pélvis renal do rim esquerdo e no ureter direito. Embora a causa exata ainda seja desconhecida, existe uma forte suspeita de um possível desbalanço nutricional, com excesso de fontes ricas em cálcio na dieta, levando ao desenvolvimento de urólitos.
Urolithiasis is a disease of the urogenital system, well documented in domestic animals and humans, and increasingly being reported in wild species, both in captivity and in the wild. However in marsupials, namely in Macropus species, is rarely documented. The present retrospective study, reports 6 cases of urolithiasis, as necropsy findings, with absence of clinical signs, in two Macropus species (Macropus rufus e Macropus rufogriseus), part of the Barcelona Zoo collection, reported between 2010 and 2015. The affected animals were all females, aged 5 to 17 years. Four of the six cases are from the Macropus rufogriseus species and the remaining two cases from the Macropus rufus species. The analyzed uroliths were composed of pure calcium carbonate. In three of the six cases, uroliths were located in the renal pelvis, bilateral; in two cases were located in the renal pelvis of the left kidney; in one case they were in the renal pelvis of the left kidney and right ureter. Although the exact cause is still unknown, there is a strong suspicion of a possible nutritional imbalance, with excess of rich calcium sources in the diet, leading to the development of uroliths.

The complement system is found in both invertebrates and vertebrates, and functions in both innate and adaptive immune responses. This thesis involves determining the presence of a complement system in non-eutherian mammals using publicly available non-eutherian genomes, followed by analysing the expression and effectiveness of the complement system in a model dasyurid, the red-tailed phascogale (Phascogale calura). Data mining was used to look for complement sequences in non-eutherian mammals, Polymerase Chain Reaction (PCR) to elucidate major complement component cDNA sequences, real-time PCR to determine the expression of those major complement components in developing phascogales, haemolytic assays to detect the presence of a functional complement system, and antimicrobial susceptibility testing to determine the antimicrobial capabilities of phascogale serum. Use of online search tools and algorithms resulted in the successful identification of 27-47 complement and complement-related sequences in gray short-tailed opossum (Monodelphis domestica), tammar wallaby (Macropus eugenii), Tasmanian devil (Sarcophilus harrisii), koala (Phascolarctos cinereus) and platypus (Ornithorhynchus anatinus) genomes. Key complement proteins, specifically involved in the Classical, Alternative and Lectin Complement pathways, were also compared with homologous human protein sequences, which then allowed for the identification of functionally important motifs and conserved sequences. The identification of complement sequences in non-eutherian genomes possibly indicate a comparable complement system to eutherian mammals, therefore the expression and functionality of the complement system was investigated in a marsupial. Real-time PCR (qPCR) has the ability to quantitate gene expression, indicating the presence of a functional gene product in a tissue. However experimental gene expression needs to be normalised against the expression of two stable reference genes. The stability of reference genes should have unregulated transcription in the various experimental conditions including differences in age, sex and tissues. Hence determining the stability of reference genes in different tissues and ages would be useful for marsupial gene expression studies. The expression of reference genes; Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH), Actin-β (ACTB), 18S rRNA (18S), 28S rRNA (28S) and Ribosomal Protein L13a (RPL13A), were investigated in liver, lung, small intestine and spleen tissues of phascogales, and at least two stable reference genes were determined per tissue. The expression stability differed according to tissue sample and age, suggesting that the analysis of reference genes according to the type of tissue and ages needs to be considered in every gene expression study. Combining the expression in juveniles and adults, GAPDH was most stable in liver and lung tissues, and 18S in small intestine and spleen tissues. The relative expression of complement component 1, subcomponent r (C1r), complement component properdin (CFP), mannan-binding lectin serine peptidase 2 (MASP2) and complement component 3 (C3) were examined in pouch young, juvenile and adult phascogales using qPCR. The complement components were chosen because of their importance in the Classical, Alternative and Lectin Complement pathways. Average complement expression of all four complement components in whole body tissues showed a significant increase in developing pouch young. However complement expression decreased in adults compared to juveniles. All juvenile and adult phascogales were males, and may indicate a link between complement expression and male semelparity. Partial cDNA sequences for C3 and MASP2 were also extracted from phascogale liver tissue using RACE-PCR, and comparisons with respective eutherian homologues revealed the conservation of functionally important sequences and motifs. This is the first time MASP2 has been suggested to be present and expressed in a marsupial, which indicates the presence of a functional Lectin Complement pathway in the phascogale. Complement proteins have been found in mammalian serum. The effectiveness of phascogale complement serum was tested using standard haemolytic assays. The presence of a Classical and Alternative Complement pathway was confirmed, with female phascogale serum being more effective at lysing foreign erythrocytes, reinforcing the possible link between the complement system and semelparity. The results obtained indicate that the function of the Classical and Alternative Complement pathways in phascogales is comparable to other vertebrates. Along with complement proteins, eutherian serum contains Acute Phase Proteins (APPs) capable of fighting bacterial infections. The presence of APPs, haptoglobin (Hp), C-reactive protein (CRP) and serum amyloid A (SAA) were identified in marsupial genomes, and expression of Hp and CRP were confirmed in phascogale tissues from birth to adulthood using PCR. Commercially available kits were used to determine the levels of Hp and SAA. Results indicate that female phascogales had higher Hp protein levels compared to males, and SAA protein levels in both males and females indicated that all phascogales sampled were clinically healthy at the time serum samples were collected. Both complement proteins and APPs contribute to the antimicrobial properties observed in serum. The introduction of adult male and female phascogale serum to bacteria resulted in the growth inhibition of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus. The effectiveness of antimicrobial properties in serum was dependent on the duration serum was introduced to bacteria at 37°C. Growth inhibition was highest for K. pneumoniae and lowest for P. aeruginosa. All four bacteria exhibited growth inhibition, confirming the antimicrobial function of phascogale serum. The results of this thesis have contributed to marsupial immunological research. Not many studies have focussed on the marsupial complement system; however this thesis has demonstrated the presence, function and effectiveness of the complement system in marsupials, and some initial research on complement in marsupial young. Marsupial young have no adaptive immune system, yet complement components were still expressed in 1-day old phascogales. In addition, the expression and levels of APPs in adult red-tailed phascogale serum have now been reported. This thesis shows that a greater emphasis should be placed on marsupial complement research, and other complement-associated components. Further research will enhance our understanding of the importance the marsupial complement system, and diseases associated with it.

The Tasmanian devil (Sarcophilus harrissi) has been threatened with extinction because of two independently arising, genetically distinct transmissible cancers (DFT1 and DFT2) that cause devil facial tumour disease (DFTD). These cancers are both caused by clonal cells of Schwann cell origin, transmitted among devils through biting during social interactions like an allograft. Fatal in almost 100% of cases, DFTD usually kills its host within 6 to 12 months after the presentation of tumour masses on facial, oral and neck regions. Since DFT1 was discovered in 1996, multiple conservation interventions have been implemented to protect the species from extinction. However, these efforts have been hindered by the lack of species-specific reagents and diagnostic tools, which is a common challenge in the study of diseases affecting non-human or non-model species. Immunological reagents are required to identify and quantify immune cells of defined phenotypes to understand how the animal immune system is affected by pathogens. For example, devils infected with DFTD show a reduction of lymphocytes, however the T lymphocyte subset that is affected by the disease is unknown due to the lack of a subset-specific antibody. Additionally, a cornerstone of disease management, including cancers, is effective diagnosis. This is especially critical in the case of DFTD due to the epidemic nature of these transmissible cancers. In this thesis, I have 1) developed a needed anti-devil CD8α monoclonal antibody (mAb) to characterise CD8+ T cells in devils; 2) characterised the proteome of extracellular vesicles derived from cultured DFTD cells to better understand DFTD cell signalling and to create a baseline for diagnostic tool development; 3) discovered a differential diagnostic protein biomarker for DFTD and 4) discovered and validated an early DFT1 biomarker using an extracellular vesicle approach. First, I developed and validated a mAb for devil CD8+ T cells. The extracellular domain of the Tasmanian devil CD8-alpha was identified and used to develop recombinant proteins for mAb production. The anti-devil CD8α mAb was validated using a transfected Chinese hamster ovary cell line expressing devil CD8 alpha protein, and peripheral blood mononuclear cells (PBMC) from captive devils by flow cytometry. I also stimulated captive devil PBMCs to analyse the production of IFNG as a surrogate measure of T cell activity and found that the majority of INFG production was by CD4-/CD8-/B- cells. Finally, I developed a sterile and portable laboratory to isolate PBMCs from wild devils in the field to immunophenotype their CD4 and CD8 T cells as proof of concept that this antibody can be utilized to assess immune competence in wild populations. Next, I employed extracellular vesicle (EV) approaches to evaluate how DFTD cancer cells may be manipulating devil physiology to aid tumorigenesis and cancer progression. EVs are nano-sized bilipid structures secreted by most cells, including cancer cells, that deliver bioactive cargo, such as a proteins, genetic material, and lipids to recipient cells. EVs play a crucial role in intercellular communication and are active participants in cancer progression. I optimised a methodology to isolate EVs derived from DFTD cultured cells to be characterised by mass spectrometry techniques. This first proteomic characterisation of EVs derived from cultured DFTD cells lead to the discovery that EVs from both DFT1 and DFT2 cell lines expressed higher levels of proteins associated with focal adhesion functions known to be linked to metastasis. Additionally, hallmark proteins of epithelial mesenchymal transition (EMT) were enriched in DFT2 EVs relative to DFT1 EVs, demonstrating the potential of EVs to detect functional differences between two clinically indistinguishable tumours, even more so than the proteome changes within the parent cancer cells. I next investigated whether the protein markers found in EVs derived from cultured cells could be used as potential differential DFTD biomarkers. I then characterised the EV proteome from 27 devil serum samples. The protein tenascin (TNC), one of the EMT hallmark proteins detected in DFT2 EVs in vitro, displayed a high predictive power to distinguish devils infected with DFT2 compared to those infected with DFT1 and healthy controls, demonstrating potential for a differential diagnosis biomarker. Finally, I analysed the EV proteome from independent discovery and validation cohorts of captive and wild Tasmanian devil serum samples (n = 87) to identify potential DFT1 biomarkers. The protein cathelicidin-3 (CATH3) was enriched in serum EVs of both devils with clinical DFTD infection and latent devils 3 to 6 months before DFTD diagnosis. CATH3 enrichment identified overt DFT1 with 87.9% sensitivity and 94.1% specificity and predicted latent DFT1 with 93.8% and 94.1% specificity, presenting a critically needed biomarker for the early detection of DFT1. This thesis has expanded the management toolbox for the conservation of the iconic Tasmanian devil in the wild. I developed the first monoclonal antibody against devil CD8α, which will be crucial to adequately analyse the cell mediated immune response in healthy, diseased, or vaccinated devils. These findings are among the very first to demonstrate clinical relevance of EVs in a wild disease system, providing a considerable step towards development of a clinical tool needed for conservation of an iconic, endangered species. The discovery and validation of an early DFT1 biomarker provides a major advance for DFTD diagnosis. The use of the preclinical biomarker I discovered will greatly enhance the capabilities of ongoing management efforts critical to ensure the viability of the remaining devil populations, such as epidemiological monitoring, maintenance of insurance populations, and deployment of vaccine and preventative measures. Finally, these results demonstrated that EV-approaches have considerable potential for other species (including humans) to provide much needed preclinical diagnostic tools that have defied previous development efforts.