Identification and characterisation of inflammatory cytokines in the Rufous hare-wallaby, Lagorchestes hirsutus

Australian marsupial populations, particularly macropods, have a high percentage of species that are threatened due to habitat destruction and competition and predation from introduced species. These already stressed populations are vulnerable to introduced and endemic pathogens, both immunologically and environmentally. Yet this vulnerability also places ethical limits on the extent of disease research that is able to be carried out. This thesis firstly investigates the applicability of using available genomes of model marsupial species, specifically the tammar wallaby (Macropus eugenii), in investigating immunological function in disease of a closely related vulnerable species, the rufous hare-wallaby (Lagorchestes hirsutus). Disease vulnerabilities in a range of marsupials highlight susceptibilities associated with T-cell function, and while other avenues have been investigated, the nature and expression of more diverse cytokines involved in T-cell mediated immune responses is a key focus for research. This thesis project began with the characterisation of expressed cytokines, IL-6, IL-10, and IFN-γ confirming their expression in the tammar wallaby and providing sequence and annotation information to further that found in published genome databases. Further to this, both IL6 and IL10 genes were found to express splicing variants in the tammar wallaby. To clarify the potential functional characteristics of both these cytokines and their isoforms, the structural nature of the predicted proteins was analysed and compared to known mammalian homologs. The information obtained for these cytokines in the tammar wallaby, was applied to the closely related but more vulnerable rufous hare-wallaby to obtain expressed sequence for these cytokines. The expression of key T-cell markers and cytokines was confirmed in the rufous-hare wallaby across a range of tissues. To further this, selected cytokines were compared semi-quantitatively to determine comparative base levels of expression to inform further quantitative assessment. The use of archival samples, particularly formalin-fixed paraffin embedded (FFPE) tissues, is an important resource in studying marsupial immunology, where threatened, or even extinct species, may be the subject of research. This project examined the use of both genomic bacterial identification and gene expression of cytokines within archival FFPE tissues as a mode to inform disease research. While the methodology has limitations, the identification of an infective agent, Mycobacterium avium, and the ability to assess the expression of cytokines associated with that agent, will inform further research in archival samples. The thesis concludes that while the sequence and structure of marsupial cytokines are comparable to their mammalian homologs, the identification of splice variants presents novel regulatory mechanisms which may affect disease outcomes. Basal expression of T-cell related cytokines and T-cell markers in rufous hare-wallaby tissues, and semi-quantitative assessment of cytokine expression is presented providing fundamental data for further comparative research. Taken together, identification of Mycobacteria in archival tissues and the associated expression of key cytokines in the rufous hare-wallaby provides a key methodological approach to assist in research for which archival FFPE tissues are available while also presenting novel disease-associated cytokine expression information.