The koala (Phascolarctos cinereus) is an iconic Australian marsupial that is the sole extant representative of the Family Phascolarctidae and forms part of the mammalian fauna in four Australian States and one Territory. There has been a significant decline in the range and distribution of the koala since European settlement, with habitat loss and its associated threats, including vehicle strikes, dog attacks and disease, continuing to reduce population numbers. Koalas provide the tourism industry and the Australian economy with revenue up to $1 billion per annum with 80 zoos and wildlife parks around the country displaying koalas. Koalas were introduced to a number of islands in southern Australia the early 20th century, in an attempt to ‘save’ them from near extinction due to habitat loss, hunting and disease. While populations on Queensland islands remain stable and require no management intervention, introduced populations on southern Australian islands show extremely low levels of genetic diversity yet have grown substantially and require constant monitoring and management to overcome overabundance and starvation associated with defoliation of food trees. The only known extant, naturally occurring, island population of koalas is on North Stradbroke Island in Southeast Queensland’s Moreton Bay. Infection with bacteria of the Family Chlamydiacae is at high prevalence in most koala populations but the resulting diseases are generally of low prevalence in koalas in Southeast Queensland and New South Wales. The role of the koala retrovirus (KoRV) in relation to infectious diseases is not fully understood, but it has been identified from all northern koala populations and the majority of southern koala populations though, like chlamydial infection, it is not usually associated with high disease prevalence. The major histocompatability complex (MHC) is a gene family in mammals that is vital for its role in disease resistance and so may play a role in disease susceptibility in koalas. This study provided detailed information about the genetic diversity and population structuring of the mainland koala population in Southeast Queensland, which is amongst the most threatened of koala populations because of anthropogenic disturbance. Microsatellite genotyping of 512 samples and mitochondrial DNA sequencing of a subset of 77 samples collected from sick, injured and dead koalas showed that in Southeast Queensland koalas had high microsatellite diversity (12.8 alleles/locus, He = 0.81) and at least 16 mitochondrial haplotypes. Population genetic analysis revealed six genetically differentiated clusters consistent with the existence of biogeographic and anthropogenic barriers like rivers and highways. The Koala Coast, an area in Southeast Queensland that provides habitat for one of the most significant natural koala populations in Australia was found to be genetically distinct from adjacent areas; the rapid decline of this population means it meets the criteria for classification as “Endangered Wildlife” under Queensland’s Nature Conservation Act 1992. Climatic patterns of warming and cooling that produced the disappearance and reappearance of rainforests is likely to have affected long term koala distribution in coastal eastern Australia – expansion into drier forests when they were available and restriction to refugia during times of rainforest dominance. The koala population in Northeast New South Wales, which has conventionally been classified as a different subspecies to the koalas in Southeast Queensland, has colonised an extensive part of the area since the destruction of the “Big Scrub” rainforest. This forest occupied about 75,000 ha before European settlement and would almost certainly have represented a natural barrier to the movement of koalas as it was unsuitable habitat due to its lack of suitable food trees. Population structuring analysis identified two major genetic clusters in the region, the northernmost of which was less differentiated from koalas sampled on the Gold Coast in Southeast Queensland than the southernmost cluster. The southern cluster, largely found around the city of Lismore, had significantly lower diversity than the northern cluster, consistent with a southward radiating colonisation pathway. Ninety-six percent (96%) of koalas in Northeast New South Wales shared the same mitochondrial DNA haplotype, which was the most common haplotype in Southeast Queensland. There was, therefore, little evidence to support the subspecies delineation of coastal koalas in Southeast Queensland and Northeast New South Wales. From a comparison between wild, mainland populations and two captive koala colonies in Southeast Queensland, genetic diversity in 106 captive koalas was found to be similar to the local wild populations (Dreamworld 9.2 alleles/locus, He = 0.75, Currumbin Wildlife Sanctuary 10.2 alleles/locus, He = 0.80). Microsatellite alleles in captive koalas were, in general, representative of the local wild populations and of similar frequencies, but the captive koalas possessed mitochondrial DNA haplotypes that were not found in the local wild koalas. Some of the original founders of the captive populations were not from Southeast Queensland so these different mitochondrial haplotypes are likely remnants of the koala genotypes at the original founders’ locations. The preservation of habitat and reduction of consequential threats to the koala’s long term survival in the wild are the most fundamental aspects of koala conservation in this region. However, the successful preservation of high genetic diversity at neutral loci in the captive colonies studied indicates that ex situ measures incorporating a captive breeding program could potentially be used as reservoirs for important genetic material should such an approach become necessary in the context of the drastic and accelerating decline towards local extinction of key Southeast Queensland koala populations. Urgent action would be essential to obtain a representative sample of current genetic diversity (both genomic and mitochondrial), given the drastic rate of decline. Island populations of organisms are expected to show reduced genetic diversity compared to their mainland counterparts because of factors such as founder effects and the increased susceptibility of small populations to the effects of genetic drift. A comparison of genetic diversity was made between the naturally occurring North Stradbroke Island koala population in Southeast Queensland and the introduced populations on St Bees, Brampton, Newry and Rabbit Islands in central Queensland with the mainland populations. As expected, the island populations had lower genetic diversity than mainland populations, however, the introduced St Bees Island population had higher mean number of alleles and expected heterozygosity (5.7 alleles/locus, He = 0.67) than the naturally occurring population on North Stradbroke Island (3.7 alleles/locus, He = 0.55). Anecdotal evidence suggests that the koala populations on Brampton, Newry and Rabbit Islands off the central Queensland coast were established by the unauthorised transfer of koalas from St Bees Island. Mitochondrial DNA haplotype analysis supports this claim however, the most likely explanations for the presence of at least four microsatellite alleles in the Brampton, Newry and Rabbit Island populations that were not found in St Bees Island koalas are that either there were additional introductions from elsewhere or that koalas were already present on these islands prior to the transfers from St Bees Island. A study on the prevalence of chlamydial and retroviral infection in captive and wild koalas showed that 100% of koalas in this study had retroviral and chlamydial infection in at least one tested swab site. None of the captive animals showed clinical signs of disease despite the high prevalence of infection. Signs of disease were evident in some wild koalas, which, in some cases, was the reason for their admission to Moggill Koala Hospital for treatment. In an attempt to understand the immunological mechanisms underlying the apparent disease resistance of koalas which usually limits latent infection progressing to clinical disease states, the MHC genes of koalas were investigated. Low variation at MHC loci is thought to increase susceptibility to infectious diseases because fewer foreign antigens are recognised. The first class II sequences identified from koalas are presented here and showed variation. Variation was also detected at exon 3 of class I, with evidence for at least three class I loci. The koala is a unique Australian marsupial that makes a nationally significant contribution to the Australian economy annually. The results of this study have important conservation and management implications for koala populations in Southeast Queensland and Northeast New South Wales - particularly declining populations in Southeast Queensland - as well as being informative on other aspects of the species’ biology and for mammalian conservation genetics, generally.

Cold-adapted (ca) live attenuated influenza vaccines (LAIVs) have been introduced as alternatives to existing inactivated influenza vaccines. The influenza A components of the FDA-approved ca LAIVs (Flumist®; Medimmune) have common internal genes derived from the donor strain A/Ann Arbor/6/60 ca and surface genes derived from current wild-type (wt) epidemic strains. The aim of this thesis was to investigate determinants of immunogenicity for reassortants of A/Ann Arbor/6/60 ca, using a range of immunological assays, including recently developed MHC tetramer techniques. From the study, the extent of viral replication in the respiratory tract of mice, the primary site of inoculation, was a key factor in determining ca vaccine immunogenicity. Replication was shown to be influenced by both viral surface Ags and the host MHC. The H3 ca reassortants CR6, CR18, CR29 and CR6-35* exhibited greater replication efficiency (as determined by their PFU:HAU ratios) than the H1 ca reassortants CR35 and CR6-35. The H3 ca reassortant CR6 caused a 3.79% loss in body weight but no losses were observed for the H1 ca reassortant CR35 and the ca H2N2 donor strain A/Ann Arbor/6/60 ca. Higher HI responses were detected after 3 weeks in groups infected with the H3 ca reassortant CR6 (GMT 80) than with the H1 reassortant CR35 (GMT 10) and the H2 ca donor strain A/Ann Arbor/6/60 ca (GMT 13). Recently developed techniques were used to evaluate specific T-cell response to ca LAIVs. Fluorescent-labelled tetramer is the key reagent for use in tetramer-based flow cytometry assays. The NP366-374 peptide of influenza A viruses comprises an immunodominant epitope that is highly conserved between subtypes. Tetramers developed for A/PR/8/34 (H1N1) were able to detect NP-specific cytotoxic T lymphocytes (CTLs) induced by A/Ann Arbor /6/60 ca (H2N2). An attempt to prepare the A/Ann Arbor/6/60 ca-specific-NP-tetramer is described. H-2Db monomers were successfully refolded with the peptide, but only 20% were able to form tetramers through biotin-streptavidin linkage, resulting in a poor capacity to stain. By contrast, an IFN-γ ICC assay developed in parallel demonstrated that peptide NP366-374 was able to restimulate A/Ann Arbor/6/60 NP ca-specific CTLs and secrete IFN-γ when tested in vitro. Specific-B and T cell responses induced in the lungs in response to infection by ca reassortants exhibited great variability that was determined by the growth characteristics of different viruses. Type I (CTL) responses were induced by low yielding ca reassortants, such as CR35 (H1N1). Viruses with enhanced growth characteristics, such as CR6 (H3N2), produced higher Type II (HA-specific Ab) responses. In addition, host factors, such as MHC type, were found to play an important role in responses to the same viruses. Susceptible mouse strains, such as C57BL/6, showed higher CTL but lower serum Ab responses than more resistant strains, such as BALB/c. Throughout this PhD project, a fine balance between the humoral and CMI, local and systemic immune responses induced by ca LAIVs was demonstrated. The need to assess local immune responses, in addition to serum antibody levels, for the evaluation of vaccine efficacy was an important conclusion of the thesis.

Thesis (Ph. D.)--University of Missouri--Columbia, 1996. / "December 1996." Typescript. Vita. Includes bibliographical references (leaves [128]-155). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 1999. / "May 1999." Typescript. Vita. Includes bibliographical references (leaves 199-250). Also available on the Internet.

Tübingen, Univ., Diss., 2001.

Tübingen, Univ., Diss., 2005.

Tübingen, Univ., Diss., 2005.

Würzburg, Univ., Diss., 2007. / Zsfassung in engl. Sprache.

University, Diss., 2001--Köln.

Universiẗat, Diss., 2002--Bonn.