Chlamydia is the most common bacterial sexually transmitted infection worldwide and it is widely acknowledged that controlling the rampant community transmission of this infection requires vaccine development. In this study, for the first time, we elucidate the long-term response to male mouse chlamydial vaccination with chlamydial major outer membrane protein (MOMP) and ISCOMATRIX (IMX) both prophylactically and in a novel therapeutic setting. Vaccination significantly reduced and, in some cases, cleared chlamydial burden from the prostates, epididymides, and testes, which correlates with high IgG and IgA tires in tissues and serum. Important markers of sperm health and fertility were protected including sperm motility and proteins associated with fertility in men. Within splenocytes, expression of IFNγ, TNFα, IL17, IL13, IL10, and TGFβ were changed by both infection and vaccination within CD4 and CD8 T cells and regulatory T cells. Within the testicular tissue, phenotypic and concentration changes were observed in macrophages and T cells (resident and transitory). This revealed some pathogenic phenotypes associated with infection and critically that vaccination allows maintenance of testicular homeostasis, likely by preventing significant influx of CD4 T cells and promoting IL10 production. Finally, we demonstrated the testes contained immature (B220+) B cells and mature (CD138+) Chlamydia-specific plasma cells. Thus, through vaccination, we can maintain the healthy function of the testes, which is vital to protection of male fertility.

Australia is home to a rich biodiversity of marsupials that are found nowhere else. Unfortunately, many of these species are currently threatened with extinction due to introduced feral predators and other anthropogenic factors. There is growing recognition that host-associated microorganisms can play important roles for animal health, with billions of dollars currently being invested into human gut microbiome research and the development of microbiome-based therapeutics to improve human health. Can microorganisms also be harnessed to stem the tide of marsupial extinctions? In this review, we provide an overview of some of the challenges facing Australia’s marsupials, and our current understanding of the microbiology of the marsupial pouch. We also propose outstanding research questions pertaining to the marsupial pouch, which, if addressed, may provide actionable knowledge and novel microbial therapies that could help stem the tide of marsupial extinctions in Australia.

This thesis investigated how microbial communities present in koala pouches impact on reproductive outcomes through the sequencing and cultivation of microorganisms from longitudinal pouch samples. The results of this thesis provide a unique insight into potential microbial drivers of neonatal mortality, which is a growing issue at several captive breeding facilities. The findings have important implications for the treatment, prevention, and management of neonatal mortality in healthy breeding koala populations.

Background Captive koala breeding programmes are essential for long-term species management. However, breeding efficacy is frequently impacted by high neonatal mortality rates in otherwise healthy females. Loss of pouch young typically occurs during early lactation without prior complications during parturition and is often attributed to bacterial infection. While these infections are thought to originate from the maternal pouch, little is known about the microbial composition of koala pouches. As such, we characterised the koala pouch microbiome across the reproductive cycle and identified bacteria associated with mortality in a cohort of 39 captive animals housed at two facilities. Results Using 16S rRNA gene amplicon sequencing, we observed significant changes in pouch bacterial composition and diversity between reproductive time points, with the lowest diversity observed following parturition (Shannon entropy — 2.46). Of the 39 koalas initially sampled, 17 were successfully bred, after which seven animals lost pouch young (overall mortality rate — 41.18%). Compared to successful breeder pouches, which were largely dominated by Muribaculaceae (phylum — Bacteroidetes), unsuccessful breeder pouches exhibited persistent Enterobacteriaceae (phylum — Proteobacteria) dominance from early lactation until mortality occurred. We identified two species, Pluralibacter gergoviae and Klebsiella pneumoniae, which were associated with poor reproductive outcomes. In vitro antibiotic susceptibility testing identified resistance in both isolates to several antibiotics commonly used in koalas, with the former being multidrug resistant. Conclusions This study represents the first cultivation-independent characterisation of the koala pouch microbiota, and the first such investigation in marsupials associated with reproductive outcomes. Overall, our findings provide evidence that overgrowth of pathogenic organisms in the pouch during early development is associated with neonatal mortality in captive koalas. Our identification of previously unreported, multidrug resistant P. gergoviae strains linked to mortality also underscores the need for improved screening and monitoring procedures aimed at minimising neonatal mortality in future.