The decline in koala (Phascolarctos cinereus) populations has been significantly driven by infectious diseases, with chlamydial disease contributing to this trend. Chlamydia pecorum is often codetected with viruses such as phascolarctid gammaherpesvirus 1 and 2 (PhaHV-1 and PhaHV-2). Koalas can also be infected with other bacteria, including Bordetella bronchiseptica, which causes sporadic respiratory disease outbreaks. Respiratory infections and respiratory disease in koalas remain under-investigated. This study reports the detection of C. pecorum, Chlamydia pneumoniae, Chlamydia psittaci, B. bronchiseptica, PhaHV-1, and PhaHV-2 in 102 samples from 49 koalas that presented to veterinary facilities in South East Queensland, Australia from 2018 to 2023. The koalas included seemingly healthy individuals (n=21), koalas with respiratory disease (n=18), and koalas with other diseases (n=10). Overall, C. pecorum was detected in 37% of koalas, C. pneumoniae in 2%, C. psittaci in 0%, B. bronchiseptica in 18%, PhaHV-1 in 41%, and PhaHV-2 in 6%. Coinfections with three agents were common, particularly in koalas with signs of disease. Among the 18 koalas with respiratory disease, one was coinfected with four agents (B. bronchiseptica, C. pecorum, PhaHV-1, and PhaHV-2), and four were coinfected with three agents (B. bronchiseptica, C. pecorum, and PhaHV-1). Additionally, six koalas had coinfections involving two agents: two with C. pecorum and PhaHV-1, two with B. bronchiseptica and PhaHV-1, one with B. bronchiseptica and C. pecorum, and one with PhaHV-1 and PhaHV-2. Analysis of the genetic diversity of infecting chlamydial strains detected in koalas with respiratory and other diseases, based on the full-length ompA gene, identified previously characterized C. pecorum and C. pneumoniae ompA genotypes, as well as a unique C. pecorum ompA genotype. This study highlights the need for incorporating these infectious agents into koala respiratory diagnostics and emphasizes the need for continued research to investigate the complexities of these infections.
Each year, hundreds of female koalas are presented to koala hospitals suffering from a range of morbidities, many of which require euthanasia for animal welfare reasons. These koalas represent a possible resource for genetic recovery by means of oocyte retrieval for genome banking or use in assisted reproductive technology. To examine the feasibility of koala oocyte recovery, this study conducted a preliminary survey of follicular activity and disease presence in fixed ovarian tissues from koala cadavers in South East Queensland. Ovarian activity and pathology were assessed by gross examination and histology. Bursal pathology was categorized into koalas with no, small (<10 mm diameter), moderate (10-20 mm diameter), or large (>20 mm diameter) sized bursae, whereas uterine pathology was diagnosed by an experienced reproductive pathologist. Antral follicles were observed in 94.4% of ovaries recovered from koalas with no bursal or uterine pathology (n = 18/44), 95.2% of the ovaries of koalas with bursal but no uterine pathology (n = 11/44), 100% of the ovaries of koalas showing only uterine pathology (n = 4/4) and 89.5% of ovaries from koalas with both bursal and uterine pathology (n = 11/44). Of the fixed ovarian tissue suitable for PCR Chlamydia detection (35/44), none were positive. As proof of concept, oocytes were also collected and evaluated from six koala cadavers within 2 h post-mortem. Although further studies are required to determine the quality and viability of the retrieved koala oocytes, our preliminary survey provides strong evidence that ovarian activity mostly continues unabated, irrespective of reproductive pathology, and that oocytes can be recovered successfully.
Gut microbiome has a profound influence on koalas' health. Yet, the relationships among the gut bacteriome, virome, antibiotic resistome, and pathogenicity throughout different stages in koala's life remain elusive. Here, we presented a metagenome-resolved survey of gut microbiome utilizing 75 fecal samples from three groups of captive koalas. The diversity of bacteriome and virome were age-dependent, predominating in adult koalas. Lytic viruses increased with age as lysogenic viruses and bacterial hosts declined, and virus-to-microbe ratios rose, revealing concomitant age-related shifts in microbial communities, though causality remains unresolved. Antibiotic resistance genes (ARGs) were more prevalent in young koalas, unlike in humans, where they accumulate with age. Two ARG-carrying pathogens, Klebsiella pneumoniae and Escherichia coli, were identified and cultured, with K. pneumoniae and E. coli predominating in young koalas. One age-dependent lytic virus infecting K. pneumoniae only detected in young koalas, and two lysogenic viruses infecting E. coli identified the in young and adult koalas. Analyses showed a positive correlation between mobile genetic elements (MGEs) and virulence factors (VFs), which facilitated the widespread dissemination of VFs and impacted health. Collectively, this study advances the understanding of gut microbiome in health, providing solutions to the treatment and management of captive koalas.
Following bushfires (also known as wildfires), impacted free-living wildlife with welfare or conservation concerns are captured and presented for veterinary assessment where possible. This study represents an in-depth retrospective analysis of the veterinary records of 259 animals in Victoria, Australia, impacted by bushfire in 2019-2020. In total, 35 different species were assessed, including 196 koalas. Multivariable analyses of 126 koalas with complete medical records identified several clinical prognostic factors affecting 6-month survival outcomes. Increased odds of negative outcomes (death or euthanasia) were associated with increasing age (tooth wear class; odds ratio 2.70 for one unit increase), lower body condition score (one-unit decrease OR 7.27), and the earlier animals were presented after the fire event (OR 0.94 for each passing day). In 83 koalas with burn injuries, negative outcomes were also associated with burns more severe than minor (85% survival for minor burns only, compared to 31% survival with moderate or severe burns), and burns to more than 10 digits (12% survival). In burnt koalas, the combination of burn severity and digital involvement appear to be important prognostic factors for long-term outcomes. These findings can support veterinarians to more accurately evaluate prognosis for bushfire-affected koalas during initial assessment and will facilitate the strategic allocation of limited treatment and rehabilitation resources to the animals most likely to recover. The scope of this study was limited to the consideration of health outcomes, with the recognition of health as just one of many factors that must inform decisions about rehabilitating injured wildlife.
The hypothalamic-pituitary-adrenal axis responds to stress by releasing the adrenocorticotropic hormone which, in turn, stimulates the release of glucocorticoids (GCs). During acute stress events, the GCs' function is to maintain homeostasis. Short-term stress events trigger psychophysiological responses which are fundamental for survival in the natural world. Chronic stress occurs when the effect of stressors persists, and GCs secretion continues. The increase of GCs interacts with receptors in the brain triggering a negative feedback loop which inhibits the secretion of ACTH, consequently down-regulating GC production. Cortisol is the main GC in most mammals, including koalas. Cortisol is metabolised by the liver and bacterial enzymes in the intestine, and its metabolites are excreted via the faeces. In a previous study, where we used the tetrahydrocorticosterone) enzyme immunoassay (aka 50c EIA) for measuring faecal cortisol metabolites (FCMs) to assess stress in koalas, we did not detect a decrease in FCM values (negative feedback) after administration of prednisolone, an exogenous GC. Using the dexamethasone suppression test (DST), this study aimed at determining the ability of the feedback loop to decrease the concentration of plasma cortisol. This was achieved by measuring the values of plasma cortisol as well as FCMs using the 50c EIA in four koalas. No cortisol suppression was observed, rather an increase in plasma cortisol concentration in all koalas. This was also reflected in the increase of FCMs. An unresponsive feedback loop, and consequent prolonged high levels of plasma cortisol, is likely to increase koalas' susceptibility to diseases and it may impact their coping mechanism in nature.
Chlamydiosis poses a significant threat to koala health and population viability. Accurate diagnosis of chlamydial infection in koalas at veterinary hospitals informs patient prognosis, biosecurity and determines treatment. However, there is no standardised approach to the diagnosis of chlamydiosis in wildlife hospitals. The performance of available diagnostic tests has not previously been rigorously assessed, and there is a lack of sufficient well-characterised reference samples. This study aimed to estimate the diagnostic accuracy of four molecular detection assays for Chlamydia pecorum infection in koalas using Bayesian latent class analysis (BLCA). Swabs were collected from koalas presented to a wildlife hospital (n = 45) and historical population surveys (n = 48). The four assays that were compared were a qPCR with melt curve that targets 16S rRNA (16SG), a probe-based qPCR assay that targets ompB, and two loop-mediated isothermal amplification (LAMP) assays, targeting MreC and a hypothetical protein (CpecG_0573). The 16SG qPCR and CpecG_0573 LAMP showed the highest overall diagnostic accuracy, with Youden's indices of 83.8 % (95 % posterior credible interval [95 % PCI]: 66.8 %, 93.4 %), and 81.5 % (95 % PCI: 62.5 %, 93.7 %) respectively. The underlying true prevalence of infection in hospitalised koalas (i.e., the latent class) was estimated to be 35.2 % (95 % PCI 21.4, 51.2 %). Combining the 16SG qPCR with the CpecG_0573 LAMP assay at this estimated infection prevalence yielded an excellent positive predictive value (PPV) > 96 %. Therefore, a testing protocol applying these tests in series is recommended for diagnosing chlamydiosis in this population of hospitalised koalas.
The koala (Phascolarctos cinereus), an iconic Australian marsupial, has experienced substantial historical and contemporary population declines. Identifying the drivers of these declines has been hindered by limited genomic data and uncertainty regarding the koala mutation rate. Here, we report a direct estimate of the koala mutation rate, based on genome sequences of four parent-offspring trios, yielding a mean of 6.12 × 10-9 mutations per base pair per generation (95% confidence interval: 5.03 to 7.45 × 10-9). Using this estimate of the mutation rate, we reconstructed the demographic history of koalas using 457 whole-genome sequences sampled across their entire range. Our results refine the estimated timing of past changes in population size, suggesting a large decline beginning ∼100 kya, before the arrival of humans in Australia. The koala population then split into five genetic populations 6 to 30 kya, which are now distributed along the east coast of Australia. We also use our estimate of the mutation rate to infer recombination maps for each koala population, confirming lower recombination rates in marsupials than in eutherian mammals. Using these estimates of population-specific recombination rates, we inferred the timing of recent population declines for koalas across all eastern states. These findings provide critical insights into the evolutionary history of koalas, while highlighting the impacts of using species-specific estimates of evolutionary rates on the inference of demographic histories. Our estimates of the genome-wide mutation rate and population-specific recombination maps for koalas provide valuable resources for future evolutionary and conservation analyses of marsupials.
Koalas are a native Australian species vulnerable to bushfire events, with 40,000 koalas estimated to have perished during the 2019-2020 summer bushfire event on Kangaroo Island. Determining the distribution and severity of burns is an important component in the treatment of burns in both human and veterinary patients. This project outlines the creation of a clinical chart to evaluate the surface area, distribution and severity of burns in koalas. This was achieved by three-dimensional computed tomographic segmentation to evaluate the surface areas of the whole koala and its external anatomy. The external anatomy was segmented into regions based on vulnerability to thermal injury. Segmentation and proportional surface area data are combined with an illustrated chart to create a chart-based triage tool. This clinical tool can be used to evaluate and document burn-affected surface area, distribution and severity to quantitatively inform prognosis and decision-making when triaging koalas during an Australian bushfire.
Abnormal vertebral column curvature is sporadically reported in koalas of the Mount Lofty Ranges, South Australia. This study evaluates the imaging features of 23 koalas from the Mount Lofty Ranges presenting with abnormal vertebral column curvature between 2015 and 2023 using digital radiography and computed tomography (CT). All images were evaluated by four reviewers to assess curve morphology, severity and Cobb angles for both scoliosis and kyphosis. For Cobb angle measurement, radiography performed similarly to CT with good agreement as measured by intraclass correlation coefficient (0.835 and 0.825 respectively) and Lin's concordance correlation coefficient (>0.85). The apex vertebra was always located between T7 and L6. For both scoliosis and kyphosis apex vertebrae, the thoracolumbar region was the most common location (8/22 and 9/19, respectively). For scoliosis, the caudal thoracic and lumbar regions were equally common (7/22 each), whereas for kyphosis, the caudal thoracic region (7/19) was more frequent than the lumbar region (3/19). Vertebral body rotation was a common component particularly in severely affected individuals, in which complex or 'S' shaped curves also occurred. Severity ranged from minimal or mild (6/23) to moderate (5/23) and severe (12/23), with simultaneous kyphosis and scoliosis present most frequently (21/23). As a result, the term, kyphoscoliosis is the most appropriate morphological description for abnormal vertebral curvature in koalas and may have pathophysiologic commonalities with human idiopathic scoliosis. This study is the first to describe imaging features of abnormal vertebral column curvature in koalas and evaluate inter-modality and interobserver agreement between radiography and CT.
Several infectious agents concurrently infect wild koalas and so, as for similar agents in other species, co-infection interactions could affect disease presentation and clinical outcomes. This study determines the frequency of circulating and mucosal Chlamydia pecorum infections along with phascolarctid herpesvirus (PhaHV), Koala retrovirus (KoRV), and trypanosome infections in 115 wild koalas admitted to wildlife hospitals in the Australian states of Queensland and New South Wales. C. pecorum, PhaHV, trypanosomes, and KoRV (endogenous subtype A and exogenous subtype D) were detected in 61.1%, 68.9%, 63.3% and 100% of the individuals sampled, respectively. The co-infection relationships identified generate hypotheses for the observed variation in disease presentations in that they resemble co-infection interactions that drive the variations in presentation and response to treatment for chlamydiosis in other species, including humans. Among koalas with chlamydiosis, PhaHV-1 mucosal shedding positively predicted euthanasia on admission, and accounting for Trypanosome irwini infection status improved the model quality. Additionally, in female koalas, the detection of mucosal PhaHV-1 and greater KoRV proviral pol loads were equal predictors of chlamydial reproductive disease. While the detection frequency of C. pecorum, PhaHV-1, PhaHV-2, and T. gilletti in circulation were low, cases with circulating C. pecorum and without mucosal C. pecorum shedding or clinical chlamydiosis were observed presenting an important consideration for future diagnostic testing. This study serves as a basis for investigating co-infection interaction pathways through mechanistic studies to determine their effect on pathogenesis of chlamydiosis, improve our understanding of host-pathogen-environment dynamics impacting the koala, and identify novel intervention and screening methods.
Koalas are the first wildlife species with a conditionally approved Chlamydia pecorum vaccine, offering a natural-host system for a pathogen whose human counterpart, Chlamydia trachomatis, lacks a licensed vaccine. Human vaccine efforts are slowed by uncertain immune correlates, asymptomatic infection, licensure-relevant disease end points, strict regulatory requirements, and limited commercial incentives, with only one candidate completing Phase 1. Koalas share these constraints, but visible ocular and urogenital disease enables clinical assessment under natural infection. Koala vaccine design integrated dominant circulating C. pecorum genotypes with a single-dose Tri-Adjuvant, evaluated for safety, immunogenicity, and clinical outcomes. Conditional approval under Australia's veterinary minor-use pathway relied on real-world evidence rather than controlled challenge studies. This opinion article highlights how natural-host evidence frameworks can inform intracellular pathogen vaccinology.
Koala retrovirus (KoRV) is a significant infectious agent impacting the health of wild and captive koala populations worldwide. While previous studies have explored its association with neoplastic diseases such as leukemia and lymphoma, its potential involvement in primary bone tumors remains unclear. This study aimed to expand our understanding of KoRV's disease spectrum by examining its potential association with primary bone tumors in koalas. Koala retrovirus proviral DNA load was analyzed in neoplastic bone using real-time quantitative polymerase chain reaction (PCR) and compared to healthy bone samples across 4 different gene targets: KoRV pol, KoRV-A env, KoRV-B env, and KoRV-D env. The relative KoRV subtype A proviral load was significantly lower in bone tumor samples (n = 14) when compared to healthy bone samples (n = 11) (P = .025), while other subtype-specific proviral loads did not differ significantly between tumor and healthy controls. In addition, we developed a novel immunohistochemistry assay to detect the KoRV capsid protein. Immunolabeling revealed KoRV capsid protein expression in all bone tumor samples (14/14, 100%), with an overall mean positive immunolabeling of 50.4% of tumor cells. The bone tumor group had a higher median H-score compared to the control group (P < .001). Among tumor subtypes, the highest mean percentage of tumor cell labeling was observed in osteosarcomas (73.0%), followed by chondrosarcomas (51.6%) and osteochondromas (38.0%). Collectively, these findings suggest that KoRV may have an important role in koala bone tumor oncogenesis, warranting further investigation into its potential as a contributing factor in tumor development.
Population bottlenecks can lead to evolutionary dead ends by eroding genetic diversity and intensifying inbreeding. Although theory predicts possible escape routes, direct observations of this process are rare. Using whole-genome data from 418 koalas, we found that populations with higher genetic diversity harbored the greatest mutational loads and had declining effective population sizes (Ne), whereas historically bottlenecked but recovering populations displayed reduced mutational load, exhibited increasing Ne, and regenerated new, rare variants. We concluded that this pattern was due to rapid demographic expansion, which reshuffled genetic variation through recombination and affected Ne more quickly than it did conventional diversity metrics. Our findings suggest that recovery of bottlenecked populations can occur through rapid demographic growth and that this can reestablish evolutionary potential in threatened populations.
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The koala (Phascolarctos cinereus) is an endangered species in parts of Australia, in part due to chlamydial infections. Treatment is difficult due to the hepatic metabolism of the koala, and the critical reliance on a gut microbiota for survival. This study aimed to identify new compounds for treatment of Chlamydia infections by screening a drug re-purposing library. Screening was conducted using an in vitro cell culture model prior to in vivo mouse infection model testing of two candidates identified from the in vitro screen. One lead, bisoprolol fumarate, showed an impact on chlamydial infection and burden in vitro and in vivo. Whilst the mechanism of action may not support progressing this lead further, the approach to screening the library and list of candidates may enable identification of other new koala treatments. This study demonstrates the potential to apply drug re-purposing to koala treatment and presents a list of candidates that could be explored further.
After more than 10 years of research, a one-shot vaccine has been developed to tackle chlamydia in one of Australia's most iconic species. Josh Loeb reports.
BACKGROUND: Species with specialised ecological niches rely heavily on specific resources or conditions, making them less resilient to habitat fragmentation and land-use changes. For specialists with limited mobility, the challenges are even greater, as they may struggle to locate new habitats for their survival. While some highly mobile species adjust their movement behaviours in human-modified environments by either avoiding areas with faster, straighter paths or adapting to forage for human-related resources, little is known about how arboreal species with low mobility adapt to urban landscapes. Koalas (Phascolarctos cinereus) are highly susceptible to the impacts of urbanisation due to their unique adaptations and reliance on tree canopy cover, which is thought to be a major factor driving population decline in the increasingly urbanised Australian coast. METHODS: In this study, we applied biotelemetry to track the movements of 72 koalas in urban and nonurban environments. We then applied hidden Markov models (HMMs) to these data to investigate how environmental factors (such as human land use), as well as biological factors (e.g., sex) and temporal cycles (e.g., time of day), influenced koala movement behaviours. RESULTS: We detected little effect of land use type on the movement behaviours of koalas in urban and nonurban landscapes, suggesting that the type of land use does not play a substantial role in how koalas shift between different movement behaviours. However, urban-dwelling koalas exhibited faster and more directed movements at night (rather than at dusk) and showed less pronounced changes in their movement behaviours across seasons than those typically observed in natural environments. CONCLUSIONS: Our findings highlight the adaptability and flexibility of koalas in modifying their movement behaviours to navigate human-modified environments. By focusing their movements during times when human activity is lower, koalas may be able to reduce the likelihood of agonistic interactions with humans. We suggest that creating low-disturbance areas in urban and peri-urban environments could allow wildlife to maintain more natural behaviours, potentially improving their overall well-being.