Koala Populations Research Articles

Using bioinformatics to investigate functional diversity: a case study of MHC diversity in koalas.

Conservation genomics can greatly improve conservation outcomes of threatened populations, including those impacted by disease. Understanding diversity within immune gene families, including the major histocompatibility complex (MHC) and toll-like receptors (TLR), is important due to the role they play in disease resilience and susceptibility. With recent advancements in sequencing technologies and bioinformatic tools, the cost of generating high-quality sequence data has significantly decreased and made it possible to investigate diversity across entire gene families in large numbers of individuals compared to investigating only a few genes or a few populations previously. Here, we use the koala as a case study for investigating functional diversity across populations. We utilised previous target enrichment data and 438 whole genomes to firstly, determine the level of sequencing depth required to investigate MHC diversity and, secondly, determine the current level of diversity in MHC genes in koala populations. We determined for low complexity, conserved genes such as TLR genes 10 × sequencing depth is sufficient to reliably genotype more than 90% of variants, whereas for complex genes such as the MHC greater than 20 × and preferably 30 × sequencing depth is required. We used whole genome data to identify 270 biallelic SNPs across 24 MHC genes as well as copy number variation (CNV) within class I and class II genes and conduct supertype analysis. Overall, we have provided a bioinformatic workflow for investigating variation in a complex immune gene family from whole genome sequencing data and determined current levels of diversity within koala MHC genes.

Immunisation of koalas against Chlamydia pecorum results in significant protection against chlamydial disease and mortality

In 2022, the Australian Government listed the koala as endangered in several states due to habitat destruction, traffic strikes, dog attacks, and Chlamydia pecorum disease. This study evaluates a 10-year assessment of a Major Outer Membrane Protein-based vaccine’s effectiveness against chlamydial disease in wild koalas from Southeast Queensland. Over a decade, 680 koalas were tracked, with five vaccine trials involving 165 koalas. While prior studies only offered up to two years of data, this study’s extended period allowed a thorough evaluation of vaccine efficacy. Results showed that vaccinated koalas had significantly lower disease incidence, with a 64% reduction in chlamydial mortality. This vaccine demonstrated positive impacts on both male and female koalas, highlighting its crucial role in conserving the Australian koala population and mitigating the threats they face.

Invasion and Amplification of Endogenous Retroviruses in Dasyuridae Marsupial Genomes.

Retroviruses are an ancient viral family that have globally coevolved with vertebrates and impacted their evolution. In Australia, a continent that has been geographically isolated for millions of years, little is known about retroviruses in wildlife, despite the devastating impacts of a retrovirus on endangered koala populations. We therefore sought to identify and characterize Australian retroviruses through reconstruction of endogenous retroviruses from marsupial genomes, in particular the Tasmanian devil due to its high cancer incidence. We screened 19 marsupial genomes and identified over 80,000 endogenous retrovirus fragments which we classified into eight retrovirus clades. The retroviruses were similar to either Betaretrovirus (5/8) or Gammaretrovirus (3/8) retroviruses, but formed distinct phylogenetic clades compared to extant retroviruses. One of the clades (MEBrv 3) lost an envelope but retained retrotranspositional activity, subsequently amplifying throughout all Dasyuridae genomes. Overall, we provide insights into Australian retrovirus evolution and identify a highly active endogenous retrovirus within Dasyuridae genomes.

Reversing the decline of threatened koala (Phascolarctos cinereus) populations in New South Wales: Using genomics to enhance conservation outcomes.

Genetic management is a critical component of threatened species conservation. Understanding spatial patterns of genetic diversity is essential for evaluating the resilience of fragmented populations to accelerating anthropogenic threats. Nowhere is this more relevant than on the Australian continent, which is experiencing an ongoing loss of biodiversity that exceeds any other developed nation. Using a proprietary genome complexity reduction-based method (DArTSeq), we generated a data set of 3239 high quality Single Nucleotide Polymorphisms (SNPs) to investigate spatial patterns and indices of genetic diversity in the koala (Phascolarctos cinereus), a highly specialised folivorous marsupial that is experiencing rapid and widespread population declines across much of its former range. Our findings demonstrate that current management divisions across the state of New South Wales (NSW) do not fully represent the distribution of genetic diversity among extant koala populations, and that care must be taken to ensure that translocation paradigms based on these frameworks do not inadvertently restrict gene flow between populations and regions that were historically interconnected. We also recommend that koala populations should be prioritised for conservation action based on the scale and severity of the threatening processes that they are currently faced with, rather than placing too much emphasis on their perceived value (e.g., as reservoirs of potentially adaptive alleles), as our data indicate that existing genetic variation in koalas is primarily partitioned among individual animals. As such, the extirpation of koalas from any part of their range represents a potentially critical reduction of genetic diversity for this iconic Australian species.

The Utility of the Koala Scat: A Scoping Review.

The use of samples or scats to provide important ecological, genetic, disease and physiology details on free-range populations is gaining popularity as an alternative non-invasive methodology. Koala populations in SE Queensland and NSW have recently been listed as endangered and continue to face anthropomorphic and stochastic environmental impacts that could potentially lead to their extinction. This scoping review examines the current and potential utility of the koala scat to contribute data relevant to the assessment of koala conservation status and decision making. Although we demonstrate that there is great potential for this methodology in providing details for both individual wild animal and population biology (distribution, abundance, sex ratio, immigration/emigration, genetic diversity, evolutionary significant unit, disease epidemiology, nutrition, reproductive status and stress physiology), the calibre of this information is likely to be a function of the quality of the scat that is sampled.

A retrospective epidemiological study of sarcoptic mange in koalas (Phascolarctos cinereus) using wildlife carer admission records

Outbreaks of sarcoptic mange are sporadically reported in koala populations across Australia, but disease characteristics (e.g., distribution across the body) remain poorly understood. In an area of Northern Victoria regular cases coming into care suggest mange may have become enzootic, and here we characterise those koala mange admission records. In 18% (n = 10) of mange affected koala reports that had a recorded outcome (n = 55), the animals died before the carers could locate them, and of the remaining 45 koalas that were alive upon carer arrival, 80% (n = 36) had to be euthanised due to severe mange. The number of admissions varied among years (highest observed in 2019), and over 60% of affected koala admissions were male. Male admissions peaked in austral spring and again in late austral summer-autumn (mating and birthing seasons), with female admissions only exhibiting the latter peak (birthing season). Fissures of the epidermis of the front paws occurred in 100% of admitted koalas, with 70% also showing these signs elsewhere on ventral surfaces or limbs. Only male koalas had signs of mange on the chest and face, and only female koalas had signs of mange on their back. Collectively, this study suggests sarcoptic mange can be a severe disease in koalas, and that male koalas may play an important role in seasonal transmission dynamics. We discuss how these findings may help inform intervention strategies.

Koala density, habitat, conservation, and response to logging in eucalyptus forest; a review and critical evaluation of call monitoring

ABSTRACT This study is the second in a series that examines the habitat requirements and response to logging of koalas (Phascolarctos cinereus) inhabiting tall eucalypt forests of north-east NSW. It presents the findings of koala population and habitat monitoring surveys in Pine Creek State Forest and Bongil Bongil National Park using a combination of call-counting and direct observation (spotlighting). The 6400 ha study area was mapped into 6 zones of increasing koala habitat quality by ground survey of forest structure and floristics on a 200 m grid. The accuracy of habitat definition and mapping was tested by stratified transect counts of koala calls and sightings over two consecutive years (1997–98). Average koala density increased steeply and significantly, from 0.02 – 0.20 koalas/hectare, with increasing mapped habitat quality based on increasing forest age, structural complexity, local food tree species diversity, history of prior koala occurrence and decreased past logging intensity. This relationship was driven primarily by breeding females, with the number of male koala calls weakly or uncorrelated with koala sightings and mapped habitat quality. Male koalas were more widely and uniformly distributed than females, including areas of low quality, plantation, and intensively logged forest. This finding explains the discrepancy between our results and those of other recent studies which concluded that koalas are tolerant of intensive logging based on modelling of calling male koalas and reliance on an untested assumption that male calling is indicative of female breeding success. Koala density in a subset of the highest quality habitat was relatively stable at 0.28 koalas/ha (3 hectares/koala) over the long term (1997–98 and 2012–2023). Key characteristics of the forest koala population, including low stable density, large home ranges, preference for high food tree diversity and locally unique food trees (including Allocasuarina torulosa and Syncarpia glomulifera), are not adequately explained by existing koala habitat models. We present a new paradigm to explain regional variation in koala distribution, habitat and foraging preferences based on variations in foliage chemistry (toxicity and nutritional value) determined by the duration and stability of local plant-koala interactions in response to past fire, hunting, predation and logging disturbance history. We hypothesize that koala density in stable forest populations is regulated at low levels by a combination of selected and induced increases in leaf toxicity and decreases in leaf nutrition that limit koala browsing to benign levels of about 1-2% of annual leaf production. Large home ranges, complex mature forest structure, high food tree diversity and a specialized or diverse gut microbiome may be essential to allow females to rotate and change food trees frequently to minimize induced toxicity and select individual leaves with sufficient nutrients to support breeding and lactation with minimal risk of predation. High density koala populations (> 0.6/ha) occur primarily in areas where koalas have been introduced or re-introduced to planted habitats and natural areas where aboriginal hunters and dingoes were historically present but are now absent, and where food trees have not been selected for resistance to koala browsing pressure.

Broad-scale acoustic monitoring of koala populations suggests metapopulation stability, but varying bellow rate, in the face of major disturbances and climate extremes.

Population trends are lacking for most threatened species, especially those that are cryptic and difficult to survey. Recent developments in passive acoustics and semi-automated call recognition provide a cost-effective option to systematically monitor populations of vocal species. We assessed recent trends for the koala Phascolarctos cinereus, an iconic marsupial, based on 7 years of acoustic monitoring across 224 forested sites. The study period overlapped with a severe drought and extensive megafires in 2019 followed by 2 years of extremely high rainfall. Dynamic occupancy modelling with a range of covariates at multiple landscape scales found that initial occupancy was related to elevation (-ve), NDVI (+ve) and previous selective harvesting (16-30-year age class; weakly +ve). Extinction probability increased with the extent of high-severity fire. Colonisation probability was related to a range of factors, with the top model showing a decrease with increasing lagged annual rainfall. However, the null model was also supported, suggesting weak associations for colonisation. Using these relationships, koala occupancy was found to be high and stable over the study period. Fire did not influence regional trends because koalas often persisted with low- to moderate-severity fire and because high-severity fire was limited to 11% of their habitat. In contrast, bellow rate varied across years, being initially low and declining immediately after the 2019 fires, with the driver of these changes unclear. Neither timber harvesting nor low-severity fire influenced koala occupancy or bellow rate. Given the extensive area of koala habitat in the region, our results point to the presence of a large population in these public forests, and in recent years, stable occupancy (albeit with site-scale reductions in density with high-severity fire). Ongoing monitoring is crucial for tracking future changes, especially with predictions of more frequent, severe forest fires due to climate change.

Koala admissions to a wildlife hospital in coastal New South Wales, Australia, over a nine-year period, 2014–2022

The koala (Phascolarctos cinereus) population in Australia has been subjected to numerous threats leading to a significant decline in their numbers. The Port Macquarie region serves as a vital habitat for these iconic marsupials. Analysing records of 1227 koalas admitted to the Port Macquarie Koala Hospital (2014–2022), this study aimed to understand admission causes, temporal trends, and risk factors. Anthropogenic activities accounted for almost half (49.7%) of all admissions with ‘dangerous area’ and ‘motor vehicle accident’ (MVA) being common aetiologies. Young koalas were more susceptible to ‘dangerous area’, ‘MVA’, and ‘dog attack’ than other age groups. Koalas admitted for suspected chlamydia were also a significant contributing factor for admissions, and was most common in young koalas. Overall, the majority of koalas were candidates for rehabilitation or release (54.3%), but mature and aged koalas had the highest odds for mortality. This study highlights the ongoing impact of anthropogenic activities on koalas, emphasises the need for public education and reporting to mitigate risks, and underscores the importance of addressing diseases like chlamydia. These results contribute to our understanding of the factors influencing koala hospital admissions and can inform conservation and management strategies for this iconic Australian species.

Novel typing scheme reveals emergence and genetic diversity of Chlamydia pecorum at the local management scale across two koala populations

To overcome shortcomings in discriminating Chlamydia pecorum strains infecting the koala (Phascolarctos cinereus) at the local level, we developed a novel genotyping scheme for this pathogen to inform koala management at a fine-scale subpopulation level. We applied this scheme to two geographically distinct koala populations in New South Wales, Australia: the Liverpool Plains and the Southern Highlands to South-west Sydney (SHSWS). Our method provides greater resolution than traditional multi-locus sequence typing, and can be used to monitor strain emergence, movement, and divergence across a range of fragmented habitats. Within the Liverpool Plains population, suspected recent introduction of a novel strain was confirmed by an absence of genetic diversity at the earliest sampling events and limited diversity at recent sampling events. Across the partially fragmented agricultural landscape of the Liverpool Plains, diversity within a widespread sequence type suggests that this degree of fragmentation may hinder but not prevent spread. In the SHSWS population, our results suggest movement of a strain from the south, where diverse strains exist, into a previously Chlamydia-free area in the north, indicating the risk of expansion towards an adjacent Chlamydia-negative koala population in South-west Sydney. In the south of the SHSWS where koala subpopulations appear segregated, we found evidence of divergent strain evolution. Our tool can be used to infer the risks of strain introduction across fragmented habitats in population management, particularly through practices such as wildlife corridor constructions and translocations.

GPS tracking reveals koalas Phascolarctos cinereus use mosaics of different forest ages after environmentally regulated timber harvesting

AbstractAn accurate picture of an animal's home range is fundamental for quantifying habitat quality and response to disturbance. When combined with remote sensing of vegetation attributes, there is potential to assess habitat selection at high resolution. We used a high‐quality GPS‐collaring data set (> 12 500 fixes) from 10 koalas Phascolarctos cinereus and a canopy height model derived from LiDAR in multiple‐use forests harvested for timber 5–10 years previously. Our aim was to assess how individual koalas use the forest mosaic created by timber harvesting by quantifying home ranges and habitat selection of different forest age/height classes created by past harvesting. We found that koalas maintained a stable home range over the average of 7 months of tracking. On average, there was 95% overlap for individuals among seasons, illustrating high site fidelity in the regenerating forest. Also, there were no apparent shifts during the intense drought of spring 2019. Male home ranges (64 ha) were three times the size of females (21 ha). Core areas were considerably smaller, ranging from 15 (male) to 6 ha (female). Three forest age/height classes were defined from LiDAR: (i) harvest – regeneration <14 m, (ii) harvest – retained trees ≥14 m and (iii) harvest – exclusion trees ≥14 m (zones excluded from harvesting for environmental protection). Home ranges covered the mosaic of forest classes and mixed models revealed no selection of forest class based on use versus availability. High site fidelity with no clear selection for forest age/height class (i.e. harvested and non‐harvested patches) confirms that resource quality in the home range remained sufficient for breeding koalas in the post‐harvest landscape. Ongoing monitoring of koalas is required to ensure that environmental protections are effective in maintaining koala populations in the face of additional disturbances from fire and climate change.

A novel multi-variate immunological approach, reveals immune variation associated with environmental conditions, and co-infection in the koala (Phascolarctos cinereus)

External signs of disease are frequently used as indicators of disease susceptibility. However, immune profiling can be a more effective indicator to understand how host responses to infection may be shaped by host, pathogen and environmental factors. To better inform wildlife health assessment and research directions, we investigated the utility of a novel multivariate immunophenotyping approach examining innate and adaptive immune responses in differing climatic, pathogen co-infection and demographic contexts across two koala (Phascolarctos cinereus) populations in New South Wales: the Liverpool Plains (LP), and Southern Highlands to South-west Sydney (SHSWS). Relative to the comparatively healthy SHSWS, the LP had greater and more variable innate immune gene expression (IL-1β, IL-6), and KoRV transcription. During extreme heat and drought, koalas from the LP displayed upregulation of a stress pathway gene and reduced adaptive immune genes expression, haematocrit and plasma protein, suggesting the possibility of environmental impacts through multiple pathways. In those koalas, KoRV transcription status, Chlamydia pecorum infection loads, and visible urogenital inflammation were not associated with immune variation, suggesting that immune markers were more sensitive indicators of real-time impacts than observed disease outcomes.

Megafires and koala occurrence: a comparative analysis of field data and satellite imagery

Megafires can have a devastating effect on koala populations. With climate change increasing habitat vulnerability to wildfires, understanding how to efficiently measure the impact of these events on koalas is essential. We analysed the relationship between the 2019-2020 megafires and the probability of koala occurrence in Mid North Coast NSW. We found that two on-field and one satellite-derived variables measuring fire severity equally explained koala occurrence. The probability of koala occurrence decreased with increasing fire severity. This supports the use of remote sensing imagery to efficiently monitor the impact of future fire events on koala populations in the region.

Contrasting segregation patterns among endogenous retroviruses across the koala population

Koalas (Phascolarctos cinereus) have experienced a history of retroviral epidemics leaving their trace as heritable endogenous retroviruses (ERVs) in their genomes. A recently identified ERV lineage, named phaCin-β, shows a pattern of recent, possibly current, activity with high insertional polymorphism in the population. Here, we investigate geographic patterns of three focal ERV lineages of increasing estimated ages, from the koala retrovirus (KoRV) to phaCin-β and to phaCin-β-like, using the whole-genome sequencing of 430 koalas from the Koala Genome Survey. Thousands of ERV loci were found across the population, with contrasting patterns of polymorphism. Northern individuals had thousands of KoRV integrations and hundreds of phaCin-β ERVs. In contrast, southern individuals had higher phaCin-β frequencies, possibly reflecting more recent activity and a founder effect. Overall, our findings suggest high ERV burden in koalas, reflecting historic retrovirus-host interactions. Importantly, the ERV catalogue supplies improved markers for conservation genetics in this endangered species.

Cryptosporidium-associated enteritis in captive koalas (Phascolarctos cinereus).

Cryptosporidium spp. sporadically infect a range of Australian native mammals including koalas, red kangaroos, eastern grey kangaroos, bilbies and brush tailed possums and can range from asymptomatic to fatal infections. Traditionally considered a disease of the young or immuno-compromised, and resulting in profuse diarrhoea in other species, here we report an atypical clinical syndrome associated with Cryptosporidium in a captive population of koalas. All affected animals were in-contact adults, and demonstrated anorexia, dehydration and abdominal pain in the absence of diarrhoea. Following euthanasia on welfare grounds, Cryptosporidium infection was confirmed postmortem in three of four symptomatic animals via faecal floatation and/or intestinal histopathology, with enteritis also diagnosed in the fourth koala. Further screening of the captive colony found the outbreak had been contained. Based on sequencing the cause of the infection was C. fayeri, but the source was undetermined. In conclusion, Cryptosporidium should be considered as a possible cause of generalised illness in koalas.

Social valuation of biodiversity relative to other types of assets at risk in wildfire.

Environmental crises, such as wildfires, can cause major losses of human life, infrastructure, biodiversity, and cultural values. In many such situations, incident controllers must make fateful choices about what to protect-and hence what to abandon. With an online representative survey of >2000 adult Australians, we investigated social attitudes to this dilemma. We used best-worst scaling to assess preferences across a set of 11 assets representing human life, infrastructure, biodiversity, and cultural values. Survey respondents overwhelmingly prioritized a single human life (best-worst score of 6647 out of possible score ranging from -10695 to 10695), even if that choice resulted in extinction of other species. Inanimate (replaceable) objects were accorded lowest priority (best-worst scores of -4655 for a shed and -3242 for a house). Among biodiversity assets, respondents prioritized protecting a population of the iconic koala (Phascolarctos cinereus) (best-worst score of 1913) ahead of preventing the extinction of a snail (score -329) and a plant species (-226). These results variably support current policy in that they emphasize the importance the community places on protection of human life, but results diverged from conventional practice in rating some biodiversity assets ahead of infrastructure. The preference for protecting a population of koalas ahead of action taken to prevent the extinction of an invertebrate and plant species corroborates previous research reporting biases in the way people value nature. If noncharismatic species are not to be treated as expendable, then the case for preventing their extinction needs to be better made to the community. Given the increasing global incidence of high-severity wildfires, further sampling of societal preferences among diverse asset types is needed to inform planning, policy, and practice relating to wildfire. Other preemptive targeted management actions (such as translocations) are needed to conserve biodiversity, especially noniconic species, likely to be imperiled by catastrophic events.

Immunohistochemical characterization of the immune cell response during chlamydial infection in the male and female koala (Phascolarctos cinereus) reproductive tract.

Chlamydiosis is one of the main causes of the progressive decline of koala populations in eastern Australia. While histologic, immunologic, and molecular studies have provided insights into the basic function of the koala immune system, the in situ immune cell signatures during chlamydial infection of the reproductive tract in koalas have not been investigated. Thirty-two female koalas and 47 males presented to wildlife hospitals with clinical signs suggestive of Chlamydia infection were euthanized with the entire reproductive tract collected for histology; immunohistochemistry (IHC) for T-cell (CD3ε, CD4, and CD8α), B-cell (CD79b), and human leukocyte antigen (HLA)-DR markers; and quantitative real-time polymerase chain reaction (rtPCR) for Chlamydia pecorum. T-cells, B-cells, and HLA-DR-positive cells were observed in both the lower and upper reproductive tracts of male and female koalas with a statistically significant associations between the degree of the inflammatory reaction; the number of CD3, CD4, CD79b, and HLA-DR positive cells; and the PCR load. CD4-positive cells were negatively associated with the severity of the gross lesions. The distribution of immune cells was also variable according to the location within the genital tract in both male and female koalas. These preliminary results represent a step forward towards further exploring mechanisms behind chlamydial infection immunopathogenesis, thus providing valuable information about the immune response and infectious diseases in free-ranging koalas.

Expanding the known distribution of phascolartid gammaherpesvirus 1 in koalas to populations across Queensland and New South Wales

Koala populations across the east coast of Australia are under threat of extinction with little known about the presence or distribution of a potential pathogen, phascolartid gammaherpesvirus 1 (PhaHV-1) across these threatened populations. Co-infections with PhaHV-1 and Chlamydia pecorum may be common and there is currently a limited understanding of the impact of these co-infections on koala health. To address these knowledge gaps, archived clinical and field-collected koala samples were examined by quantitative polymerase chain reaction to determine the distribution of PhaHV-1 in previously untested populations across New South Wales and Queensland. We detected PhaHV-1 in all regions surveyed with differences in detection rate between clinical samples from rescued koalas (26%) and field-collected samples from free-living koalas (8%). This may reflect increased viral shedding in koalas that have been admitted into care. We have corroborated previous work indicating greater detection of PhaHV-1 with increasing age in koalas and an association between PhaHV-1 and C. pecorum detection. Our work highlights the need for continued surveillance of PhaHV-1 in koala populations to inform management interventions, and targeted research to understand the pathogenesis of PhaHV-1 and determine the impact of infection and co-infection with C. pecorum.

Koalas in space and time: Lessons from 20 years of vehicle‐strike trends and hot spots in South East Queensland

AbstractThe localized clustering of wildlife vehicle‐strike in space and time, often called ‘hot spots’, are commonly used to guide placement of road mitigation installations. We interrogated koala vehicle‐strike data across a 20‐year period between 1997 and 2016, examining trends in identified hot spots. We found that hot spots were not static and decline or emerge in line with the resultant pressures (e.g., from development and associated traffic increases) being exerted on koala populations within the surrounding landscape. We suggest that there is likely to be a relationship between hot spots waning over time and localized extinction of koalas. We feel strategies that solely focus on hot spot intervention will do little to halt and reverse the significant decline in the koala population across South East Queensland because they are almost always retrospective. It is imperative that regional wildlife movement solutions, around and across roads, are appropriately planned and implemented ahead of time (i.e., during initial construction/developmental expansion), if they are to serve as effective mitigation for remaining local koala population across South East Queensland. We caution against recommending mitigation based solely on historical vehicle‐strike data without considering contemporary population, movement and behavioural data.

Genetic markers of Chlamydia pecorum virulence in ruminants support short term host-pathogen evolutionary relationships in the koala, Phascolarctos cinereus

In ruminants infected with Chlamydia pecorum, shorter lengths of coding tandem repeats (CTR) within two genes, the inclusion membrane protein (incA) and Type III secretor protein (ORF663), have been previously associated with pathogenic outcomes. In other chlamydial species, the presence of a chlamydial plasmid has been linked to heightened virulence, and the plasmid is not ubiquitous in C. pecorum across the koala's range. We therefore investigated these three markers: incA, ORF663 and C. pecorum plasmid, as potential indicators of virulence in two koala populations in New South Wales with differing expression of urogenital chlamydiosis; the Liverpool Plains and one across the Southern Highlands and South-west Sydney (SHSWS). We also investigated the diversity of these loci within strains characterised by the national multi-locus sequence typing (MLST) scheme. Although CTR lengths of incA and ORF663 varied across the populations, they occurred only within previously described pathogenic ranges for ruminants. This suggests a relatively short-term host-pathogen co-evolution within koalas and limits the utility of CTR lengths for incA and ORF663 as virulence markers in the species. However, in contrast to reports of evolution of C. pecorum towards lower virulence, as indicated by longer CTR lengths in ruminants and swine, CTR lengths for ORF663 appeared to be diverging towards less common shorter CTR lengths within strains recently introduced to koalas in the Liverpool Plains. We detected the plasmid across 90% and 92% of samples in the Liverpool Plains and SHSWS respectively, limiting its utility as an indicator of virulence. It would be valuable to examine the CTR lengths of these loci across koala populations nationally. Investigation of other hypervariable loci may elucidate the evolutionary trajectory of virulence in C. pecorum induced disease in koalas. Profiling of virulent strains will be important in risk assessments for strain movement to naïve or susceptible populations through translocations and wildlife corridor construction.