The CARD9 Gene in Koalas (Phascolarctos cinereus): Does It Play a Role in the Cryptococcus-Koala Interaction?

Cryptococcus is a genus of fungal pathogens that can infect and cause disease in a range of host species and is particularly prominent in koalas (Phascolarctos cinerus). Like other host species, koalas display a range of outcomes upon exposure to environmental Cryptococcus, from external nasal colonization to asymptomatic invasive infection and, in rare cases, severe clinical disease resulting in death. Host factors contributing to these varied outcomes are poorly understood. Due to their close relationship with eucalypt trees (a key environmental niche for Cryptococcus gattii) and suspected continual exposure to the pathogen, koalas provide a unique opportunity to examine host susceptibility in natural infections. Caspase recruitment domain-containing protein 9 (CARD9) is a key intracellular signaling protein in the fungal innate immune response. Humans with mutations in CARD9 succumb to several different severe and chronic fungal infections. This study is the first to sequence and explore CARD9 variation in multiple koalas using Sanger sequencing. Four CARD9 exons were successfully sequenced in 22 koalas from a New South Wales, Australia population. We found minimal variation between koalas across all four exons, an observation that was also made when CARD9 sequences were compared between koalas and six other species, including humans and mice. Ten single-nucleotide polymorphisms (SNP) were identified in this study and explored in the context of cryptococcal exposure outcomes. While we did not find any significant association with variation in cryptococcal outcomes, we found a high degree of conservation between species at several SNP loci that requires further investigation. The findings from this study lay the groundwork for further investigations of CARD9 and Cryptococcus both in koalas and other species, and highlight several considerations for future studies.

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.

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-1ß, 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.

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.