Association of maternal genetics with the gut microbiome and eucalypt diet selection in captive koalas.

Background: Koalas, an Australian arboreal marsupial, depend on eucalypt tree leaves for their diet. They selectively consume only a few of the hundreds of available eucalypt species. Since the koala gut microbiome is essential for the digestion and detoxification of eucalypts, their individual differences in the gut microbiome may lead to variations in their eucalypt selection and eucalypt metabolic capacity. However, research focusing on the relationship between the gut microbiome and differences in food preferences is very limited. We aimed to determine whether individual and regional differences exist in the gut microbiome of koalas as well as the mechanism by which these differences influence eucalypt selection. Methods: Foraging data were collected from six koalas and a total of 62 feces were collected from 15 koalas of two zoos in Japan. The mitochondrial phylogenetic analysis was conducted to estimate the mitochondrial maternal origin of each koala. In addition, the 16S-based gut microbiome of 15 koalas was analyzed to determine the composition and diversity of each koala's gut microbiome. We used these data to investigate the relationship among mitochondrial maternal origin, gut microbiome and eucalypt diet selection. Results and Discussion: This research revealed that diversity and composition of the gut microbiome and that eucalypt diet selection of koalas differs among regions. We also revealed that the gut microbiome alpha diversity was correlated with foraging diversity in koalas. These individual and regional differences would result from vertical (maternal) transmission of the gut microbiome and represent an intraspecific variation in koala foraging strategies. Further, we demonstrated that certain gut bacteria were strongly correlated with both mitochondrial maternal origin and eucalypt foraging patterns. Bacteria found to be associated with mitochondrial maternal origin included bacteria involved in fiber digestion and degradation of secondary metabolites, such as the families Rikenellaceae and Synergistaceae. These bacteria may cause differences in metabolic capacity between individual and regional koalas and influence their eucalypt selection. Conclusion: We showed that the characteristics (composition and diversity) of the gut microbiome and eucalypt diet selection of koalas differ by individuals and regional origins as we expected. In addition, some gut bacteria that could influence eucalypt foraging of koalas showed the relationships with both mitochondrial maternal origin and eucalypt foraging pattern. These differences in the gut microbiome between regional origins may make a difference in eucalypt selection. Given the importance of the gut microbiome to koalas foraging on eucalypts and their strong symbiotic relationship, future studies should focus on the symbiotic relationship and coevolution between koalas and the gut microbiome to understand individual and regional differences in eucalypt diet selection by koalas.

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.

Molecular epidemiological investigation of Cryptococcus spp. carried by captive koalas (Phascolarctos cinereus) in Japan.

Cryptococcus neoformans and Cryptococcus gattii cause cryptococcosis, a systemic mycosis that infects a wide range of species. Recent molecular biological investigations have allowed for the genotyping of these species, providing more detailed information on their pathogenicity and infection routes. Koalas (Phascolarctos cinereus) are frequently colonized by Cryptococcus spp., but molecular epidemiological studies have yet to be conducted in Japan. Here, we conducted multi-locus sequence typing (MLST) analysis on Cryptococcus spp. colonization isolates obtained from all koalas kept in seven parks across Japan. Out of 46 koalas examined, 10 (22%) were positive for C. gattii and 3 (6.5%) were positive for C. neoformans. All C. gattii isolates belonged to molecular type VGI and were either sequence type (ST) 51 or a novel ST, and all C. neoformans isolates belonged to molecular type VNI and ST23. Despite the frequent movement of koalas between parks, the STs were relatively park-specific, suggesting that the floor of the rearing barns is a source of infection and may act as a reservoir. MLST analysis confirmed that C. gattii was transported, established, and spread by koalas in areas where C. gattii was not originally present. MLST analysis is considered useful in assessing the pathogenicity and tracing the transmission routes of Cryptococcus spp. carried by koalas.IMPORTANCEThis is the first study to conduct a multi-locus sequence typing analysis on Cryptococcus spp. carried by captive koalas in Japan. Cryptococcosis remains a globally high-fatality fungal infection in humans, and captive koalas are known to carry a high percentage of Cryptococcus spp. Through this research, the molecular types and transmission routes of Cryptococcus spp. carried by koalas have been elucidated, revealing the potential role of enclosure flooring as a reservoir. It has been confirmed that Cryptococcus gattii, which is not endemic in Japan, has become established through koalas and is spreading to new individuals in Japan. This study is believed to provide valuable insights into koala conservation and contribute to the One Health approach for Cryptococcosis, a zoonotic infection.

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.

A retrospective study on antibacterial treatments for koalas infected with Chlamydia pecorum.

Chlamydiosis remains the leading infectious disease and is one of the key factors responsible for the dramatic reduction of koala populations in South-East Queensland (SEQ) and New South Wales (NSW) regions of Australia. Possible infection outcomes include blindness, infertility, painful cystitis, and death if left untreated. Studies have reported the treatment efficacy of chloramphenicol and doxycycline, which are the two most commonly administered treatments in diseased koalas, in clinical settings. However, none have directly compared the treatment efficacy of these antibacterials on koala survival. A retrospective study was essential to identify any relationships between the demographical information, and the animals' responses to the current treatment regimens. Associations were explored between six explanatory (sex; maturity; location; clinical signs, treatment; treatment duration) and two outcome variables (survival; post-treatment PCR). Results showed that female koalas had a statistical trend of lower odds of surviving when compared to males (OR = 0.36, p = 0.05). Koalas treated with chloramphenicol for ≥ 28 days had greater odds of surviving than when treated for < 28 days (OR = 8.8, p = 0.02), and those koalas administered doxycycline had greater odds of testing PCR negative when compared to chloramphenicol treatments (OR = 5.45, p = 0.008). There was no difference between the antibacterial treatments (chloramphenicol, doxycycline, and mixed/other) and the survival of koalas. Female koalas had greater odds of exhibiting UGT signs only (OR = 4.86, p < 0.001), and also greater odds of having both ocular and UGT clinical signs (OR = 5.29, p < 0.001) when compared to males. Of the koalas, 28.5% initially had no clinical signs but were PCR positive for C. pecorum. This study enables further understanding of the complex nature between chlamydial infection and response to antibacterial treatment.

LONEPINELLA SP. ISOLATED FROM WOUND INFECTIONS OF KOALAS.

We describe two cases of wound infections of koalas (Phascolarctos cinereus), one wild and one captive, in which Lonepinella-like organisms were involved. The wild adult koala was captured with bite wound injuries, as part of a koala population management program in Queensland, Australia. In both cases, there was evidence of physical trauma causing the initial wound. The captive koala suffered injury from the cage wire, and the wild koala had injuries suggestive of intermale fighting. Gram-negative bacteria isolated from both cases proved to be challenging to identify using routine diagnostic tests. The wound in the captive koala yielded a pure culture of an organism shown by whole genome sequence (WGS) analysis to be a member of the genus Lonepinella, but not a member of the only formally described species, L. koalarum. The wound of the wild koala yielded a mixed culture of Citrobacter koseri, Enterobacter cloacae and an organism shown by WGS analysis to be Lonepinella, but again not Lonepinella koalarum. Both cases were difficult to treat; the captive koala eventually had to have the phalanges amputated, and the wild koala required removal of the affected claw. The two Lonepinella isolates from these cases have a close relationship to an isolate from a human wound caused by a koala bite and may represent a novel species within the genus Lonepinella. Wound infections in koalas linked to Lonepinella have not been reported previously. Wildlife veterinarians need to be aware of the potential presence of Lonepinella-like organisms when dealing with wound infections in koalas, and the inability of commercial kits and systems to correctly identify the isolates.

Characterisation of the koala (Phascolarctos cinereus) pouch microbiota in a captive population reveals a dysbiotic compositional profile associated with neonatal mortality.

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. Video Abstract.

Animal Age Affects the Gut Microbiota and Immune System in Captive Koalas (Phascolarctos cinereus).

Gut microbiota is one of the major elements in the control of host health. However, the composition of gut microbiota in koalas has rarely been investigated. Here, we performed 16S rRNA gene sequencing to determine the individual and environmental determinants of gut microbiota diversity and function in 35 fecal samples collected from captive koalas. Meanwhile, blood immune-related cytokine levels were examined by quantitative reverse transcription-PCR to initially explore the relationship between the gut microbiota and the immune system in koalas. The relative abundance of many bacteria, such as Lonepinella koalarum, varies at different ages in koalas and decreases with age. Conversely, Ruminococcus flavefaciens increases with age. Moreover, bacterial pathways involved in lipid metabolism, the biosynthesis of other secondary metabolites, and infectious disease show a significant correlation with age. Age affects the relationship between the microbiota and the host immune system. Among them, the gut microbiota of subadult and aged koalas was closely correlated with CD8ß and CD4, whereas adult koalas were correlated with CLEC4E. We also found that sex, reproductive status, and living environment have little impact on the koala gut microbiota and immune system. These results shed suggest age is a key factor affecting gut microbiota and immunity in captive koalas and thus provide new insight into its role in host development and the host immune system. IMPORTANCE Although we have a preliminary understanding of the gut microbiota of koalas, we lack insight into which factors potentially impact captive koalas. This study creates the largest koala gut microbiota data set in China to date and describes several factors that may affect gut microbiota and the immune system in captive koalas, highlighting that age may be a key factor affecting captive koalas. Moreover, this study is the first to characterize the correlation between gut microbiota and cytokines in koalas. Better treatment strategies for infectious disorders may be possible if we can better understand the interactions between the immune system and the microbiota.

Begerowomyces aurantius sp. nov., a novel yeast isolated from koalas' habitat in a Japanese zoological park.

Koalas are iconic mammals indigenous to Australia. These rare animals and their habitats are occasionally associated with pathogenic fungi, including species of Cryptococcus, and consequently, monitoring the mycobiota of areas inhabited by koalas is of considerable importance. In this report, we describe a novel basidiomycetous yeast isolated from a site in Kanazawa Zoo, Japan, associated with captive koalas. Swab samples were collected from koala breeding environments, from which we isolated a novel unencapsulated yeast characterized by ovoid to ellipsoidal cells (3.2-4.9 × 3.5-5 µm). These cells were observed to undergo polar budding and grow as parent bud pairs, with an optimal growth temperature of 28°C. Colonies grown on yeast extract peptone dextrose agar at 28°C have a characteristic coral pink color. On the basis of physiological, morphological, and molecular characters, the new species was placed in the genus Begerowomyces, and the name Begerowomyces aurantius JCM33898T(LSEM1333T=CBS16241T) is proposed.

A targeted approach to investigating immune genes of an iconic Australian marsupial.

Disease is a contributing factor to the decline of wildlife populations across the globe. Koalas, iconic yet declining Australian marsupials, are predominantly impacted by two pathogens, Chlamydia and koala retrovirus. Chlamydia is an obligate intracellular bacterium and one of the most widespread sexually transmitted infections in humans worldwide. In koalas, Chlamydia infections can present as asymptomatic or can cause a range of ocular and urogenital disease signs, such as conjunctivitis, cystitis and infertility. In this study, we looked at differences in response to Chlamydia in two northern populations of koalas using a targeted gene sequencing of 1209 immune genes in addition to genome-wide reduced representation data. We identified two MHC Class I genes associated with Chlamydia disease progression as well as 25 single nucleotide polymorphisms across 17 genes that were associated with resolution of Chlamydia infection. These genes are involved in the innate immune response (TLR5) and defence (TLR5, IFNγ, SERPINE1, STAT2 and STX4). This study deepens our understanding of the role that genetics plays in disease progression in koalas and leads into future work that will use whole genome resequencing of a larger sample set to investigate in greater detail regions identified in this study. Elucidation of the role of host genetics in disease progression and resolution in koalas will directly contribute to better design of Chlamydia vaccines and management of koala populations which have recently been listed as "endangered."

Genetic diversity of Chlamydia pecorum detected in sheep flocks from Mexico.

Chlamydia pecorum, an obligate intracellular bacterium, is associated with reproductive and systemic diseases in sheep, goats, pigs, cattle, and koalas. The main conditions include polyarthritis, conjunctivitis, enteritis, pneumonia, encephalomyelitis, orchitis, placentitis, and abortion. Even though there are several studies showing that C. pecorum infections are widely spread in the world, in Mexico there are no reports. During 2016, as part of a sheep restocking program in Mexico, sheep were imported from New Zealand. Briefly after their arrival in the herds in the State of Mexico, these sheep presented abortions during the last third of gestation. A total of 62 sheep vaginal swabs that had presented abortion from different municipalities of the State of Mexico were collected. Bacterial isolation was performed using L929 mouse fibroblasts, and molecular identification was achieved by 23S rRNA (Chlamydiaceae family) and ompA gene (species-specific) real-time polymerase chain reaction (PCR). In addition, the 16S rRNA subunit and ompA gene were amplified and sequenced. Seven of 62 samples were positive for C. pecorum by bacterial isolation, 23S rRNA, and ompA gene real-time PCR. The 16S rRNA subunit and ompA gene amplicons were purified and the nucleotide sequence was determined in both directions. The consensus sequences homology search was performed using BLASTn analysis and showed a 100% of homology with the C. pecorum 16S rRNA subunit and 99% with the C. pecorum ompA gene. The population structure analyses using ompA gene demonstrated 15 genetic populations or clusters of 198 sequences from GenBank and our sequences were in a particular genetic structure corresponding to genotype "O." Herein, we describe the presence of C. pecorum in sheep imported from New Zealand into Mexico. Genetic analysis of the ompA gene showed that the isolates belong to genotype O and are related to strains isolated from sheep, cattle, and koalas.

In vitro transmission of Chlamydia using naturally infected koala (Phascolarctos cinereus) semen.

Transmission of Chlamydia pecorum infection has generally been assumed to be via the urogenital route and in an attempt to confirm this we investigated an in vitro method of Chlamydia infection using naturally infected koala semen to inoculate a cell line and attempt to estimate C. pecorum infectious load. A total of 57% of 122 koala semen samples had low C. pecorum copy number or no burden, while 18% of semen samples contained >10000 inclusion-forming units/mL, as determined by quantitative polymerase chain reaction. In vitro inoculation of a McCoy cell line resulted in successful infection from 4% of semen samples where C. pecorum burden was >105 inclusion-forming units/mL. Our preliminary study suggests that transmission of C. pecorum infectious dose may be restricted to peak bacterial shedding in semen associated with recent infection. Here, we report venereal transmission of C. pecorum in koala semen is possible; however, we speculate that antimicrobial factors and innate immune function receptors associated with semen may inhibit chlamydial growth. These mechanisms have yet to be reported in marsupial semen.

Characterization of the juvenile koala gut microbiome across wild populations.

In this study we compared the faecal microbiomes of wild joey koalas (Phascolarctos cinereus) to those of adults, including their mothers, to establish whether gut microbiome maturation and inheritance in the wild is comparable to that seen in captivity. Our findings suggest that joey koala microbiomes slowly shift towards an adult assemblage between 6 and 12 months of age, as the microbiomes of 9-month-old joeys were more similar to those of adults than those of 7-month-olds, but still distinct. At the phylum level, differences between joeys and adults were broadly consistent with those in captivity, with Firmicutes increasing in relative abundance over the joeys' development and Proteobacteria decreasing. Of the fibre-degrading genes that increased in abundance over the development of captive joeys, those involved in hemicellulose and cellulose degradation, but not pectin degradation, were also generally found in higher abundance in adult wild koalas compared to 7-month-olds. Greater maternal inheritance of the faecal microbiome was seen in wild than in captive koalas, presumably due to the more solitary nature of wild koalas. This strong maternal inheritance of the gut microbiome could contribute to the development of localized differences in microbiome composition, population health and diet through spatial clustering of relatives.

Maternal inheritance of the koala gut microbiome and its compositional and functional maturation during juvenile development.

The acquisition and maturation of the gastrointestinal microbiome is a crucial aspect of mammalian development, particularly for specialist herbivores such as the koala (Phascolarctos cinereus). Joey koalas are thought to be inoculated with microorganisms by feeding on specialized maternal faeces (pap). We found that compared to faeces, pap has higher microbial density, higher microbial evenness and a higher proportion of rare taxa, which may facilitate the establishment of those taxa in joey koalas. We show that the microbiomes of captive joey koalas were on average more similar to those of their mothers than to other koalas, indicating strong maternal inheritance of the faecal microbiome, which can lead to intergenerational gut dysbiosis when the mother is ill. Directly after pap feeding, the joey koalas' microbiomes were enriched for milk-associated bacteria including Bacteroides fragilis, suggesting a conserved role for this species across mammalian taxa. The joeys' microbiomes then changed slowly over 5 months to resemble those of adults by 1 year of age. The relative abundance of fibrolytic bacteria and genes involved in the degradation of plant cell walls also increased in the infants over this time, likely in response to an increased proportion of Eucalyptus leaves in their diets.

Koala cathelicidin PhciCath5 has antimicrobial activity, including against Chlamydia pecorum.

Devastating fires in Australia over 2019-20 decimated native fauna and flora, including koalas. The resulting population bottleneck, combined with significant loss of habitat, increases the vulnerability of remaining koala populations to threats which include disease. Chlamydia is one disease which causes significant morbidity and mortality in koalas. The predominant pathogenic species, Chlamydia pecorum, causes severe ocular, urogenital and reproductive tract disease. In marsupials, including the koala, gene expansions of an antimicrobial peptide family known as cathelicidins have enabled protection of immunologically naïve pouch young during early development. We propose that koala cathelicidins are active against Chlamydia and other bacteria and fungi. Here we describe ten koala cathelicidins, five of which contained full length coding sequences that were widely expressed in tissues throughout the body. Focusing on these five, we investigate their antimicrobial activity against two koala C. pecorum isolates from distinct serovars; MarsBar and IPTaLE, as well as other bacteria and fungi. One cathelicidin, PhciCath5, inactivated C. pecorum IPTaLE and MarsBar elementary bodies and significantly reduced the number of inclusions compared to the control (p<0.0001). Despite evidence of cathelicidin expression within tissues known to be infected by Chlamydia, natural PhciCath5 concentrations may be inadequate in vivo to prevent or control C. pecorum infections in koalas. PhciCath5 also displayed antimicrobial activity against fungi and Gram negative and positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Electrostatic interactions likely drive PhciCath5 adherence to the pathogen cell membrane, followed by membrane permeabilisation leading to cell death. Activity against E. coli was reduced in the presence of 10% serum and 20% whole blood. Future modification of the PhciCath5 peptide to enhance activity, including in the presence of serum/blood, may provide a novel solution to Chlamydia infection in koalas and other species.

MOLECULAR IDENTIFICATION OF MEMBERS OF THE FAMILY PASTEURELLACEAE FROM THE ORAL CAVITY OF KOALAS (PHASCOLARCTOS CINEREUS) AND THEIR RELATIONSHIP WITH ISOLATES FROM KOALA BITE WOUNDS IN HUMANS.

A total of 22 Pasteurellaceae isolates obtained from the oral cavity of koalas (Phascolarctos cinereus) at different wildlife centers in Australia were investigated using amplification and sequencing of two housekeeping genes, rpoA and recN. The available sequences from the Lonepinella koalarum type strain (ACM3666T) and the recent isolates of Lonepinella-like bacteria obtained from human infected wounds associated with koala bites were also included. Phylogenetic analysis was performed on the concatenated rpoA-recN genes and genome relatedness was calculated based on the recN sequences. The oral cavity isolates, the koala bite wound isolates, and L. koalarum ACM3666T resulted in four clusters (Clusters 1-4). Clusters 1-3 were clearly not members of the genus Lonepinella. Cluster 1 was closely related to the genus Fredericksenia, and Clusters 2 and 3 appeared to be novel genera. Cluster 4 consisted of three subclusters: Cluster 4a with one koala bite wound isolate and L. koalarum ACM3666T, Cluster 4b with three oral cavity isolates and two Lonepinella-like wound isolates, and Cluster 4c with three nearly identical oral cavity isolates that may represent a different species within the genus Lonepinella. The rich Pasteurellaceae population, including potential novel taxa in the oral cavity of koalas supports an important role of these highly adapted microorganisms in the physiology of koalas. Moreover, the pathogenic potential of Lonepinella-like species is an important consideration when investigating infected koala bites in humans.

A 29-year retrospective analysis of koala rescues in New South Wales, Australia.

The koala (Phascolarctos cinereus) is currently listed by both the IUCN and the Australian Governments' Threatened Species Scientific Committee as vulnerable to extinction with an overall decreasing population trend. It is unknown exactly how many koalas remain in the wild, but it is known that habitat fragmentation and bushfires have ultimately contributed to the decline of the koala all over Australia. This novel study is a retrospective analysis of data over a 29-year period (1989-2018) using records for 12,543 sightings and clinical care admissions for wild koalas from the major koala hot-spots (Port Stephens, port Macquarie and Lismore) in New South Wales, Australia. This study aims to understand the long-term patterns and trends of key stressors that are contributing to the decline of koalas in New South Wales, and the synergic interactions of factors such as rescue location, sex and age of the koala, and if their decline is influenced progressively by year. The main findings of this retrospective analysis indicated that between all 3 rescue sites, the most common prognosis was disease, the most common disease was signs of chlamydia, and the most common outcome was release. The location where the highest number of koalas were found prior to being reported as sighted or admitted into clinical care was within the regional area of Lismore. Furthermore, sex was not a discriminating factor when it came to prognosis or outcome, but age was significant. Finally, incidents of disease were found to increase over long-term, whereas release decreased over time and euthanasia increased. The wealth of data available to us and the retrospective analysis enabled us in a way to 'zoom out' and reveal how the key environmental stressors have fluctuated spatially and temporally. In conclusion, our data provides strong evidence of added pressures of increased human population growth in metropolitan zones, which increases risks of acute environmental trauma and proximate stressors such as vehicle collisions and dog-attacks as well as increased sightings of virtually healthy koalas found in exposed environments. Thus our 'zoom out' approach provides support that there is an urgent need to strengthen on-ground management, bushfire control regimes, environmental planning and governmental policy actions that should hopefully reduce the proximate environmental stressors in a step wise approach. This will ensure that in the next decade (beyond 2020), NSW koalas will hopefully start to show reversed trends and patterns in exposure to environmental trauma and disease, and population numbers will return towards recovery and stability.

Clinical comparison of five anti-chlamydial antibiotics in koalas (Phascolarctos cinereus).

Chlamydiosis is the most significant infectious disease of koalas (Phascolarctos cinereus). It is primarily a systemic sexually transmitted disease caused by Chlamydia pecorum and was responsible for 46% of the 2348 koala admissions to Australia Zoo Wildlife Hospital between 2013 and 2017. Treatment of chlamydiosis in koalas is complicated by three major factors. Firstly, koalas rely on bacterial fermentation of their high fibre diet making antibiotic therapy a risk. Secondly, they possess efficient metabolic pathways for the excretion of plant toxins and potentially of therapeutic agents. Thirdly, wild koalas, often present to rehabilitation facilities with chronic and severe disease. Traditional anti-chlamydial antibiotics used in other species may cause fatal dysbiosis in koalas or be excreted before they can be effective. We compared five anti-chlamydial antibiotics, azithromycin, chloramphenicol, doxycycline, enrofloxacin and florfenicol, which were used to treat 86 wild koalas with chlamydiosis presented to Australia Zoo Wildlife Hospital under consistent conditions of nutrition, housing, husbandry and climate. Response to treatment was assessed by recovery from clinical signs, and clearance of detectable Chlamydia via quantitative PCR. Doxycycline was the most effective anti-chlamydial antibiotic of the five, producing a 97% cure rate, followed by chloramphenicol (81%), enrofloxacin (75%), florfenicol (66%) and azithromycin (25%). The long-acting injectable preparation of doxycycline was well tolerated by koalas when administered via the subcutaneous route, and the weekly dosing requirement is a major advantage when treating wild animals. These findings indicate that doxycycline is the current drug of choice for the treatment of chlamydiosis in koalas, with chloramphenicol being the best alternative.

Helping koalas battle disease - Recent advances in Chlamydia and koala retrovirus (KoRV) disease understanding and treatment in koalas.

The iconic Australian marsupial, the koala (Phascolarctos cinereus), has suffered dramatic population declines as a result of habitat loss and fragmentation, disease, vehicle collision mortality, dog attacks, bushfires and climate change. In 2012, koalas were officially declared vulnerable by the Australian government and listed as a threatened species. In response, research into diseases affecting koalas has expanded rapidly. The two major pathogens affecting koalas are Chlamydia pecorum, leading to chlamydial disease and koala retrovirus (KoRV). In the last eight years, these pathogens and their diseases have received focused study regarding their sources, genetics, prevalence, disease presentation and transmission. This has led to vast improvements in pathogen detection and treatment, including the ongoing development of vaccines for each as a management and control strategy. This review will summarize and highlight the important advances made in understanding and combating C. pecorum and KoRV in koalas, since they were declared a threatened species. With complementary advances having also been made from the koala genome sequence and in our understanding of the koala immune system, we are primed to make a significant positive impact on koala health into the future.

Inbreeding and disease avoidance in a free-ranging koala population.

Habitat destruction and fragmentation are increasing globally, forcing surviving species into small, isolated populations. Isolated populations typically experience heightened inbreeding risk and associated inbreeding depression and population decline; although individuals in these populations may mitigate these risks through inbreeding avoidance strategies. For koalas, as dietary specialists already under threat in the northern parts of their range, increased habitat fragmentation and associated inbreeding costs are of great conservation concern. Koalas are known to display passive inbreeding avoidance through sex-biased dispersal, although population isolation will reduce dispersal pathways. We tested whether free-ranging koalas display active inbreeding avoidance behaviours. We used VHF tracking data, parentage reconstruction, and veterinary examination results to test whether free-ranging female koalas avoid mating with (a) more closely related males; and (b) males infected with sexually transmitted Chlamydia pecorum. We found no evidence that female koalas avoid mating with relatively more related available mates. In fact, as the relatedness of potential mates increases, so did inbreeding events. We also found no evidence that female koalas can avoid mating with males infected with C. pecorum. The absence of active inbreeding avoidance mechanisms in koalas is concerning from a conservation perspective, as small, isolated populations may be at even higher risk of inbreeding depression than expected. At risk koala populations may require urgent conservation interventions to augment gene flow and reduce inbreeding risks. Similarly, if koalas are not avoiding mating with individuals with chlamydial disease, populations may be at higher risk from disease than anticipated, further impacting population viability.