Dynamics of Antimicrobial Resistance Carriage in Koalas (Phascolarctos Cinereus) and Pteropid Bats (Pteropus Poliocephalus) Before, During and After Wildfires.

In the 2019-2020 summer, wildfires decimated the Australian bush environment and impacted wildlife species, including koalas (Phascolarctos cinereus) and grey headed flying fox pups (Pteropid bats, Pteropus poliocephalus). Consequently, hundreds of koalas and thousands of bat pups entered wildlife hospitals with fire-related injuries/illness, where some individuals received antimicrobial therapy. This study investigated the dynamics of antimicrobial resistance (AMR) in pre-fire, fire-affected and post-fire koalas and Pteropid bat pups. PCR and DNA sequencing were used to screen DNA samples extracted from faeces (koalas and bats) and cloacal swabs (koalas) for class 1 integrons, a genetic determinant of AMR, and to identify integron-associated antibiotic resistance genes. Class 1 integrons were detected in 25.5% of koalas (68 of 267) and 59.4% of bats (92 of 155). Integrons contained genes conferring resistance to aminoglycosides, trimethoprim and beta-lactams. Samples were also screened for blaTEM (beta-lactam) resistance genes, which were detected in 2.6% of koalas (7 of 267) and 25.2% of bats (39 of 155). Integron occurrence was significantly higher in fire-affected koalas in-care compared to wild pre-fire koalas (P < 0.0001). Integron and blaTEM occurrence were not significantly different in fire-affected bats compared to pre-fire bats (P > 0.05), however, their occurrence was significantly higher in fire-affected bats in-care compared to wild fire-affected bats (P < 0.0001 and P = 0.0488 respectively). The observed shifts of AMR dynamics in wildfire-impacted species flags the need for judicious antibiotic use when treating fire-affected wildlife to minimise unwanted selective pressure and negative treatment outcomes associated with carriage of resistance genes and antibiotic resistant bacteria.

Carriage of antibiotic resistance genes to treatments for chlamydial disease in koalas (Phascolarctos cinereus): A comparison of occurrence before and during catastrophic wildfires.

Growing reports of diverse antibiotic resistance genes in wildlife species around the world symbolises the extent of this global One Health issue. The health of wildlife is threatened by antimicrobial resistance in situations where wildlife species develop disease and require antibiotics. Chlamydial disease is a key threat for koalas in Australia, with infected koalas frequently entering wildlife hospitals and requiring antibiotic therapy, typically with chloramphenicol or doxycycline. This study investigated the occurrence and diversity of target chloramphenicol and doxycycline resistance genes (cat and tet respectively) in koala urogenital and faecal microbiomes. DNA was extracted from 394 urogenital swabs and 91 faecal swabs collected from koalas in mainland Australia and on Kangaroo Island (KI) located 14 km off the mainland, before (n = 145) and during (n = 340) the 2019-2020 wildfires. PCR screening and DNA sequencing determined 9.9% of samples (95%CI: 7.5% to 12.9%) carried cat and/or tet genes, with the highest frequency in fire-affected KI koalas (16.8%) and the lowest in wild KI koalas sampled prior to fires (6.5%). The diversity of cat and tet was greater in fire-affected koalas (seven variants detected), compared to pre-fire koalas (two variants detected). Fire-affected koalas in care that received antibiotics had a significantly higher proportion (p < 0.05) of cat and/or tet genes (37.5%) compared to koalas that did not receive antibiotics (9.8%). Of the cat and/or tet positive mainland koalas, 50.0% were Chlamydia-positive by qPCR test. Chloramphenicol and doxycycline resistance genes in koala microbiomes may contribute to negative treatment outcomes for koalas receiving anti-chlamydial antibiotics. Thus a secondary outcome of wildfires is increased risk of acquisition of cat and tet genes in fire-affected koalas that enter care, potentially exacerbating the already significant threat of chlamydial disease on Australia's koalas. This study highlights the importance of considering impacts to wildlife health within the One Health approach to AMR and identifies a need for greater understanding of AMR ecology in wildlife.

Cutaneous and Respiratory Lesions in Bushfire-Affected Koalas.

In the wake of increasingly frequent bushfires emerging as a threat to wildlife worldwide, koalas have notably been the most rescued species in Australia. However, our understanding of burns and their severity in koalas is limited; hence, this study investigated the histopathological features and depth of burns in koala skin, as well as the presence of smoke-induced respiratory tract damage. In four bushfire-affected koalas that had been euthanised on welfare grounds, skin burns in various body regions were scored based on clinical appearance as superficial, partial thickness, or full thickness. Histological sections of affected regions of skin were assessed as Grades I-IV and showed that furred regions on the ear margins and dorsum were histologically more severe, at Grade III, compared with the clinical score. There was a similar finding for footpad burns, which were the most common body region affected. In the respiratory tract, pulmonary oedema and congestion were evident in all koalas. Overall, the results highlight that cutaneous burn lesions on furred and palmar/plantar surfaces can have higher severity based on the burn depth than is clinically apparent. Therefore, there is a need to consider this when developing treatment plans and establishing prognosis for burnt koalas at triage, as well as that a high likelihood of pulmonary oedema exists.

Individuality and stability of the koala (Phascolarctos cinereus) faecal microbiota through time.

Gut microbiota studies often rely on a single sample taken per individual, representing a snapshot in time. However, we know that gut microbiota composition in many animals exhibits intra-individual variation over the course of days to months. Such temporal variations can be a confounding factor in studies seeking to compare the gut microbiota of different wild populations, or to assess the impact of medical/veterinary interventions. To date, little is known about the variability of the koala (Phascolarctos cinereus) gut microbiota through time. Here, we characterise the gut microbiota from faecal samples collected at eight timepoints over a month for a captive population of South Australian koalas (n individuals = 7), and monthly over 7 months for a wild population of New South Wales koalas (n individuals = 5). Using 16S rRNA gene sequencing, we found that microbial diversity was stable over the course of days to months. Each koala had a distinct faecal microbiota composition which in the captive koalas was stable across days. The wild koalas showed more variation across months, although each individual still maintained a distinct microbial composition. Per koala, an average of 57 (±16) amplicon sequence variants (ASVs) were detected across all time points; these ASVs accounted for an average of 97% (±1.9%) of the faecal microbial community per koala. The koala faecal microbiota exhibits stability over the course of days to months. Such knowledge will be useful for future studies comparing koala populations and developing microbiota interventions for this regionally endangered marsupial.

Genome Reference Assembly for Bottlenecked Southern Australian Koalas.

Koala populations show marked differences in inbreeding levels and in the presence or absence of the endogenous Koala retrovirus (KoRV). These genetic differences among populations may lead to severe disease impacts threatening koala population viability. In addition, the recent colonization of the koala genome by KoRV provides a unique opportunity to study the process of retroviral adaptation to vertebrate genomes and the impact this has on speciation, genome structure, and function. The genome build described here is from an animal from the bottlenecked Southern population free of endogenous and exogenous KoRV. It provides a more contiguous genome build than the previous koala reference derived from an animal from a more outbred Northern population and is the first koala genome from a KoRV polymerase-free animal.

Health and Diseases of Koalas.

The koala (Phascolarctos cinereus) is an arboreal marsupial that is found throughout much of eastern and southeastern Australia, and it relies primarily on eucalypt trees for food, water and shelter [...].

TESTIS ABNORMALITIES IN A POPULATION OF SOUTH AUSTRALIAN KOALAS (PHASCOLARCTOS CINEREUS).

Testis asymmetry, in which the testes in an individual differ in size, has recently been reported in koalas (Phascolarctos cinereus) in the Mount Lofty Ranges population of South Australia. We describe the morphology and histology of both testes from affected individuals in this population (n=56) and the parameters of koalas with normal-sized testes based on age and breeding season (n=56). Morphologic measurements included testis weight, length, width, and volume; histologic parameters included seminiferous tubule diameter, seminiferous epithelial height, and seminiferous tubule (interstitial tissue ratio and presence or absence of spermatozoa). Of the 56 koalas with intraindividual variation in testes size, 47 were classified as asymmetric and nine as microtestes. For koalas with asymmetric testes, all morphologic parameters were significantly decreased in the smaller testes compared with normal-sized testes, but for the histologic parameters, only seminiferous tubule diameter was significantly less. Histopathologic examination of the asymmetric testes showed 38 with normal parenchyma histologically indistinguishable between intraindividual testes, four with degeneration and atrophy, and three with hypoplasia, whereas examination of microtestes showed degeneration and atrophy in seven, hypoplasia in one, and aplasia in one. No association of testis size difference with Chlamydia pecorum infection was found in a subset of animals. For the 56 koalas with normal-sized testes, morphologic parameters were found to increase with age, and juvenile and young adults were found to have smaller seminiferous tubule diameters than adults. No differences were found between testes of koalas in the breeding and nonbreeding season. Overall, these findings indicate that testis asymmetry in koalas from the Mount Lofty Ranges population is common but not associated with decreased function, except where testis malformations such as hypoplasia or aplasia occur or when parenchyma has been disrupted by acquired disease.

An Analysis of Demographic and Triage Assessment Findings in Bushfire-Affected Koalas (Phascolarctos cinereus) on Kangaroo Island, South Australia, 2019-2020.

In the 2019-2020 Australian bushfires, Kangaroo Island, South Australia, experienced catastrophic bushfires that burnt approximately half the island, with an estimated 80% of the koala population lost. During and after the event, rescued koalas were triaged at a designated facility and a range of initial data were recorded including rescue location and date, sex, estimation of age, body condition and hydration, and assessment of burn severity (n = 304 records available). Koalas were presented to the triage facility over a span of 10 weeks, with 50.2% during the first 14 days of the bushfire response, the majority of which were rescued from regions of lower fire severity. Burns were observed in 67.4% of koalas, with the majority (60.9%) classified as superficial burns, primarily affecting the limbs and face. Poor body condition was recorded in 74.6% of burnt koalas and dehydration in 77.1%. Negative final outcomes (death or euthanasia, at triage or at a later date) occurred in 45.6% of koalas and were significantly associated with higher mean burn score, maximum burn severity, number of body regions burnt, poor body condition score, and dehydration severity. The findings of this retrospective study may assist clinicians in the field with decision making when triaging koalas in future fire rescue efforts.

Molecular Diagnosis of Koala Retrovirus (KoRV) in South Australian Koalas (Phascolarctos cinereus).

Koala retrovirus, a recent discovery in Australian koalas, is endogenised in 100% of northern koalas but has lower prevalence in southern populations, with lower proviral and viral loads, and an undetermined level of endogenisation. KoRV has been associated with lymphoid neoplasia, e.g., lymphoma. Recent studies have revealed high complexity in southern koala retroviral infections, with a need to clarify what constitutes positive and negative cases. This study aimed to define KoRV infection status in Mount Lofty Ranges koalas in South Australia using RNA-seq and proviral analysis (n = 216). The basis for positivity of KoRV was deemed the presence of central regions of the KoRV genome (gag 2, pol, env 1, and env 2) and based on this, 41% (89/216) koalas were positive, 57% (124/216) negative, and 2% inconclusive. These genes showed higher expression in lymph node tissue from KoRV positive koalas with lymphoma compared with other KoRV positive koalas, which showed lower, fragmented expression. Terminal regions (LTRs, partial gag, and partial env) were present in SA koalas regardless of KoRV status, with almost all (99.5%, 215/216) koalas positive for gag 1 by proviral PCR. Further investigation is needed to understand the differences in KoRV infection in southern koala populations.

Identification and Prevalence of Phascolarctid Gammaherpesvirus Types 1 and 2 in South Australian Koala Populations.

To determine Phascolarctid gammaherpesviruses (PhaHV) infection in South Australian koala populations, 80 oropharyngeal swabs from wild-caught and 87 oropharyngeal spleen samples and swabs from euthanased koalas were tested using two specific PCR assays developed to detect PhaHV-1 and PhaHV-2. In wild-caught koalas, active shedding of PhaHV was determined by positive oropharyngeal samples in 72.5% (58/80) of animals, of which 44.8% (26/58) had PhaHV-1, 20.7% (12/58) PhaHV-2 and 34.5% (20/58) both viral subtypes. In the euthanased koalas, systemic infection was determined by positive PCR in spleen samples and found in 72.4% (63/87) of koalas. Active shedding was determined by positive oropharyngeal results and found in 54.0% (47/87) of koalas. Koalas infected and actively shedding PhaHV-1 alone, PhaHV-2 alone or shedding both viral subtypes were 48.9% (23/47), 14.9% (7/47) and 36.2% (17/47), respectively. Only 45.9% (40/87) were not actively shedding, of which 40.0% (16/40) of these had systemic infections. Both wild-caught and euthanased koalas actively shedding PhaHV-2 were significantly more likely to be actively shedding both viral subtypes. Active shedding of PhaHV-2 had a significant negative correlation with BCS in the euthanased cohort, and active shedding of PhaHV-1 had a significant positive relationship with age in both wild-caught and euthanased cohorts.

Field immobilization using alfaxalone and alfaxalone-medetomidine in free-ranging koalas (Phascolarctos cinereus): a randomized comparative study.

OBJECTIVE: To characterize and compare two intramuscular drug protocols using alfaxalone and alfaxalone-medetomidine combination for the field immobilization of free-ranging koalas. STUDY DESIGN: Blinded, randomized, comparative field study. ANIMALS: A total of 66 free-ranging koalas from the Mount Lofty Ranges, South Australia. METHODS: Koalas were randomly allocated into two groups. Group A animals were given alfaxalone alone at 3.5 mg kg-1. Group AM animals were given alfaxalone 2 mg kg-1 and medetomidine 40 µg kg-1, reversed with atipamezole at 0.16 mg kg-1. Blinded operators recorded heart rate (HR), respiratory rate (fR), cloacal temperature, depth of sedation and times to: first effect, sedation suitable for clinical interventions, first arousal and full recovery. Data were analysed using independent t test, Mann-Whitney U test, chi-square analysis and log-rank test at 5% level of significance. RESULTS: Suitable immobilization for clinical examination and sample collection was achieved in all animals. In groups A and AM, median time to working depth was 6.5 minutes (range: 3.4-15) and 8.1 minutes (range: 4.3-24) and time to complete recovery was 66 minutes (range: 12-138) and 34 minutes (range: 4-84), respectively, following reversal. Time to first effect was significantly shorter in group A (p = 0.013), whereas time to full arousal was significantly shorter in group AM (p = 0.007) probably due to the administration of atipamezole. Maximum HR was 117 ± 28 beats minute-1 in group A, which was a significant increase from baseline values (p < 0.0001), whereas group AM showed a significant tachypnoea of 67 ± 25 (normal fR 10-15; p < 0.0001). CONCLUSIONS AND CLINICAL RELEVANCE: Both the protocols produced immobilization, enabling clinical examination and sample collection; however, protocol AM was more suitable for field work due to shorter recovery times.

Koala retrovirus viral load and disease burden in distinct northern and southern koala populations.

Koala retrovirus (KoRV) displays features of both an endogenous and exogenous virus and is linked to neoplasia and immunosuppression in koalas. This study explores the apparent differences in the nature and impact of KoRV infection between geographically and genetically separated "northern" and "southern" koala populations, by investigating the disease status, completeness of the KoRV genome and the proviral (DNA) and viral (RNA) loads of 71 northern and 97 southern koalas. All northern animals were positive for all KoRV genes (gag, pro-pol and env) in both DNA and RNA forms, whereas many southern animals were missing one or more KoRV genes. There was a significant relationship between the completeness of the KoRV genome and clinical status in this population. The proviral and viral loads of the northern population were significantly higher than those of the southern population (P < 0.0001), and many provirus-positive southern animals failed to express any detectable KoRV RNA. Across both populations there was a positive association between proviral load and neoplasia (P = 0.009). Potential reasons for the differences in the nature of KoRV infection between the two populations are discussed.

CHANGES IN ANTIOXIDANT CAPACITY OF KOALAS (PHASCOLARCTOS CINEREUS) BASED ON HEALTH STATUS AND MEASUREMENT OF THE ANTIOXIDANT ASCORBATE IN EUCALYPT LEAVES.

Antioxidants have a crucial role in protecting the body from oxidative stress, which would otherwise result in cellular damage and possibly predispose animals to disease. The antioxidant capacity of koalas (Phascolarctos cinereus) and its association with health or disease status is currently unknown. Ascorbate, a dietary antioxidant, has previously been identified in a few eucalypt species eaten by koalas. This study aimed to determine 1) differences between the antioxidant capacity of healthy and diseased koalas, and 2) concentration of the antioxidant ascorbate in Eucalyptus spp. leaves eaten by koalas. To determine differences in antioxidant capacity of koalas, plasma samples from clinically healthy koalas in Kangaroo Island, South Australia (SA) (n = 23), euthanized koalas with oxalate nephrosis from Mount Lofty Ranges, SA (n = 11), and euthanized koalas with chlamydiosis from Moggill, Queensland (n = 11) were analyzed for the three antioxidants α-tocopherol, ascorbate, and retinol and for two measures of antioxidant capacity, ferric reducing ability of plasma (FRAP), and trolox equivalent antioxidant capacity (TEAC). The thiobarbituric acid reactive substance (TBARS) measured formation of oxidants, and an oxidative stress index (OSI) was calculated by TBARS/(TEAC + FRAP). Ascorbate concentration was measured in dietary eucalypt leaves from Mount Lofty Ranges and Moggill. Results showed that in diseased Mount Lofty Ranges and Moggill koalas, plasma α-tocopherol concentrations were significantly lower, and ascorbate, TBARS, and OSI was significantly higher compared with clinically healthy koalas from Kangaroo Island. Ascorbate was high in eucalypt leaves, particularly young leaves from the Mount Lofty Ranges. This study showed that disease was associated with some measures of poor antioxidant capacity in koalas and also found that ascorbate is high in the dietary eucalypts of koalas.

Genetic diversity of Koala retrovirus env gene subtypes: insights into northern and southern koala populations.

Koala retrovirus (KoRV) is a recently endogenized retrovirus associated with neoplasia and immunosuppression in koala populations. The virus is known to display sequence variability and to be present at varying prevalence in different populations, with animals in southern Australia displaying lower prevalence and viral loads than northern animals. This study used a PCR and next-generation sequencing strategy to examine the diversity of the KoRV env gene in both proviral DNA and viral RNA forms in two distinct populations representative of the 'northern' and 'southern' koala genotypes. The current study demonstrated that the full range of KoRV subtypes is present across both populations, and in both healthy and sick animals. KoRV-A was the predominant proviral subtype in both populations, but there was marked diversity of DNA and RNA subtypes within individuals. Many of the northern animals displayed a higher RNA viral diversity than evident in their proviral DNA, indicating relatively higher replication efficiency of non-KoRV-A subtypes. The southern animals displayed a lower absolute copy number of KoRV than the northern animals as reported previously and a higher preponderance of KoRV-A in individual animals. These discrepancies in viral replication and diversity remain unexplained but may indicate relative protection of the southern population from KoRV replication due to either viral or host factors and may represent an important protective effect for the host in KoRV's ongoing entry into the koala genome.

Chlamydia pecorum prevalence in South Australian koala (Phascolarctos cinereus) populations: Identification and modelling of a population free from infection.

Chlamydia pecorum is an established and prevalent infection that produces severe clinical disease in many koala populations, contributing to dramatic population declines. In wild South Australian koala populations, C. pecorum occurrence and distribution is unknown. Here, C. pecorum-specific real-time quantitative PCR (qPCR) was applied to ocular and urogenital swabs from targeted surveys of wild koalas from the mainland Mount Lofty Ranges (MLR) (n = 75) and Kangaroo Island (KI) (n = 170) populations. Historical data from 13,081 KI koalas (1997-2018) provided additional evidence for assessing the absence of C. pecorum infection. In the MLR population, 46.7% (CI: 35.1-58.6%) of koalas were C. pecorum positive by qPCR but only 4% had grade 3 clinical disease. MLR koala fertility was significantly reduced by C. pecorum infection; all reproductively active females (n = 16) were C. pecorum negative, whereas 85.2% of inactive females (n = 23) were positive (P < 0.001). KI koalas were C. pecorum negative and the population was demonstrated to be free of C. pecorum infection with 95% confidence. C. pecorum is a real threat for the sustainability of the koala and KI is possibly the last isolated, large C. pecorum-free population remaining in Australia. These koalas could provide a safeguard against this serious disease threat to an iconic Australian species.