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.

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.

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.

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.

Pulmonary Actinomycosis in South Australian Koalas (Phascolarctos cinereus).

Pneumonia has been reported in both free-ranging and captive koalas and a number of causative agents have been described. Between 2016 and 2019, 16 free-ranging and 1 captive koala (Phascolarctos cinereus) from the Mount Lofty Ranges of South Australia were identified with pyogranulomatous lobar pneumonia, which involved the left caudal lobe in 14/17 (82%) cases. Within lesions, numerous gram-positive or gram-variable, non-acid-fast filamentous bacteria were observed in association with Splendore-Hoeppli phenomenon. Culture yielded growth of anaerobic bacteria, which were unidentifiable by MALDI-TOF-MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) analysis in 5/5 cases. Sequencing of the bacterial 16S rRNA gene identified a novel Actinomyces species in 4 samples, confirming a diagnosis of pulmonary actinomycosis. Concurrent examination of resin lung casts from healthy koalas suggested greater laminar flow of air to the left caudal lung lobe in koalas. Actinomyces spp. have been reported as commensals of the oral microbiome in other species, and an association with similar pulmonary lesions in other species. Considering the predilection for involvement of the left caudal lung lobe, aspiration is suggested as the likely cause in some cases of pulmonary actinomycosis in koalas. Pulmonary actinomycosis has not been previously described in koalas and further work needs to be undertaken in order to classify this organism within the Actinomyces genus.

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.