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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 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 (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.

Purpose. Koala retrovirus (KoRV-A) is 100 % prevalent in northern Australian (Queensland and New South Wales) koala populations, where KoRV-B has been associated with Chlamydia pecorum disease and the development of lymphosarcoma. In southern populations (Victoria and South Australia), KoRV-A is less prevalent and KoRV-B has not been detected in Victoria, while the current prevalence in South Australian populations is unknown but is thought to be low. This study aimed to determine (i) the prevalence of KoRV in the two largest South Australian koala populations [Kangaroo Island (KI) and Mount Lofty Ranges (MLR)], (ii) KoRV subtype and (iii) if an association between KoRV and C. pecorum exists.Methodology. Wild koalas were sampled in KI (n = 170) between 2014 and 2017 and in MLR (n = 75) in 2016. Clinical examinations were performed, with blood collected for KoRV detection and typing by PCR.Results. KoRV prevalence was 42.4 % [72/170, 95 % confidence interval (CI): 34.9-49.8 %] in KI and 65.3 % (49/ 75, 95 % CI: 54.6-76.1 %) in MLR. Only KoRV-A, and not KoRV-B, was detected in both populations. In MLR, there was no statistical association between KoRV and C. pecorum infection (P = 0.740), or KoRV and C. pecorum disease status (P=0.274), although KoRV-infected koalas were more likely to present with overt C. pecorum disease than subclinical infection (odds ratio: 3.15, 95 % CI: 0.91-5.39).Conclusion. KoRV-A is a prevalent pathogen in wild South Australian koala populations. Future studies should continue to investigate KoRV and C. pecorum associations, as the relationship is likely to be complex and to differ between the northern and southern populations.

Historical hunting pressures on koalas in the southern part of their range in Australia have led to a marked genetic bottleneck when compared with their northern counterparts. There are a range of suspected genetic disorders such as testicular abnormalities, oxalate nephrosis and microcephaly reported at higher prevalence in these genetically restricted southern animals. This paper reports analysis of differential expression of genes from RNAseq of lymph nodes, SNPs present in genes and the fixation index (population differentiation due to genetic structure) of these SNPs from two populations, one in south east Queensland, representative of the northern genotype and one in the Mount Lofty Ranges South Australia, representative of the southern genotype. SNPs that differ between these two populations were significantly enriched in genes associated with brain diseases. Genes which were differentially expressed between the two populations included many associated with brain development or disease, and in addition a number associated with testicular development, including the androgen receptor. Finally, one of the 8 genes both differentially expressed and with a statistical difference in SNP frequency between populations was SLC26A6 (solute carrier family 26 member 6), an anion transporter that was upregulated in SA koalas and is associated with oxalate transport and calcium oxalate uroliths in humans. Together the differences in SNPs and gene expression described in this paper suggest an underlying genetic basis for several disorders commonly seen in southern Australian koalas, supporting the need for further research into the genetic basis of these conditions, and highlighting that genetic selection in managed populations may need to be considered in the future.

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

Koala retrovirus (KoRV) infection shows differences in prevalence and load between northern and southern Australian koala populations; however, the effect of this on diseases such as lymphoma and chlamydial disease is unclear. This study compared clinicopathological findings, haematology and splenic lymphoid area of KoRV-positive koalas from northern (Queensland [Qld], n = 67) and southern (South Australia [SA], n = 92) populations in order to provide further insight into KoRV pathogenesis. Blood was collected for routine haematology and for measurement of KoRV proviral load by quantitative polymerase chain reaction (qPCR). Plasma samples were assessed for KoRV viral load by reverse transcriptase qPCR and conjunctival and cloacal swabs were collected for measurement of the load of Chlamydia pecorum (qPCR). During necropsy examination, spleen was collected for lymphoid area analysis. Lymphoma was morphologically similar between the populations and occurred in koalas with the highest KoRV proviral and viral loads. Severe ocular chlamydial disease was observed in both populations, but urinary tract disease was more severe in Qld, despite similar C. pecorum loads. No associations between KoRV and chlamydial disease severity or load were observed, except in SA where viral load correlated positively with chlamydial disease severity. In both populations, proviral and viral loads correlated positively with lymphocyte and metarubricyte counts and correlated negatively with erythrocyte and neutrophil counts. Splenic lymphoid area was correlated positively with viral load. This study has shown further evidence for KoRV-induced oncogenesis and highlighted that lymphocytes and splenic lymphoid tissue may be key sites for KoRV replication. However, KoRV infection appears to be highly complex and continued investigation is required to fully understand its pathogenesis.

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 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.

Koala retrovirus (KoRV) infects the majority of Australia’s koalas (Phascolarctos cinereus) and has been linked to several life-threatening diseases such as lymphoma and leukemia, as well as Chlamydia and thus poses a threat to the continued survival of this species. While quarantine and antiretroviral drug treatment are possible control measures, they are impractical, leaving vaccination as the only realistic option. In this study, we examined the effect of a recombinant envelope protein-based anti-KoRV vaccine in two groups of South Australian koalas: KoRV infected or KoRV free. We report a successful vaccination response in the koalas with no vaccine-associated side effects. The vaccine induced a significant humoral immune response as well as the production of neutralizing antibodies in both groups of koalas. We also identified B-cell epitopes that were differentially recognized in KoRV-infected versus KoRV-free koalas following vaccination. Importantly, we also showed that vaccination had a therapeutic effect on koalas infected exogenously with KoRV by reducing their circulating viral load. Together, this study highlights the possibility of successfully developing a vaccine against KoRV infection in koalas., Koala retrovirus: Hopes for an effective vaccine A vaccine candidate for Koala retrovirus elicits a protective antibody response and reduces the viral load in already-infected koalas. Koala retrovirus (KoRV), first identified in the last 20 years, is a life-threatening, endemic pathogen affecting Australian koalas. In pursuit of an effective KoRV vaccine, the University of the Sunshine Coast’s Peter Timms led a group of Australian scientists to develop a candidate based on the transmembrane section of the virus’ envelope protein. The six koalas vaccinated in the study all generated a strong antibody response to the envelope protein, and a strong neutralizing antibody response was reported during in vitro tests. Vaccinated koalas with pre-existing KoRV infection benefited from an average 79% reduction in viral load when measured 12 weeks after vaccination. Further research should be prioritized to provide much-needed protection to Australia’s koalas.

To better understand host and immune response to diseases, gene expression studies require identification of reference genes with stable expression for accurate normalisation. This study describes the identification and testing of reference genes with stable expression profiles in koala lymph node tissues across two genetically distinct koala populations. From the 25 most stable genes identified in transcriptome analysis, 11 genes were selected for verification using reverse transcription quantitative PCR, in addition to the commonly used ACTB and GAPDH genes. The expression data were analysed using stable genes statistical software - geNorm, BestKeeper, NormFinder, the comparative ΔCt method and RefFinder. All 13 genes showed relative stability in expression in koala lymph node tissues, however Tmem97 and Hmg20a were identified as the most stable genes across the two koala populations.

Koala retrovirus (KoRV) infection, thought to be associated with lymphoid neoplasia, and Chlamydia pecorum-related ocular and urogenital disease are both highly prevalent in eastern Australian koala (Phascolarctos cinereus) populations. However, in South Australian koalas, little is known about KoRV infection and C. pecorum-associated disease. We report the first South Australian case of lymphoma in a KoRV-A-positive female koala also affected by severe reproductive chlamydiosis. The koala was from the Mount Lofty Ranges population and was presented with hindlimb lameness. Clinical examination identified right stifle crepitus, enlarged superficial lymph nodes and paraovarian cysts. Necropsy examination revealed extensive cartilage degeneration and loss over the medial femoral condyle, solid femoral bone marrow, mesenteric and ovarian tumours, paraovarian cysts and purulent metritis. Histopathology confirmed lymphoma in the bone marrow, mesenteric lymph nodes and ovary, with infiltration and parenchymal effacement in the pancreas, adrenal glands and other tissues. Lymphoma, KoRV and chlamydiosis are being investigated further in this population.

Infection with the cat-borne parasite Toxoplasma gondii has been detected in numerous Australian marsupials and can lead to severe disease (toxoplasmosis) in some cases. The seroprevalence of Toxoplasma on Kangaroo Island, South Australia has been reported to be higher than the South Australian mainland in macropods, cats, and sheep, suggesting an increased risk of infection on this island. However, Toxoplasma seroprevalence in small- and medium-sized terrestrial mammals was almost zero on the island and did not differ from that on the mainland. We surveyed Toxoplasma seroprevalence in koala (Phascolarctos cinereus) populations on the island and on the mainland and assessed their risk of infection and their role in the life cycle of Toxoplasma. All screened koalas from the island (n = 94) and the mainland (n = 63) were seronegative. This represents the largest Toxoplasma seroprevalence survey in this species and provided sufficient evidence to confidently demonstrate freedom from parasite exposure in both island and mainland populations at the time of the survey. Because koalas are extensively arboreal and predominately consume tree foliage, they appear to be at negligible risk of Toxoplasma infection. Furthermore, as koalas are rarely consumed by cats, we suggest that they have a minor role in the parasite's life cycle.

There is a belief that the administration of nitrous oxide (N 2 O) increases hearing acuity. This increase can be interpreted as hearing low intensity sounds more loudly. This study examined auditory threshold levels, acoustic impedance, acoustic perception, memory, and discrimination to determine if hearing was altered by end-tidal 10% or 20% nitrous oxide. Subjects also qualitatively interpreted three intensity levels using a subjective intensity test. Observations were made in the breathing mediums of room air in the control stages (Stages 1 and 4), compared to N 2 O and supplemental oxygen (Stages 2 and 3), in 16 human subjects in a quiet room. Breathing end-tidal 10% and 20% N 2 O significantly increased middle ear pressure, and produced a significant effect on the subjective interpretation of a sound's intensity. There was no significant effect on auditory threshold at a range of frequencies. It appears that 10% or 20% N 2 O inhalation does not lead to the commonly held view of increased hearing acuity as measured in terms of auditory threshold. Rather, at these levels of N 2 O inhalation, subjects experience a state similar to a pre-sleep stage, whereby the hearing diminishes but remains active for loud intensity sounds.