Differential and defective transcription of koala retrovirus indicates the complexity of host and virus evolution.

Koala retrovirus (KoRV) is unique amongst endogenous (inherited) retroviruses in that its incorporation to the host genome is still active, providing an opportunity to study what drives this fundamental process in vertebrate genome evolution. Animals in the southern part of the natural range of koalas were previously thought to be either virus-free or to have only exogenous variants of KoRV with low rates of KoRV-induced disease. In contrast, animals in the northern part of their range universally have both endogenous and exogenous KoRV with very high rates of KoRV-induced disease such as lymphoma. In this study we use a combination of sequencing technologies, Illumina RNA sequencing of 'southern' (south Australian) and 'northern' (SE QLD) koalas and CRISPR enrichment and nanopore sequencing of DNA of 'southern' (South Australian and Victorian animals) to retrieve full-length loci and intregration sites of KoRV variants. We demonstrate that koalas that tested negative to the KoRV pol gene qPCR, used to detect replication-competent KoRV, are not in fact KoRV-free but harbour defective, presumably endogenous, 'RecKoRV' variants that are not fixed between animals. This indicates that these populations have historically been exposed to KoRV and raises questions as to whether these variants have arisen by chance or whether they provide a protective effect from the infectious forms of KoRV. This latter explanation would offer the intriguing prospect of being able to monitor and selectively breed for disease resistance to protect the wild koala population from KoRV-induced disease.

Pathological Findings in Koala Retrovirus-positive Koalas (Phascolarctos cinereus) from Northern and Southern Australia.

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.

Haematological reference intervals of wild southern Australian koalas (Phascolarctos cinereus).

OBJECTIVE: Current haematology reference intervals (RIs) for koalas were developed in northern Australian koalas, using low numbers and/or individuals of unknown Chlamydia pecorum and koala retrovirus (KoRV) status. This study developed haematological RIs for wild, clinically healthy southern Australian koalas of known C. pecorum and KoRV infection status and investigated the effects of population, age and sex. METHODS: Haematological RIs were determined for 138 clinically healthy South Australian koalas (Mount Lofty Ranges [MLR], n = 68; Kangaroo Island, n = 70) examined in April 2016 and February 2017, respectively. C. pecorum and KoRV status were determined by PCR. RESULTS: RIs for southern koala haematological parameters were established for all koalas based on the finding that there were limited differences in haematological values in koalas with subclinical C. pecorum or KoRV infections (P > 0.05), except KoRV-infected koalas had a lower haematocrit than noninfected koalas. MLR koalas had significantly lower erythrocyte mass and leucocyte counts than Kangaroo Island koalas. Young koalas had significantly lower haemoglobin, haematocrit and higher mean cellular haemoglobin concentration and lymphocyte counts than adult koalas. MLR male koalas had elevated erythrocyte, leucocyte and neutrophil counts compared with MLR females. CONCLUSION: The haematological RIs developed in this study are based on a large number of clinically healthy koalas, where subclinical C. pecorum and KoRV infections had no effect on haematological values and will be a valuable tool during clinical examination and disease investigation by veterinarians and researchers Australia-wide.

Periodontal disease in free-ranging koalas (Phascolarctos cinereus) from the Mount Lofty Ranges, South Australia, and its association with koala retrovirus infection.

BACKGROUND: In northern Australian koala populations (Queensland and New South Wales), periodontal disease (gingivitis and periodontitis) is common while koala retrovirus subtype A is endogenous, with other subtypes transmitted exogenously. Koala retrovirus has been hypothesised to cause immune suppression and may predispose koalas to diseases caused by concurrent infections. In southern Australia populations (Victoria and South Australia) periodontal disease has not been investigated, and koala retrovirus is presumably exogenously transmitted. This study described oral health in South Australian koalas and investigated if an association between periodontal disease and koala retrovirus exists. METHODS: Oral health was examined for wild-caught koalas from the Mount Lofty Ranges (n = 75). Koala retrovirus provirus was detected in whole blood using nested PCR and proviral load determined with qPCR. Periodontal disease severity was recorded and used to calculate the Final Oral Health Index (0-normal, 24-severe).Results Periodontal disease was observed in 84% (63/75) of koalas; 77% had gingivitis (58/75) and 65% (49/75) had periodontitis. The average Final Oral Health Index was 5.47 (s.d 3.13). Most cases of periodontal disease were associated with the incisors. Koala retrovirus-infected koalas were more likely to present with periodontitis (p = 0.042) and the Final Oral Health Index was negatively correlated with proviral load (ρ = -0.353, p = 0.017). CONCLUSION: South Australian koalas had a high prevalence of gingivitis and periodontitis. Periodontal disease was more prevalent in the incisors. Exogenous koala retrovirus infection may also facilitate the development of periodontitis by modulation of the immune response to concurrent oral bacterial infections.

Malocclusions in the koala (Phascolarctos cinereus).

Malocclusions are a misalignment or incorrect positioning of the teeth when the upper and lower jaws close. These are poorly described in the koala and can result in irregular mastication which can have lifelong effects on body condition and oral health. A total of 370 koalas from two populations in Queensland (295) and one in South Australia (75) were examined for malocclusions. The prevalence of malocclusions in South Australian free-ranging koalas, captive Queensland koalas and Queensland free-ranging koalas was 39% (44), 30% (29) and 22% (29) respectively. Four types of malocclusion were identified based on severity of misalignment of the incisor/canine region, types 1, 2, 3 and 4. Maxillary overbite measurements of the molariform teeth were determined and these anisognathic values were then used to describe malocclusions within familial relationships in captive colonies. Captive koalas with a malocclusion had narrower mandibular width that ranged between 0.5 and 1% less than the normal measurements. The specific malocclusions reported in this study affected individuals by leading to tooth rotation, mobility and erosion with inefficient mastication of food and vegetation compaction. These changes increased the oral cavity pathology, by placing animals at risk of periodontal disease. There was evidence of familial links to malocclusion types in captive animals. Therefore captive breeding recommendations should consider known koala malocclusion traits to minimise their effect on future generations.

No Evidence of Toxoplasma Gondii Exposure in South Australian Koalas (Phascolarctos cinereus).

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.

Induction of neutralizing antibody response against koala retrovirus (KoRV) and reduction in viral load in koalas following vaccination with recombinant KoRV envelope protein.

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.

Identification of stable reference genes for quantitative PCR in 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.

Lymphoma, Koala Retrovirus Infection and Reproductive Chlamydiosis in a Koala (Phascolarctos cinereus).

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.