Northern Koalas Research Articles

Koala retrovirus infection and disease in South Australian koala (Phascolarctos cinereus) populations. In Proceedings of the Second Koala Retrovirus Workshop, ed. D. E. Alquezar-Planas, D. P. Higgins, C. L. Singleton, and A. D. Greenwood

Koala retrovirus (KoRV) infection, endogenous in all northern koalas (Phascolarctos cinereus), has been found to occur at lower, but increasing, prevalence in the Kangaroo Island and Mount Lofty Ranges koala populations in South Australia. Proviral and viral loads are also lower than in Queensland koalas, which may be due to exogenous spread of infection, or may be related to the variable presence of viral genes and fragmented expression that has been found in positive Mount Lofty Ranges koalas. However, high proviral loads and full expression across the KoRV genome in South Australian koalas has been found in individuals with neoplasia, particularly lymphoma, which can be as extensive and as severe as that observed in northern koalas. KoRV-A is the predominant subtype and no association with chlamydial status has been found except that high viral loads correlate with severity of chlamydiosis. Based on the complexity of KoRV infections in South Australian koalas, further research is needed to understand the differences in transmission and pathogenesis that occur.

Incidence, trends, and significance of putative koala retrovirus-associated diseases in monitored wild koala populations in southeast Queensland. In Proceedings of the Second Koala Retrovirus Workshop, ed. D. E. Alquezar-Planas, D. P. Higgins, C. L. Singleton, and A. D. Greenwood

Research indicates that northern koalas (Phascolarctos cinereus) are ubiquitously infected with koala retrovirus (KoRV). There is increasing evidence linking KoRV with neoplasia and a range of disorders associated with immunodeficiency, conditions observed at high rates in captive colonies, and sick koalas presenting to wildlife hospitals. However, less is known about the occurrence of these putative KoRV-associated diseases in wild populations. We analysed health data collected at the veterinary examinations of 691 koalas inhabiting three monitored wild koala populations in southeast Queensland between 2013 and 2020. At initial presentation, neoplasia and AIDS-like syndrome were detected at a prevalence of 1.16% (8/691; 95% CI 0.5–2.19%). Longitudinal data from koalas recruited into the monitoring programmes and receiving one or more subsequent examination revealed an incidence rate of 3.5 cases/100 koalas/year (95% CI 2.35–4.9). These findings indicate that a relatively small proportion of the populations studied were affected by these putative KoRV-associated diseases. However, the impact on individuals was severe, with high associated mortality in the diseased cohort. Furthermore, northern koala populations endure multiple threats, suffering severe declines in recent decades. We propose that the significance of putative KoRV-associated diseases on these populations should be considered within this context and that further research into the interactions between KoRV and other drivers of decline is warranted.

Geographic patterns of koala retrovirus genetic diversity, endogenization, and subtype distributions

Koala retrovirus (KoRV) subtype A (KoRV-A) is currently in transition from exogenous virus to endogenous viral element, providing an ideal system to elucidate retroviral-host coevolution. We characterized KoRV geography using fecal DNA from 192 samples across 20 populations throughout the koala's range. We reveal an abrupt change in KoRV genetics and incidence at the Victoria/New South Wales state border. In northern koalas, pol gene copies were ubiquitously present at above five per cell, consistent with endogenous KoRV. In southern koalas, pol copies were detected in only 25.8% of koalas and always at copy numbers below one, while the env gene was detected in all animals and in a majority at copy numbers above one per cell. These results suggest that southern koalas carry partial endogenous KoRV-like sequences. Deep sequencing of the env hypervariable region revealed three putatively endogenous KoRV-A sequences in northern koalas and a single, distinct sequence present in all southern koalas. Among northern populations, env sequence diversity decreased with distance from the equator, suggesting infectious KoRV-A invaded the koala genome in northern Australia and then spread south. The exogenous KoRV subtypes (B to K), two novel subtypes, and intermediate subtypes were detected in all northern koala populations but were strikingly absent from all southern animals tested. Apart from KoRV subtype D, these exogenous subtypes were generally locally prevalent but geographically restricted, producing KoRV genetic differentiation among northern populations. This suggests that sporadic evolution and local transmission of the exogenous subtypes have occurred within northern Australia, but this has not extended into animals within southern Australia.

Koala retrovirus load and non-A subtypes are associated with secondary disease among wild northern koalas.

Koala Retrovirus (KoRV) has been associated with neoplasia in the vulnerable koala (Phascolarctos cinereus). However, there are conflicting findings regarding its association with secondary disease. We undertook a large-scale assessment of how the different KoRV subtypes and viral load are associated with Chlamydia pecorum infection and a range of disease pathologies in 151 wild koalas admitted for care to Currumbin Wildlife Hospital, Australia. Viral load (KoRV pol copies per ml of plasma) was the best predictor of more disease pathologies than any other KoRV variable. The predicted probability of a koala having disease symptoms increased from 25% to over 85% across the observed range of KoRV load, while the predicted probability of C. pecorum infection increased from 40% to over 80%. We found a negative correlation between the proportion of env deep sequencing reads that were endogenous KoRV-A and total KoRV load. This is consistent with suppression of endogenous KoRV-A, while the exogenous KoRV subtypes obtain high infection levels. Additionally, we reveal evidence that the exogenous subtypes are directly associated with secondary disease, with the proportion of reads that were the endogenous KoRV-A sequence a negative predictor of overall disease probability after the effect of KoRV load was accounted for. Further, koalas that were positive for KoRV-D or KoRV-D/F were more likely to have urogenital C. pecorum infection or low body condition score, respectively, irrespective of KoRV load. By contrast, our findings do not support previous findings that KoRV-B in particular is associated with Chlamydial disease. Based on these findings we suggest that koala research and conservation programs should target understanding what drives individual differences in KoRV load and limiting exogenous subtype diversity within populations, rather than seeking to eliminate any particular subtype.

Completing the Genome Sequence of Chlamydia pecorum Strains MC/MarsBar and DBDeUG: New Insights into This Enigmatic Koala (Phascolarctos cinereus) Pathogen.

Chlamydia pecorum, an obligate intracellular pathogen, causes significant morbidity and mortality in livestock and the koala (Phascolarctos cinereus). A variety of C. pecorum gene-centric molecular studies have revealed important observations about infection dynamics and genetic diversity in both koala and livestock hosts. In contrast to a variety of C. pecorum molecular studies, to date, only four complete and 16 draft genomes have been published. Of those, only five draft genomes are from koalas. Here, using whole-genome sequencing and a comparative genomics approach, we describe the first two complete C. pecorum genomes collected from diseased koalas. A de novo assembly of DBDeUG_2018 and MC/MarsBar_2018 resolved the chromosomes and chlamydial plasmids each as single, circular contigs. Robust phylogenomic analyses indicate biogeographical separation between strains from northern and southern koala populations, and between strains infecting koala and livestock hosts. Comparative genomics between koala strains identified new, unique, and shared loci that accumulate single-nucleotide polymorphisms and separate between northern and southern, and within northern koala strains. Furthermore, we predicted novel type III secretion system effectors. This investigation constitutes a comprehensive genome-wide comparison between C. pecorum from koalas and provides improvements to annotations of a C. pecorum reference genome. These findings lay the foundations for identifying and understanding host specificity and adaptation behind chlamydial infections affecting koalas.

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

Simple SummaryKoala retrovirus (KoRV) is a significant threat to koalas across Australia. Koalas in northern koala populations (from New South Wales and Queensland) have KoRV inserted into their DNA and inherited to their offspring. Southern koala populations (from Victoria and South Australia) have KoRV infection spread through close contact of koalas. As such, there are koalas within South Australia that are not infected with KoRV. Accurate diagnosis of the infection of each koala is therefore fundamental for disease studies. Previous studies have shown differences in prevalence of different KoRV genes in the Mount Lofty Ranges Koala population; therefore, clarification is necessary. This study uses a large cohort (n = 216) and defines the diagnostic regions of the KoRV genome within the South Australian population. Using multiple molecular techniques, it demonstrates strong evidence for two clear groupings of koalas: KoRV positive and KoRV negative. Within this study, a population of 41% were shown to be KoRV positive and 57% were KoRV negative, with 2% inconclusive. This differentiation is of great importance when examining the clinical importance of KoRV infection within southern koalas.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.

Koalas vaccinated against Koala retrovirus respond by producing increased levels of interferon-gamma

Koala retrovirus (KoRV) is believed to be in an active state of endogenization into the koala genome. KoRV is present as both an endogenous and exogenous infection in all koalas in northern Australia. KoRV has been linked to koala pathologies including neoplasia and increased susceptibility to Chlamydia. A KoRV vaccine recently trialled in 10 northern koalas improved antibody response and reduced viral load. This communication reports the expression of key immune genes underlining the innate and adaptive immune response to vaccination in these northern koalas. The results showed that prior to vaccination, IL-8 was expressed at the highest levels, with at least 200-fold greater expression compared to other cytokines, while CD8 mRNA expression was significantly higher than CD4 mRNA expression level. Interferon-γ was up-regulated at both 4- and 8-weeks post-vaccination while IL-8 was down-regulated at 8-weeks post-vaccination.

Koala translocations and Chlamydia: Managing risk in the effort to conserve native species

Translocation of the koala (Phascolarctos cinereus) has been a controversial measure that has been utilised for over a century in the southern states of Australia. Recently, translocation has been suggested as an option in the management of some declining northern koala populations. Infectious diseases present within donor and recipient populations are important factors that must be considered when planning any wildlife translocation. In the koala, infectious diseases, caused by the bacterial pathogen Chlamydia pecorum, are one of the key threats to koala conservation. In recent years, significant progress has been made in understanding the biology and epidemiology of koala C. pecorum infections revealing complex patterns of infection and disease and the potential for ‘spill-over’ from C. pecorum infected livestock. Here, in light of this new epidemiological data, we provide a discussion of risk assessment and management in the context of enzootic chlamydial infections. We conclude that without careful investigation and management, significant risk of pathogen transfer is likely, especially for larger and more distant translocations. Thus, for such a programme to be appropriate, they must: 1) perform adequate molecular screening methods for Chlamydia at both donor and recipient sites; 2) implement risk mitigation strategies that avoid transmission of Chlamydia genotypes that are not enzootic to the recipient site; and 3) assess and mitigate risk associated with potential transmission between koalas and livestock. Standardised and comprehensive veterinary procedures are crucial in the assessment and management of disease and infection transmission risk, and telemetric monitoring is essential for the post-release monitoring of both translocated and resident koalas at the recipient site and subsequent evaluation of programme success.

Identification of unusual Chlamydia pecorum genotypes in Victorian koalas (Phascolarctos cinereus) and clinical variables associated with infection.

Chlamydia pecorum infection is a threat to the health of free-ranging koalas (Phascolarctos cinereus) in Australia. Utilizing an extensive sample archive we determined the prevalence of C. pecorum in koalas within six regions of Victoria, Australia. The ompA genotypes of the detected C. pecorum were characterized to better understand the epidemiology of this pathogen in Victorian koalas. Despite many studies in northern Australia (i.e. Queensland and New South Wales), prior Chlamydia studies in Victorian koalas are limited. We detected C. pecorum in 125/820 (15 %) urogenital swabs, but in only one ocular swab. Nucleotide sequencing of the molecular marker C. pecorum ompA revealed that the majority (90/114) of C. pecorum samples typed were genotype B. This genotype has not been reported in northern koalas. In general, Chlamydia infection in Victorian koalas is associated with milder clinical signs compared with infection in koalas in northern populations. Although disease pathogenesis is likely to be multifactorial, the high prevalence of genotype B in Victoria may suggest it is less pathogenic. All but three koalas had C. pecorum genotypes unique to southern koala populations (i.e. Victoria and South Australia). These included a novel C. pecorum ompA genotype and two genotypes associated with livestock. Regression analysis determined that significant factors for the presence of C. pecorum infection were sex and geographical location. The presence of 'wet bottom' in males and the presence of reproductive tract pathology in females were significantly associated with C. pecorum infection, suggesting variation in clinical disease manifestations between sexes.

Koala retrovirus in free-ranging populations—prevalence

The prevalence of koala retrovirus (KoRV) provirus (DNA) and the average number of proviral insertions per cell vary in different free-ranging koala (Phascolarctos cinereus) populations across Australia. Populations in the northern states of Queensland and New South Wales have 100% proviral prevalence and mean proviral copy number of 140-165 per cell. In contrast, the proviral prevalence in the southern states of Victoria and South Australia differs among populations, with a mean prevalence in these states' mainland populations of 73% and 38%, respectively and with the prevalence on southern island populations ranging from 0-50%. The proviral load in southern populations is comparatively low, with some populations having an average of less than 1 proviral copy per cell. The KoRV RNA load in plasma shows a similar discordance between northern and southern populations, with consistently high loads in northern koalas (103 to 1010 RNA copies per ml plasma), and loads ranging from 0 to 102 copies per ml in southern KoRV provirus-positive koalas. The variation in KoRV proviral prevalence and the disparity in proviral and viral loads between northern and southern koalas may reflect different types of infection in the two populations (endogenous versus exogenous). Alternatively, it is possible that KoRV has been present for a longer time period in northern populations resulting in differences in the host-virus relationship.

Establishing priorities for research on the epidemiology of koala retrovirus (KoRV) in koalas (Phascolarctos cinereus)

This manuscript summarizes the break-out session held on the epidemiology of disease expression of koala retrovirus (KoRV) in koalas (Phascolarctos cinereus) at the Koala Conservation Workshop: The koala and its retroviruses: implications for sustainability and survival held at San Diego Zoo, April 17–18, 2013. The goals of this break-out session were to develop and prioritize specific research goals related to KoRV epidemiology, to identify actions, and to determine the responsible parties and timelines. Identified areas for epidemiologic research include studies in both wild and captive populations. For wild populations, baseline estimates of incidence and prevalence that account for potential biases in surveillance are needed. Landscape-level studies that determine whether KoRV contributes to the decline or stability of wild populations are also a priority. Captive populations with high-quality health data and management records can provide opportunities to identify factors associated with disease expression. These populations may also be pivotal in understanding the clinical importance of different KoRV subtypes. Witte, Carmel L. 2014. Establishing priorities for research on the epidemiology of koala retrovirus (KoRV) in koalas (Phascolarctos cinereus). In The Koala and its Retroviruses: Implications for Sustainability and Survival, ed. Geoffrey W. Pye, Rebecca N. Johnson and Alex D. Greenwood. Technical Reports of the Australian Museum, Online 24: 61–63. Relevant to any epidemiologic study are the two important questions: What do we already know and what are the current gaps in knowledge? What do we know? There are koala populations with low KoRV prevalence and with no disease expression. The Kangaroo Island population is a good example of lower KoRV prevalence with little disease expression (Simmons et al., 2012). There are also populations with high KoRV prevalence with little disease expression (e.g., St. Bees Island) (Tarlinton et al., 2006; Bill Ellis, pers. comm. 2014). There appears to be a difference in prevalence and disease expression between populations of northern koalas versus southern koalas (Simmons et al., 2012). There also appears to be similar prevalence of KoRV with widely differing prevalence of disease expression between southeastern and central Queensland koala populations (Simmons et al., 2013; Amber Gillett and Sean FitzGibbon pers. comm. 2013). One challenge in Australia is that there have only been a few studies where sampling and testing was limited and opportunistic. Gaps in knowledge. Currently the important gaps in knowledge are: Does KoRV causes disease in koalas and if so, is it associated with declines? What diseases (e.g., neoplasia) are caused by KoRV in koalas? Are there environmental, social, or other triggers for disease expression? What role do the exogenous and endogenous variants of KoRV play in causing disease? Does KoRV viral load increase with age?

GASTROINTESTINAL TORSIONS AND INTUSSUSCEPTION IN NORTHERN KOALAS (PHASCOLARCTOS CINEREUS) AT SAN DIEGO ZOO (1976–2012)

The recent classification as threatened status of the northern koala (Phascolarctos cinereus) by the Australian Government highlights the importance of the conservation and health management of this iconic Australian marsupial. This case series describes gastrointestinal torsion and intussusception in six northern koalas (three males, three females, 2-11 yr old) at the San Diego Zoo from 1976 to 2012. Two koalas died shortly after presentation. Diagnoses of ileocecal intussusception, resulting from enteritis in one case and cecal torsion in the other, were made at postmortem examination. One koala died 4 days after an exploratory laparotomy, with negative findings, and an acute double colonic intussusception was diagnosed at postmortem examination. Two small intestinal mesenteric torsion and one proximal colon mesenteric torsion cases were successfully corrected surgically. In the case of colonic mesenteric torsion, the koala had recurrent clinical signs 2 wk later, and a second surgery requiring resection and anastomosis of ischemic jejunum was performed, with the koala dying shortly afterward. One koala with small intestinal torsion had a recurrence of torsion 22 mo later and subsequently died. The koala with the second case of small intestinal torsion remains alive 14 mo postsurgical correction. All six koalas presented with signs of colic that included anorexia, lethargy, depression, acute abdominal distension, abdominal stretching, decreased fecal output, open-mouth gasping, or a combination of symptoms. Abdominal radiographs may show stacked gastrointestinal linear gas patterns and contrast stasis. Prevalence of torsion and intussusception is low at this institution (2%), although recurrence in individuals is common (50%) and overall survival is poor (83%), which emphasizes the importance of timely recognition, surgical correction, and postoperative management. While inciting etiologies were unable to be determined in these cases, monitoring generalized gastrointestinal health and differing Eucalyptus sp. effects on individual koala's gastrointestinal function, parasite control, and stress minimization through standardized husbandry practices are likely important.

Shoulder Dysplasia in Koalas (Phascolarctos cinereus) at San Diego Zoo

A radiographic study documented shoulder dysplasia (n = 43), with varying degrees of malformation of the supraglenoid and infraglenoid tubercles and the coracoid process, shallowing or loss of the glenoid cavity, flattening or loss of the humeral head, malformation of the greater and lesser tubercles, loss of the intertubercle groove, and humeral diaphyseal abnormalities, in northern koalas (Phascolarctos cinereus) in the San Diego Zoo (San Diego, California, USA) colony. Retrospectively, historic radiographs (n = 38) were examined where available. Prospectively, three standard views (lateral extended arm, ventrodorsal cranially positioned arms, and ventrodorsal caudally positioned arms) were imaged (n = 25). In all radiographs, shoulders were graded as normal, or mildly, moderately, or severely dysplastic. Although affected koalas typically do not exhibit clinical signs, degenerative joint disease may develop and clinical signs treated with nonsteroidal anti-inflammatory drugs. Where shoulder and hip radiographs were both available (n = 60), 92% of individuals had correlation between the degree of shoulder and hip dysplasia.

Noninvasive Monitoring of Reproductive Activity Based on Fecal Progestagen Profiles and Sexual Behavior in Koalas, Phascolarctos cinereus1

Studies on the reproductive endocrinology of koalas have been performed mainly by using blood samples; however, in practice it is difficult to collect blood periodically because koalas are easily stressed. The purposes of the present study were to establish a noninvasive endocrine monitoring technique and to investigate the reproductive physiology of female koalas. Feces were collected from female northern and southern koalas, and progestagen was extracted from lyophilized fecal samples and determined by enzyme immunoassay. In nonpregnant northern and southern koalas, fecal progestagen markedly increased after copulation and remained high for 36.3 +/- 2.5 days and 38.9 +/- 1.4 days (luteal phase, mean +/- SEM), respectively. Mean (+/-SEM) progestagen levels (6.34 +/- 0.49 microg/g) during the luteal phase in northern koalas were significantly higher than in southern koalas (4.19 +/- 0.24 microg/g). Fecal progestagen in parturient northern koalas remained high for 36.2 +/- 1.9 days (gestation period, 34.1 +/- 0.3 days). In northern koalas, the mean levels and profiles of progestagen during pregnancy (6.44 +/- 0.37 microg/g) were consistent with those during nonpregnancy after copulation (6.34 +/- 0.49 microg/g). The duration of behavioral estrus in northern koalas was 13.5 +/- 0.9 days without copulation. In contrast, when estrous females mated, the estrous sign disappeared just after copulation. The mean (+/-SEM) length of the estrous cycle in northern koalas, as determined by behavioral estrus intervals, was 33.5 +/- 2.2 days without the luteal phase and 69.2 +/- 7.6 days with the luteal phase. Fecal progestagen analysis is a helpful and noninvasive tool to monitor ovulatory activity in northern and southern koalas and could help us to understand the reproductive activity of koalas by the combination approach with behavioral estrus.

Hip Dysplasia in Koalas (Phascolarctos cinereus) at the San Diego Zoo

A retrospective/prospective radiographic study documented 55 cases of moderate to severe hip dysplasia, with varying degrees of shallowing of the acetabulum, flattening or loss of the femoral head, widening or loss of the femoral neck, and femoral diaphyseal abnormalities in northern koalas (Phascolarctos cinereus) in the San Diego Zoo (San Diego, California, USA) collection. For the retrospective study, historic radiographs were examined when availble. For the prospective study, three standard views (ventrodorsal extended leg, ventrodorsal frog leg, and lateral extended leg) were used. A scoring system was developed using four areas (acetabulum, femoral head, femoral neck, and femur) and ranges of 0 to 5 (0 = not affected to 5 = severely affected) for each area, creating a total score out of 40. Scores were graded as follows: 0-2 = normal dysplasia; 3-6 = mild dysplasia; 10-19 = moderate dysplasia; and 20-40 = severe dysplasia. Thirty koalas were graded as severe, 25 koalas as moderate, and 38 koalas as excellent or mild. Affected koalas may or may not demonstrate gait abnormalities. Mild to severe degenerative joint disease may develop and symptoms may be alleviated with glucosamine/chondroitin sulfate and nonsteroidal anti-inflammatory drugs. The etiology of hip dysplasia in koalas is not currently understood.