We describe two cases of wound infections of koalas (Phascolarctos cinereus), one wild and one captive, in which Lonepinella-like organisms were involved. The wild adult koala was captured with bite wound injuries, as part of a koala population management program in Queensland, Australia. In both cases, there was evidence of physical trauma causing the initial wound. The captive koala suffered injury from the cage wire, and the wild koala had injuries suggestive of intermale fighting. Gram-negative bacteria isolated from both cases proved to be challenging to identify using routine diagnostic tests. The wound in the captive koala yielded a pure culture of an organism shown by whole genome sequence (WGS) analysis to be a member of the genus Lonepinella, but not a member of the only formally described species, L. koalarum. The wound of the wild koala yielded a mixed culture of Citrobacter koseri, Enterobacter cloacae and an organism shown by WGS analysis to be Lonepinella, but again not Lonepinella koalarum. Both cases were difficult to treat; the captive koala eventually had to have the phalanges amputated, and the wild koala required removal of the affected claw. The two Lonepinella isolates from these cases have a close relationship to an isolate from a human wound caused by a koala bite and may represent a novel species within the genus Lonepinella. Wound infections in koalas linked to Lonepinella have not been reported previously. Wildlife veterinarians need to be aware of the potential presence of Lonepinella-like organisms when dealing with wound infections in koalas, and the inability of commercial kits and systems to correctly identify the isolates.

Habitat destruction and fragmentation are increasing globally, forcing surviving species into small, isolated populations. Isolated populations typically experience heightened inbreeding risk and associated inbreeding depression and population decline; although individuals in these populations may mitigate these risks through inbreeding avoidance strategies. For koalas, as dietary specialists already under threat in the northern parts of their range, increased habitat fragmentation and associated inbreeding costs are of great conservation concern. Koalas are known to display passive inbreeding avoidance through sex-biased dispersal, although population isolation will reduce dispersal pathways. We tested whether free-ranging koalas display active inbreeding avoidance behaviours. We used VHF tracking data, parentage reconstruction, and veterinary examination results to test whether free-ranging female koalas avoid mating with (a) more closely related males; and (b) males infected with sexually transmittedChlamydia pecorum. We found no evidence that female koalas avoid mating with relatively more related available mates. In fact, as the relatedness of potential mates increases, so did inbreeding events. We also found no evidence that female koalas can avoid mating with males infected withC. pecorum. The absence of active inbreeding avoidance mechanisms in koalas is concerning from a conservation perspective, as small, isolated populations may be at even higher risk of inbreeding depression than expected. At risk koala populations may require urgent conservation interventions to augment gene flow and reduce inbreeding risks. Similarly, if koalas are not avoiding mating with individuals with chlamydial disease, populations may be at higher risk from disease than anticipated, further impacting population viability.

An understanding of the threats to threatened species in urban and peri-urban areas is essential to develop successful management approaches. Dog attacks are considered to be a major contributor to koala (Phascolarctos cinereus) mortalities in peri-urban areas of north-eastern Australia. Predation could be due to either domestic dogs or wild dogs (dingoes and dingo-domestic dog hybrids), gentically-identifiable groups of Canis familiaris. Here, we aimed to use genetic sampling methods to determine or verify the identity, number and successful removal of canid predators of koalas in a peri-urban environment in south-eastern Queensland. Genetic samples were taken from the remains of 12 koalas suspected to have died from predation. Canine genotypes were present on 11 of 12 predated koalas (similar to 92%) and were from wild dogs, not domestic dogs. Most koalas had only one canine genotype identified, suggesting they were killed by a single dog. Our results show that DNA samples collected from deceased prey species can be used to identify the predator, and distinguish between closely-related species, and hybrids of the two. Genetic methods confirmed the identification of the predator obtained through conventional necropsy and support growing evidence that wild dog predation is a significant cause of koala mortality in this region. Strategies to reduce predation on koalas should therefore focus on reducing the impact of free-ranging wild dog populations. This approach is important to identify and target those canids responsible for predation of threatened prey populations, particularly where multiple predators are present and/or predator removals may be controversial.

1. Management actions designed to mitigate development or anthropogenic impacts on species of conservation concern are often implemented without quantifying the benefit to the species. It is often unclear what combinations and intensities of management actions are required to achieve meaningful conservation outcomes. We investigate whether disease and predator control can reverse population-declines of koalas (Phascolarctos cinereus). 2. Based on longitudinal monitoring of the epidemiological and demographic status of over 500 animals over 4 years, coupled with an intensive disease and predator management programme, we use survival analyses to estimate annual age-specific survival rates and population growth, and simulations to quantify the benefit of these actions. 3. Predation and disease accounted for 63% and 29% of mortality, respectively, across all years, with wild dog (dingoes or dingo-hybrids: Canis dingo, C. dingo x Canis familiaris), carpet pythons (Morelia spilota) and domestic dogs (C. familiaris) accounting for 82%, 14% and 4% of confirmed predation mortalities, respectively. In the first 2 years, before disease and dog control had major impact, the population was declining rapidly with annual growth rates of 0.66 and 0.90. In the third and fourth years, after interventions had been fully implemented, the population growth rate had increased to 1.08 and 1.20. The intrinsic survival rate of joeys was 71.2% (excluding deaths resulting from the death of the mother). Adult survival rates varied as a function of sex, age and year. 4. Even in a declining koala population, management actions can achieve meaningful conservation outcomes (population growth rates greater than one). However, benefits may be short-lived in the absence of longer term strategies to manage threats. This work also identifies wild dogs as a major threat to koalas, highlighting the need to better understand how wild dog impacts vary in space and time. 5. Policy implications. Offsetting policy that addresses habitat loss alone may achieve little or no meaningful benefit to declining koalas populations. Management must address suites of threats affecting these populations and ensure that the cumulative effects of these actions achieve positive population growth rates.

Chlamydia pecorum in koalas (Phascolarctos cinereus) is considered a sexually transmitted infection. Analysis of samples from koala joeys (<1 yr) suggested that mother-toyoung direct transmission was also occurring. Further, evidence suggested that joeys from vaccinated mothers were less likely to contract infections than joeys with unvaccinated mothers.

Developing a vaccine against Chlamydia is key to combating widespread mortalities and morbidities associated with this infection in koalas (Phascolarctos cinereus). In previous studies, we have shown that two or three doses of a Recombinant Major Outer Membrane Protein (rMOMP) antigen-based vaccine, combined with immune stimulating complex (ISC) adjuvant, results in strong cellular and humoral immune responses in koalas. We have also separately evaluated a single dose vaccine, utilising a tri-adjuvant formula that comprises polyphosphazine based poly I: C and host defense peptides, with the same antigen. This formulation also produced strong cellular and humoral immune responses in captive koalas. In this current study, we directly compared the host immune responses of two sub-groups of wild Chlamydia negative koalas in one population vaccinated with the rMOMP protein antigen and adjuvanted with either the ISC or tri-adjuvant formula. Overall, both adjuvants produced strong Chlamydia-specific cellular (IFN-γ and IL-17A) responses in circulating PBMCs as well as MOMP-specific and functional, in vitro neutralising antibodies. While the immune responses were similar, there were adjuvant-specific immune differences between the two adjuvants, particularly in relation to the specificity of the MOMP epitope antibody responses.

The development of a vaccine is a key strategy to combat the widespread and debilitating effects of chlamydial infection in koalas. One such vaccine in development uses recombinant chlamydial major outer membrane protein (rMOMP) as an antigen and has shown promising results in several koala trials. Previous chlamydial vaccine studies, primarily in the mouse model, suggest that both cell-mediated and antibody responses will be required for adequate protection. Recently, the important protective role of antibodies has been highlighted. In our current study, we conducted a detailed analysis of the antibody-mediated immune response in koalas that are either (a) naturally-infected, and/or (b) had received an rMOMP vaccine. Firstly, we observed that naturally-infected koalas had very low levels of Chlamydia pecorum-specific neutralising antibodies. A strong correlation between low IgG total titers/neutralising antibody levels, and higher C. pecorum infection load was also observed in these naturally-infected animals. In vaccinated koalas, we showed that the vaccine was able to boost the humoral immune response by inducing strong levels of C. pecorum-specific neutralising antibodies. A detailed characterisation of the MOMP epitope response was also performed in naturally-infected and vaccinated koalas using a PepScan epitope approach. This analysis identified unique sets of MOMP epitope antibodies between naturally-infected non-protected and diseased koalas, versus vaccinated koalas, with the latter group of animals producing a unique set of specific epitope-directed antibodies that we demonstrated were responsible for the in vitro neutralisation activity. Together, these results show the importance of antibodies in chlamydial infection and immunity following vaccination in the koala.

Traditionally, culling has been the expedient, most common, and in many cases, the only tool used to control free-ranging kangaroo populations. We applied a reproductive control program to a population of eastern grey kangaroos confined to a golf course in South East Queensland. The program aimed to reduce fecundity sufficiently for the population to decrease over time so that overgrazing of the fairways and the frequency of human–animal conflict situations were minimised. In 2003, 92% of the female kangaroos above 5 kg bodyweight were implanted with the GnRH agonist deslorelin after darting with a dissociative anaesthetic. In 2007, 86% of the females above 5 kg were implanted with deslorelin and also 87% of the males above 5 kg were sterilised by either orchidectomy or vasectomy. In 2005, 2008 and 2009, the population was censused to assess the effect of each treatment. The 2003 deslorelin program resulted in effective zero population growth for approximately 2.5 years. The combined deslorelin–surgery program in 2007 reduced the birth rate from 0.3 to 0.06%/year for 16 months, resulting in a 27% population reduction by November 2009. The results were consistent with implants conferring contraception to 100% of implanted females for at least 12 months. The iatrogenic mortality rates for each program were 10.5% and 4.9%, respectively, with 50% of all mortalities due to darting-related injuries, exertional myopathy/hyperthermia or recovery misadventure. The short term sexual and agonistic behaviour of the males was assessed for the 2007 program: no significant changes were seen in adult males given the vasectomy procedure, while sexual behaviours’ were decreased in adult males given the orchidectomy procedure. It is concluded that female reproduction was effectively controlled by implantation with deslorrelin and male reproductive behaviour was reduced by orchidectomy, which together achieved population control.

A wide range of serious, and oftentimes fatal, conditions has been observed in both free-living and captive populations of koalas (Phascolarctos cinereus) and are attributed, perhaps prematurely, to the koala retrovirus (KoRV). These maladies include lymphoma, leukaemia, and other bone marrow conditions, and the so-called koala AIDS. A variety of other conditions that involve disordered growth of cells and tissues, altered or inappropriate immune responses, and degenerative conditions may also be consequences of insertional mutagenesis, or other pathogenic mechanisms associated with KoRV infection. The list of potential KoRV-associated pathologies continues to grow, as more thorough and consistent approaches to clinical assessment and diagnosis are applied to wild and captive koalas.

Chlamydial infections in koalas can cause life-threatening diseases leading to blindness and sterility. However, little is known about the systemic spread of chlamydiae in the inner organs of the koala, and data concerning related pathological organ lesions are limited. The aim of this study was to perform a thorough investigation of organs from 23 koalas and to correlate their histopathological lesions to molecular chlamydial detection. To reach this goal, 246 formalin-fixed and paraffin embedded organ samples from 23 koalas were investigated by histopathology, Chlamydiaceae real-time PCR and immunohistochemistry, ArrayTube Microarray for Chlamydiaceae species identification as well as Chlamydiales real-time PCR and sequencing. By PCR, two koalas were positive for Chlamydia pecorum whereas immunohistochemical labelling for Chlamydiaceae was detected in 10 tissues out of nine koalas. The majority of these (n = 6) had positive labelling in the urogenital tract related to histopathological lesions such as cystitis, endometritis, pyelonephritis and prostatitis. Somehow unexpected was the positive labelling in the gastrointestinal tract including the cloaca as well as in lung and spleen indicating systemic spread of infection. Uncultured Chlamydiales were detected in several organs of seven koalas by PCR, and four of these suffered from plasmacytic enteritis of unknown aetiology. Whether the finding of Chlamydia-like organisms in the gastrointestinal tract is linked to plasmacytic enteritis is unclear and remains speculative. However, as recently shown in a mouse model, the gastrointestinal tract might play a role being the site for persistent chlamydial infections and being a source for reinfection of the genital tract.

The genome of koala retrovirus (KoRV) has striking similarity to the gibbon ape leukemia virus (GALV) genome, suggesting the two viruses may share a common ancestor. Screening of DNA from a range of potential hosts of this putative ancestor virus revealed retroviral sequence from a grassland melomys (Melomys burtoni) that was closely related to sequence of both KoRV and GALV. This novel virus has been named Melomys burtoni retrovirus (MbRV). As grassland melomys and koalas share habitat, it is possible that there has been cross-species transmission of virus in the past. Although a causative relationship between KoRV infection and disease in koalas is yet to be confirmed, koala populations with a high prevalence of KoRV infection have a higher incidence of diseases characteristic of retroviruses (cancer and immunosuppression) than populations with low KoRV-prevalence. Not all KoRV-infected koalas develop clinical disease. This variation in disease expression may result from differences in proviral (DNA) insertion sites among koalas, genetic variability of KoRV in different individuals or from variation in host genetics.

The genome of koala retrovirus (KoRV) has striking similarity to the gibbon ape leukemia virus (GALV) genome, suggesting the two viruses may share a common ancestor. Screening of DNA from a range of potential hosts of this putative ancestor virus revealed retroviral sequence from a grassland melomys (Melomys burtoni) that was closely related to sequence of both KoRV and GALV. This novel virus has been named Melomys burtoni retrovirus (MbRV). As grassland melomys and koalas share habitat, it is possible that there has been cross-species transmission of virus in the past. Although a causative relationship between KoRV infection and disease in koalas is yet to be confirmed, koala populations with a high prevalence of KoRV infection have a higher incidence of diseases characteristic of retroviruses (cancer and immunosuppression) than populations with low KoRVprevalence. Not all KoRV-infected koalas develop clinical disease. This variation in disease expression may result from differences in proviral (DNA) insertion sites among koalas, genetic variability of KoRV in different individuals or from variation in host genetics. Simmons, Greg, Joanne Meers, Daniel T. W. Clarke, Paul R. Young, Kiersten Jones, Jon J. Hanger, Jo Loader, and Jeff J. McKee. 2014. The origins and ecological impact of koala retrovirus. 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: 31–33. Gammaretroviruses (RV) infect a large range of vertebrate hosts, and are causative agents of a number of diseases including lymphoid tumours and immunosuppression (Bendinelli et al., 1985; Rosenberg & Jolicoeur, 1997). Koala retrovirus (KoRV) is a relatively newly discovered retrovirus which is widespread throughout wild koala (Phascolarctos cinereus) populations in Australia (Hanger et al., 2000; Simmons et al., 2012). KoRV is of particular interest because it is the only known retrovirus currently undergoing a process of active endogenization in its host (Tarlinton et al., 2006). Koalas are known to suffer a high incidence of both chlamydiosis and cancer, and the high prevalence of KoRV has been suggested as a possible aetiological agent for immunosuppression and cancer in these animals (Tarlinton et al., 2008). Koala numbers in the wild have declined alarmingly since the beginning of European colonization and their geographic range has been significantly reduced. While the reasons for this decline are multi factorial, the high prevalence of KoRV and its apparent association with other diseases in koalas is a serious cause for concern. 32 Technical Reports of the Australian Museum, Online (2014) No. 24

Background: Many koala populations around Australia are in serious decline, with a substantial component of this decline in some Southeast Queensland populations attributed to the impact of Chlamydia. A Chlamydia vaccine for koalas is in development and has shown promise in early trials. This study contributes to implementation preparedness by simulating vaccination strategies designed to reverse population decline and by identifying which age and sex category it would be most effective to target.Methods: We used field data to inform the development and parameterisation of an individual-based stochastic simulation model of a koala population endemic with Chlamydia. The model took into account transmission, morbidity and mortality caused by Chlamydia infections. We calibrated the model to characteristics of typical Southeast Queensland koala populations. As there is uncertainty about the effectiveness of the vaccine in real-world settings, a variety of potential vaccine efficacies, half-lives and dosing schedules were simulated.Results: Assuming other threats remain constant, it is expected that current population declines could be reversed in around 5-6 years if female koalas aged 1-2 years are targeted, average vaccine protective efficacy is 75%, and vaccine coverage is around 10% per year. At lower vaccine efficacies the immunological effects of boosting become important: at 45% vaccine efficacy population decline is predicted to reverse in 6 years under optimistic boosting assumptions but in 9 years under pessimistic boosting assumptions. Terminating a successful vaccination programme at 5 years would lead to a rise in Chlamydia prevalence towards pre-vaccination levels.Conclusion: For a range of vaccine efficacy levels it is projected that population decline due to endemic Chlamydia can be reversed under realistic dosing schedules, potentially in just 5 years. However, a vaccination programme might need to continue indefinitely in order to maintain Chlamydia prevalence at a sufficiently low level for population growth to continue. (C) 2014 Elsevier Ltd. All rights reserved.

Chlamydial infections represent a major threat to the long-term survival of the koala and a successful vaccine would provide a valuable management tool. Vaccination however has the potential to enhance inflammatory disease in animals exposed to a natural infection prior to vaccination, a finding in early human and primate trials of whole cell vaccines to prevent trachoma. In the present study, we vaccinated both healthy koalas as well as clinically diseased koalas with a multi-subunit vaccine consisting of Chlamydia pecorum MOMP and NrdB mixed with immune stimulating complex as adjuvant. Following vaccination, there was no increase in inflammatory pathological changes in animals previously infected with Chlamydia. Strong antibody (including neutralizing antibodies) and lymphocyte proliferation responses were recorded in all vaccinated koalas, both healthy and clinically diseased. Vaccine induced antibodies specific for both vaccine antigens were observed not only in plasma but also in ocular secretions. Our data shows that an experimental chlamydial vaccine is safe to use in previously infected koalas, in that it does not worsen infection-associated lesions. Furthermore, the prototype vaccine is effective, as demonstrated by strong levels of neutralizing antibody and lymphocyte proliferation responses in both healthy and clinically diseased koalas. Collectively, this work illustrates the feasibility of developing a safe and effective Chlamydia vaccine as a tool for management of disease in wild koalas.

Complex interactions between Chlamydia pecorum infection, the immune response and disease exist in the koala. We used quantitative polymerase chain reaction to investigate the relationship between C. pecorum infectious load and ocular and urogenital tract disease. Chlamydia pecorum shedding was generally higher in animals with chronic, active disease than in animals with inactive disease. The absence of ocular disease was generally associated with low levels of shedding, but relatively high levels of shedding in the urogenital tract were detected in some koalas without clinical disease signs. These results suggest a complex disease pathogenesis and clinical course in C. pecorum-infected koalas.