Infection In Koalas Research Articles

Epidemiology, Transmission Mode, and Pathogenesis of Chlamydia pecorum Infection in Koalas (Phascolarctos cinereus): An Overview.

Chlamydial infections pose a significant threat to koala populations. Chlamydia pecorum (C. pecorum) remains the major chlamydial species affecting koala health, both in the wild and in captivity, and chlamydial infections are considered important factors affecting the long-term survival of koalas. A clear understanding of chlamydial infections, including the epidemiology, transmission mode, pathogenesis, immune response, control, and prevention thereof, is essential for improving the management of chlamydial infections in koalas. In this study, we discuss the important advances made in our understanding of C. pecorum infection in koalas, focusing on the epidemiology of chlamydial infections, and the transmission, pathogenesis, immune response, and control strategies for chlamydial infection, with the aim of improving koala health and achieving effective conservation strategies.

Plethora of New Marsupial Genomes Informs Our Knowledge of Marsupial MHC Class II.

The major histocompatibility complex (MHC) plays a vital role in the vertebrate immune system due to its role in infection, disease and autoimmunity, or recognition of "self". The marsupial MHC class II genes show divergence from eutherian MHC class II genes and are a unique taxon of therian mammals that give birth to altricial and immunologically naive young providing an opportune study system for investigating evolution of the immune system. Additionally, the MHC in marsupials has been implicated in disease associations, including susceptibility to Chlamydia pecorum infection in koalas. Due to the complexity of the gene family, automated annotation is not possible so here we manually annotate 384 class II MHC genes in 29 marsupial species. We find losses of key components of the marsupial MHC repertoire in the Dasyuromorphia order and the Pseudochiridae family. We perform PGLS analysis to show the gene losses we find are true gene losses and not artifacts of unresolved genome assembly. We investigate the associations between the number of loci and life history traits, including lifespan and reproductive output in lineages of marsupials and hypothesize that gene loss may be linked to the energetic cost and tradeoffs associated with pregnancy and reproduction. We found support for litter size being a significant predictor of the number of DBA and DBB loci, indicating a tradeoff between the energetic requirements of immunity and reproduction. Additionally, we highlight the increased susceptibility of Dasyuridae species to neoplasia and a potential link to MHC gene loss. Finally, these annotations provide a valuable resource to the immunogenetics research community to move forward and further investigate diversity in MHC genes in marsupials.

Investigation of koala retrovirus in captive koalas with pneumonia and comparative analysis of subtype distribution.

This study focused on the involvement of koala retrovirus (KoRV) in pneumonia in koalas. Three deceased pneumonic koalas from a Japanese zoo were examined in this study. Hematological and histopathological findings were assessed, and KoRV proviral DNA loads in the blood and tissues were compared with those of eight other KoRV-infected koalas from different zoos. Demographic data and routine blood profiles were collected, and blood and tissue samples were analyzed to rule out concurrent infections in pneumonic koalas. KoRV subtyping and measurement of the KoRV proviral DNA load were performed by polymerase chain reaction (PCR) using specific primers targeting the pol and env genes. The results showed that the koalas had histopathologically suppurative and fibrinous pneumonia. Chlamydiosis was not detected in any of the animals. PCR analysis revealed KoRV-A, -B, and -C infections in all koalas, except for animals K10-11, which lacked KoRV-B. Significant variations in the proviral DNA loads of these KoRV subtypes were observed in all tissues and disease groups. Most tissues showed reduced KoRV loads in koalas with pneumonia, except in the spleen, which had significantly higher loads of total KoRV (2.54 × 107/µg DNA) and KoRV-A (4.74 × 107/µg DNA), suggesting potential immunosuppression. This study revealed the intricate dynamics of KoRV in various tissues, indicating its potential role in koala pneumonia via immunosuppression and opportunistic infections. Analysis of the levels of KoRV proviral DNA in different tissues will shed light on viral replication and the resulting pathogenesis in future studies.

LONEPINELLA SP. ISOLATED FROM WOUND INFECTIONS OF KOALAS.

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.

Development of diagnostic and point of care assays for a gammaherpesvirus infecting koalas.

The recent listing of koala populations as endangered across much of their range has highlighted the need for better management interventions. Disease is a key threat to koala populations but currently there is no information across the threatened populations on the distribution or impact of a gammaherpesvirus, phascolarctid gammaherpesvirus 1 (PhaHV-1). PhaHV-1 is known to infect koalas in southern populations which are, at present, not threatened. Current testing for PhaHV-1 involves lengthy laboratory techniques that do not permit quantification of viral load. In order to better understand distribution, prevalence and impacts of PhaHV-1 infections across koala populations, diagnostic and rapid point of care tests are required. We have developed two novel assays, a qPCR assay and an isothermal assay, that will enable researchers, clinicians and wildlife managers to reliably and rapidly test for PhaHV-1 in koalas. The ability to rapidly diagnose and quantify viral load will aid quarantine practices, inform translocation management and guide research into the clinical significance and impacts of PhaHV-1 infection in koalas.

Toll-Like Receptor and Cytokine Responses to Infection with Endogenous and Exogenous Koala Retrovirus, and Vaccination as a Control Strategy.

Koala populations are currently declining and under threat from koala retrovirus (KoRV) infection both in the wild and in captivity. KoRV is assumed to cause immunosuppression and neoplastic diseases, favoring chlamydiosis in koalas. Currently, 10 KoRV subtypes have been identified, including an endogenous subtype (KoRV-A) and nine exogenous subtypes (KoRV-B to KoRV-J). The host’s immune response acts as a safeguard against pathogens. Therefore, a proper understanding of the immune response mechanisms against infection is of great importance for the host’s survival, as well as for the development of therapeutic and prophylactic interventions. A vaccine is an important protective as well as being a therapeutic tool against infectious disease, and several studies have shown promise for the development of an effective vaccine against KoRV. Moreover, CRISPR/Cas9-based genome editing has opened a new window for gene therapy, and it appears to be a potential therapeutic tool in many viral infections, which could also be investigated for the treatment of KoRV infection. Here, we discuss the recent advances made in the understanding of the immune response in KoRV infection, as well as the progress towards vaccine development against KoRV infection in koalas.

Koala cathelicidin PhciCath5 has antimicrobial activity, including against Chlamydia pecorum.

Devastating fires in Australia over 2019–20 decimated native fauna and flora, including koalas. The resulting population bottleneck, combined with significant loss of habitat, increases the vulnerability of remaining koala populations to threats which include disease. Chlamydia is one disease which causes significant morbidity and mortality in koalas. The predominant pathogenic species, Chlamydia pecorum, causes severe ocular, urogenital and reproductive tract disease. In marsupials, including the koala, gene expansions of an antimicrobial peptide family known as cathelicidins have enabled protection of immunologically naïve pouch young during early development. We propose that koala cathelicidins are active against Chlamydia and other bacteria and fungi. Here we describe ten koala cathelicidins, five of which contained full length coding sequences that were widely expressed in tissues throughout the body. Focusing on these five, we investigate their antimicrobial activity against two koala C. pecorum isolates from distinct serovars; MarsBar and IPTaLE, as well as other bacteria and fungi. One cathelicidin, PhciCath5, inactivated C. pecorum IPTaLE and MarsBar elementary bodies and significantly reduced the number of inclusions compared to the control (p<0.0001). Despite evidence of cathelicidin expression within tissues known to be infected by Chlamydia, natural PhciCath5 concentrations may be inadequate in vivo to prevent or control C. pecorum infections in koalas. PhciCath5 also displayed antimicrobial activity against fungi and Gram negative and positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Electrostatic interactions likely drive PhciCath5 adherence to the pathogen cell membrane, followed by membrane permeabilisation leading to cell death. Activity against E. coli was reduced in the presence of 10% serum and 20% whole blood. Future modification of the PhciCath5 peptide to enhance activity, including in the presence of serum/blood, may provide a novel solution to Chlamydia infection in koalas and other species.

Toll-Like Receptor Expression Profiles in Koala (Phascolarctos cinereus) Peripheral Blood Mononuclear Cells Infected with Multiple KoRV Subtypes.

Simple SummaryKoala retrovirus (KoRV) is a major pathogen of koala. Toll-like receptors (TLRs) are important innate immune component that are evolutionary conserved and play a crucial role in the early defense against invading pathogens. The expression profile of TLRs in KoRV infection in koalas is not characterized yet. Therefore, in this study, we characterized TLR expression patterns in koalas infected with KoRV-A only vs. KoRV-A with KoRV-B and/or -C. Using qRT-PCR, we measured TLR2–10 and TLR13 mRNA expression in peripheral blood mononuclear cells (PBMCs) and/or tissues from captive koalas in Japanese zoos. We observed variations in TLR expression in koalas with a range of subtype infection profiles (KoRV-A only vs. KoRV-A with KoRV-B and/or -C). The findings of this study might improve our current understanding of koala’s immune response to KoRV infection.Toll-like receptors (TLRs), evolutionarily conserved pattern recognition receptors, play an important role in innate immunity by recognizing microbial pathogen-associated molecular patterns. Koala retrovirus (KoRV), a major koala pathogen, exists in both endogenous (KoRV-A) and exogenous forms (KoRV-B to J). However, the expression profile of TLRs in koalas infected with KoRV-A and other subtypes is yet to characterize. Here, we investigated TLR expression profiles in koalas with a range of subtype infection profiles (KoRV-A only vs. KoRV-A with KoRV-B and/or -C). To this end, we cloned partial sequences for TLRs (TLR2–10 and TLR13), developed real-time PCR assays, and determined TLRs mRNA expression patterns in koala PBMCs and/or tissues. All the reported TLRs for koala were expressed in PBMCs, and variations in TLR expression were observed in koalas infected with exogenous subtypes (KoRV-B and KoRV-C) compared to the endogenous subtype (KoRV-A) only, which indicates the implications of TLRs in KoRV infection. TLRs were also found to be differentially expressed in koala tissues. This is the first report of TLR expression profiles in koala, which provides insights into koala’s immune response to KoRV infection that could be utilized for the future exploitation of TLR modulators in the maintenance of koala health.

Longitudinal study of wild koalas (Phascolarctos cinereus) reveals chlamydial disease progression in two thirds of infected animals

Chlamydial disease threatens many of Australia’s koala populations, and yet our understanding of chlamydial epidemiology and disease dynamics in koalas is limited by a lack of comprehensive, longitudinal population studies. To address this, we utilised longitudinal samples from a large-scale population study of wild koalas in south-east Queensland, to follow chlamydial infections over time and to investigate some of the drivers of disease progression. Our findings show, firstly, that almost two thirds of chlamydial infections progressed to disease, challenging the notion that chlamydial infections in koalas commonly remain chronic and asymptomatic. Secondly, disease progression at the urogenital tract site was associated with infection load, and urogenital tract shedding was significantly higher when koalas acquired a new infection. Thirdly, chronic chlamydial exposure was not necessary for pathogenic sequelae to develop, such as infertility and mortality. Fourthly, ompA-characterised strain sub-types may reflect tissue tropisms and pathogenicity, and the chlamydial status of some chronically infected koalas may be explained by reinfections with novel genotypes. Finally, successful antimicrobial treatment provided only short-term protection against reinfection and disease progression in susceptible koalas. These findings highlight the importance of identifying and preventing chlamydial infections in koalas, informing new population management strategies and research priorities.

Chlamydia pecorum Infection in the Male Reproductive System of Koalas ( Phascolarctos cinereus).

Chlamydiosis is the most documented and serious disease of koalas, characterized by ocular, urinary, and reproductive lesions. Since little attention has been paid to the pathological effects of this infection in the male reproductive system, we aimed to determine the incidence and severity of reproductive pathology associated with chlamydial infection in male koalas submitted to koala hospitals in southeast Queensland. The entire reproductive tract from 62 sexually mature male koalas not suitable for rehabilitation was evaluated and 677 tissue samples were collected for histology, immunohistochemistry (IHC), and real-time polymerase chain reaction (qPCR). Lymphoplasmacytic inflammation was observed in 178 of 677 (26.3%) tissue samples from the upper and lower reproductive tract, mainly in the prostatic, penile, and membranous urethra. IHC was positive for the chlamydial antigen in 19 of 451 normal samples (4.2%) and 46 of 178 samples with inflammation (25.8%), located within the cytoplasm of epithelial cells of the epididymis, vas deferens, prostate, bulbourethral glands, and the prostatic membranous and penile urethra. Chlamydia pecorum was detected via qPCR in 319 of 451 normal samples (70.7%) and 159 of 178 samples with inflammation (89.3%), with the highest incidence in the penile urethra, prostate, membranous urethra, and bulbourethral glands. This study suggests that Chlamydia infection in the male reproductive tract is more widespread than originally thought. Furthermore, the male reproductive tract might be a reservoir for persistent chlamydial infections in koalas, with important implications for prophylactic strategies and epidemiology.

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.

KOALA RETROVIRUS: A REVIEW

Koala retrovirus (KoRV) is a gammaretrovirus that has been identified in both captive and free-ranging koalas ( Phascolarctos cinereus ) with variable geographic distribution in Australia. KoRV is capable of both exogenous and endogenous transmission, which provides an interesting research platform for scientists to study active retrovirus endogenization into a host genome and offers veterinary scientists an opportunity to examine the clinical consequences of KoRV infection in koalas. Causation between KoRV and frequently recognized clinical conditions associated with immune suppression and neoplasia in koalas has not been definitively established, however research continues to evaluate a potential association. Three KoRV variants, KoRV-A, KoRV-B, and KoRV-J, have been the most thoroughly described and preliminary evidence suggests KoRV variability may be fundamental in host pathogenicity. In addition to reviewing what is currently known about KoRV, this article discusses treatment, management, and future research directions.

Antibody and Cytokine Responses of Koalas (Phascolarctos cinereus) Vaccinated with Recombinant Chlamydial Major Outer Membrane Protein (MOMP) with Two Different Adjuvants.

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.

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.

Characterisation of CD4 T cells in healthy and diseased koalas (Phascolarctos cinereus) using cell-type-specific monoclonal antibodies

The koala (Phascolarctos cinereus) is an arboreal herbivorous marsupial that is an Australian icon. Koalas in many parts of Australia are under multiple threats including habitat destruction, dog attacks, vehicular accidents, and infectious diseases such as Chlamydia spp. and the koala retrovirus (KoRV), which may contribute to the incidence of lymphoma and leukaemia in this species. Due to a lack of koala-specific immune reagents and assays there is currently no way to adequately analyse the immune response in healthy, diseased or vaccinated animals. This paper reports the production and characterisation of the first anti-koala CD4 monoclonal antibody (mAb). The koala CD4 gene was identified and used to develop recombinant proteins for mAb production. Fluorochrome-conjugated anti-CD4 mAb was used to measure the levels of CD4+ lymphocytes collected from koala spleens (41.1%, range 20–45.1%) lymph nodes (36.3%, range 19–55.9%) and peripheral blood (23.8%, range 17.3–35%) by flow cytometry. Biotin-conjugated anti-CD4 mAb was used for western blot to determine an approximate size of 52 kDa for the koala CD4 molecule and used in immunohistochemistry to identify CD4+ cells in the paracortical region and germinal centres of spleen and lymph nodes. Using the anti-CD4 mab we showed that CD4 cells from vaccinated, but not control, koalas proliferated following in vitro stimulation with UV-inactivated Chlamydia pecorum and recombinant chlamydial antigens. Since CD4+ T cells have been shown to play a pivotal role in clearing chlamydial infection in both human and mouse infections, using this novel antibody will help determine the role CD4+ T cells play in protection against chlamydial infection in koalas and also enhance our knowledge of how KoRV affects the koala immune system.

Humoral immune responses in koalas (Phascolarctos cinereus) either naturally infected with Chlamydia pecorum or following administration of a recombinant chlamydial major outer membrane protein vaccine

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.

Interleukin 17A is an immune marker for chlamydial disease severity and pathogenesis in the koala (Phascolarctos cinereus)

The koala (Phascolarctos cinereus) is an iconic Australian marsupial species that is facing many threats to its survival. Chlamydia pecorum infections are a significant contributor to this ongoing decline. A major limiting factor in our ability to manage and control chlamydial disease in koalas is a limited understanding of the koala’s cell-mediated immune response to infections by this bacterial pathogen. To identify immunological markers associated with chlamydial infection and disease in koalas, we used koala-specific Quantitative Real Time PCR (qrtPCR) assays to profile the cytokine responses of Peripheral Blood Mononuclear Cells (PBMCs) collected from 41 koalas with different stages of chlamydial disease. Target cytokines included the principal Th1 (Interferon gamma; IFNγ), Th2 (Interleukin 10; IL10), and pro-inflammatory cytokines (Tumor Necrosis Factor alpha; TNFα). A novel koala-specific IL17A qrtPCR assay was also developed as part of this study to quantitate the gene expression of this Th17 cytokine in koalas. A statistically significant higher IL17A gene expression was observed in animals with current chlamydial disease compared to animals with asymptomatic chlamydial infection. A modest up-regulation of pro-inflammatory cytokines, such as TNFα and IFNγ, was also observed in these animals with signs of current chlamydial disease. IL10 gene expression was not evident in the majority of animals from both groups. Future longitudinal studies are now required to confirm the role played by cytokines in pathology and/or protection against C. pecorum infection in the koala.

KoRV and Chlamydia: are they co-culprits?

There are two main infectious disease threats for the koala; Chlamydia and KoRV. A major question is whether or not KoRV predisposes koalas to more severe chlamydial disease. In the only study to date that has examined co-infections, KoRV load (as determined by qPCR) and chlamydial load (as determined by qPCR) and chlamydial disease were examined in wild koalas. While there was a statistically significant correlation between Chlamydia infection load and Chlamydia clinical disease score, there was no significant correlation between KoRV load and either Chlamydia infection load or Chlamydia clinical disease score, however the groups were not ideally constructed and hence additional comparisons are needed. If KoRV does predispose koalas to more severe chlamydial disease, one would expect it to do this via an effect on the koala immune system. A series of Chlamydia vaccine trials in captive as well as wild koalas are showing that koalas in fact appear to make perfectly normal antibody and cytokine responses to vaccine antigens, even if they have high circulating KoRV loads, arguing against an immune suppressive effect by KoRV. Timms, Peter. 2014. KoRV and Chlamydia: are they co-culprits? 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: 89–90. In Australia, wild koala (Phascolarctos cinereus) populations in many areas, particularly Queensland and NSW, are declining for many reasons. One of the main causes of these declines is infection and disease due to Chlamydia (Polkinghorne et al., 2013). While Chlamydia cause similar disease syndromes in their non-koala hosts, the koala seems to have a higher than expected level of disease. This raises the question as to whether or not KoRV is somehow contributing to chlamydial disease. This brief overview will focus first on what we know about Chlamydia in koalas and then look at the very limited data regarding KoRV and Chlamydia. Overview of Chlamydia Chlamydia is an obligate intracellular bacterium with a unique two-phase developmental cycle. Immunity to chlamydial infections requires both a strong, neutralising antibody response as well as an interferon-gamma directed T cell response. Of the nine species present in the genus Chlamydia, two, C. pecorum and C. pneumoniae, cause infections in koalas (Jackson et al., 1999; Deveraux et al., 2003). The frequency of chlamydial infections (measured by a range of techniques, but utilizing PCR mostly of late) varies between populations, ranging from nil (on just a few island populations) to 90% in several populations (Polkinghorne et al., 2013). Disease levels also vary, but usually represent 25% or so of the infection level at any time point sampled. Animals are infected at ocular and urogenital sites mainly. Of the two chlamydial species, C. pecorum is by far the most common and is thought to be the species responsible for the symptoms observed (Glassick et al., 1996). Even though it is C. pecorum that is responsible for most infection and disease in koalas, there is considerable genetic diversity between sub-strains (Jackson et al., 1997). A range of gene markers have been used to assess C. pecorum strain diversity and while there are some minor differences, they all show that the various koala C. pecorum genotypes cluster together, but show considerable strain diversity.

Prevention and treatment of koala retrovirus (KoRV) infection: lessons from studies of AIDS viruses in nonhuman primate models

The presence of multiple retroviruses in koalas (Phascolarctos cinereus), including viruses with exogenous infectious forms that may be associated with malignant disease manifestations, poses challenges for both management of captive populations and species preservation in the wild. The development of antiretroviral medications (ARV) for the treatment of human immunodeficiency virus (HIV) infection is one of the triumphs of modern medicine, and many of these drugs have relatively broad antiretroviral activity, suggesting they might be active against koala retroviruses (KoRVs). However, accumulating experience with the use of these medications in non-human primate (NHP) models of HIV infection and acquired immune deficiency syndrome (AIDS) points out several caveats and provides guidance in attempting to use anti-HIV drugs in the treatment of retroviral infection in nonhuman species. This manuscript reviews that experience from the perspective of potential use of ARVs for prevention and treatment of KoRV infection. Lifson, Jeffrey D. 2014. Prevention and treatment of koala retrovirus (KoRV) infection: lessons from studies of AIDS viruses in nonhuman primate models. 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: 65–69. The koala (Phascolarctos cinereus) represents a fascinating instance of retrovirus/host species interactions, with geographically high prevalence of an endogenizing retrovirus, provisionally designated koala retrovirus-A (KoRV-A), that is also found in exogenous, pathogenic forms, along with a more recently described distinct exogenous related virus, provisionally designated KoRV-B, that utilizes a different cellular receptor and is associated with malignant hematologic manifestations (Avila-Arcos et al., 2013; Canfield et al., 1988; Hanger et al., 2000; Oliveira et al., 2007; Shojima et al., 2013; Simmons et al., 2012; Stoye, 2006; Tarlinton et al., 2005, 2006, 2008). These viruses represent a management problem for captive populations, and a challenge for species preservation in the wild. The development of antiretroviral drugs for the treatment of HIV infection has dramatically improved both survival and quality of life for HIV infected individuals, and the relatively broad antiretroviral activity of many of these drugs suggest they may also be active against retroviruses affecting non-human species, such as KoRVs (Oliveira et al., 2007). However accumulating experience with the use of anti-HIV drugs in NHP models highlights important considerations and potential limitations to such use that may help inform efforts to use anti-HIV drugs for the treatment of KoRV infection in koalas (Del Prete & Lifson, 2013). Factors to consider include potency against the target virus (compared to HIV), drug delivery, pharmacokinetics, toxicity and sustainability of treatment. Perhaps the most important consideration is the relationship between the mechanism(s) of action and targets of the drugs considered in relation to the underlying pathogenesis of the disease process of concern.

Leukemogenesis by murine leukemia viruses: lessons for koala retrovirus (KoRV)

Murine leukemia viruses (MuLVs) are the prototypical gammaretroviruses, and they have been extensively studied with regard to how they cause disease. Leukemogenesis by two MuLVs is reviewed here: the endogenous Akv MuLV of AKR mice, and exogenous Moloney MuLV. Important features of MuLV leukemogenesis include the in vivo generation of envelope recombinants (MCFs) through recombination with endogenous MuLVs, and induction of preleukemic changes typified by splenic hyperplasia secondary to bone marrow defects. Studies of MuLV leukemogenesis help to frame virological questions about how koala retrovirus (KoRV) may induce neoplastic or other diseases in koalas. Fan, Hung. 2014. Leukemogenesis by murine leukemia viruses: lessons for koala retrovirus (KoRV). 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: 83–88. The discovery of koala retrovirus (KoRV) in free-ranging and captive koalas (Phascolarctos cinereus) has been viewed with concern and interest. The primary concern is that KoRV-associated disease such as neoplasms, while yet to be conclusively proven to be KoRV caused, could increase the threats to survival of these animals. In the scientific community there is interest for several reasons: KoRV may be associated with lymphoma in koalas, it appears to be recently introduced into this species, and endogenization is an ongoing process. KoRV infection in koalas may provide an opportunity to study introduction and spread of a gammaretrovirus into a new host species and its accompanying effects. This process has happened in other species, notably mice, but in the more distant past, so some of the processes can only be deduced. At the same time, information learned from the relationship of murine gammaretroviruses and their hosts may provide lessons for understanding the potential relationships of KoRV and disease in koalas. The recent discovery of a second KoRV (KoRV-B) that may be associated with leukemogenicity (Xu et al., 2013) has similarities to oncogenesis in murine leukemia viruses (MuLVs). Leukemogenesis by MuLVs will be summarized here and possible implications to KoRV pathogenesis will be pointed out. Murine leukemia viruses MuLVs were first discovered in inbred mouse strains that had high incidences of leukemia. These studies resulted in isolation of several MuLV strains that cause leukemias of different hematopoietic lineages. For instance Moloney MuLV (M-MuLV) and Gross MuLV induce T-lymphoma, while Friend (F-MuLV) and Rauscher MuLV (R-MuLV) induce erythroleukemia and myeloid leukemia (Fan, 1997). These are the predominant MuLVs used in studies of MuLV leukemogenesis. They are prototypical retroviruses of the gammaretrovirus family. MuLVs can be classified into types based on their envelope proteins and the kinds of cells that they infect, determined by the cell surface proteins that they bind. The leukemogenic MuLVs are mostly ecotropic; they infect cells of mice and rats, but they do not infect most