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.

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.

High-throughput immunogenetic typing of koalas suggests possible link between MHC alleles and cancers.

Characterizing the allelic diversity within major histocompatibility complex (MHC) genes is an important way of determining the potential genetic resilience of a population to infectious and ecological pressures. For the koala (Phascolarctos cinereus), endemic diseases, anthropogenic factors and climate change are all placing increased pressure on this vulnerable marsupial. To increase the ability of researchers to study MHC genetics in koalas, this study developed and tested a high-throughput immunogenetic profiling methodology for targeting MHC class I UA and UC genes and MHC class II DAB, DBB, DCB and DMB genes in a population of 82 captive koalas. This approach was validated by comparing the determined allelic profiles from 36 koala family units (18 dam-sire-joey units and 18 parent-joey pairs), finding 96% overall congruence within family profiles. Cancers are a significant cause of morbidity in koalas and the risk factors remain undetermined. Our analysis of this captive population revealed several novel MHC alleles, including a potential link between the DBB*03 allele and a risk of developing cancer. This method offers a reliable, high-throughput protocol for expanded study into koala immunogenetics.

Koala immunogenetics and chlamydial strain type are more directly involved in chlamydial disease progression in koalas from two south east Queensland koala populations than koala retrovirus subtypes.

Chlamydial disease control is increasingly utilised as a management tool to stabilise declining koala populations, and yet we have a limited understanding of the factors that contribute to disease progression. To examine the impact of host and pathogen genetics, we selected two geographically separated south east Queensland koala populations, differentially affected by chlamydial disease, and analysed koala major histocompatibility complex (MHC) genes, circulating strains of Chlamydia pecorum and koala retrovirus (KoRV) subtypes in longitudinally sampled, well-defined clinical groups. We found that koala immunogenetics and chlamydial genotypes differed between the populations. Disease progression was associated with specific MHC alleles, and we identified two putative susceptibility (DCb 03, DBb 04) and protective (DAb 10, UC 01:01) variants. Chlamydial genotypes belonging to both Multi-Locus Sequence Typing sequence type (ST) 69 and ompA genotype F were associated with disease progression, whereas ST 281 was associated with the absence of disease. We also detected different ompA genotypes, but not different STs, when long-term infections were monitored over time. By comparison, KoRV profiles were not significantly associated with disease progression. These findings suggest that chlamydial genotypes vary in pathogenicity and that koala immunogenetics and chlamydial strains are more directly involved in disease progression than KoRV subtypes.

Koala retrovirus epidemiology, transmission mode, pathogenesis, and host immune response in koalas (Phascolarctos cinereus): a review.

Koala retrovirus (KoRV) is a major threat to koala health and conservation. It also represents a series of challenges across the fields of virology, immunology, and epidemiology that are of great potential interest to any researcher in the field of retroviral diseases. KoRV is a gammaretrovirus that is present in both endogenous and exogenous forms in koala populations, with a still-active endogenization process. KoRV may induce immunosuppression and neoplastic conditions such as lymphoma and leukemia and play a role in chlamydiosis and other diseases in koalas. KoRV transmission modes, pathogenesis, and host immune response still remain unclear, and a clear understanding of these areas is critical for devising effective preventative and therapeutic strategies. Research on KoRV is clearly critical for koala conservation. In this review, we provide an overview of the current understanding and future challenges related to KoRV epidemiology, transmission mode, pathogenesis, and host immune response and discuss prospects for therapeutic and preventive vaccines.

Vaccination of koalas during antibiotic treatment for Chlamydia-induced cystitis induces an improved antibody response to Chlamydia pecorum.

Chlamydia infection and disease are endemic in free-ranging koalas. Antibiotics remain the front line treatment for Chlamydia in koalas, despite their rates of treatment failure and adverse gut dysbiosis outcomes. A Chlamydia vaccine for koalas has shown promise for replacing antibiotic treatment in mild ocular Chlamydia disease. In more severe disease presentations that require antibiotic intervention, the effect of vaccinating during antibiotic use is not currently known. This study investigated whether a productive immune response could be induced by vaccinating koalas during antibiotic treatment for Chlamydia-induced cystitis. Plasma IgG antibody levels against the C. pecorum major outer membrane protein (MOMP) dropped during antibiotic treatment in both vaccinated and unvaccinated koalas. Post-treatment, IgG levels recovered. The IgG antibodies from naturally-infected, vaccinated koalas recognised a greater proportion of the MOMP protein compared to their naturally-infected, unvaccinated counterparts. Furthermore, peripheral blood mononuclear cell gene expression revealed an up-regulation in genes related to neutrophil degranulation in vaccinated koalas during the first month post-vaccination. These findings show that vaccination of koalas while they are being treated with antibiotics for cystitis can result in the generation of a productive immune response, in the form of increased and expanded IgG production and host response through neutrophil degranulation.

Helping koalas battle disease - Recent advances in Chlamydia and koala retrovirus (KoRV) disease understanding and treatment in koalas.

The iconic Australian marsupial, the koala (Phascolarctos cinereus), has suffered dramatic population declines as a result of habitat loss and fragmentation, disease, vehicle collision mortality, dog attacks, bushfires and climate change. In 2012, koalas were officially declared vulnerable by the Australian government and listed as a threatened species. In response, research into diseases affecting koalas has expanded rapidly. The two major pathogens affecting koalas are Chlamydia pecorum, leading to chlamydial disease and koala retrovirus (KoRV). In the last eight years, these pathogens and their diseases have received focused study regarding their sources, genetics, prevalence, disease presentation and transmission. This has led to vast improvements in pathogen detection and treatment, including the ongoing development of vaccines for each as a management and control strategy. This review will summarize and highlight the important advances made in understanding and combating C. pecorum and KoRV in koalas, since they were declared a threatened species. With complementary advances having also been made from the koala genome sequence and in our understanding of the koala immune system, we are primed to make a significant positive impact on koala health into the future.

Therapeutic effect of a Chlamydia pecorum recombinant major outer membrane protein vaccine on ocular disease in koalas (Phascolarctos cinereus).

Chlamydia pecorum is responsible for causing ocular infection and disease which can lead to blindness in koalas (Phascolarctos cinereus). Antibiotics are the current treatment for chlamydial infection and disease in koalas, however, they can be detrimental for the koala's gastrointestinal tract microbiota and in severe cases, can lead to dysbiosis and death. In this study, we evaluated the therapeutic effects provided by a recombinant chlamydial major outer membrane protein (MOMP) vaccine on ocular disease in koalas. Koalas with ocular disease (unilateral or bilateral) were vaccinated and assessed for six weeks, evaluating any changes to the conjunctival tissue and discharge. Samples were collected pre- and post-vaccination to evaluate both humoral and cell-mediated immune responses. We further assessed the infecting C. pecorum genotype, host MHC class II alleles and presence of koala retrovirus type (KoRV-B). Our results clearly showed an improvement in the clinical ocular disease state of all seven koalas, post-vaccination. We observed increases in ocular mucosal IgA antibodies to whole C. pecorum elementary bodies, post-vaccination. We found that systemic cell-mediated immune responses to interferon-γ, interleukin-6 and interleukin-17A were not significantly predictive of ocular disease in koalas. Interestingly, one koala did not have as positive a clinical response (in one eye primarily) and this koala was infected with a C. pecorum genotype (E') that was not used as part of the vaccine formula (MOMP genotypes A, F and G). The predominant MHC class II alleles identified were DAb*19, DAb*21 and DBb*05, with no two koalas identified with the same genetic sequence. Additionally, KoRV-B, which is associated with chlamydial disease outcome, was identified in two (29%) ocular diseased koalas, which still produced vaccine-induced immune responses and clinical ocular improvements post-vaccination. Our findings show promise for the use of a recombinant chlamydial MOMP vaccine for the therapeutic treatment of ocular disease in koalas.

Koala immunology and infectious diseases: How much can the koala bear?

Infectious diseases are contributing to the decline of the iconic Australian marsupial, the koala (Phascolarctos cinereus). Infections with the obligate intracellular bacteria, Chlamydia pecorum, cause debilitating ocular and urogenital-tract disease while the koala-retrovirus (KoRV) has been implicated in host immunosuppression and exacerbation of chlamydial pathogenesis. Although histological studies have provided insight into the basic architecture of koala immune tissues, our understanding of the koala immune response to infectious disease has been limited, until recently, by a lack of species-specific immune reagents. Recent advances in the characterisation of key immune genes have focused on advancing our understanding of the immune response to Chlamydia infection, revealing commonalities in disease pathologies and immunity between koalas and other hosts and paving the way for the development of a koala Chlamydia vaccine. This review summarises these recent findings and highlights key aspects of the koala immune system requiring further attention with particular regard to their most prominent infectious diseases.

Immunization of a wild koala population with a recombinant Chlamydia pecorum Major Outer Membrane Protein (MOMP) or Polymorphic Membrane Protein (PMP) based vaccine: New insights into immune response, protection and clearance.

We assessed the effects of two different single-dose anti-Chlamydia pecorum (C. pecorum) vaccines (containing either Major Outer Membrane Protein (3MOMP) or Polymorphic Membrane Protein (Pmp) as antigens) on the immune response of a group of wild koalas. Both vaccines elicited a systemic humoral response as seen by the production of anti-chlamydial IgG antibodies in more than 90% of vaccinated koalas. A mucosal immune response was also observed, with an increase in Chlamydia-specific mucosal IgG and/or IgA antibodies in some koalas post-vaccination. Both vaccines elicited a cell-mediated immune response as measured by the production of the cytokines IFN-γ and IL-17 post-vaccination. To determine the level of protection provided by the vaccines under natural conditions we assessed C. pecorum infection loads and chlamydial disease status of all vaccinated koalas pre- and post-vaccination, compared to a non-vaccinated cohort from the same habitat. The MOMP vaccinated koalas that were infected on the day of vaccination showed significant clearance of their infection at 6 months post-vaccination. In contrast, the number of new infections in the PMP vaccine was similar to the control group, with some koalas progressing to disease. Genotyping of the ompA gene from the C. pecorum strains infecting the vaccinated animals, identified genetic variants of ompA-F genotype and a new genotype ompA-O. We found that those animals that were the least well protected became infected with strains of C. pecorum not covered by the vaccine. In conclusion, a single dose vaccine formulated with either recombinant PmpG or MOMP can elicit both cell-mediated and humoral (systemic and mucosal) immune responses, with the MOMP vaccine showing clearance of infection in all infected koalas. Although the capability of our vaccines to stimulate an adaptive response and be protective needs to be fully evaluated, this work illustrates the necessity to combine epitopes most relevant to a large panel of variable strains with an efficient adjuvant.

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.

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.

Immunomics of the koala (Phascolarctos cinereus).

The study of the koala transcriptome has the potential to advance our understanding of its immunome--immunological reaction of a given host to foreign antigens--and to help combat infectious diseases (e.g., chlamydiosis) that impede ongoing conservation efforts. We used Illumina sequencing of cDNA to characterize genes expressed in two different koala tissues of immunological importance, blood and spleen. We generated nearly 600 million raw sequence reads, and about 285 million of these were subsequently assembled and condensed into ~70,000 subcomponents that represent putative transcripts. We annotated ~16% of these subcomponents and identified those related to infection and the immune response, including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), major histocompatibility complex (MHC) genes, and koala retrovirus (KoRV). Using phylogenetic analyses, we identified 29 koala genes in these target categories and report their concordance with currently accepted gene groups. By mapping multiple sequencing reads to transcripts, we identified 56 putative SNPs in genes of interest. The distribution of these SNPs indicates that MHC genes (34 SNPs) are more diverse than KoRV (12 SNPs), TLRs (8 SNPs), or RLRs (2 SNPs). Our sequence data also indicate that KoRV sequences are highly expressed in the transcriptome. Our efforts have produced full-length sequences for potentially important immune genes in koala, which should serve as targets for future investigations that aim to conserve koala populations.

Comparison of subcutaneous versus intranasal immunization of male koalas (Phascolarctos cinereus) for induction of mucosal and systemic immunity against Chlamydia pecorum.

Chlamydia pecorum infections are debilitating in the koala, contributing significantly to morbidity and mortality, with current antibiotic treatments having minimal success and adversely affecting gut microflora. This, combined with the sometimes-asymptomatic nature of the infection, suggests that an efficacious anti-chlamydial vaccine is required to control chlamydial infections in the koala. To date vaccination studies have focused primarily on female koalas, however, given the physiological differences between male and female reproductive tracts, we tested the efficacy of a vaccine in 12 captive male koalas. We evaluated the potential of both subcutaneous and intranasal vaccine delivery to elicit mucosal immunity in male koalas. Our results showed that both intranasal and subcutaneous delivery of a vaccine consisting of C. pecorum major outer membrane protein (MOMP) and the adjuvant immunostimulating complex (ISC) induced significant immune responses in male koalas. Subcutaneous immunization elicited stronger cell-mediated responses in peripheral blood lymphocytes (PBL), and greater plasma antibody levels whereas the intranasal immunization elicited stronger humoral responses in urogenital tract (UGT) secretions. This is the first time a Chlamydia vaccine has been tested in the male koala and the first assessment of a mucosal vaccination route in this species. Our results suggest that vaccination of male koalas can elicit mucosal immunity and could contribute to the long-term survivability of wild populations of the koala.

Vaccination of koalas (Phascolarctos cinereus) with a recombinant chlamydial major outer membrane protein adjuvanted with poly I:C, a host defense peptide and polyphosphazine, elicits strong and long lasting cellular and humoral immune responses.

Chlamydial infections are wide spread in koalas across their range and a solution to this debilitating disease has been sought for over a decade. Antibiotics are the currently accepted therapeutic measure, but are not an effective treatment due to the asymptomatic nature of some infections and a low efficacy rate. Thus, a vaccine would be an ideal way to address this infectious disease threat in the wild. Previous vaccine trials have used a three-dose regimen; however this is very difficult to apply in the field as it would require multiple capture events, which are stressful and invasive processes for the koala. In addition, it requires skilled koala handlers and a significant monetary investment. To overcome these challenges, in this study we utilized a polyphosphazine based poly I:C and a host defense peptide adjuvant combined with recombinant chlamydial major outer membrane protein (rMOMP) antigen to induce long lasting (54 weeks) cellular and humoral immunity in female koalas with a novel single immunizing dose. Immunized koalas produced a strong IgG response in plasma, as well as at mucosal sites. Moreover, they showed high levels of C. pecorum specific neutralizing antibodies in the plasma as well as vaginal and conjunctival secretions. Lastly, Chlamydia-specific lymphocyte proliferation responses were produced against both whole chlamydial elementary bodies and rMOMP protein, over the 12-month period. The results of this study suggest that a single dose rMOMP vaccine incorporating a poly I:C, host defense peptide and polyphosphazine adjuvant is able to stimulate both arms of the immune system in koalas, thereby providing an alternative to antibiotic treatment and/or a three-dose vaccine regime.

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.

Vaccination of koalas with a recombinant Chlamydia pecorum major outer membrane protein induces antibodies of different specificity compared to those following a natural live infection.

Chlamydial infection in koalas is common across the east coast of Australia and causes significant morbidity, infertility and mortality. An effective vaccine to prevent the adverse consequences of chlamydial infections in koalas (particularly blindness and infertility in females) would provide an important management tool to prevent further population decline of this species. An important step towards developing a vaccine in koalas is to understand the host immune response to chlamydial infection. In this study, we used the Pepscan methodology to identify B cell epitopes across the Major Outer Membrane Protein (MOMP) of four C. pecorum strains/genotypes that are recognized, either following (a) natural live infection or (b) administration of a recombinant MOMP vaccine. Plasma antibodies from the koalas naturally infected with a C. pecorum G genotype strain recognised the epitopes located in the variable domain (VD) four of MOMP G and also VD4 of MOMP H. By comparison, plasma antibodies from an animal infected with a C. pecorum F genotype strain recognised epitopes in VD1, 2 and 4 of MOMP F, but not from other genotype MOMPs. When Chlamydia-free koalas were immunised with recombinant MOMP protein they produced antibodies not only against epitopes in the VDs but also in conserved domains of MOMP. Naturally infected koalas immunised with recombinant MOMP protein also produced antibodies against epitopes in the conserved domains. This work paves the way for further refinement of a MOMP-based Chlamydia vaccine that will offer wide cross-protection against the variety of chlamydial infections circulating in wild koala populations.

Genetic diversity of Chlamydia pecorum strains in wild koala locations across Australia and the implications for a recombinant C. pecorum major outer membrane protein based vaccine.

The long term survival of the koala (Phascolarctos cinereus) is at risk due to a range of threatening processes. A major contributing factor is disease caused by infection with Chlamydia pecorum, which has been detected in most mainland koala populations and is associated with ocular and genital tract infections. A critical aspect for the development of vaccines against koala chlamydial infections is a thorough understanding of the prevalence and strain diversity of C. pecorum infections across wild populations. In this study, we describe the largest survey (403 koalas from eight wild populations and three wildlife hospitals) examining the diversity of C. pecorum infections. 181 of the 403 koalas tested (45%) positive for C. pecorum by species-specific quantitative PCR with infection rates ranging from 20% to 61% in the eight wild populations sampled. The ompA gene, which encodes the chlamydial major outer membrane protein (MOMP), has been the major target of several chlamydial vaccines. Based on our analysis of the diversity of MOMP amino types in the infected koalas, we conclude that, (a) there exists significant diversity of C. pecorum strains in koalas, with 10 distinct, full length C. pecorum MOMP amino types identified in the 11 koala locations sampled, (b) despite this diversity, there are predicted T and B cell epitopes in both conserved as well as variable domains of MOMP which suggest cross-amino type immune responses, and (c) a recombinant MOMP-based vaccine consisting of MOMP "F" could potentially induce heterotypic protection against a range of C. pecorum strains.

Preliminary characterisation of tumor necrosis factor alpha and interleukin-10 responses to Chlamydia pecorum infection in the koala (Phascolarctos cinereus).

Debilitating infectious diseases caused by Chlamydia are major contributors to the decline of Australia's iconic native marsupial species, the koala (Phascolarctos cinereus). An understanding of koala chlamydial disease pathogenesis and the development of effective strategies to control infections continue to be hindered by an almost complete lack of species-specific immunological reagents. The cell-mediated immune response has been shown to play an influential role in the response to chlamydial infection in other hosts. The objective of this study, hence, was to provide preliminary data on the role of two key cytokines, pro-inflammatory tumour necrosis factor alpha (TNFα) and anti-inflammatory interleukin 10 (IL10), in the koala Chlamydia pecorum response. Utilising sequence homology between the cytokine sequences obtained from several recently sequenced marsupial genomes, this report describes the first mRNA sequences of any koala cytokine and the development of koala specific TNFα and IL10 real-time PCR assays to measure the expression of these genes from koala samples. In preliminary studies comparing wild koalas with overt chlamydial disease, previous evidence of C. pecorum infection or no signs of C. pecorum infection, we revealed strong but variable expression of TNFα and IL10 in wild koalas with current signs of chlamydiosis. The description of these assays and the preliminary data on the cell-mediated immune response of koalas to chlamydial infection paves the way for future studies characterising the koala immune response to a range of its pathogens while providing reagents to assist with measuring the efficacy of ongoing attempts to develop a koala chlamydial vaccine.