Novel molecular markers of Chlamydia pecorum genetic diversity in the koala (Phascolarctos cinereus)

Background Chlamydia pecorum is an obligate intracellular bacterium and the causative agent of reproductive and ocular disease in several animal hosts including koalas, sheep, cattle and goats. C. pecorum strains detected in koalas are genetically diverse, raising interesting questions about the origin and transmission of this species within koala hosts. While the ompA gene remains the most widely-used target in C. pecorum typing studies, it is generally recognised that surface protein encoding genes are not suited for phylogenetic analysis and it is becoming increasingly apparent that the ompA gene locus is not congruent with the phylogeny of the C. pecorum genome. Using the recently sequenced C. pecorum genome sequence (E58), we analysed 10 genes, including ompA, to evaluate the use of ompA as a molecular marker in the study of koala C. pecorum genetic diversity. Results Three genes (incA, ORF663, tarP) were found to contain sufficient nucleotide diversity and discriminatory power for detailed analysis and were used, with ompA, to genotype 24 C. pecorum PCR-positive koala samples from four populations. The most robust representation of the phylogeny of these samples was achieved through concatenation of all four gene sequences, enabling the recreation of a "true" phylogenetic signal. OmpA and incA were of limited value as fine-detailed genetic markers as they were unable to confer accurate phylogenetic distinctions between samples. On the other hand, the tarP and ORF663 genes were identified as useful "neutral" and "contingency" markers respectively, to represent the broad evolutionary history and intra-species genetic diversity of koala C. pecorum. Furthermore, the concatenation of ompA, incA and ORF663 sequences highlighted the monophyletic nature of koala C. pecorum infections by demonstrating a single evolutionary trajectory for koala hosts that is distinct from that seen in non-koala hosts. Conclusions While the continued use of ompA as a fine-detailed molecular marker for epidemiological analysis appears justified, the tarP and ORF663 genes also appear to be valuable markers of phylogenetic or biogeographic divisions at the C. pecorum intra-species level. This research has significant implications for future typing studies to understand the phylogeny, genetic diversity, and epidemiology of C. pecorum infections in the koala and other animal species.

Vaccination of healthy and diseased koalas (Phascolarctos cinereus) with a Chlamydia pecorum multi-subunit vaccine : evaluation of immunity and pathology

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.

Comparison of antigen detection and quantitative PCR in the detection of chlamydial infection in koalas (Phascolarctos cinereus)

The gold standard method for detecting chlamydial infection in domestic and wild animals is PCR, but the technique is not suited to testing animals in the field when a rapid diagnosis is frequently required. The objective of this study was to compare the results of a commercially available enzyme immunoassay test for Chlamydia against a quantitative Chlamydia pecorum-specific PCR performed on swabs collected from the conjunctival sac, nasal cavity and urogenital sinuses of naturally infected koalas (Phascolarctos cinereus). The level of agreement for positive results between the two assays was low (43.2%). The immunoassay detection cut-off was determined as approximately 400 C. pecorum copies, indicating that the test was sufficiently sensitive to be used for the rapid diagnosis of active chlamydial infections.

Antigenic specificity of a monovalent versus polyvalent MOMP based Chlamydia pecorum vaccine in koalas (Phascolarctos cinereus)

Chlamydia continues to be a major pathogen of koalas. The bacterium is associated with ocular, respiratory and urogenital tract infections and a vaccine is considered the best option to limit the decline of mainland koala populations. Over the last 20 years, efforts to develop a chlamydial vaccine in humans have focussed on the use of the chlamydial major outer membrane protein (MOMP). Potential problems with the use of MOMP-based vaccines relate to the wide range of genetic diversity in its four variable domains. In the present study, we evaluated the immune response of koalas vaccinated with a MOMP-based C. pecorum vaccine formulated with genetically and serologically diverse MOMPs. Animals immunised with individual MOMPs developed strong antibody and lymphocyte proliferation responses to both homologous as well as heterologous MOMP proteins. Importantly, we also showed that vaccine induced antibodies which effectively neutralised various heterologous strains of koala C. pecorum in an in vitro assay. Finally, we also demonstrated that the immune responses in monovalent as well as polyvalent MOMP vaccine groups were able to recognise whole chlamydial elementary bodies, illustrating the feasibility of developing an effective MOMP based C. pecorum vaccine that could protect against a range of strains.

Molecular characterisation and expression analysis of interferon gamma in response to natural chlamydia infection in the koala, phascolarctos cinereus

Interferon gamma (IFNγ) is a key Th1 cytokine, with a principal role in the immune response against intracellular organisms such as Chlamydia. Along with being responsible for significant morbidity in human populations, Chlamydia is also responsible for wide spread infection and disease in many animal hosts, with reports that many Australian koala subpopulations are endemically infected. An understanding of the role played by IFNγ in koala chlamydial diseases is important for the establishment of better prophylactic and therapeutic approaches against chlamydial infection in this host. A limited number of IFNγ sequences have been published from marsupials and no immune reagents to measure expression have been developed. Through preliminary analysis of the koala transcriptome, we have identified the full coding sequence of the koala IFNγ gene. Transcripts were identified in spleen and lymph node tissue samples. Phylogenetic analysis demonstrated that koala IFNγ is closely related to other marsupial IFNγ sequences and more distantly related to eutherian mammals. To begin to characterise the role of this important cytokine in the koala's response to chlamydial infection, we developed a quantitative real time PCR assay and applied it to a small cohort of koalas with and without active chlamydial disease, revealing significant differences in expression patterns between the groups. Description of the IFNγ sequence from the koala will not only assist in understanding this species' response to its most important pathogen but will also provide further insight into the evolution of the marsupial immune system

Investigation of genetic diversity and development of vaccine for Chlamydia pecorum infections in koala (Phascolarctos cinereus)

Many wild koala populations in Australia continue to experience serious declines due to factors such as disease caused by Chlamydia. This thesis is the first of its kind to investigate diversity of the chlamydial infections in wild koala populations across Australia and has made significant progress towards the development of a vaccine for koalas. The findings in this study have demonstrated that it is feasible to develop a safe and effective recombinant vaccine against Chlamydia in both disease free as well as severely diseased koalas. Most importantly, this study is also first of its kind to evaluate a multi-component vaccine that should be effective against the range of Chlamydia pecorum strains circulating in both captive as well as wild koala populations.

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.

Conjunctivitis associated with Chlamydia pecorum in three koalas (Phascolarctos cinereus) in the Mount Lofty Ranges, South Australia

Chlamydiosis is a significant factor contributing to the decline of koala (Phascolarctos cinereus) populations in Australia but has not previously been reported in South Australia. We describe conjunctivitis in three wild koalas from South Australia, with Chlamydia pecorum identified by quantitative PCR.

Characterisation of cytokine response to Chlamydia pecorum infection in the koala, Phascolarctos cinereus

This thesis aimed at identifying cytokine markers associated with chlamydial infection and disease in koalas which is facing many threats to its survival, Chlamydia pecorum infections being a major one. To identify immunological markers associated with chlamydial infection and disease in koalas, key cytokines such as TNF alpha, IL10, IFN gamma and IL17A were cloned and sequenced and subsequently developed Quantitative Real Time PCR (qrtPCR) assays. The thesis provides preliminary data on the role of these cytokines in koala chlamydial disease and further longitudinal studies are required to confirm the role played by cytokines in pathology and protection against C. pecorum infection in the koala.

A transcriptome resource for the koala (Phascolarctos cinereus) : insights into koala retrovirus transcription and sequence diversity

Background The koala, Phascolarctos cinereus, is a biologically unique and evolutionarily distinct Australian arboreal marsupial. The goal of this study was to sequence the transcriptome from several tissues of two geographically separate koalas, and to create the first comprehensive catalog of annotated transcripts for this species, enabling detailed analysis of the unique attributes of this threatened native marsupial, including infection by the koala retrovirus. Results RNA-Seq data was generated from a range of tissues from one male and one female koala and assembled de novo into transcripts using Velvet-Oases. Transcript abundance in each tissue was estimated. Transcripts were searched for likely protein-coding regions and a non-redundant set of 117,563 putative protein sequences was produced. In similarity searches there were 84,907 (72%) sequences that aligned to at least one sequence in the NCBI nr protein database. The best alignments were to sequences from other marsupials. After applying a reciprocal best hit requirement of koala sequences to those from tammar wallaby, Tasmanian devil and the gray short-tailed opossum, we estimate that our transcriptome dataset represents approximately 15,000 koala genes. The marsupial alignment information was used to look for potential gene duplications and we report evidence for copy number expansion of the alpha amylase gene, and of an aldehyde reductase gene. Koala retrovirus (KoRV) transcripts were detected in the transcriptomes. These were analysed in detail and the structure of the spliced envelope gene transcript was determined. There was appreciable sequence diversity within KoRV, with 233 sites in the KoRV genome showing small insertions/deletions or single nucleotide polymorphisms. Both koalas had sequences from the KoRV-A subtype, but the male koala transcriptome has, in addition, sequences more closely related to the KoRV-B subtype. This is the first report of a KoRV-B-like sequence in a wild population. Conclusions This transcriptomic dataset is a useful resource for molecular genetic studies of the koala, for evolutionary genetic studies of marsupials, for validation and annotation of the koala genome sequence, and for investigation of koala retrovirus. Annotated transcripts can be browsed and queried at http://koalagenome.org

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 (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.