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IntroductionThis study employs bulk RNA sequencing, PCR, and ELISA assays to analyze the pathological factors affecting the outcomes of C. pecorum ocular infections in koalas. It investigates the immune responses and gene expression profiles associated with various stages of koala ocular chlamydiosis.MethodsA cohort of 114 koalas from Queensland, Australia were assessed, with 47% displaying clinical signs of ocular disease. Animals were classified into three cohorts: acute active disease (G1), chronic active disease (G2), and chronic inactive disease (G3), along with subclinical Chlamydia pecorum positive (H2) and healthy (H1) cohorts.ResultsAnalysis of clinical, microbiological, humoral immune and cellular immune biomarkers revealed varying chlamydial loads and anti-chlamydial IgG levels across disease grades, with a negative correlation observed between ocular chlamydial load and anti-chlamydial IgG. Koala ocular mucosa gene expression analysis from 27 koalas identified shared expression pathways across disease cohorts, with a significant upregulation of IFNγ expression and tryptophan metabolism in all disease stages.DiscussionThese findings help elucidate immune response dynamics and molecular pathways underlying koala ocular chlamydiosis, providing insights crucial for disease management strategies.

The obligate intracellular bacterial pathogen Coxiella burnetii has been identified in a few species of marine mammals, some of which are showing population declines. It has been hypothesized that C. burnetii in marine mammals is a distinct genotype that varies significantly from the typical terrestrial genotypes. It appears to lack an IS1111. Isolates originating from Australian marine animals have a distinctly non-Australian profile of multiple-locus variable-number tandem-repeat analysis (MLVA). Extracted Coxiella DNA of Australian fur seal placental origin was sequenced using the Novaseq platform. Illumina 150 bp paired-end reads were filtered and trimmed with Trimgalore. The microbial community present in the sequenced genome was evaluated with Kraken and Bracken software using the NCBI database. A phylogenetic analysis was performed using 1131 core genes. Core genes were identified using Panaroo and inputted into Iqtree to determine the maximum-likelihood tree. A second phylogenetic tree was created using Rickettsiella grylii and using seven housekeeping genes. Results were compared with the C. burnetii Nine Mile RSA439 virulent genome. This new Australian marine mammal isolate of Coxiella (PG457) appears to be a novel genotype that lacks IS1111 and has a distinct MLVA signature (ms26, ms27, ms28, ms30, and ms31). The presence of genes for multiple virulence factors appears to give this genotype sufficient pathogenicity for it to be considered a possible causative agent of abortion in Australian fur seals as well as a potential zoonotic risk.

Abstract Background Wildlife species carry a remarkable diversity of trypanosomes. The detection of trypanosome infection in native Australian fauna is central to understanding their diversity and host-parasite associations. The implementation of total RNA sequencing (meta-transcriptomics) in trypanosome surveillance and diagnosis provides a powerful methodological approach to better understand the host species distribution of this important group of parasites. Methods We implemented a meta-transcriptomic approach to detect trypanosomes in a variety of tissues (brain, liver, lung, skin, gonads) sampled from native Australian wildlife, comprising four marsupials (koala, Phascolarctos cinereus; southern brown bandicoot, Isoodon obesulus; swamp wallaby, Wallabia bicolor; bare-nosed wombat, Vombatus ursinus), one bird (regent honeyeater, Anthochaera phrygia) and one amphibian (eastern dwarf tree frog, Litoria fallax). Samples corresponded to both clinically healthy and diseased individuals. Sequencing reads were de novo assembled into contigs and annotated. The evolutionary relationships among the trypanosomatid sequences identified were determined through phylogenetic analysis of 18S rRNA sequences. Results We detected trypanosome sequences in all six species of vertebrates sampled, with positive samples in multiple organs and tissues confirmed by PCR. Phylogenetic analysis indicated that the trypanosomes infecting marsupials were related to those previously detected in placental and marsupial mammals, while the trypanosome in the regent honeyeater grouped with avian trypanosomes. In contrast, we provide the first evidence for a trypanosome in the eastern dwarf tree frog that was phylogenetically distinct from those described in other amphibians. Conclusions To our knowledge, this is the first meta-transcriptomic analysis of trypanosomes in native Australian wildlife, expanding the known genetic diversity of these important parasites. We demonstrated that RNA sequencing is sufficiently sensitive to detect low numbers of Trypanosoma transcripts and from diverse hosts and tissues types, thereby representing an effective means to detect trypanosomes that are divergent in genome sequence.

Chlamydiae are obligate intracellular bacterial pathogens of humans. Infections in animals are also widespread with some species, such as Chlamydia psittaci, long recognised as a serious threat to human health. Critical to the public health response of any zoonotic disease outbreaks is reliable and up-to-date information on the epidemiology of the target pathogen. Aided by advances in the use of quantitative PCR, molecular typing and culture-independent genomic studies, significant recent work has highlighted an expanded diversity and host range of chlamydial pathogens in animals. New and unexpected cases of chlamydial zoonoses have now been recently documented in Australia and elsewhere, emphasising the importance of multi-disciplinary ‘One Health' collaboration and the use of standardised methods to detect and characterise chlamydial pathogens in humans and animals.

Introduction: Dirofilaria repens is a vector-borne filaroid helminth of carnivorous animals, primarily domesticated dogs. Humans are considered to be accidental hosts in which D. repens rarely reach sexual maturity but induce local inflammation, mainly in subcutaneous and ocular tissues. Methods: In the current study, we present the detection of multiple adults of D. repens, endosymbiont Wolbachia sp. and microfilariae by molecular analysis in peripheral tissues and bloodstream of a human host. A subsequent meta-analysis of published literature identified 21 cases of human infection with adult D. repens producing microfilariae. Results: Within the study population, there were 13 (59.09%) males, eight (36.36%) females and, in one (4.55%) case, sex was not reported. A total of 11 (50.00%) cases had subcutaneous dirofilariasis, six (27.27%) had ocular dirofiliariasis, with single cases (4.55% each) of genital, mammary, lymphatic and a combination of subcutaneous and pulmonary dirofilariasis described. In one (4.55%) case, the primary anatomical site of adult D. repens could not be found. D. repens microfilariae were detected in the local tissue (local microfilariasis) in 11 (50.00%) cases and the peripheral blood (microfilaremia) in 11 (50.50%) cases. Final identification of D. repens microfilariae was based on morphological detection in 14 (63.64%) cases, and molecular detection in eight (36.36%) cases. Conclusion: The results of this study suggest that humans may act as a final host for D. repens, however its role as a source of D. repens infection is less clear.

Abstract Island populations can represent genetically distinct and evolutionarily important lineages relative to mainland conspecifics. However, phenotypic divergence of island populations does not necessarily reflect genetic divergence, particularly for lineages inhabiting islands periodically connected during Pleistocene low sea stands. Marine barriers may also not be solely responsible for any divergence that is observed. Here, we investigated genetic divergence among and within the three phenotypically distinct subspecies of bare‐nosed wombats (Vombatus ursinus) in south‐east Australia that are presently—but were not historically—isolated by marine barriers. Using genome‐wide single nucleotide polymorphisms, we identified three genetically distinct groups (mainland Australia, Bass Strait island, and Tasmania) corresponding to the recognized subspecies. However, isolation by distance was observed in the Tasmanian population, indicating additional constraints on gene flow can contribute to divergence in the absence of marine barriers, and may also explain genetic structuring among fragmented mainland populations. We additionally confirm origins and quantify the genetic divergence of an island population 46 years after the introduction of 21 individuals from the Vulnerable Bass Strait subspecies. In the light of our findings, we make recommendations for the maintenance of genetic variation and fitness across the species range.

Introduction: Human dirofilariasis is a disease historically linked to the Mediterranean area. For the last few decades, however, Dirofilaria nematodes have been spreading, both in terms of prevalence and the geographical expansion in non-endemic areas. Currently, cases of human dirofilariasis are recorded in more than 40 countries worldwide. Croatia is considered an endemic area of the Adriatic basin. Methods: In a nationwide investigation, new and previously published cases of human dirofilariasis in Croatia were analyzed. Results: Since 1996, 30 cases of human dirofilariosis were reported in Croatia. A total of 14 (46,67%) cases were from the coastal and 16 (53,33%) from continental regions of the country. Based on anatomical location, 13 (43,33%) cases were subcutaneous, 12 (40%) were ocular and five (16,67%) occurred in the reproductive organs. In all 30 cases, Dirofilaria repens was identified as the causative agent. Conclusions: An increase in air temperature as climate change, changes in mosquito fauna, high prevalence of D. repens in dogs and limited use of chemoprophylaxis are possible risk factors for Dirofilaria infection in the Croatian population. Since reporting to epidemiological services is not mandatory in this country, the real number of human dirofilariasis cases is probably significantly higher than published. This emphasizes the need for mandatory reporting of human cases and surveillance of Dirofilaria infection in dogs and mosquitoes in Croatia, following the “One Health” concept.

Abstract Background Chlamydia (C.) gallinacea is a recently identified bacterium that mainly infects domestic chickens. Demonstration of C. gallinacea in human atypical pneumonia suggests its zoonotic potential. Its prevalence in chickens exceeds that of C. psittaci, but genetic and genomic research on C. gallinacea is still at the beginning. In this study, we conducted whole-genome sequencing of C. gallinacea strain JX-1 isolated from an asymptomatic chicken, and comparative genomic analysis between C. gallinacea strains and related chlamydial species. Results The genome of C. gallinacea JX-1 was sequenced by single-molecule, real-time technology and is comprised of a 1,059,522-bp circular chromosome with an overall G + C content of 37.93% and sequence similarity of 99.4% to type strain 08-1274/3. In addition, a plasmid designated pJX-1, almost identical to p1274 of the type strain, except for two point mutations, was only found in field strains from chicken, but not in other hosts. In contrast to chlamydial species with notably variable polymorphic membrane protein (pmp) genes and plasticity zone (PZ), these regions were conserved in both C. gallinacea strains. There were 15 predicted pmp genes, but only B, A, E1, H, G1 and G2 were apparently intact in both strains. In comparison to chlamydial species where the PZ may be up to 50 kbp, C. gallinacea strains displayed gene content reduction in the PZ (14 kbp), with strain JX-1 having a premature STOP codon in the cytotoxin (tox) gene, while tox gene is intact in the type strain. In multilocus sequence typing (MLST), 15 C. gallinacea STs were identified among 25 strains based on cognate MLST allelic profiles of the concatenated sequences. The type strain and all Chinese strains belong to two distinct phylogenetic clades. Clade of the Chinese strains separated into 14 genetically distinct lineages, thus revealing considerable genetic diversity of C. gallinacea strains in China. Conclusions In this first detailed comparative genomic analysis of C. gallinacea, we have provided evidence for substantial genetic diversity among C. gallinacea strains. How these genetic polymorphisms affect C. gallinacea biology and pathogenicity should be addressed in future studies that focus on phylogenetics and host adaption of this enigmatic bacterial agent.

Abstract Background Chlamydia suis is an important, globally distributed, highly prevalent and diverse obligate intracellular pathogen infecting pigs. To investigate the prevalence and genetic diversity of C. suis in China, 2,137 nasal, conjunctival, and rectal swabs as well as whole blood and lung samples of pigs were collected in 19 regions from ten provinces of China in this study. Results We report an overall positivity of 62.4% (1,334/2,137) of C. suis following screening by Chlamydia spp. 23S rRNA-based FRET-PCR and high-resolution melting curve analysis and confirmatory sequencing. For C. suis-positive samples, 33.3 % of whole blood and 62.5% of rectal swabs were found to be positive for the C. suis tetR(C) gene, while 13.3% of whole blood and 87.0% of rectal swabs were positive for the C. suis tet(C) gene. Phylogenetic comparison of partial C. suis ompA gene sequences revealed significant genetic diversity in the C. suis strains. This genetic diversity was confirmed by C. suis-specific multilocus sequence typing (MLST), which identified 26 novel sequence types among 27 examined strains. Tanglegrams based on MLST and ompA sequences provided evidence of C. suis recombination amongst the strains analyzed. Conclusions Genetically highly diverse C. suis strains are exceedingly prevalent in pigs. As it stands, the potential pathogenic effect of C. suis on pig health and production of C. suis remains unclear and will be the subject of further investigations. Further study is also required to address the transmission of C. suis between pigs and the risk of 'spill-over' and 'spill-back' of infections to wild animals and humans.

Piglet castration results in acute pain and stress to the animal. There is a critical need for effective on-farm methods of pain mitigation. Local anaesthesia using Tri-Solfen® (Animal Ethics Pty Ltd., Melbourne, Australia), a topical local anaesthetic and antiseptic formulation instilled to the wound during surgery, is a newly evolving on-farm method to mitigate castration pain. To investigate the efficacy of Tri-Solfen®, instilled to the wound during the procedure, to alleviate subsequent castration-related pain in neonatal piglets, we performed a large, negatively controlled, randomised field trial in two commercial pig farms in Europe. Piglets (173) were enrolled and randomised to undergo castration with or without Tri-Solfen®, instilled to the wound immediately following skin incision. A 30 s wait period was then observed prior to completing castration. Efficacy was investigated by measuring pain-induced motor and vocal responses during the subsequent procedure and post-operative pain-related behaviour in treated versus untreated piglets. There was a significant reduction in nociceptive motor and vocal response during castration and in the post-operative pain-related behaviour response in Tri-Solfen®-treated compared to untreated piglets, in the first 30 min following castration. Although not addressing pain of skin incision, Tri-Solfen® is effective to mitigate subsequent acute castration-related pain in piglets under commercial production conditions.

Abstract Background Debilitating skin infestations caused by the mite, Sarcoptes scabiei, have a profound impact on human and animal health globally. In Australia, this impact is evident across different segments of Australian society, with a growing recognition that it can contribute to rapid declines of native Australian marsupials. Cross-host transmission has been suggested to play a significant role in the epidemiology and origin of mite infestations in different species but a chronic lack of genetic resources has made further inferences difficult. To investigate the origins and molecular epidemiology of S. scabiei in Australian wildlife, we sequenced the mitochondrial genomes of S. scabiei from diseased wombats (Vombatus ursinus) and koalas (Phascolarctos cinereus) spanning New South Wales, Victoria and Tasmania, and compared them with the recently sequenced mitochondrial genome sequences of S. scabiei from humans. Results We found unique S. scabiei haplotypes among individual wombat and koala hosts with high sequence similarity (99.1% - 100%). Phylogenetic analysis of near full-length mitochondrial genomes revealed three clades of S. scabiei (one human and two marsupial), with no apparent geographic or host species pattern, suggestive of multiple introductions. The availability of additional mitochondrial gene sequences also enabled a re-evaluation of a range of putative molecular markers of S. scabiei, revealing that cox1 is the most informative gene for molecular epidemiological investigations. Utilising this gene target, we provide additional evidence to support cross-host transmission between different animal hosts. Conclusions Our results suggest a history of parasite invasion through colonisation of Australia from hosts across the globe and the potential for cross-host transmission being a common feature of the epidemiology of this neglected pathogen. If this is the case, comparable patterns may exist elsewhere in the ‘New World’. This work provides a basis for expanded molecular studies into mange epidemiology in humans and animals in Australia and other geographic regions.

Abstract Background Classification of Babesia parasites has traditionally relied on morphological differentiation based on piroplasm size and shape. Molecular typing has subsequently revealed a more complex taxonomy for these piroplasms than previously thought. To evaluate the factors that influence the morphology of Babesia species upon microscopic examination and hence, their taxonomic classification, we performed detailed characterizations of piroplasms from archival and prospective collections of cytological samples of dogs with piroplasmosis before and after death. Merozoite morphology and time of parasite disappearance following imidocarb dipropionate was also investigated. Methods The study was divided into a (i) review of archived cytological slides from confirmed cases of canine piroplasmosis, and (ii) a prospective study of smears and tissue imprints from 15 recently necropsied dogs. The latter group could be further sub-divided into a non-treated group and an imidocarb dipropionate-treated group. Exact times of treatment before death were reviewed. Additional blood smears prepared from the live dogs and taken before therapy were also evaluated in the latter group. Parasite burden per each slide was determined in both studies. The shape and size of merozoites were described from blood smears taken while the dogs were alive and from different organs during necropsy. The results of all measurements were statistically analyzed. Results The morphology and size of merozoites from live dogs corresponded to that of previously described ‘large’ Babesia. The morphology and size of merozoites were significantly different (P

Abstract Background Babesia spp. and Theileria spp. are important emerging causes of disease in dogs. Alongside these domesticated hosts, there is increasing recognition that these piroplasms can also be found in a range of wild animals with isolated reports describing the presence of these pathogen in foxes (Vulpes vulpes) and captive grey wolves (Canis lupus). The prevalence and impact of these infections in free-ranging populations of canids are unknown. To gain a better insight into the epidemiology and pathogenesis of piroplasm infections in free-ranging grey wolves, pathological and molecular investigations into captive and free-ranging grey wolves in Croatia were performed. Results The carcasses of 107 free-ranging wolves and one captive wolf were the subjects of post-mortem investigations and sampling for molecular studies. A blood sample from one live captured wolf for telemetric tracking was also used for molecular analysis. PCR amplification targeting the 18S RNA gene revealed that 21 of 108 free-ranging wolves and one captive animal were positive for Theileria/Babesia DNA. Subsequent sequencing of a fragment of the 18S RNA gene revealed that 7/22 animals were positive for Babesia canis while the other amplified sequence were found to be identical with corresponding 18S rDNA sequences of Theileria capreoli isolated from wild deer (15/22). Haematological and cytological analysis revealed the presence of signet-ring shaped or pear-shaped piroplasms in several animals with the overall parasite burden in all positive animals assessed to be very low. Pathological investigation of the captive animal revealed fatal septicemia as a likely outcome of hemolytic anaemia. There was little or no evidence of hemolytic disease consistent with babesiosis in other animals. Conclusion Importantly, the presence of B. canis in free-ranging grey wolves has not been described before but has been reported in a single fox and domestic dogs only. That B. canis infections cause disease in dogs but have little impact on wolf health possibly suggests that the wolf is the natural and the domestic dog is a secondary host. Surprisingly, the frequent finding of Theileria capreoli in wolves suggests that this Theileria species is not restricted to ungulates (cervids) but commonly infects also this carnivore species. Nevertheless, the potential role that these asymptomatically infected animals may play in the dispersal of these pathogens to susceptible sympatric species such as domesticated dogs requires further investigation.

Chlamydia are a genus of successful obligate intracellular pathogens spread across humans, wildlife, and domesticated animals. The most common species reported in livestock in this genus are Chlamydia abortus, Chlamydia psittaci, Chlamydia suis, and Chlamydia pecorum. Chlamydial infections trigger a series of inflammatory disease-related sequelae including arthritis, conjunctivitis, pneumonia, and abortion. Other bacteria in the phylum Chlamydiae have also been reported in livestock and wildlife but their impact on animal health is less clear. Control of chlamydial infections relies on the use of macrolides, fluoroquinolones, and tetracyclines. Tetracycline resistance (TETR) reported for porcine C. suis strains in association with the use of tetracycline feed is a potentially significant concern given experimental evidence highlighting that the genetic elements inferring TETR may be horizontally transferred to other chlamydial species. As documented in human Chlamydia trachomatis infections, relapse of infections, bacterial shedding post-antibiotic treatment, and disease progression despite chlamydial clearance in animals have also been reported. The identification of novel chlamydiae as well as new animal hosts for previously described chlamydial pathogens should place a renewed emphasis on basic in vivo studies to demonstrate the efficacy of existing and new antimicrobial treatment regimes. Building on recent reviews of antimicrobials limited to C. trachomatis and C. suis, this review will explore the use of antimicrobials, the evidence and factors that influence the treatment failure of chlamydial infections in animals and the future directions in the control of these important veterinary pathogens.

Increasing human population size and the concomitant expansion of urbanisation significantly impact natural ecosystems and native fauna globally. Successful conservation management relies on precise information on the factors associated with wildlife population decline, which are challenging to acquire from natural populations. Wildlife Rehabilitation Centres (WRC) provide a rich source of this information. However, few researchers have conducted large-scale longitudinal studies, with most focussing on narrow taxonomic ranges, suggesting that WRC-associated data remains an underutilised resource, and may provide a fuller understanding of the anthropogenic threats facing native fauna. We analysed admissions and outcomes data from a WRC in Queensland, Australia Zoo Wildlife Hospital, to determine the major factors driving admissions and morbidity of native animals in a region experiencing rapid and prolonged urban expansion. We studied 31,626 admissions of 83 different species of native birds, reptiles, amphibians, marsupials and eutherian mammals from 2006 to 2017. While marsupial admissions were highest (41.3%), admissions increased over time for all species and exhibited seasonal variation (highest in Spring to Summer), consistent with known breeding seasons. Causes for admission typically associated with human influenced activities were dominant and exhibited the highest mortality rates. Car strikes were the most common reason for admission (34.7%), with dog attacks (9.2%), entanglements (7.2%), and cat attacks (5.3%) also high. Admissions of orphaned young and overt signs of disease were significant at 24.6% and 9.7%, respectively. Mortality rates were highest following dog attacks (72.7%) and car strikes (69.1%) and lowest in orphaned animals (22.1%). Our results show that WRC databases offer rich opportunities for wildlife monitoring and provide quantification of the negative impacts of human activities on ecosystem stability and wildlife health. The imminent need for urgent, proactive conservation management to ameliorate the negative impacts of human activities on wildlife is clearly evident from our results.

Tri-Solfen® is a combination topical anaesthetic and antiseptic solution containing lidocaine, bupivacaine, adrenaline and cetrimide. Applied to wounds, it is reported to reduce the pain experienced by calves following thermocautery disbudding. While lidocaine and bupivacaine are widely used in medicine, conflicting data exist on the impact of these compounds when applied directly to the surgical wound. To investigate the safety of Tri-Solfen® applied to thermocautery disbudding wounds of calves, experiments were performed to measure (i) the safety of Tri-Solfen® (including in overdose situations); and (ii) the impact of Tri-Solfen® application at recommended doses on disbudding wound healing under field conditions. Haematological, biochemical and urinalysis parameters did not show clinically significant differences between placebo and Tri-Solfen® groups (1×, 3× and 5× dose). No adverse health impacts were reported. Histopathological analysis of wounds noted a reduction in bacterial colonies in Tri-Solfen®-treated wounds. Under field conditions, no negative impacts on wound healing were noted. Conversely, there was reduced incidence of abnormal wounds, with an associated trend toward improved average daily gain at days 11–12 in Tri-Solfen®-treated animals. These data are considered to support the safety of topical anaesthesia, as formulated in Tri-Solfen®, to the thermocautery disbudding wound in calves.

Emerging Microbes & Infections (2017) 6, e7; doi:10.1038/emi.2016.135; published online 15 February 2017

Analgesic products for piglet castration are critically needed. This requires extensive animal experimentation such as to meet regulatory-required proof of efficacy. At present, there are no validated methods of assessing pain in neonatal piglets. This poses challenges for investigators to optimize trial design and to meet ethical obligations to minimize the number of animals needed. Pain in neonatal piglets may be subtle, transient, and/or variably expressed and, in the absence of validated methods, investigators must rely on using a range of biochemical, physiological and behavioural variables, many of which appear to have very low (or unknown) sensitivity or specificity for documenting pain, or pain-relieving effects. A previous systematic review of this subject was hampered by the high degree of variability in the literature base both in terms of methods used to assess pain and pain mitigation, as well as in outcomes reported. In this setting we provide a narrative review to assist in determining the optimal methods currently available to detect piglet pain during castration and methods to mitigate castration-induced pain. In overview, the optimal outcome variables identified are nociceptive motor and vocal response scores during castration and quantitative sensory-threshold response testing and pain-associated behaviour scores following castration.

Background The globally distributed epidermal ectoparasite, Sarcoptes scabiei, is a serious health and welfare burden to at-risk human and animal populations. Rapid and sensitive detection of S. scabiei infestation is critical for intervention strategies. While direct microscopy of skin scrapings is a widely utilised diagnostic method, it has low sensitivity. PCR, alternatively, has been shown to readily detect mite DNA even in microscopy-negative skin scrapings. However, a limitation to the latter method is the requirements for specialised equipment and reagents. Such resources may not be readily available in regional or remote clinical settings and are an important consideration in diagnosis of this parasitic disease. Methodology A Loop Mediated Isothermal Amplification (LAMP) assay targeting the ITS-2 gene for S. scabiei was developed and evaluated on clinical samples from various hosts, previously screened with conventional S. scabies-specific PCR. Species specificity of the newly developed LAMP assay was tested against a range of DNA samples from other arthropods. The LAMP assays were performed on a real-time fluorometer as well as thermal cycler to evaluate an end-point of detection. Using skin scrapings, a rapid sample processing method was assessed to eliminate extensive processing times involved with DNA extractions prior to diagnostic assays, including LAMP. Results The S. scabiei LAMP assay was demonstrated to be species-specific and able to detect DNA extracted from a single mite within a skin scraping in under 30 minutes. Application of this assay to DNA extracts from skin scrapings taken from a range of hosts revealed 92.3% congruence (with 92.50% specificity and 100% sensitivity) to the conventional PCR detection of S. scabiei. Preliminary results have indicated that diagnostic outcome from rapidly processed dry skin scrapings using our newly developed LAMP is possible in approximately 40 minutes. Discussion We have developed a novel, rapid and robust molecular assay for detecting S. scabiei infesting humans and animals. Based on these findings, we anticipate that this assay will serve an important role as an ancillary diagnostic tool at the point-of-care, complementing existing diagnostic protocols for S. scabiei.

Koalas (Phascolarctos cinereus) are arboreal marsupials native to Australia that eat a specialized diet of almost exclusively eucalyptus leaves. Microbes in koala intestines are known to break down otherwise toxic compounds, such as tannins, in eucalyptus leaves. Infections by Chlamydia, obligate intracellular bacterial pathogens, are highly prevalent in koala populations. If animals with Chlamydia infections are received by wildlife hospitals, a range of antibiotics can be used to treat them. However, previous studies suggested that koalas can suffer adverse side effects during antibiotic treatment. This study aimed to use 16S rRNA gene sequences derived from koala feces to characterize the intestinal microbiome of koalas throughout antibiotic treatment and identify specific taxa associated with koala health after treatment. Although differences in the alpha diversity were observed in the intestinal flora between treated and untreated koalas and between koalas treated with different antibiotics, these differences were not statistically significant. The alpha diversity of microbial communities from koalas that lived through antibiotic treatment versus those who did not was significantly greater, however. Beta diversity analysis largely confirmed the latter observation, revealing that the overall communities were different between koalas on antibiotics that died versus those that survived or never received antibiotics. Using both machine learning and OTU (operational taxonomic unit) co-occurrence network analyses, we found that OTUs that are very closely related to Lonepinella koalarum, a known tannin degrader found by culture-based methods to be present in koala intestines, was correlated with a koala’s health status. This is the first study to characterize the time course of effects of antibiotics on koala intestinal microbiomes. Our results suggest it may be useful to pursue alternative treatments for Chlamydia infections without the use of antibiotics or the development of Chlamydia-specific antimicrobial compounds that do not broadly affect microbial communities.

Background Chlamydia pecorum is a globally significant livestock pathogen causing pathology and production losses. The on-farm infection and serological dynamics and the relevance of existing diagnostic tools for diagnosing C. pecorum in livestock remains poorly characterized. In this study, we characterized the antigen and antibody dynamics of this pathogen in a longitudinal study of prime lamb production, utilizing the infection focused C. pecorum-specific 16S rRNA qPCR assay and serology based chlamydial Complement fixation Test (CFT). Methods The study consisted of 76 Border Leicester mixed sex lambs (39 females and 37 males) that were sampled bimonthly from 2–10 months of age in a commercial farm operating in Central NSW, Australia. Blood/plasma was analysed for CFT antibodies, and swabs from conjunctival, rectal and vaginal sites were analysed for C. pecorum shedding using qPCR. We assessed the temporal and overall dynamics of C. pecorum in lambs, including detailed description and comparison of qPCR and CFT, the timing of first detection by either diagnostic method, the lag between infection and antibody response; and the distribution of qPCR load and CFT antibody titre over time. Results Over the study period, C. pecorum was highly prevalent (71.0% by qPCR, 92.1% by CFT, 96.0% by both), with 21.1% (16/76) lambs shedding ≥1, 000 qPCR copies/µl (denoted as high shedders). C. pecorum shedding (as evidence of infection) were first observed at two months of age (14.4%) with a significant peak of infection occurring at six months of age (34.2%), whereas seroconversions peaked at eight months of age (81.5%). 52.6% of C. pecorum qPCR and CFT positive lambs became qPCR negative by 10 months of age, indicating clearance of chlamydial infection. Although CFT is utilised for on-farm detection of active infection, we confirm that it lagged behind qPCR detection (average lag 1.7 ± 2.1 months) and that the proportion of qPCR positives simultaneously identified by CFT was low with 2/11 (18.1%), 0/13, 17/25 (68.0%), 5/7 (71.4%) and 1/10 (10.0%) concurrent seroconversions occurring at two, four, six, eight and 10 months of age, respectively. Discussion This work reveals rapid rates of C. pecorum infection and widespread exposure during lamb production. The comparison of molecular and serological diagnostic agreement longitudinally, supports the use of qPCR as an important ancillary tool for the detection of active infections in conjunction with chlamydial CFT for routine veterinary diagnostics. Development of rapid Point-of-Care (POC) tools for diagnosing active infection would be valuable for producers and veterinarians.

BACKGROUND:Chlamydia infects multiple sites within hosts, including the gastrointestinal tract (GIT). In certain hosts, gastrointestinal infection is linked to treatment avoidance and self-infection at disease susceptible sites. GIT C. pecorum has been detected in livestock and koalas, however GIT prevalence rates within the koala are yet to be established. METHODS:Paired conjunctival, urogenital and rectal samples from 33 koalas were screened for C. pecorum and C. pecorum plasmid using 16S rRNA and CDS5-specific quantitative PCR assays, respectively. Amplicon sequencing of 359 bp ompA fragment was used to identify site-specific genotypes. RESULTS:The overall C. pecorum prevalence collectively (healthy and clinically diseased koalas) was 51.5%, 57.6% and 42.4% in urogenital, conjunctival and gastrointestinal sites, respectively. Concurrent urogenital and rectal Chlamydia was identified in 14 koalas, with no cases of GIT only Chlamydia shedding. The ompA genotype G dominated the GIT positive samples, and genotypes A and E' were dominant in urogenital tract (UGT) positive samples. Increases in C. pecorum plasmid per C. pecorum load (detected by PCR) showed clustering in the clinically diseased koala group (as assessed by scatter plot analysis). There was also a low correlation between plasmid positivity and C. pecorum infected animals at any site, with a prevalence of 47% UGT, 36% rectum and 40% faecal pellet. CONCLUSIONS:GIT C. pecorum PCR positivity suggests that koala GIT C. pecorum infections are common and occur regularly in animals with concurrent genital tract infections. GIT dominant genotypes were identified and do not appear to be related to plasmid positivity. Preliminary results indicated a possible association between C. pecorum plasmid load and clinical UGT disease.

Sarcoptic mange, caused by the parasitic mite Sarcoptes scabiei, causes a substantive burden of disease to humans, domestic animals and wildlife, globally. There are many effects of S. scabiei infection, culminating in the disease which hosts suffer. However, major knowledge gaps remain on the pathogenic impacts of this infection. Here, we focus on the bare-nosed wombat host (Vombatus ursinus) to investigate the effects of mange on: (i) host heat loss and thermoregulation, (ii) field metabolic rates, (iii) foraging and resting behaviour across full circadian cycles, and (iv) fatty acid composition in host adipose, bone marrow, brain and muscle tissues. Our findings indicate that mange-infected V. ursinus lose more heat to the environment from alopecia-affected body regions than healthy individuals. Additionally, mange-infected individuals have higher metabolic rates in the wild. However, these metabolic demands are difficult to meet, because infected individuals spend less time foraging and more time inactive relative to their healthy counterparts, despite being outside of the burrow for longer. Lastly, mange infection results in altered fatty acid composition in adipose tissue, with increased amounts of omega-6 acids, and decreased amounts of omega-3 acids, a consequence of chronic cutaneous inflammation and inhibition of anti-inflammatory responses. These findings highlight the interactions of mange-induced physiological and behavioural changes, and have implications for the treatment and rehabilitation of infected individuals.

At a campus in a low socioeconomic (SES) area, our University allows enrolled nurses entry into the second year of a Bachelor of Nursing, but attrition is high. Using the factors, described by Yorke and Thomas (2003) to have a positive impact on the attrition of low SES students, we developed strategies to prepare the enrolled nurses for the pharmacology and bioscience units of a nursing degree with the aim of reducing their attrition. As a strategy, the introduction of review lectures of anatomy, physiology and microbiology, was associated with significantly reduced attrition rates. The subsequent introduction of a formative website activity of some basic concepts in bioscience and pharmacology, and a workshop addressing study skills and online resources, were associated with a further reduction in attrition rates of enrolled nursing students in a Bachelor of Nursing

Background Chlamydia psittaci and Chlamydia pecorum are important veterinary pathogens, with the former also being responsible for zoonoses, and the latter adversely affecting koala populations in Australia and livestock globally. The rapid detection of these organisms is still challenging, particularly at the point-of-care (POC). In the present study, we developed and evaluated rapid, sensitive and robust C. psittaci-specific and C. pecorum-specific Loop Mediated Isothermal Amplification (LAMP) assays for detection of these pathogens. Methods and Materials The LAMP assays, performed in a Genie III real-time fluorometer, targeted a 263 bp region of the C. psittaci-specific Cps_0607 gene or a 209 bp region of a C. pecorum-specific conserved gene CpecG_0573, and were evaluated using a range of samples previously screened using species-specific quantitative PCRs (qPCRs). Species-specificity for C. psittaci and C. pecorum LAMP targets was tested against DNA samples from related chlamydial species and a range of other bacteria. In order to evaluate pathogen detection in clinical samples, C. psittaci LAMP was evaluated using a total of 26 DNA extracts from clinical samples from equine and avian hosts, while for C. pecorum LAMP, we tested a total of 63 DNA extracts from clinical samples from koala, sheep and cattle hosts. A subset of 36 C. pecorum samples was also tested in a thermal cycler (instead of a real-time fluorometer) using newly developed LAMP and results were determined as an end point detection. We also evaluated rapid swab processing (without DNA extraction) to assess the robustness of these assays. Results Both LAMP assays were demonstrated to species-specific, highly reproducible and to be able to detect as little as 10 genome copy number/reaction, with a mean amplification time of 14 and 24 min for C. psittaci and C. pecorum, respectively. When testing clinical samples, the overall congruence between the newly developed LAMP assays and qPCR was 92.3% for C. psittaci (91.7% sensitivity and 92.9% specificity); and 84.1% for C. pecorum (90.6% sensitivity and 77.4% specificity). For a subset of 36 C. pecorum samples tested in a thermal cycler using newly developed LAMP, we observed 34/36 (94.4%) samples result being congruent between LAMP performed in fluorometer and in thermal cycler. Rapid swab processing method evaluated in this study also allows for chlamydial DNA detection using LAMP. Discussion In this study, we describe the development of novel, rapid and robust C. psittaci-specific and C. pecorum-specific LAMP assays that are able to detect these bacteria in clinical samples in either the laboratory or POC settings. With further development and a focus on the preparation of these assays at the POC, it is anticipated that both tests may fill an important niche in the repertoire of ancillary diagnostic tools available to clinicians.

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.

Chlamydia pecorum is a globally recognised livestock pathogen due to the significant clinical and economic impact it poses to livestock producers. Routine serological diagnosis is through a complement fixation test (CFT), which is often criticised for cross-reactivity, poor sensitivity and specificity. Although serology remains the preferred method in veterinary diagnostic laboratories, serological assays based on surface antigens of C. pecorum have not been established until now. In this study, we evaluated the use of two chlamydial recombinant protein antigens (PmpG and MOMP-G) by a direct IgG ELISA method for detection of ovine anti-chlamydial antibodies. Using the Pepscan method we then identified B cell epitopes across PmpG and MOMP-G proteins, in lambs with (a) naturally occurring asymptomatic C. pecorum infections (b) C. pecorum-associated polyarthritis and (c) recombinant PmpG and MOMP-G vaccine. Plasma IgG antibodies to PmpG in natural infection of lambs were detected earlier in infection than CFT and served as an acute phase marker. Antibodies to MOMP-G IgG were significantly heightened in lambs with C. pecorum-associated polyarthritis. PmpG and MOMP-G specific B-cell epitope mapping revealed epitope responses in immunised lambs cluster with some of the epitope responses in naturally infected lambs. B-cell epitope mapping further revealed that lambs with polyarthritis recognised several unique PmpG (50% frequency, peptide 8, 25, 40, 41 and 50) and MOMP (50% frequency, peptide 50) epitopes in comparison to asymptomatic infections. The findings of this study will have implications towards improved serodiagnosis of C. pecorum infections in livestock and inform the downstream development of alternative peptide-based antigens for future C. pecorum vaccine studies.

Chlamydial disease continues to be one of the main factors threatening the long-term survival of the koala (Phascolarctos cinereus). Despite this, large epidemiological studies of chlamydial infection and disease in wild koala populations are lacking. A better understanding of the prevalence, transmission and pathogenesis is needed to improve control measures, such as the development of vaccines. We investigated the prevalence of Chlamydia pecorum infection and disease in 160 koalas in a peri-urban wild population in Queensland, Australia and found that 31% of koalas were Chlamydia PCR positive and 28% had clinically detectable chlamydial disease. Most infections were at the urogenital site (27%; both males and females) with only 14% at the ocular site. Interestingly, we found that 27% (4/15) of koalas considered to be sexually immature (9-13 months) were already infected with C. pecorum, suggesting that a significant percentage of animals are infected directly from their mother. Ocular infection levels were less prevalent with increasing age (8% in koalas older than 4 years), whereas the prevalence of urogenital tract infections remained high into older age (26% in koalas older than 4 years), suggesting that, after mother-to-young transmission, C. pecorum is predominantly a sexually transmitted infection. While 28% of koalas in this population had clinically detectable chlamydial disease (primarily urogenital tract disease), many PCR positive koalas had no detectable disease and importantly, not all diseased animals were PCR positive. We also observed higher chlamydial loads in koalas who were C. pecorum infected without clinical disease than in koalas who were C. pecorum infected with clinical disease. These results shed light on the potential mechanisms of transmission of C. pecorum in koalas and also guide future control measures, such as vaccination.

Koalas (Phascolarctos cinereus) are native Australian marsupials whose populations are in decline from a range of threats. Infectious diseases caused by the bacterium Chlamydia pecorum and other pathogens are of particular concern. We analysed 26 poly-A selected RNA-sequencing libraries from a data set designed to study the immune response of koalas to ocular chlamydial infection. Using virus discovery techniques, we identified the coding-complete genome sequence of a novel picorna-like virus, denoted Burpengary virus, that was most common in south-east Queensland. Notably, abundance measurements of the virus across all 26 libraries revealed an inverse relationship between abundance and ocular disease in koalas, suggesting that the co-infection of Burpengary virus and Chlamydia pecorum is inhibited.

Diseases associated with Chlamydia pecorum infection are a major cause of decline in koala populations in Australia. While koalas in care can generally be treated, a vaccine is considered the only option to effectively reduce the threat of infection and disease at the population level. In the current study, we vaccinated 30 free-ranging koalas with a prototype Chlamydia pecorum vaccine consisting of a recombinant chlamydial MOMP adjuvanted with an immune stimulating complex. An additional cohort of 30 animals did not receive any vaccine and acted as comparison controls. Animals accepted into this study were either uninfected (Chlamydia PCR negative) at time of initial vaccination, or infected (C. pecorum positive) at either urogenital (UGT) and/or ocular sites (Oc), but with no clinical signs of chlamydial disease. All koalas were vaccinated/sampled and then re-released into their natural habitat before re-capturing and re-sampling at 6 and 12 months. All vaccinated koalas produced a strong immune response to the vaccine, as indicated by high titres of specific plasma antibodies. The incidence of new infections in vaccinated koalas over the 12-month period post-vaccination was slightly less than koalas in the control group, however, this was not statistically significant. Importantly though, the vaccine was able to significantly reduce the infectious load in animals that were Chlamydia positive at the time of vaccination. This effect was evident at both the Oc and UGT sites and was stronger at 6 months than at 12 months post-vaccination. Finally, the vaccine was also able to reduce the number of animals that progressed to disease during the 12-month period. While the sample sizes were small (statistically speaking), results were nonetheless striking. This study highlights the potential for successful development of a Chlamydia vaccine for koalas in a wild setting.

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 Australian northern quoll is an important predatory marsupial carnivore that is currently endangered due to inappropriate fire regimes, predation, and the spread of invasive cane toads. The microbiota of Australian marsupials has not been extensively studied, but is thought to play a role in their health. This study provides an initial characterization of the cloacal microbiota of the northern quoll, as well as other marsupials including possums and kangaroos which were opportunistically sampled. The northern quoll cloaca microbiota was dominated by Enterococcus and Lactobacillus and had a relatively high proportion of members of the Proteobacteria phylum, which has been observed in other carnivorous marsupials. The diversity and structure of the microbiota was not influenced by presence of Chlamydiales which are intracellular bacteria and potential pathogens. The microbiota of the other marsupials was quite varied, which may be related to their health status. Characterization of the northern quoll microbiota will help to better understand the biology of this endangered animal.

Toll-like receptors (TLRs) play a crucial role in the early defence against invading pathogens, yet our understanding of TLRs in marsupial immunity is limited. Here, we describe the characterisation of nine TLRs from a koala immune tissue transcriptome and one TLR from a draft sequence of the koala genome and the subsequent development of an assay to study genetic diversity in these genes. We surveyed genetic diversity in 20 koalas from New South Wales, Australia and showed that one gene, TLR10 is monomorphic, while the other nine TLR genes have between two and 12 alleles. 40 SNPs (16 non-synonymous) were identified across the ten TLR genes. These markers provide a springboard to future studies on innate immunity in the koala, a species under threat from two major infectious diseases.

Translocation of E. coli across the gut epithelium can result in fatal sepsis in post-surgical patients. In vitro and in vivo experiments have identified the existence of a novel pathotype of translocating E. coli (TEC) that employs an unknown mechanism for translocating across epithelial cells to the mesenteric lymph nodes and the blood stream in both humans and animal models. In this study the genomes of four TEC strains isolated from the mesenteric lymph nodes of a fatal case of hospitalised patient (HMLN-1), blood of pigs after experimental shock (PC-1) and after non-lethal haemorrhage in rats (KIC-1 and KIC-2) were sequenced in order to identify the genes associated with their adhesion and/or translocation. To facilitate the comparison, the genomes of a non-adhering, non-translocating E. coli (46-4) and adhering but non-translocating E. coli (73-89) were also sequenced and compared. Whole genome comparison revealed that three (HMLN-1, PC-1 and KIC-2) of the four TEC strains carried a genomic island that encodes a Type 6 Secretion System that may contribute to adhesion of the bacteria to gut epithelial cells. The human TEC strain HMLN-1 also carried the invasion ibeA gene, which was absent in the animal TEC strains and is likely to be associated with host-specific translocation. Phylogenetic analysis revealed that the four TEC strains were distributed amongst three distinct E. coli phylogroups, which was supported by the presence of phylogroup specific fimbriae gene clusters. The genomic comparison has identified potential genes that can be targeted with knock-out experiments to further characterise the mechanisms of E. coli translocation.

Six consecutively hatched cohorts and one cohort of pre-hatch eggs of farmed barramundi (Lates calcarifer) from south Australia were examined for Chlamydia-like organisms associated with epitheliocystis. To identify and characterise the bacteria, 59 gill samples and three pre-hatch egg samples were processed for histology, in situ hybridisation and 16S rRNA amplification, sequencing and comprehensive phylogenetic analysis. Cases of epitheliocystis were observed microscopically and characterised by membrane-enclosed basophilic cysts filled with a granular material that caused hypertrophy of the epithelial cells. In situ hybridisation with a Chlamydiales-specific probe lead to specific labelling of the epitheliocystis inclusions within the gill epithelium. Two distinct but closely related 16S rRNA chlamydial sequences were amplified from gill DNA across the seven cohorts, including from pre-hatch eggs. These genotype sequences were found to be novel, sharing 97.1 - 97.5% similarity to the next closest 16S rRNA sequence, Ca. Similichlamydia latridicola, from Australian striped trumpeter. Comprehensive phylogenetic analysis of these genotype sequences against representative members of the Chlamydiales order and against other epitheliocystis agents revealed these Chlamydia-like organisms to be novel and taxonomically placed them within the recently proposed genus Ca. Similichlamydia. Following Fredricks and Relman's molecular postulates and based on these observations, we propose the epitheliocystis agents of barramundi to be known as "Candidatus Similichlamydia laticola" (sp. nov.).

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.

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.

Growing evidence suggests that a novel member of the Chlamydiales order, Waddlia chondrophila, is a potential agent of miscarriage in humans and abortion in ruminants. Due to the lack of genetic tools to manipulate chlamydia, genomic analysis is proving to be the most incisive tool in stimulating investigations into the biology of these obligate intracellular bacteria. 454/Roche and Solexa/Illumina technologies were thus used to sequence and assemble de novo the full genome of the first representative of the Waddliaceae family, W. chondrophila. The bacteria possesses a 2'116'312 bp chromosome and a 15'593 bp low-copy number plasmid that might integrate into the bacterial chromosome. The Waddlia genome displays numerous repeated sequences indicating different genome dynamics from classical chlamydia which almost completely lack repetitive elements. Moreover, W. chondrophila exhibits many virulence factors also present in classical chlamydia, including a functional type III secretion system, but also a large complement of specific factors for resistance to host or environmental stresses. Large families of outer membrane proteins were identified indicating that these highly immunogenic proteins are not Chlamydiaceae specific and might have been present in their last common ancestor. Enhanced metabolic capability for the synthesis of nucleotides, amino acids, lipids and other co-factors suggests that the common ancestor of the modern Chlamydiales may have been less dependent on their eukaryotic host. The fine-detailed analysis of biosynthetic pathways brings us closer to possibly developing a synthetic medium to grow W. chondrophila, a critical step in the development of genetic tools. As a whole, the availability of the W. chondrophila genome opens new possibilities in Chlamydiales research, providing new insights into the evolution of members of the order Chlamydiales and the biology of the Waddliaceae.