Chlamydial Infection In Koalas Research Articles

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

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

Preliminary analyses of tryptophan, kynurenine, and the kynurenine: Tryptophan ratio in plasma, as potential biomarkers for systemic chlamydial infections in koalas.

Chlamydiosis is the major infectious disease responsible for significant morbidity and mortality in free-living koalas. Recently, it was reported that 28.5% of koalas infected with chlamydiosis were presented with no overt clinical signs. Identification and quantification of changes in plasma biomarkers' fluctuations have the potential to enhance C. pecorum detection and facilitate the monitoring of therapeutic efficacy of antibiotics to treat this disease in koalas. Therefore, concentrations of the essential amino acid tryptophan, tryptophan's metabolite kynurenine, and the kynurenine:tryptophan ratio were quantified by high-performance liquid chromatography in the plasma of clinically normal koalas (n = 35), koalas identified with chlamydial disease (n = 35) and koalas that had other non-chlamydial co-morbidities (n = 10). Results showed that there was a significant difference between the clinically normal versus diseased, and clinically normal versus 'other' (both p < 0.001) in kynurenine plasma concentrations and kynurenine:tryptophan ratio; and also between the clinically normal and diseased in tryptophan plasma concentrations (p = 0.001). Proposed reference ranges of tryptophan, kynurenine, and kynurenine:tryptophan ratio in koalas are: 4.27-10.4 μg/mL, 0.34-1.23 μg/mL, and 0.05-0.22, respectively. Proposed optimal cut-off points to differentiate between clinically normal and diseased are: ≤ 4.75 μg/mL (tryptophan), ≥ 0.88 μg/mL (kynurenine), and ≥ 0.12 (kynurenine:tryptophan); and ≤ 7.67 μg/mL (tryptophan), ≥ 1.18 μg/mL (kynurenine), and ≥ 0.16 (kynurenine:tryptophan) to differentiate between released/recovered and euthanised of the diseased/'other' koalas. Significant differences in haematological and biochemical analytes were in the plasma globulins between the clinically normal and diseased koalas (p = 0.01), and in alkaline phosphatase between the clinically normal and 'other' koalas (p = 0.03). Although these potential biomarkers, especially tryptophan, may not be specific for detecting C. pecorum from the rest of the population, kynurenine and the kynurenine:tryptophan ratio may have a role in identifying unhealthy koalas from the clinically normal ones, irrespective of the underlying cause.

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

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

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

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

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.

Chlamydiaceae and Chlamydia-like organisms in the koala (Phascolarctos cinereus)—Organ distribution and histopathological findings

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

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.

Recent advances in understanding the biology, epidemiology and control of chlamydial infections in koalas

The koala (Phascolarctos cinereus) is recognised as a threatened wildlife species in various parts of Australia. A major contributing factor to the decline and long-term viability of affected populations is disease caused by the obligate intracellular bacteria, Chlamydia. Two chlamydial species infect the koala, Chlamydia pecorum and Chlamydia pneumoniae, and have been reported in nearly all mainland koala populations. Chlamydial infections of koalas are associated with ocular infections leading to blindness and genital tract infections linked to infertility, among other serious clinical manifestations. Diagnosis can be based on clinical presentation alone, however, it is complicated by the observation that many koala chlamydial infections occur with no overt signs of clinical disease. Instead, accurate diagnosis requires detailed clinical assessment and confirmatory testing by a range of PCR-based assays. Antibiotic treatment for koala chlamydial infection is possible, however, results on its success are mixed. A more practical solution for the protection of diseased populations is the application of a koala Chlamydia vaccine, with recent trials indicating promising results. Interestingly, molecular epidemiology studies of koala C. pecorum infections and recent comparative genomic analyses of koala C. pneumoniae have revealed potential differences in their origin that will have wider ramifications for our understanding of human chlamydial infections and host adaptation of the chlamydiae. This review summarises changes to the taxonomy of koala chlamydial infections and recent advances in our understanding of the epidemiology, diagnosis, treatment, control and evolution of Chlamydia infections in this iconic wildlife species.

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.

Epizootiology of Chlamydia infections in two free-range koala populations

The prevalence of Chlamydia pecorum and Chlamydia pneumoniae infections in two free-range koala populations was assessed using genus-specific PCR combined with species-specific DNA probe hybridisation. Population A had a very high overall level of chlamydial infection (85%) with significantly more of these infections being due to C. pecorum (73%) compared to C. pneumoniae (24%). The second population had a much lower prevalence of infection (10%) with equal levels of both species. An important finding of this study was that, while five of 24 C. pecorum-infected koalas had clinical signs of the disease (both ocular and urogenital sites), none out of seven C. pneumoniae-infected koalas had signs of clinical disease. This suggests that C. pecorum may be the more pathogenic of the two chlamydial species infecting this host. The level of infection (assessed by intensity of the specific hybridisation signal) also differed between chlamydial species, with C. pecorum infections ranging from low to high grade whereas C. pneumoniae infections were always low grade. When the age of infected koalas was examined, 58% of young, sexually immature koalas were found to have C. pecorum infections, increasing to 100% of koalas in the older age groups. This suggests that, in this population at least, young koalas are readily infected with C. pecorum from their mothers. While the infection levels with C. pneumoniae were too low to be statistically significant, again, sexually immature koalas were found to be infected. The recent separation of chlamydial infections in koalas into two species is beginning to indicate different epizootiologies for koala C. pecorum compared to koala C. pneumoniae.

Outer Membrane Protein 2 Gene Sequences Indicate that Chlamydia pecorum and Chlamydia pneumoniae Cause Infections in Koalas

Chlamydial infection in koalas causes conjunctivitis, rhinitis, urinary tract disease and female infertility. The chlamydial strains responsible have previously been designated as Chlamydia psittaci and have been subdivided into two types, I and II. In the present study we determined the DNA sequence of a 530 bp segment of the omp 2 gene from seven koala chlamydial strains, the type strain of C. pecorum, and the horse strain N16, and compared these with previously reported omp 2 gene sequences from 10 other chlamydial strains. Omp 2 sequence data clearly separated the seven koala strains into two genetic groups. Koala omp 2 group A corresponds to the previous type I designation and includes a conjunctival strain and a systemic disease-causing strain. The koala group A omp 2 sequence is 99% similar to the human C. pneumoniae IOL-207 strain and also 99% similar to the horse strain, N16. We propose that these koala strains be referred to as koala C. pneumoniae in future. Koala omp 2 group B corresponds to the previous koala type II designation and includes three urogenital tract strains plus two ocular strains. The koala group B omp 2 sequence is 99% similar to the C. pecorum type strain, VR628, while being only 77% similar to the EAE C. psittaci strain and only 71% similar to the koala group A strains. We propose that these koala strains be referred to as koala C. pecorum in future. The fact that two different species of Chlamydia infect koalas reinforces the observation that chlamydial species are not host restricted and makes us rethink the epidemiology of chlamydial disease in this marsupial.

Chlamydial infection in koalas under relatively little alienation pressure

Chlamydial infection in koalas under relatively little alienation pressure