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.

Chlamydia pecorum is a mucosal infection, which causes debilitating disease of the urinary tract, reproductive tract and ocular sites of koalas (Phascolarctos cinereus). While antibiotics are available for treatment, they are detrimental to the koalas’ gastrointestinal tract microflora leaving the implementation of a vaccine as an ideal option for the long-term management of koala populations. We have previously reported on the successes of an anti-chlamydial recombinant major outer membrane protein (rMOMP) vaccine however, recombinant protein based vaccines are not ideal candidates for scale up from the research level to small-medium production level for wider usage. Peptide based vaccines are a promising area for vaccine development, because peptides are stable, cost effective and easily produced. In this current study, we assessed, for the first time, the immune responses to a synthetic peptide based anti-chlamydial vaccine in koalas. Five healthy male koalas were vaccinated with two synthetic peptides derived from C. pecorum MOMP and another five healthy male koalas were vaccinated with full length recombinant C. pecorum MOMP (genotype G). Systemic (IgG) and mucosal (IgA) antibodies were quantified and pre-vaccination levels compared to post-vaccination levels (12 and 26 weeks). MOMP-peptide vaccinated koalas produced Chlamydia-specific IgG and IgA antibodies, which were able to recognise not only the genotype used in the vaccination, but also MOMPs from several other koala C. pecorum genotypes. In addition, IgA antibodies induced at the ocular site not only recognised recombinant MOMP protein but also, whole native chlamydial elementary bodies. Interestingly, some MOMP-peptide vaccinated koalas showed a stronger and more sustained vaccine-induced mucosal IgA antibody response than observed in MOMP-protein vaccinated koalas. These results demonstrate that a synthetic MOMP peptide based vaccine is capable of inducing a Chlamydia-specific antibody response in koalas and is a promising candidate for future vaccine development.

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

Background C. psittaci has recently emerged as an equine abortigenic pathogen causing significant losses to the Australian Thoroughbred industry, while Equine herpesvirus-1 (EHV-1) is a well-recognized abortigenic agent. Diagnosis of these agents is based on molecular assays in diagnostic laboratories. In this study, we validated C. psittaci and newly developed EHV-1 Loop Mediated Isothermal Amplification (LAMP) assays performed in a real-time fluorometer (rtLAMP) against the reference diagnostic assays. We also evaluated isothermal amplification using commercially available colorimetric mix (cLAMP), and SYBR Green DNA binding dye (sgLAMP) for “naked eye” end-point detection when testing ‘real-world’ clinical samples. Finally, we applied the C. psittaci LAMP assays in two pilot Point-of-Care (POC) studies in an equine hospital. Results The analytical sensitivity of C. psittaci and EHV-1 rt-, and colorimetric LAMPs was determined as one and 10 genome equivalents per reaction, respectively. Compared to reference diagnostic qPCR assays, the C. psittaci rtLAMP showed sensitivity of 100%, specificity of 97.5, and 98.86% agreement, while EHV-1 rtLAMP showed 86.96% sensitivity, 100% specificity, and 91.43% agreement. When testing rapidly processed clinical samples, all three C. psittaci rt-, c-, sg-LAMP assays were highly congruent with each other, with Kappa values of 0. 906 for sgLAMP and 0. 821 for cLAMP when compared to rtLAMP. EHV-1 testing also revealed high congruence between the assays, with Kappa values of 0.784 for cLAMP and 0.638 for sgLAMP when compared to rtLAMP. The congruence between LAMP assays and the C. psittaci or EHV-1 qPCR assays was high, with agreements ranging from 94.12 to 100% for C. psittaci, and 88.24 to 94.12% for EHV-1, respectively. At the POC, the C. psittaci rt- and c-LAMP assays using rapidly processed swabs were performed by technicians with no prior molecular experience, and the overall congruence between the POC C. psittaci LAMPs and the qPCR assays ranged between 90.91–100%. Conclusions This study describes reliable POC options for the detection of the equine pathogens: C. psittaci and EHV-1. Testing ‘real-world’ samples in equine clinical setting, represents a proof-of-concept that POC isothermal diagnostics can be applied to rapid disease screening in the equine industry.