Testing the effectiveness of genetic monitoring using genetic non-invasive sampling.

Effective conservation requires accurate data on population genetic diversity, inbreeding, and genetic structure. Increasingly, scientists are adopting genetic non-invasive sampling (gNIS) as a cost-effective population-wide genetic monitoring approach. gNIS has, however, known limitations which may impact the accuracy of downstream genetic analyses. Here, using high-quality single nucleotide polymorphism (SNP) data from blood/tissue sampling of a free-ranging koala population (n = 430), we investigated how the reduced SNP panel size and call rate typical of genetic non-invasive samples (derived from experimental and field trials) impacts the accuracy of genetic measures, and also the effect of sampling intensity on these measures. We found that gNIS at small sample sizes (14% of population) can provide accurate population diversity measures, but slightly underestimated population inbreeding coefficients. Accurate measures of internal relatedness required at least 33% of the population to be sampled. Accurate geographic and genetic spatial autocorrelation analysis requires between 28% and 51% of the population to be sampled. We show that gNIS at low sample sizes can provide a powerful tool to aid conservation decision-making and provide recommendations for researchers looking to apply these techniques to free-ranging systems.

Koala Retrovirus in Northern Australia Shows a Mixture of Stable Endogenization and Exogenous Lineage Diversification within Fragmented Koala Populations.

The koala population in northern Australia has become increasingly fragmented due to natural and man-made barriers and interventions. This situation has created a unique opportunity to study both endogenous and exogenous koala retrovirus (KoRV). To determine the impact that population isolation has had on KoRV diversity in Queensland, 272 koalas from six fragmented koala populations were profiled for their KoRV provirus across two natural biogeographical barriers (the St Lawrence Gap and the Brisbane Valley Barrier), one man-made geographical barrier (the city of Brisbane) and two translocation events (the single movement of koalas to an island and the repeated movement of koalas into a koala sanctuary). Analysis revealed that all koalas tested were KoRV-A positive, with 90 - 96% of the detected KoRV provirus from each koala representing a single, likely endogenous, KoRV-A strain. The next most abundant proviral sequence was a defective variant of the dominant KoRV-A strain, accounting for 3 - 10% of detected provirus. The remaining KoRV provirus represented expected exogenous strains of KoRV and included geographically localized patterns of KoRV-B, -C, -D, -F, -G, and -I. These results indicate that lineage diversification of exogenous KoRV is actively ongoing. In addition, comparison of KoRV provirus within known dam-sire-joey family groups from the koala sanctuary revealed that joeys consistently had KoRV proviral patterns more similar to their dams than their sires in KoRV-B, -C and -D provirus composition. Collectively, this study highlights both the consistency of endogenous KoRV and the diversity of exogenous KoRV across the fragmented koala populations in northern Australia.IMPORTANCE KoRV infection has become a permanent part of koalas in northern Australia. With KoRV presence and abundance linked to more severe chlamydial disease and neoplasia in these koalas, understanding how KoRV exists throughout an increasingly fragmented koala population is a key first step in designing conservation and management strategies. This survey of KoRV provirus in Queensland koalas indicates that endogenous KoRV provirus is ubiquitous and consistent throughout the state while exogenous KoRV provirus is diverse and distinct in fragmented koala populations. Understanding the prevalence and impact of both endogenous and exogenous KoRV will be needed to ensure a future for all koala populations.

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

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

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.

Predation by carpet pythons (Morelia spilota) is an important cause of mortality in a free-living koala (Phascolarctos cinereus) population in South East Queensland.

Koalas (Phascolarctos cinereus) are experiencing significant declines across the northern part of their range. However, unbiased, population-level estimates of mortality are rarely reported, as it's difficult to quantify causes of mortality robustly in this cryptic species. We aimed to determine the relative importance of carpet python (Morelia spilota) predation in a free-living koala population and describe the characteristic pathological findings during necropsy. In total, 503 koalas were captured, underwent veterinary examination and telemetric tagging, and were monitored after release over a four-year period. Mortalities were detected when activity data reported by K-Tracker® biotelemetry collars indicated low or zero activity, or during routine field monitoring events. Experienced koala veterinarians performed thorough, standardised necropsy examinations on retrieved carcasses to determine causes of death. The three, sometimes subtle, cardinal signs used to definitively diagnose carpet python-caused deaths of koalas were a U-shaped primary bite site, slicking of the fur by python saliva (particularly around the face), and diffuse, uniform pulmonary congestion. We found that carpet pythons were important predators of koalas, second only to wild dogs (dingoes and dingo hybrids (Canis familiaris dingo)), accounting for 11.6% of predation deaths and 7.2% of total deaths. Less than half (38%) of the koalas killed by carpet pythons were ingested. Though carpet pythons are known predators of koalas, their relative importance as a cause of mortality hasn't previously been recognised. Population viability analyses and conservation management plans benefit from robust cause-of-death data collected during longitudinal monitoring studies, requiring telemetry methods that facilitate rapid detection of mortalities.

Changes in Endogenous and Exogenous Koala Retrovirus Subtype Expression over Time Reflect Koala Health Outcomes.

Koala retrovirus (KoRV) is unique in that it exists as both an exogenous and actively endogenizing gamma retrovirus of koalas. While nine subtypes of KoRV have been recognized, focused study of these subtypes in koalas over time and with different health outcomes has been lacking. Therefore, in this study, three wild koala cohorts were established and monitored to examine KoRV proviral and expression data from koalas that either remained healthy over time, began healthy before developing chlamydial cystitis, or presented with chlamydial cystitis and were treated with antibiotics. Deep sequencing of the proviral KoRV envelope gene revealed KoRV-A, -B, -D, and -F to be the major subtypes in this population and allowed for subtype-specific assays to be created. Quantification of KoRV transcripts revealed that KoRV-D expression mirrored the total KoRV expression levels (106 copies/ml of plasma), with KoRV-A and KoRV-F expression being ∼10-fold less and KoRV-B expression being ∼100-fold less, when detected. Strikingly, there was significantly higher expression of KoRV-D in healthy koalas than in koalas that developed chlamydial cystitis, with healthy koalas expressing a major KoRV-D/minor KoRV-A profile, whereas koalas that developed cystitis had variable KoRV expression profiles. Total anti-KoRV IgG antibody levels were found not to correlate with the expression of total KoRV or any individual KoRV subtype. Finally, KoRV expression was consistent between systemic and mucosal body sites and during antibiotic treatment. Collectively, this gives a comprehensive picture of KoRV dynamics during several important koala health states.IMPORTANCE The long-term survival of the koala is under serious threat, with this iconic marsupial being declared "vulnerable" by the Australian Government and officially listed as a threatened species. KoRV is clearly contributing to the overall health status of koalas, and research into this virus has been lacking detailed study of the multiple subtypes at both the proviral and expressed viral levels over time. By designing new subtype-specific assays and following well-defined koala cohorts over time, this study has generated a new more complete picture of KoRV and its relationship to koala health outcomes in the wild. Only by building a comprehensive picture of KoRV during both koala health and disease can we bring meaningful koala health interventions into better focus.

Optimising the short and long-term clinical outcomes for koalas (Phascolarctos cinereus) during treatment for chlamydial infection and disease.

Koalas (Phascolarctos cinereus) have suffered severe declines in the northern extent of their range due to a variety of threats, including habitat destruction, trauma from cars and dogs, climate change and importantly, disease. The most significant pathogen in koalas is Chlamydia pecorum, which causes inflammation and fibrosis at mucosal sites, resulting in blindness, infertility and death in severe cases. Chlamydia treatment can be problematic in koalas as the response to treatment is often poor in chronic cases and antimicrobial choice is limited. Thus, chlamydial disease is a severely threatening process for koala conservation. We investigated the short and long-term clinical outcomes for 167 koalas with Chlamydia that underwent capture, telemetric monitoring and intensive veterinary management as part of a large-scale population management program in South East Queensland. Chlamydia treatments included the standard regimen of daily subcutaneous chloramphenicol injections (60mg/kg) for 14 to 28-days, and a variety of non-standard regimens such as topical antimicrobials only (for ocular disease), surgical treatment only (for bilateral reproductive tract disease), and other antimicrobials/treatment lengths. To assess these regimens we analysed clinical records, field monitoring data and swab samples collected from the urogenital tract and ocular conjunctiva. Overall, in contrast to other studies, treatment was generally successful with 86.3% of treated koalas released back into the wild. The success of treatment rose to 94.8% however, when the standard treatment regimen was employed. Further, 100% of koalas that were also treated with surgical ovariohysterectomy (n = 12) remained healthy for a median of 466 days of post-treatment monitoring, demonstrating the benefits of surgical treatment. Previous studies reported 45-day chloramphenicol regimens, but the shorter standard regimen still achieved microbiological cure and reduces the risk of negative sequelae associated with treatment and/or captivity and treatment costs. Despite these positive clinical outcomes, alternatives to chloramphenicol are warranted due to its decreasing availability.

Chlamydia pecorum gastrointestinal tract infection associations with urogenital tract infections in the koala (Phascolarctos cinereus).

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.

Molecular Dynamics and Mode of Transmission of Koala Retrovirus as It Invades and Spreads through a Wild Queensland Koala Population

The recent acquisition of a novel retrovirus (KoRV) by koalas (Phascolarctos cinereus) has created new opportunities for retroviral research and new challenges for koala conservation. There are currently two major subtypes of KoRV: KoRV-A, which is believed to be endogenous only in koalas from the northern part of Australia, and KoRV-B, which appears to be exogenous. Understanding and management of these subtypes require population level studies of their prevalence and diversity, especially when coinfected in the same population, and investigations of their modes of transmission in the wild. Toward this end, we studied a wild Queensland koala population of 290 animals over a 5-year period and investigated the prevalence, diversity and mode of transmission of KoRV-A and KoRV-B. We found KoRV-A to have an infection level of 100% in the population, with all animals sharing the same dominant envelope protein sequence. In contrast, the KoRV-B infection prevalence was only 24%, with 21 different envelope protein sequence variants found in the 83 KoRV-B-positive animals. Linked to severe disease outcomes, a significant association between KoRV-B positivity and both chlamydial disease and neoplasia was found in the population. Transmission of KoRV-B was found at a rate of 3% via adult-to-adult contact per year, while there was a 100% rate of KoRV-B-positive mothers transmitting the virus to their joeys. Collectively, these findings demonstrate KoRV-B as the pathogenic subtype in this wild koala population and inform future intervention strategies with subtype variation and transmission data. IMPORTANCE KoRV represents a unique opportunity to study a relatively young retrovirus as it goes through its molecular evolution in both an endogenous form and a more recently evolved exogenous form. The endogenous form, KoRV-A, now appears to have stably and completely established itself in Northern Australian koala populations and is progressing south. Conversely, the exogenous form, KoRV-B, is undergoing continuous mutation and spread in the north and, as yet, has not reached all southern koala populations. We can now link KoRV-B to neoplasia and chlamydial disease in both wild and captive koalas, making it an imminent threat to this already vulnerable species. This work represents the largest study of koalas in a wild population with respect to KoRV-A/KoRV-B-infected/coinfected animals and the linkage of this infection to chlamydial disease, neoplasia, viral evolution, and spread.

Epidemiology of chlamydial infection and disease in a free-ranging koala (Phascolarctos cinereus) population.

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.

Microbiota composition of the koala (Phascolarctos cinereus) ocular and urogenital sites, and their association with Chlamydia infection and disease.

Disease caused by Chlamydia pecorum is characterised by ocular and urogenital infections that can lead to blindness and infertility in koalas. However, koalas that are infected with C. pecorum do not always progress to disease. In other host systems, the influence of the microbiota has been implicated in either accelerating or preventing infections progressing to disease. This study investigates the contribution of koala urogenital and ocular microbiota to Chlamydia infection and disease in a free ranging koala population. Using univariate and multivariate analysis, it was found that reproductive status in females and sexual maturation in males, were defining features in the koala urogenital microbiota. Changes in the urogenital microbiota of koalas is correlated with infection by the common pathogen, C. pecorum. The correlation of microbiota composition and C. pecorum infection is suggestive of members of the microbiota being involved in the acceleration or prevention of infections progressing to disease. The analysis also suggests that multiple microbes are likely to be associated with this process of disease progression, rather than a single organism. While other Chlamydia-like organisms were also detected, they are unlikely to contribute to chlamydial disease as they are rare members of the urogenital and ocular microbiota communities.

Infection with koala retrovirus subgroup B (KoRV-B), but not KoRV-A, is associated with chlamydial disease in free-ranging koalas (Phascolarctos cinereus).

The virulence of chlamydial infection in wild koalas is highly variable between individuals. Some koalas can be infected (PCR positive) with Chlamydia for long periods but remain asymptomatic, whereas others develop clinical disease. Chlamydia in the koala has traditionally been studied without regard to coinfection with other pathogens, although koalas are usually subject to infection with koala retrovirus (KoRV). Retroviruses can be immunosuppressive, and there is evidence of an immunosuppressive effect of KoRV in vitro. Originally thought to be a single endogenous strain, a new, potentially more virulent exogenous variant (KoRV-B) was recently reported. We hypothesized that KoRV-B might significantly alter chlamydial disease outcomes in koalas, presumably via immunosuppression. By studying sub-groups of Chlamydia and KoRV infected koalas in the wild, we found that neither total KoRV load (either viraemia or proviral copies per genome), nor chlamydial infection level or strain type, was significantly associated with chlamydial disease risk. However, PCR positivity with KoRV-B was significantly associated with chlamydial disease in koalas (p = 0.02961). This represents an example of a recently evolved virus variant that may be predisposing its host (the koala) to overt clinical disease when co-infected with an otherwise asymptomatic bacterial pathogen (Chlamydia).

A Prototype Recombinant-Protein Based Chlamydia pecorum Vaccine Results in Reduced Chlamydial Burden and Less Clinical Disease in Free-Ranging Koalas (Phascolarctos cinereus).

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.