Expanding the known distribution of phascolartid gammaherpesvirus 1 in koalas to populations across Queensland and New South Wales.

Koala populations across the east coast of Australia are under threat of extinction with little known about the presence or distribution of a potential pathogen, phascolartid gammaherpesvirus 1 (PhaHV-1) across these threatened populations. Co-infections with PhaHV-1 and Chlamydia pecorum may be common and there is currently a limited understanding of the impact of these co-infections on koala health. To address these knowledge gaps, archived clinical and field-collected koala samples were examined by quantitative polymerase chain reaction to determine the distribution of PhaHV-1 in previously untested populations across New South Wales and Queensland. We detected PhaHV-1 in all regions surveyed with differences in detection rate between clinical samples from rescued koalas (26%) and field-collected samples from free-living koalas (8%). This may reflect increased viral shedding in koalas that have been admitted into care. We have corroborated previous work indicating greater detection of PhaHV-1 with increasing age in koalas and an association between PhaHV-1 and C. pecorum detection. Our work highlights the need for continued surveillance of PhaHV-1 in koala populations to inform management interventions, and targeted research to understand the pathogenesis of PhaHV-1 and determine the impact of infection and co-infection with C. pecorum.

An evaluation of DNA sample source and molecular markers to determine gender in the short-beaked echidna (Tachyglossus aculeatus).

The short-beaked echidna is sexually monomorphic such that gender identification without veterinary intervention is challenging. The aim of this study was to evaluate and compare the most optimal noninvasive genetic source by extracting echidna genomic DNA (gDNA) from fecal scats, plucked hair, and quills to perform genetic sex testing using a range of molecular markers. Sex determination of 14 captive short-beaked echidnas was determined by amplifying isolated DNA from noninvasive samples, targeting two Y chromosome (male-specific) genes (mediator complex subunit 26 Y-gametologue [CRSPY] and anti-Müllerian hormone Y-gametologue [AMHY]), in addition to four confirmed sex-specific RADseq markers. Results of noninvasive samples were compared with blood samples and clinical records. Receiver operating characteristic curves were used to assess accuracy of sex determination of markers for each sample type. The gender of the echidnas was successfully identified on 75% of occasions using fecal samples, 90.6% occasions using hair, and 84.6% occasions with quills. Overall, the male-specific RADseq markers accurately identified the sex of echidnas with all sample types for 90% of animals; compared with 81.5% using CRSPY, and 82.0% using AMHY to identify sex. Collection of hair, quills, and feces provides a useful alternative to invasively collected samples, however, the accuracy of results depends on sample type and genetic marker selected. We found gender determination in the short-beaked echidna was most accurate using four male-specific RADseq markers on gDNA isolated from blood and hair. The noninvasive genetic sexing techniques documented here will inform and facilitate husbandry and genetic management of captive echidna populations.

Chlamydiosis and cystic dilatation of the ovarian bursa in the female koala (Phascolarctos cinereus): Novel insights into the pathogenesis and mechanisms of formation.

Infection with Chlamydia pecorum is one of the main causes of progressive decline of koala (Phascolarctos cinereus) populations in Eastern Australia. Pathological changes associated with the chlamydial infection in the genital tract of female and male koalas have been widely described with reports of acute and chronic lymphoplasmacytic inflammation and the description of the cystic dilatation of the ovarian bursa. Although these disease manifestations can result in severe chronic inflammation, structural changes and even sterility, only limited data is currently available on the organism's distribution and associated histopathological and ultrastructural changes within the upper genital tract of affected females. This study examined the pathogenesis of the most common pathological lesion associated with chlamydiosis in female koalas, the cystic dilation of the ovarian bursa starting from the evidence that Chlamydia spp. induces disruption of the intercellular junctions in the epithelium of the reproductive organs in humans. Histology, immunohistochemistry (IHC) and transmission electron microscopy (TEM) were performed to evaluate the structural features and the expression of epithelial cell and cellular junctions' markers in affected bursae from 39 Chlamydia-infected female koalas. Epithelial cells from the ovarian bursae of one affected animal examined by transmission electron microscopy showed severe widening of the intercellular space, as morphologic evidence of disrupted permeability of the epithelial barrier. The epithelial cell-cell junctions markers E-cadherin, ß-catenin and ZO-1 expressions were significantly reduced in samples from cystic bursae when compared to normal tissue samples (P < 0.0001). On the other end, a significantly higher expression of the proliferation marker Ki67 was observed in cystic bursae compared to control samples (P < 0.0001). As these proteins are required to maintain epithelial functional integrity and cell-cell adhesive interactions, their loss may permanently impair and affect female koala fertility and suggest the molecular basis of the pathogenesis of the cystic accumulation of bursal fluid within this tissue.

In vitro transmission of Chlamydia using naturally infected koala (Phascolarctos cinereus) semen.

Transmission of Chlamydia pecorum infection has generally been assumed to be via the urogenital route and in an attempt to confirm this we investigated an in vitro method of Chlamydia infection using naturally infected koala semen to inoculate a cell line and attempt to estimate C. pecorum infectious load. A total of 57% of 122 koala semen samples had low C. pecorum copy number or no burden, while 18% of semen samples contained >10000 inclusion-forming units/mL, as determined by quantitative polymerase chain reaction. In vitro inoculation of a McCoy cell line resulted in successful infection from 4% of semen samples where C. pecorum burden was >105 inclusion-forming units/mL. Our preliminary study suggests that transmission of C. pecorum infectious dose may be restricted to peak bacterial shedding in semen associated with recent infection. Here, we report venereal transmission of C. pecorum in koala semen is possible; however, we speculate that antimicrobial factors and innate immune function receptors associated with semen may inhibit chlamydial growth. These mechanisms have yet to be reported in marsupial semen.

Investigation of pathology associated with Chlamydia pecorum infection in the male reproductive tract, and the effect on spermatogenesis and semen quality in the koala (Phascolarctos cinereus).

There is growing evidence that Chlamydia pecorum infection of the male koala reproductive tract causes inflammation and pathology of the urogenital tract. Previous studies have revealed that male koalas exhibiting severe clinical signs of urogenital chlamydial disease had an increased incidence of sperm DNA fragmentation and abnormal sperm morphology, suggestive of chronic exposure to C. pecorum infection and/or inflammation in the testis and epididymis, with residual pathology and lesions disrupting spermatogenesis and maturation of spermatozoa. This study specifically aimed to determine whether pathology associated with chlamydial infection in different regions of the male koala reproductive tract had an adverse effect on classical seminal parameters, sperm DNA quality and endocrine function (testosterone secretion) of naturally infected males. Semen from 58 sexually mature male koalas deemed not suitable for rehabilitation or treatment was assessed, in addition to undertaking a GnRH challenge to determine the androgenic capacity of the testis. Following euthanasia, tissue samples from testes, epididymis and prostate were evaluated for histopathology and real time polymerase chain reaction (qPCR). A significant difference in sperm concentration was observed between males with unilateral and bilateral testicular atrophy and C. pecorum infection (P = 0.011); and between males with unilateral atrophy and C. pecorum infection in one testis and bilateral normal testes with no C. pecorum infection (P = 0.008). No significant association was found for any other semen parameters when categorised by histopathology and C. pecorum tissue presence within the testes, epididymis and prostate. Plasma testosterone concentrations did not significantly differ between testicular histopathology diagnosis and/or C. pecorum infection status. This study suggests Chlamydia infection and inflammation may not be the predominant reason of disruption to spermatogenesis in the wild koala but rather testicular degeneration and atrophy, irrespective of Chlamydia infection, appears to be the primary reason of decreased sperm concentration.

Proteomic analysis of koala (phascolarctos cinereus) spermatozoa and prostatic bodies.

The aims of this study were to investigate the proteome of koala spermatozoa and that of the prostatic bodies with which they interact during ejaculation. For this purpose, spermatozoa and prostatic bodies were fractionated from the semen of four male koalas and analysed by HPLC MS/MS. This strategy identified 744 sperm and 1297 prostatic body proteins, which were subsequently attributed to 482 and 776 unique gene products, respectively. Gene ontology curation of the sperm proteome revealed an abundance of proteins mapping to the canonical sirtuin and 14-3-3 signalling pathways. By contrast, protein ubiquitination and unfolded protein response pathways dominated the equivalent analysis of proteins uniquely identified in prostatic bodies. Koala sperm proteins featured an enrichment of those mapping to the functional categories of cellular compromise/inflammatory response, whilst those of the prostatic body revealed an over-representation of molecular chaperone and stress-related proteins. Cross-species comparisons demonstrated that the koala sperm proteome displays greater conservation with that of eutherians (human; 93%) as opposed to reptile (crocodile; 39%) and avian (rooster; 27%) spermatozoa. Together, this work contributes to our overall understanding of the core sperm proteome and has identified biomarkers that may contribute to the exceptional longevity of koala spermatozoa during ex vivo storage.

The effect of Chlamydia infection on koala (Phascolarctos cinereus) semen quality.

Although it is well established that chlamydial disease renders female koalas infertile, there has been limited research on its effects on male koala fertility, specifically sperm quality. This study determined whether chlamydial infection adversely affects semen quality of naturally infected koalas and spermatozoa recovered from Chlamydia negative koalas co-incubated in vitro with C. pecorum elementary bodies (EBs). Semen from 102 south-east Queensland sexually mature wild koalas exhibiting varying degrees of chlamydiosis and clinical signs of disease were assessed for semen quality and compared to 11 clinically healthy, Chlamydia-free captive male koalas. For in vitro studies, semen samples were collected from 6 Chlamydia-free captive koalas, and co-incubated over 24 h with high and low concentrations of C. pecorum EBs and sperm quality assessed. Wild koalas displaying severe signs of clinical disease with C. pecorum present in the semen had significantly greater sperm DNA damage (P = 0.0267). The total % of morphologically abnormal spermatozoa was highest in wild koalas that had severe signs of clinical disease but whose semen was negative for C. pecorum (P = 0.0328). This apparent contradiction is possibly associated with wild males having resolved the infection but still possessing underlining reproductive pathology. A higher incidence of loose head spermatozoa occurred in semen of wild koalas not infected with C. pecorum compared to those that were C. pecorum infected (P = 0.026). In vitro incubation of semen with C. pecorum significantly decreased sperm motility and viability over 24 h.

EPIDEMIOLOGY OF CHLAMYDIA-INDUCED REPRODUCTIVE DISEASE IN MALE KOALAS (PHASCOLARCTOS CINEREUS) FROM SOUTHEAST QUEENSLAND, AUSTRALIA AS ASSESSED FROM PENILE URETHRAL SWABS AND SEMEN.

Declining population sizes of koalas (Phascolarctos cinereus) in SE Queensland (QLD), Australia can partially be attributed to chlamydiosis, with the majority of epidemiological studies focusing on the prevalence of infection and associated pathology in female koalas, with lesser attention given to males. We aimed to explore the epidemiology of Chlamydia pecorum infection in the male urogenital tract from wild (hospitalized and free-ranging) koalas in SE QLD. Although 67% of male koalas were infected with C. pecorum in their urogenital tract and 55% were shedding the organism in their semen, only a third of the males sampled presented with overt signs of urogenital disease. Infection with C. pecorum was lower in populations from rural locations, compared with periurban locations, with a corresponding low association between urogenital infection and clinical disease. The presence of C. pecorum in penile urethral swabs was a good predictor of the presence of C. pecorum in semen, with a significant correlation (P=0.006) in 58% of males. In contrast, the C. pecorum load in penile urethral swabs was not a good predictor of the C. pecorum load in semen, with no significant correlation. In addition, 57% of male koalas had large numbers of bacterial copy numbers in the penile urethra (upper quartile) and 40% shedding into semen with no overt signs of disease. Investigation of the association of C. pecorum infection, body condition score, and age revealed that the highest incidence of urogenital infection occurred in males with the lowest body score (1 out of 10). Furthermore, 63% of sexually mature male koalas (>2 yr old) had urethral infections and 50% had C. pecorum in their semen. Our study suggested that the role of chlamydia in male koala infertility has been previously underestimated.

Rapid point-of-care diagnostics for the detection of Chlamydia pecorum in koalas (Phascolarctos cinereus) using loop-mediated isothermal amplification without nucleic acid purification.

Infectious disease, predominately chlamydiosis, contributes significantly to the decline in health of wild koala (Phascolarctos cinereus) populations in some regions of Australia. In this study, we describe the development and evaluation of a simple, sensitive, and specific loop-mediated isothermal amplification (LAMP) assay for the detection of Chlamydia pecorum in koalas as a point-of-care diagnostic tool that can be used in any wildlife hospital and in the field on specialized instrumentation. A set of primers targeting a 188-bp region of the C. pecorum genome was designed. 100% specificity of the LAMP assay was revealed by demonstrating no cross-reactivity with 33 nontarget pathogens, and complete correlation with qPCR results for 43 clinical swabs collected opportunistically from wildlife hospitals. In sensitivity evaluations, the technique successfully detected serial dilutions of extracted C. pecorum DNA with a detection limit of 44 IFU/ml.

Developing noninvasive methodologies to assess koala population health through detecting Chlamydia from scats.

Wildlife diseases are a recognized driver of global biodiversity loss, have substantial economic impacts, and are increasingly becoming a threat to human health. Disease surveillance is critical but remains difficult in the wild due to the substantial costs and potential biases associated with most disease detection methods. Noninvasive scat surveys have been proposed as a health monitoring methodology to overcome some of these limitations. Here, we use the known threat of Chlamydia disease to the iconic, yet vulnerable, koala Phascolarctos cinereus to compare three methods for Chlamydia detection in scats: multiplex quantitative PCR, next generation sequencing, and a detection dog specifically trained on scats from Chlamydia-infected koalas. All three methods demonstrated 100% specificity, while sensitivity was variable. Of particular interest is the variable sensitivity of these diagnostic tests to detect sick individuals (i.e., not only infection as confirmed by Chlamydia-positive swabs, but with observable clinical signs of the disease); for koalas with urogenital tract disease signs, sensitivity was 78% with quantitative PCR, 50% with next generation genotyping and 100% with the detection dog method. This may be due to molecular methods having to rely on high-quality DNA whereas the dog most likely detects volatile organic compounds. The most appropriate diagnostic test will vary with disease prevalence and the specific aims of disease surveillance. Acknowledging that detection dogs might not be easily accessible to all, the future development of affordable and portable "artificial noses" to detect diseases from scats in the field might enable cost-effective, rapid and large-scale disease surveillance.

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.

Development and application of two multiplex real-time PCR assays for detection and speciation of bacterial pathogens in the koala.

Infectious diseases have contributed to the decline in the health of koala ( Phascolarctos cinereus) populations in the wild in some regions of Australia. Herein we report the development and validation of 2 multiplex real-time PCR (rtPCR) panels for the simultaneous detection of Mycoplasma spp., Ureaplasma spp., Bordetella bronchiseptica, and Chlamydia, including speciation and quantification of Chlamydia, in ocular, reproductive, and nasal swab samples in addition to semen and male urogenital and reproductive tissues, from koalas. Each rtPCR panel was developed for use as a single-tube reaction using pathogen-specific primers and fluorescently labeled probe sets. DNA extracted from reference strains and isolates was used for validation of sequence gene targets for the multiplex rtPCR panels. Each panel was shown to be sensitive and specific in detecting and differentiating the bacterial pathogens. The multiplex rtPCR panels were used to screen clinical samples from free-ranging and hospitalized koalas for multiple pathogens simultaneously. The multiplex rtPCR will improve turnaround time compared to individual-pathogen rtPCR methods used, to date, for confirmation of diagnosis and will provide the wildlife clinician with the ability to make treatment decisions more rapidly.

Ultrasonographic assessment of the male koala (Phascolarctos cinereus) reproductive tract.

Studies documenting the application of ultrasonography to depict normal and pathological changes in koalas (Phascolarctos cinereus), especially in the male, are scarce. Sixty-two wild koalas were used in this study to define ultrasonographic protocols and features for the assessment of the male koala reproductive tract. Testis, epididymis and spermatic cord were examined using a hockey stick transducer. The normal koala testis showed a homogeneous echogenicity and an obvious hyper-echoic band corresponding to the tunica albuginea. The cauda epididymis was characterised by hypo- and hyper-echoic regions and was most effectively imaged in sagittal section. The koala prostate was assessed using a micro-curved transducer positioned midline, caudal to the bladder. On transverse section, it showed distinct margins and a well-defined internal structure, although the prostatic urethra was not apparent on most scans. To image the bulbourethral glands (BGs), the hockey stick transducer was placed lateral to the cloaca. BGIII was located just below the skin, while BGII was located deeper than BGIII. BGI was too small and not sufficiently echogenic to be detected. The ultrasonographic appearance of the BGs was similar to that of the testes but with more obvious hypo-echoic stippling. This comprehensive review of the ultrasonographic appearance of normal male koala reproductive tract can be used by veterinarians and others, in zoos or those working with wild koalas, during assessment of the reproductive tract of male koalas in relation to seasonal changes in accessory gland function or for the pathological investigation of reproductive lesions and infertility problems.

Haematological and biochemical reference intervals for three species of hydrophiine sea snakes (Hydrophis curtus, H. elegans and H. peronii) in Australia.

This study presents the first set of comprehensive reference intervals (RIs) for plasma biochemistry and haematology for three species of sea snakes common to the Indo-Pacific waters of Australia. In total 98 snakes, composed of Hydrophis curtus (n= 60), H. elegans (n = 27) and H. peronii (n = 11), were captured, clinically examined and had venous blood samples collected. All snakes were deemed healthy and in good to excellent body condition with snout to vent lengths of 40.7-73.9 cm (H. curtus), 68.9-131.4 cm (H. elegans) and 55.0-83.0 cm (H. peronii), respectively. Lymphocyte numbers, alkaline phosphatase, alanine aminotransferase, creatine kinase and lactate dehydrogenase levels were species-dependent. All other parameters are presented as a single range for the three species. Gender ratio was evenly distributed for H. curtus and H. elegans, but 8/11 (73%) of H. peronii were males. No significant differences were detected between males and females for any of the measured blood parameters. Lymph contamination was considered and accounted for. Although only three species of sea snakes are represented in this study, the RIs generated may be useful in the clinical assessment of other sea snake species.