Pulmonary Actinomycosis in South Australian Koalas (Phascolarctos cinereus).

Pneumonia has been reported in both free-ranging and captive koalas and a number of causative agents have been described. Between 2016 and 2019, 16 free-ranging and 1 captive koala (Phascolarctos cinereus) from the Mount Lofty Ranges of South Australia were identified with pyogranulomatous lobar pneumonia, which involved the left caudal lobe in 14/17 (82%) cases. Within lesions, numerous gram-positive or gram-variable, non-acid-fast filamentous bacteria were observed in association with Splendore-Hoeppli phenomenon. Culture yielded growth of anaerobic bacteria, which were unidentifiable by MALDI-TOF-MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) analysis in 5/5 cases. Sequencing of the bacterial 16S rRNA gene identified a novel Actinomyces species in 4 samples, confirming a diagnosis of pulmonary actinomycosis. Concurrent examination of resin lung casts from healthy koalas suggested greater laminar flow of air to the left caudal lung lobe in koalas. Actinomyces spp. have been reported as commensals of the oral microbiome in other species, and an association with similar pulmonary lesions in other species. Considering the predilection for involvement of the left caudal lung lobe, aspiration is suggested as the likely cause in some cases of pulmonary actinomycosis in koalas. Pulmonary actinomycosis has not been previously described in koalas and further work needs to be undertaken in order to classify this organism within the Actinomyces genus.

Genetic diversity in the plasticity zone and the presence of the chlamydial plasmid differentiates Chlamydia pecorum strains from pigs, sheep, cattle, and koalas.

BACKGROUND: Chlamydia pecorum is a globally recognised pathogen of livestock and koalas. To date, comparative genomics of C. pecorum strains from sheep, cattle and koalas has revealed that only single nucleotide polymorphisms (SNPs) and a limited number of pseudogenes appear to contribute to the genetic diversity of this pathogen. No chlamydial plasmid has been detected in these strains despite its ubiquitous presence in almost all other chlamydial species. Genomic analyses have not previously included C. pecorum from porcine hosts. We sequenced the genome of three C. pecorum isolates from pigs with differing pathologies in order to re-evaluate the genetic differences and to update the phylogenetic relationships between C. pecorum from each of the hosts. METHODS: Whole genome sequences for the three porcine C. pecorum isolates (L1, L17 and L71) were acquired using C. pecorum-specific sequence capture probes with culture-independent methods, and assembled in CLC Genomics Workbench. The pairwise comparative genomic analyses of 16 pig, sheep, cattle and koala C. pecorum genomes were performed using several bioinformatics platforms, while the phylogenetic analyses of the core C. pecorum genomes were performed with predicted recombination regions removed. Following the detection of a C. pecorum plasmid, a newly developed C. pecorum-specific plasmid PCR screening assay was used to evaluate the plasmid distribution in 227 C. pecorum samples from pig, sheep, cattle and koala hosts. RESULTS: Three porcine C. pecorum genomes were sequenced using C. pecorum-specific sequence capture probes with culture-independent methods. Comparative genomics of the newly sequenced porcine C. pecorum genomes revealed an increased average number of SNP differences (~11 500) between porcine and sheep, cattle, and koala C. pecorum strains, compared to previous C. pecorum genome analyses. We also identified a third copy of the chlamydial cytotoxin gene, found only in porcine C. pecorum isolates. Phylogenetic analyses clustered porcine isolates into a distinct clade, highlighting the polyphyletic origin of C. pecorum in livestock. Most surprising, we also discovered a plasmid in the porcine C. pecorum genome. Using this novel C. pecorum plasmid (pCpec) sequence, a) we developed a pCpec screening assay to evaluate the plasmid distribution in C. pecorum from different hosts; and b) to characterise the pCpec sequences from available previously sequenced C. pecorum genome data. pCpec screening showed that the pCpec is common in all hosts of C. pecorum, however not all C. pecorum strains carry pCpec. CONCLUSIONS: This study provides further insight into the complexity of C. pecorum epidemiology and novel genomic regions that may be linked to host specificity. C. pecorum plasmid characterisation may aid in improving our understanding of C. pecorum pathogenesis across the variety of host species this animal pathogen infects.

Prevalence and clinical significance of koala retrovirus in two South Australian koala (Phascolarctos cinereus) populations.

PURPOSE: Koala retrovirus (KoRV-A) is 100  % prevalent in northern Australian (Queensland and New South Wales) koala populations, where KoRV-B has been associated with Chlamydia pecorum disease and the development of lymphosarcoma. In southern populations (Victoria and South Australia), KoRV-A is less prevalent and KoRV-B has not been detected in Victoria, while the current prevalence in South Australian populations is unknown but is thought to be low. This study aimed to determine (i) the prevalence of KoRV in the two largest South Australian koala populations [Kangaroo Island (KI) and Mount Lofty Ranges (MLR)], (ii) KoRV subtype and (iii) if an association between KoRV and C. pecorum exists. METHODOLOGY: Wild koalas were sampled in KI ( n =170) between 2014 and 2017 and in MLR ( n =75) in 2016. Clinical examinations were performed, with blood collected for KoRV detection and typing by PCR. RESULTS: KoRV prevalence was 42.4  % [72/170, 95 % confidence interval (CI): 34.9-49.8  %] in KI and 65.3  % (49/75, 95 % CI: 54.6-76.1  %) in MLR. Only KoRV-A, and not KoRV-B, was detected in both populations. In MLR, there was no statistical association between KoRV and C. pecorum infection (P =0.740), or KoRV and C. pecorum disease status ( P=0.274), although KoRV-infected koalas were more likely to present with overt C. pecorum disease than subclinical infection (odds ratio: 3.15, 95 % CI: 0.91-5.39). CONCLUSION: KoRV-A is a prevalent pathogen in wild South Australian koala populations. Future studies should continue to investigate KoRV and C. pecorum associations, as the relationship is likely to be complex and to differ between the northern and southern populations.

PREVALENCE AND PATHOLOGIC FEATURES OF CHLAMYDIA PECORUM INFECTIONS IN SOUTH AUSTRALIAN KOALAS (PHASCOLARCTOS CINEREUS).

Chlamydia pecorum infection is highly prevalent in many koala ( Phascolarctos cinereus ) populations in the eastern states of Australia, causing ocular and urogenital tract disease. In contrast, the current prevalence of chlamydiosis in South Australian (SA) koalas is largely unknown, with few reports of clinical cases. We examined 65 SA rescued wild koalas at necropsy and collected ocular and urogenital swabs for the detection of C. pecorum by PCR. We detected C. pecorum in ocular or urogenital swabs from 57 koalas (88%), and 34 koalas were positive at both ocular and urogenital sites. Clinically overt chlamydial disease was present in only 12 (21%) positive koalas. Gross lesions were often externally inapparent as they affected the urogenital tract (n=5), and 24 infected koalas had microscopically evident lesions only. Lesions were predominantly mild and included conjunctivitis, cystitis, and urethritis. Reproductive tract disease was infrequently observed. We detected C. pecorum in 16 (28%) koalas with no evidence of chlamydial disease, suggesting the presence of subclinical carriers in this population. Based on these findings, chlamydiosis has a higher occurrence in SA koala populations than previously thought, but is most often mild and does not always result in overt clinical disease; inapparent and subclinical infections appear common. Further studies of the prevalence in wild-caught SA koalas are needed along with research into the host and bacterial factors that may influence disease outcome in these animals.

Plasma biochemistry and urinalysis variables of koalas (Phascolarctos cinereus) with and without oxalate nephrosis.

BACKGROUND: Oxalate nephrosis is a highly prevalent disease in the Mount Lofty Ranges koala population in South Australia, but associated clinicopathologic findings remain undescribed. OBJECTIVES: The aims of this study were to determine plasma biochemical and urinalysis variables, particularly for renal function and urinary crystal morphology and composition, in koalas with oxalate nephrosis. METHODS: Blood and urine samples from Mount Lofty Ranges koalas with oxalate nephrosis were compared with those unaffected by renal oxalate crystal deposition from Mount Lofty and Kangaroo Island, South Australia and Moggill, Queensland. Plasma and urine biochemistry variables were analyzed using a Cobas Bio analyzer, and urinary oxalate by high-performance liquid chromatography. Urinary crystal composition was determined by infrared spectroscopy and energy dispersive X-ray analysis. RESULTS: Azotemia (urea > 6.6 mmol/L, creatinine > 150 µmol/L) was found in 93% of koalas with oxalate nephrosis (n = 15). All azotemic animals had renal insufficiency (urine specific gravity [USG] < 1.035), and in 83%, USG was < 1.030. Koalas with oxalate nephrosis were hyperoxaluric compared with Queensland koalas (P < .01). Urinary crystals from koalas with oxalate nephrosis had atypical morphology and were composed of calcium oxalate. Mount Lofty Ranges koalas unaffected by renal oxalate crystal deposition had renal insufficiency (43%), although only 14% had USG < 1.030 (n = 7). Unaffected Mount Lofty Ranges and Kangaroo Island koalas were hyperoxaluric compared with Queensland koalas (P < .01). CONCLUSIONS: Koalas with oxalate nephrosis from the Mount Lofty Ranges had renal insufficiency, hyperoxaluria, and pathognomonic urinary crystals. The findings of this study will aid veterinary diagnosis of this disease.