Research, Connect, Protect



Digestion, nutrition & metabolism

A survey of pesticide accumulation in a specialist feeder, the koala (Phascolarctos cinereus)

Marschner, C, Higgins, DP & Krockenberger, MB 2017, Bulletin of Environmental Contamination and Toxicology, vol. 99, no. 1, p. 303-307.

This study is the first to examine exposure to and effects of pesticides in koalas. In all tissue samples from koalas that inhabited areas impacted by horticultural, agricultural or urban development, pesticide levels were consistently below the limit of detection. This finding suggests that, for these koalas, chronic ongoing or acute exposure to pesticides was unlikely.

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Administration of a 5HT3 receptor antagonist increases the intake of diets containing Eucalyptus secondary metabolites by marsupials

Lawler, IR, Foley, WJ, Pass, GJ & Eschler, BM 1998, Journal of Comparative Physiology B, vol. 168, no. 1, pp. 611-618.

Jensenone, an acylphloroglucinol derivative that is a secondary metabolite in Eucalyptus, was found to cause common ringtail and brushtail possums to regulate their food intake to consume only a defined maximum amount. Injecting jensenone into possums’ stomachs reduced their food consumption so as not to exceed a maximum dosage, although individuals responded variably. After injecting ondansetron, an antagonist of serotonin 5HT3 receptors, the maximum amount of food able to be consumed increased significantly, suggesting that jensenone’s negative effects occur by acting on 5HT3 serotonin receptors, thereby causing ‘nausea’ and food aversion.

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An investigation of streptococcal flora in faeces of koalas

Osawa, RO 1991, Journal of Wildlife Management, vol. 55, no. 4, pp. 623-627.

The occurrence, counts and biochemical characteristics of streptococcal flora in koala faeces differ according to sex and living environment. Female koalas have approximately ten times more of the tannin-protein complex degrading (T-PCD) Streptococcus bovis bacterium in their faeces than males. Strains of T-PCD S. bovis were not only more dominant among the faecal streptococcal of free-ranging koalas than of captive koalas but were also biochemically unique between each group. The enterococci S. faecalis and S. faecium were found only in the faeces of captive koalas, indicating that the bacteria were spread from humans or other animals.

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Bark chewing reveals a nutrient limitation of leaves for a specialist folivore

Au, J, Youngentob, KN, Clark, RH, Phillips, R & Foley, WJ 2017, Journal of Mammalogy, vol. 98, no. 4, pp. 1185-1192.

Koalas of the subalpine Monaro region in New South Wales appear to have developed the unusual adaptation of chewing the sodium-rich bark of Eucalyptus mannifera to meet their nutritional requirements within a landscape that is otherwise lacking in the mineral micronutrient.

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Characterisation of gram-negative anaerobic strains, isolated from koala faeces, which exhibit satellite growth and pleomorphism

Osawa, RO, Fujusawa, T & Mitsuoka, T 1992, Systematic and Applied Microbiology, vol. 15, no. 1, pp. 628-635.

As part of a series of studies investigating the characteristics of koala faecal microflora, three strains of anaerobic gram-negative bacteria which demonstrated satellitism around colonies of Escherichia coli are described here.

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Cloning and expression of koala (Phascolarctos cinereus) liver cytochrome P450 CYP4A15

Ngo, SNT, McKinnon, RA & Stupans, I 2006, Gene, vol. 376, p. 123-132.

Digestion of eucalyptus oil compounds, or monoterpenes, is thought to be done by liver enzymes called cytochromes P450 (CYP). In the past it has been reported that koala liver has increased hydroxylation of lauric acid, a fatty acid, which is indicative of CYP4A enzyme activity. The molecular and enzymatic characteristics of CYP4A in koala liver are reported in this study.

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Complete genome sequence of the Streptococcus gallolyticus subsp. gallolyticus Strain DSM 16831

Grimm, I, Dumke, J, Vollmer, T, Hinse, D, Rückert, C, Kalinowski, J, Knabbe, C & Dreier, J 2017, Genome Announcements, vol. 5, no. 16,  e00108-17.

The complete genome sequence of Streptococcus gallolyticus subsp. gallolyticus strain DSM 16831, found in koala faeces and noted for its low virulence compared to other isolates, has been determined and reported here.

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Composition of preferred and rejected Eucalyptus browse offered to captive koalas, Phascolarctos cinereus (Marsupialia)

Ullrey, DE, Robinson, PT & Whetter, PA 1981, Australian Journal of Zoology, vol. 29, pp. 839-846.

Browse from eleven species of Eucalyptus tree shoots or mature limbs were offered to captive koalas at the San Diego Zoo, California and subsequently sorted into ‘preferred’ or ‘rejected’ species. Analyses revealed that, compared to rejected browse, preferred browse contained significantly higher crude protein, remaining proximate fraction, potassium and phosphorus concentrations and significantly lower neutral detergent fibre, acid detergent fibre, ether extract, calcium, iron, selenium, permanganate lignin, and gross energy concentrations.

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Cytochrome P450 4A, peroxisomal enzymes and nicotinamide cofactors in koala liver

Ngo, S, Kong, S, Kirlich, A, McKinnon, RA & Stupans, I 2000, Comparative Biochemistry and Physiology Part C, vol. 127, no. 1, pp. 327-334.

Microsomal lauric acid hydroxylase activity and cyanide-insensitive palmitoyl coenzyme A oxidative (CIPCO) activity were examined in koala, tammar wallaby and rat liver. The microsomal lauric acid hydroxylation was higher in the koala than either the wallaby or rat, whereas CIPCO activity was completely absent in the koala. Hepatic nicotinamide cofactors were observed for all three species, with nicotinamide-adenine dinucleotide (NAD) levels and the ratio of NAD to NAD phosphate (NADP) higher in koalas than either of the other species. A cloned cDNA for CYP4A from the koala was found to be ~70% similar to human CYP4A11, prompting its naming as CYP4A15.

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Dental microwear texture analysis of extant koalas: clarifying causal agents of microwear

Hedberg, C & DeSantis, LRG 2016, Journal of Zoology, vol. 301, no. 1, pp. 206-214.

The dental microwear of the koala is consistent with that of other tough object feeders and reflects its dietary composition and behaviours. The most likely factors influencing microwear patterns in the koala are the properties of food consumed, abrasion during mastication and the ingestion of grit.

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Detection of Helicobacter species in the gastrointestinal tract of ringtail possum and koala: Possible influence of diet, on the gut microbiota

Coldham, T, Rose, K, O’Rourke, J, Neilan, BA, Dalton, H, Lee, A & Mitchell, H 2013, Veterinary Microbiology, vol. 166, pp. 429-437.

Cultures from the liver and distinct regions of the gastrointestinal tract (GIT) revealed no Helicobacter species to be present in studied koalas. Deoxyribonucleic acid (DNA) extraction from stomach mucus scrapings and homogenised livers enabled gene sequencing of the 16S rRNA gene which was later identified to be of Helicobacter species. Examination of liver and intestine sections using histopathological methods further showed that the presence of Helicobacter was not related to infection.

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Dietary specialisation and Eucalyptus species preferences in Queensland koalas (Phascolarctos cinereus)

Higgins, AL, Bercovitch, FB, Tobey, JR & Hamlin Andrus, C 2011, Zoo Biology, vol. 30, no. 1, pp. 52-58.

Koalas appear to demonstrate individual dietary preferences for different species of Eucalyptus and an overall preference for particular species; however, the factors driving these preferences are not yet understood.

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Digestion and nitrogen metabolism in the koala, Phascolarctos cinereus

Harrop, CJF & Degabriele, R 1976, Australian Journal of Zoology, vol. 24, no. 1, pp. 201-215.

The koala’s digestive tract is shown here to be well-suited to the consumption of fibrous Eucalyptus leaves. The caecum, the sac at the beginning of the large intestine that stores food in herbivores and allows bacteria to break down cellulose, was large, as would be expected for a specialised mammalian herbivore. Koalas maintained a positive but low nitrogen level over the experimental period, with digestibility of nitrogen lower in winter, requiring more nitrogen to be consumed. This was demonstrated by mean nitrogen balances and apparent digestible nitrogen intakes not being significantly different between summer and winter, and the daily dietary nitrogen intakes differing significantly between seasons (0.426 g/kg W0.75 in summer and 0.493 g/kg W0.75 in winter).

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Effects of acidic and alkaline treatments on tannic acid and its binding property to protein

Osawa, R & Walsh, TP 1993, Journal of Agricultural and Food Chemistry, vol. 41, no. 1, pp. 704-707.

Tannin-protein complexes that must be broken down in the gut of the koala dissociated under highly acidic and highly alkaline conditions in vitro in an environment designed to simulate the marsupial’s stomach.

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Foliage of Eucalyptus punctata and the maintenance nitrogen requirements of koalas, Phascolarctos cinereus

Cork, SJ 1986, Australian Journal of Zoology, vol. 34, pp. 17-23.

In an analysis of foliage fed to captive koalas, winter foliage was found to have a lower nitrogen content than summer foliage; however, this was compensated for by the koalas eating more during winter. Nitrogen balance in the koalas was, therefore, not significantly different between the seasons. Similarly, the excretion of nitrogen did not change seasonally but the non-dietary losses of nitrogen in the faeces were higher in the winter compared to summer.

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Formation of a clear zone on tannin-treated brain heart infusion agar by a Streptococcus sp. isolated from faeces of koalas

Osawa, RO 1990, Applied and Environmental Microbiology, vol. 56, no. 3, pp. 829-831.

A strain of tannin-protein complex degrading bacterium was identified in the faeces of koalas. Colonies of the isolate formed a clear zone on a tannin-treated brain heart infusion agar and were characterised as Streptococcus bovis biotype I.

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Hepatic nuclear receptor PPARα in the koala (Phascolarctos cinereus): Cloning and molecular characterisation

Ngo, SNT, McKinnon, RA & Stupans, I 2007, Comparative Biochemistry and Physiology, part C, vol. 146, pp. 375-382.

Peroxisome proliferator-activated receptor α, or PPARα, is a protein that is involved in lipid homeostasis. It controls gene expression of certain enzymes, such as CYP4As, which play a role in digesting eucalyptus leaves. The molecular characteristics of PPARα in koala liver are reported in this study.

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Identification and cloning of two forms of liver peroxisomal fatty Acyl CoA Oxidase from the koala (Phascolarctos cinereus)

Ngo, SNT, McKinnon, RA & Stupans, I 2003, Gene, vol. 309, no. 1, pp. 91-99.

Reverse transcription polymerase chain reaction was used to amplify two sections of koala liver acyl CoA oxidase (AOX) cDNA named AOX1 and AOX2. The two cDNAs were 2039 bp each, encoding for proteins of 662 amino acids in length. Transfection of these cDNAs into Cos-7 cells resulted in cells with palmitoyl-CoA oxidase activity. Apparent Km values (an inverse measure of affinity) were the same order of magnitude as rat and human AOX enzymes. Northern analysis found a more intense AOX mRNA band for koala liver than human and rat, and genomic DNA southern blot analysis found a single <14 kb AOX gene fragment in all three species. It is therefore likely that the lack of peroxisomal cyanide-insensitive palmitoyl-CoA oxidation activity in koala hepatic tissue is due to enzymes downstream of AOX or deficiencies in mitochondrial β-oxidation enzymes.

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Ingestion and excretion of Eucalyptus punctata D. C. and its essential oil by the koala, Phascolarctos cinereus (Goldfuss)

Eberhard, IH, McNamara, J, Pearse, RJ & Southwell, IA 1975, Australian Journal of Zoology, vol. 23, pp. 169-179.

This study describes a feeding trial during which ingestion and excretion by four captive koalas fed an exclusive diet of Eucalyptus punctata punctata was examined. Findings showed that 60% of leaf dry matter was digestible and of the volatile oil ingested, an insignificant proportion was excreted, with 7-30% passing through to the faeces and 1% in the urine.

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Investigation of the koala (Phascolarctos cinereus) hindgut microbiome via 16S pyrosequencing

Barker, CJ, Gillet, A, Polkinghorne, A & Timms, P 2013, Veterinary Microbiology, vol. 167, pp. 554-564.

Little is known about the diversity and composition of microorganisms involved in the koala’s digestive process. Consequently, this study performed rRNA gene pyrosequencing of caecum, colon and faecal samples from two wild koalas. Findings reveal a diverse and highly complex ecosystem, with considerable variation of microorganisms at the genus level between individuals.

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Levels of trace elements in the liver and diet of free-living koalas, Phascolarctos cinereus (Goldfuss)

McOrist, S & Thomas, KW 1984, Journal of Wildlife Diseases, vol. 20, no. 3, pp. 220-225.

In free-living koalas in Victoria, reference values were determined for a number of different trace elements in the liver and blood plasma. The findings were comprehensive, ultimately revealing suboptimal copper levels in both the plasma and liver, especially in older koalas. No significant trends with age were identified for cobalt, zinc or manganese levels.

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Lonepinella koalarum gen. nov., sp. nov., a new tannin-protein complex degrading bacterium

Osawa, R, Rainey, F, Fujisawa, T, Lang, E, Busse, HJ, Walsh, TP & Stackebrandt, E 1995, Systematic and Applied Microbiology, vol. 18, no. 1, pp. 368-373.

Osawa and colleagues explored the phylogeny of tannin-protein complex (T-PC)-degrading bacteria within faecal samples from koalas, which led to the proposal of a new genus and species of a bacterium named Lonepinella koalarum after the place at which it was discovered, Lone Pine Koala Sanctuary.

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Metabolism of tannin-protein complex by facultatively anaerobic bacteria isolated from koala faeces

Osawa, R, Walsh, TP & Cork, SJ 1993, Biodegradation, vol. 4, no. 1, pp. 91-99.

Isolates of tannin-protein complex-degrading enterobacteria (T-PCDE) and Streptococcus bovis biotype I from koala faeces can degrade protein complexed with the hydrolysable tannin gallotannin, but cannot degrade protein complexed with the condensed tannin quebracho. These bacteria could also metabolise gallic acid to pyrogallol, as could the faecal isolates Klebsiella pneumoniae and K. oxytoca, although these strains could not degrade tannin-protein complexes (T-PCs). None of the strains examined were able to further degrade pyrogallol to phloroglucinol, although pyrogallol did not occur in fresh koala faecal samples.

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Metabolites of dietary 1,8-cineole in the male koala (Phascolarctos cinereus)

Boyle, R, McLean, S, Foley, W, Davies, NW, Peacock, EJ & Moore, B 2001, Comparative Biochemistry and Physiology Part C, vol. 129, pp. 385-396.

Koalas detoxify and eliminate 1,8-cineole, a Eucalyptus monoterpene, through oxidation into several metabolites. The average 1,8-cineole intake of six male koalas was 2.4 ± 1.1 mmol/kg, of which 1.3 ± 0.4 and 1.4 ± 0.4 was recovered free and total, respectively, in urine and faeces in various forms. The identities and amounts of seven metabolites were found: 9- and 7-hydroxycineole, 9- and 7-cineolic acid, 7-hydroxy-9-cineolic acid, 9-hydroxy-7-cineolic acid and 7,9-dicineolic acid. Hydroxy-cineolic acids comprised 85% of the metabolic profile, with 7-hydroxy-9-cineolic acid accounting for 77% by itself, followed by 10% 7,9-dicineolic acid.

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Microbial studies of the intestinal microflora of the koala, Phascolarctos cinereus. II. Pap, a special maternal faeces consumed by juvenile koalas

Osawa, R, Blanshard, WH & O’Callaghan, PG 1993, Australian Journal of Zoology, pp. 611-620.

The presence of tannin-protein-complex-degrading enterobacteria (T-PCDE) present in ‘pap’, a faecal substance ingested by juvenile koalas, was found to be a prerequisite to obtaining protein from tannin-rich eucalyptus leaves. Microflora present in the faeces of female koalas during the production of pap was found to have a higher water content and pH (81.8%, 7.0 respectively) than faeces produced prior to the beginning of the pap feeding process (54.6-56.4%, 5.5). Counts of viable T-PCDE were found to be higher in pap while normal faeces contained less, with some samples testing negative for T-PCDE altogether.

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Microbiological studies of the intestinal microflora of the koala, Phascolarctos cinereus. I. Colonisation of the caecal wall by tannin-protein-complex-degrading enterobacteria

Osawa, R, Bird, PS, Harbrow, DJ, Ogimoto, K & Seymour, GJ 1993, Australian Journal of Zoology, vol. 41, no. 1, pp. 599-609.

Tannin-protein-complex-degrading enterobacteria (T-PCDE) normally occur in the bacterial layer bound to the wall of the caecum in koalas but are dangerously susceptible to antibiotic treatment. T-PCDE are suggested to maintain a symbiotic relationship with the host whereby the bacteria generate health benefits for the koala in exchange for its extensive colonisation of the intricate caecal wall.

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Multifarious activities of cellulose degrading bacteria from Koala (Phascolarctos cinereus) faeces 

Singh, S, Thavamani, P, Megharaj, M & Naidu, R 2015, Journal of Animal Science and Technology, vol. 57, no. 3, pp. 1-6. 

  The most common bacteria found in koala faeces are Bacillus sp. and Pseudomonas sp. after isolation using carboxymethylcellulose-Congo red agar and hydrolytic enzyme activity screening in vitro. There were high activity levels for several enzymes, including xylanase, lignin peroxidase and amylase, with lower concentrations of endoglucanase activity. The isolates were also able to grow in the presence of phenanthrene, suggesting possible applications in bioremediation.

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Nutrients, antinutrients and leaf selection by captive koalas (Phascolarctos cinereus)

Hume, ID, & Esson C 1993, Australian Journal of Zoology, vol. 41, pp. 379-92.

Eucalyptus foliage selection by captive koalas is driven by a suite of variables rather than a single factor.  The present study revealed that foliage most preferred by captive koalas had higher volatile monoterpenes, which are the most aromatic oil component, and lower sesquiterpenes in their essential oils.  Moreover, the most preferred foliage had higher nitrogen to fibre ratio, lower condensed tannin content and higher ratio of nitrogen to condensed tannin content than rejected foliage.

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Occurrence of tannin-protein complex degrading Streptococcus sp. in faeces of various animals

Osawa, R & Sly, LI 1992, Systematic and Applied Microbiology, vol. 15, no. 1, pp. 144-147.

Strains of the tannin-protein complex (T-PC) degrading Streptococcus bovis bacterium occur more commonly in browsing and omnivorous animals than in grazing and carnivorous animals. S. bovis bacteria dominates the streptococcal flora in the faeces of koalas (59.9% of streptococcal flora) and ringtail possums (59.1% of streptococcal flora), which are both animals with diets consisting primarily of tannin-rich eucalypt leaves.

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Phenotypic characterisation of CO2-requiring strains of Streptococcus bovis from koalas

Osawa, R & Sly, LI 1991, Applied and Environmental Microbiology, vol. 57, no. 10, pp. 3037-3039.

Of six strains of the bacterium Streptococcus bovis sampled from koala faeces, two strains that were previously reported as anaerobic were found to be able to grow in oxygen-reduced air that was supplemented with carbon dioxide. The unique characteristic in these aerobic strains was likely the presence of the enzyme β-N-acetylglucosaminidase.

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Seasonal variation in water flux, field metabolic rate and food consumption of free-ranging koalas (Phascolarctos cinereus)

Ellis, WAH, Melzer, A, Green, B, Newgrain, K, Hindell, MA & Carrick, FN 1995, Australian Journal of Zoology, vol. 43, pp. 59-68.

Male free-ranging koalas in central Queensland, Australia were found to have field metabolic rates (corrected for mass) that exhibited a variation of 0.382 MJ kg0.75 per day in the winter and 0.329 MJ kg0.75 per day during the summer. Measurements of influx of water in the same koalas were 59.9 mL kg0.8 per day during the summer and 50.8 kg0.8 per day in the winter. This water influx had a positive correlation with the moisture values in the consumed food. Water influx in the winter was lower in Springsure koalas compared to those from Victoria. Feeding rates were discovered to be higher in the winter compared to summer.

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Selective medium for enumeration of tannin-protein complex-degrading Streptococcus spp. in faeces of koalas

Osawa, RO & Mitsuoka, T 1990, Applied and Environmental Microbiology, vol. 56, no. 11, pp. 3609-3611.

The authors of this study developed a selective agar plate medium that can successfully enumerate strains of tannin-protein complex-degrading Streptococcus bovis bacteria in faecal samples from koalas.

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Spectrometric prediction of secondary metabolites and nitrogen in fresh Eucalyptus foliage: towards remote sensing of the nutritional quality of foliage for leaf-eating marsupials

Ebbers, MJH, Wallis, IR, Dury, S, Floyd, R & Foley, WJ  2002, Australian Journal of Botany, vol. 50, no. 1, pp. 761-768.

Near-infrared reflectance spectroscopy was used to obtain spectra from whole fresh and dry leaves for E. melliodora and E. globulus, two common Eucalyptus species, to determine concentration of total nitrogen, 1,8 cineole (a terpene) and sideroxylonal A (a phenolic antifeedant compound). This technique was successful, with the water absorbance peaks not obscuring the peaks on 1,8 cineole’s spectrum and obscuring some non-vital peaks on nitrogen and sideroxylonal A’s spectra, meaning that their concentrations could be reliably predicted. The concentrations for E. globulus were less accurate than for E. melliodora, likely due to wax on the leaf surface interfering in the experiment.

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Tannin-protein complex-degrading enterobacteria isolated from the alimentary tracts of koalas and a selective medium for their enumeration

Osawa, RO 1992, Applied and Environmental Microbiology, vol. 58, no. 5, pp. 1754-1759.

Strains of tannin-protein complex degrading (T-PCD) bacteria were identified in the faeces and caecum of the koala. These enterobacteria were facultatively anaerobic, gram-negative, pleomorphic, nonmotile bacilli, which are characteristics indicating that the strains fell within the Enterobacteriaceae family. All strains formed distinctly separate colonies after incubation on a tannin-treated agar plate and were also resistant to the antibiotic vancomycin. These characteristics informed the development of a selective agar plate medium that could be used to isolate and identify T-PCD enterobacteria from koala faeces while simultaneously restricting the growth of the common T-PCD bacterium Streptococcus bovis.

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Testosterone dehydrogenase activity in koala liver: characterisation of cofactor and steroid substrate differences

Stupans, I, Kong, S, Kirlich, A, McKinnon, RA & Murray, M 2000, Comparative Biochemistry and Physiology, part C, pp. 245-250.

The activity of testosterone dehydrogenase, or 17b-HSD, in the liver was evaluated in koalas, tammar wallabies, rats and humans. The activity was higher in koalas than wallabies, but was similar when NAD, an enzyme cofactor, was used in assays. Enzyme kinetics showed testosterone dehydrogenase mostly uses NADP as a cofactor in the liver of the koala, and that this pathway is utilised to a lesser extent in wallabies, human and rats than it is in koalas.

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The use of faecal cuticle examination to determine the dietary composition of koalas

Ellis, W, Carrick, F, Lundgren, P, Veary, A & Cohen, B 1999, Australian Zoologist, vol. 31, no. 1, pp. 127-133.

Eucalyptus leaves ingested by captive koalas were present in faecal pellets up to 154 hours after consumption. Dietary composition examination utilising the ‘faecal cuticle analysis’ technique was deemed an appropriate method to identify and estimate quantities of different Eucalyptus spp. in a koala’s diet.

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Variation in koala microbiomes within and between individuals: effect of body region and captivity status

Alfano, N, Courtiol, A, Vielgrader, H, Timms, P, Roca, AL & Greenwood, AD 2015, Nature: Scientific Reports, vol. 5, no. 10189.

Sequencing microbes’ 16S ribosomal RNA (rRNA) directly from the sample without culture is an accurate way to assess the relationship between the microbe and the host. The bacterial community, or the microbiome, in the eyes and digestive system of the koala were evaluated here. Captive koala microbiomes mostly resembled those of other animals and wild koalas. Unexpectedly, the microbiome of the koala eye contained many bacteria belonging to the Phyllobacteriaceae family.

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Visual reading method for detection of bacterial tannase

Osawa, R & Walsh, TP 1993, Applied and Environmental Microbiology, vol. 59, no. 4, pp. 1251-1252.

The authors of this study develop and describe a new visual reading method for detecting the presence of tannase in cultures of bacteria that can degrade the tannin-protein complexes present in eucalypts, the main food source for the koala.

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