Crystalloid inclusions in the Sertoli cell of the koala,Phascolarctos cinereus (Marsupialia)

Summary Crystalloid inclusions are a common feature in the basal region of Sertoli cells in the koala, Phascolarctos cinereus. Generally located near the nucleus, they are non membrane-bounded, slender rectangular structures composed of tubules which are orientated at right angles to the long axis of the crystalloid and regularly arranged in rows parallel to this long axis. The tubules in adjacent rows are offset from one another at definite angles and extensively interconnected by filaments. Neither the composition nor function of the crystalloids has been determined, but their association with tonofilaments and the presence of ribosomes in the vicinity suggests that they are most likely proteinaceous.The authors would like to thank Mr. D. Harbrow, Mr. S. Brown and Ms. B. Canty for technical assistance; the Queensland National Parks and Wildlife Service and the Victorian Ministry of Conservation (Fisheries and Wildlife Division) for providing permits to work on this protected species; the staff of the Lone Pine Koala Sanctuary, Brisbane, for their assistance in obtaining animals. This work was supported by a grant from the Australian Research Grants Committee, Number DI-77/15525     

Some aspects of the immune response of koalas (Phascolarctos cinereus) and in vitro neutralization of chlamydia psittaci (koala strains)

Abstract Western-blot analysis was used to study the reaction of koala antisera, two specific polyclonal antibodies and one monoclonal antibody, with chlamydial antigens in koalas infected with Chlamydia psittaci . The koala sera recognized four C. psittaci surface antigens, corresponding to the major outer membrane protein (39.5 kDa), 31 kDa protein, 18 kDa protein and lipopolysaccharide. The S25-23 LPS specific monoclonal antibody inhibited chlamydial infection (55–67%) with both koala strains (type I and type II). Both koala antiserum and rabbit polyclonal antibodies against either type of chlamydia significantly reduced the number of infected cells resulting from type II infections at a dilution of 1 in 20. Rabbit antiserum against type II was effective in neutralizing infection by type II elementary bodies, but was less effective against type I infection. In addition, no koala antiserum was effective in neutralizing type I infection.     

Streptobacillary pleuritis in a koala (Phascolarctos cinereus).

A case of Streptobacillus moniliformis pleuritis in a koala (Phascolarctos cinereus) is reported. Lesions were granulomatous in nature. S. moniliformis was recovered in pure culture, and found by experimental inoculation to be pathogenic for mice but not for a rat.     

Possible antimicrobial compounds from the pouch of the koala, Phascolarctos cinereus

The antimicrobial activity of secretions of the pouch of the koala, Phascolarctos cinereus has been documented in thepresence and absence of young. These secretions have been analysed by polyacrylamide gel electrophoresis, high performanceliquid chromatography and mass spectrometry (MS) including time of flight (TOF), MS-MS and Edman sequencing. Gel electrophoresis of reduced samples revealed the presence of a four polypeptides in active and inactive secretions. The sequence data obtained from all four do not appear to be either defensin or cathelicidin-like peptides and show little homology with knownantimicrobial protein/peptide sequence available on public accessdatabases. It is proposed that marsupials regulate microbialpopulations in the pouch via cleavage of large, inactive molecules to produce small active peptides.     

Possible antimicrobial compounds from the pouch of the koala,Phascolarctos cinereus

Summary The antimicrobial activity of secretions of the pouch of the koala,Phaseolarctos cinereus has been documented in the presence and absence of young. These secretions have been analysed by polyacrylamide gel electrophoresis, high performance liquid chromatography and mass spectrometry (MS) including time of flight (TOF), MS-MS and Edman sequencing. Gel electrophoresis of reduced samples revealed the presence of a four polypeptides in active and inactive secretions. The sequence data obtained from all four do not appear to be either defensin or cathelicidin-like peptides and show little homology with known antimicrobial protein/peptide sequence available on public access databases. It is proposed that marsupials regulate microbial populations in the pouch via cleavage of large, inactive molecules to produce small active peptides.     

Microflora of the pouch of the koala (Phascolarctos cinereus).

Microflora of the pouch epithelium of 17 female koalas (Phascolarctos cinereus) were examined in relation to their current reproductive status and recent reproductive history. No microbial growth was observed in pouch swabs from 13 of 17 (76%) koalas, including four females without young, seven with pouch young and two with back young (i.e. permanently emerged from the pouch). Growth of bacteria or yeasts was observed in pouch swabs from four koalas, each of which had experienced mortality of its pouch young during the current breeding season. Seven species of microorganisms were isolated, including Pseudomonas aeruginosa, Serratia marcescens and Enterococcus faecalis. Based on the absence of microflora in the majority of females examined, we propose that the pouch epithelium normally provides a hostile environment for microbial colonization.     

Chemical characterization of milk oligosaccharides of the koala (Phascolarctos cinereus)

Previous structural characterizations of marsupial milk oligosaccharides had been performed in only two macropod species, the tammar wallaby and the red kangaroo. To clarify the homology and heterogeneity of milk oligosaccharides among marsupial species, which could provide information on their evolution, the oligosaccharides of the koala milk carbohydrate fraction were characterized in this study. Neutral and acidic oligosaccharides were separated from the carbohydrate fraction of milk of the koala, a non-macropod marsupial, and characterized by ¹H-nuclear magnetic resonance spectroscopy. The structures of the neutral saccharides were found to be Gal(β1-4)Glc (lactose), Gal(β1-3)Gal(β1-4)Glc (3′-galactosyllactose), Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc (3′,3″-digalactosyllactose), Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I) and Gal(β1-3){Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)}Gal(β1-4)Glc (fucosyl lacto-N-novopentaose I), while those of the acidic saccharides were Neu5Ac(α2-3)Gal(β1-4)Glc (3′-SL), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-4)Gal (sialyl 3′-galactosyllactose), Neu5Ac(α2-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose a), Gal(β1-3)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose b), Gal(β1-3)[Neu5Ac(α2-3)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose c), and Neu5Ac(α2-3)Gal(β1-3){Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)}Gal(β1-4)Glc (fucosyl sialyl lacto-N-novopentaose a). The neutral oligosaccharides, other than fucosyl lacto-N-novopentaose I, a novel hexasaccharide, had been found in milk of the tammar wallaby, a macropod marsupial, while the acidic oligosaccharides, other than fucosyl sialyl lacto-N-novopentaose a had been identified in milk carbohydrate of the red kangaroo. The presence of fucosyl oligosaccharides is a significant feature of koala milk, in which it differs from milk of the tammar wallaby and the red kangaroo.     

Dietary aluminium and renal failure in the koala (Phascolarctos cinereus)

The study investigated the link between the potentially nephrotoxic levels of aluminium ingested in the natural diet of eucalypt leaves by koalas in the Adelaide Hills, South Australia, and the high incidence of renal failure in koalas within this habitat. Routine histology of kidney specimens revealed no pathologies at the light microscopic level and contrasted sharply with the clinical signs of renal failure. However staining with solochrome azurine and Perl's Prussian blue showed aluminium was present in some proximal convoluted tubules in all specimens. Aluminium was also found in bone samples. The presence of aluminium in bone and kidney tissues was confirmed using electron dispersive x-ray analysis with transmission and scanning electron microscopy. Ultrastructural changes, including a decrease in lysosomal numbers, were seen in proximal convoluted tubules and these changes were shown to coincide with the presence of aluminium. No aluminium was found in koalas that died from causes other than renal failure. It was concluded that renal failure in the koalas of the Adelaide Hills is characterised by the presence of aluminium in the kidneys and bone and it is probably related to the high levels of aluminium in their restricted diet of eucalypt leaves. However, it is not known if the presence of aluminium is the cause or effect of the renal failure. The study is the first account where aluminium ingested as part of the natural diet of mammals has been shown to accumulate in the animal and be implicated with nephrotoxicity.     

Nitrogen and the koala (Phascolarctos cinereus): some indirect evidence

Indirect evidence provides additional support for the hypothesis that the level of available nitrogenous food is the major limiting influence in the abundance of the koala. It appears that koalas may select for low fibre content which is usually correlated with high nitrogen levels of leaves.     

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

The in vivo metabolic fate of 1,8-cineole was investigated in six male koalas. Koalas were fed ad lib a diet of Eucalyptus cephalocarpa leaf with a 1,8-cineole concentration of 2.53+/-0.70% dry mass of leaf, corresponding to a 1,8-cineole intake of 2.4+/-1.1 mmol/kg (3.1+/-1.3 g). Urine and faeces were collected for 24 h and metabolites identified by GC-MS and LC-MS. Metabolites were quantified before and after hydrolysis with beta-glucuronidase to give free and total levels, respectively. Fractional recovery of ingested 1,8-cineole was 1.3+/-0.4 and 1.4+/-0.4 (mean+/-S.D.) for free and total measurements, respectively. Seven metabolites were identified and quantified: 9- and 7-hydroxycineole, 9- and 7-cineolic acid, 7-hydroxy-9-cineolic acid, 9-hydroxy-7-cineolic acid and 7,9-dicineolic acid. The hydroxycineolic acids dominated the metabolite profile (85%). 7,9-Dicineolic acid, a novel metabolite of 1,8-cineole, accounted for almost 10% of the recovered dose making it the second most abundant metabolite after 7-hydroxy-9-cineolic acid (77%). Together, the less oxidised metabolites, the hydroxycineoles and cineolic acid, accounted for only 5% of the cineole consumed. Significant conjugation only occurred with four minor, less oxidised, alcohol and carboxylic acid metabolites. We have shown that the koala detoxifies and eliminates 1,8-cineole primarily by extensive oxidation without utilising conjugation pathways.     

Jaw movements from microwear on the molar teeth of the koala Phascolarctos cinereus

To study the direction of jaw movements in the koala from wear facets on the molar teeth by scanning electron microscopy, gold coated epoxy resin replicas from the right maxillary and mandibular tooth quadrants were examined from 12 koala skulls. The progressive development and location of facets, the orientation of striae on them and directional data were recorded and transferred from electron micrographs to superimposable transparencies.
Polished facets with laterally oriented striations developed on the cristids and cristae progressively into dentine, where Greaves' effect indicated that the direction of the chewing stroke was labiolingual. Polished and pitted facets, aligned and striated in the parasagittal plane, occurred on the smooth interactive enamel surfaces of maxillary and mandibular cusps.
Labiolingual transit of the crislids over the cristae, with a slight anteromedial shift, was inferred to be the predominant chewing stroke on the working side with no contralateral balancing contact. A propalinal isognathous movement in which successive cusps made contact was also deduced.
Previous concepts of koala chewing and tooth wear were confirmed and amplified, and these may have application to studies of extinct marsupial jaw mechanisms.     

Studies of the oestrous cycle, oestrus and pregnancy in the koala (Phascolarctos cinereus)

As an integral part of the development of an artificial insemination programme in the captive koala, female reproductive physiology and behaviour were studied. The oestrous cycle in non-mated and mated koalas was characterized by means of behavioural oestrus, morphology of external genitalia and changes in the peripheral plasma concentrations of oestradiol and progestogen. The mean (+/- SEM) duration of the non-mated oestrous cycle and duration of oestrus in 12 koalas was 32.9 +/- 1.1 (n = 22) and 10.3 +/- 0.9 (n = 24) days, respectively. Although the commencement of oestrous behaviour was associated with increasing or high concentrations of oestradiol, there were no consistent changes in the morphology or appearance of the clitoris, pericloacal region, pouch or mammary teats that could be used to characterize the non-mated cycle. As progestogen concentrations remained at basal values throughout the interoestrous period, non-mated cycles were considered non-luteal and presumed anovulatory. After mating of the 12 koalas, six females gave birth with a mean (+/- SEM) gestation of 34.8 +/- 0.3 days, whereas the remaining six non-parturient females returned to oestrus 49.5 +/- 1. 0 days later. After mating, oestrous behaviour ceased and the progestogen profile showed a significant increase in both pregnant and non-parturient females, indicating that a luteal phase had been induced by the physical act of mating. Progestogen concentrations throughout the luteal phase of the pregnant females were significantly higher than those of non-parturient females. Parturition was associated with a decreasing concentration of progestogen, which was increased above that of basal concentrations until 7 days post partum.     

Phylogeographic differentiation in the mitochondrial control region in the koala, Phascolarctos cinereus (Goldfuss 1817)

The koala, Phascolarctos cinereus, is a geographically widespread species endemic to Australia, with three currently recognized subspecies: P.c. adustus, P.c. cinereus, and P.c. victor. Intraspecific variation in the mitochondrial DNA (mtDNA) control region was examined in over 200 animals from 16 representative populations throughout the species’ range. Eighteen different haplotypes were defined in the ≈ 860 bp mtDNA control region, as determined by heteroduplex analysis/temperature gradient gel electrophoresis (HDA/TGGE). Any single population typically possessed only one or two haplotypes yielding an average within-population haplotypic diversity of 0.180 ± 0.003, and nucleotide diversity of 0.16%. Overall, mtDNA control region sequence diversity between populations averaged 0.67%, and ranged from 0% to 1.56%. Nucleotide divergence between populations averaged 0.51%, and ranged from 0% to 1.53%. Neighbour-joining methods revealed limited phylogenetic distinction between geographically distant populations of koalas, and tentative support for a single evolutionarily significant unit (ESU). This is consistent with previous suggestions that the morphological differences formalized by subspecific taxonomy may be interpreted as clinal variation. Significant differentiation in mtDNA-haplotype frequencies between localities suggested that little gene flow currently exists among populations. When combined with microsatellite analysis, which has revealed substantial differentiation among koala populations, we conclude that the appropriate short-term management unit (MU) for koalas is the local population.