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Collaborative Research


Lone Pine Koala Sanctuary is dedicated to the research of native species and continues to collaborate with Universities and the Government.  This page highlights some of the papers published resulting from these efforts.  For more information on this or other research, e-mail us at This e-mail address is being protected from spambots. You need JavaScript enabled to view it



Johnston, S. D., O'Callaghan, P, Nilsson, K, Tzipori, G., Strong, H. and Curlewis, J. D. (2003). Semen-induced luteal phase in the koala. In: Martin and G.B., Reproduction, Fertility and Development. Society for Reproductive Biology Thirty-fourth Annual Conference, Melbourne, Victoria, (92-92). 14-17 September 2003.

Abstract: The koala ovulates in response to mating. The purpose of this study was to document the LH surge induced by copulation and to investigate the potential roles of mechanical stimulation of the urogenital sinus and deposition of semen in induction of the luteal phase. In experiment 1, serial blood samples from four koalas that underwent normal mating showed elevated concentrations of LH approximately 24-32 h post-coitus. There was no corresponding elevation in LH in koalas (n = 4) that were exposed to the presence of a male but received no physical contact. In experiment 2, koalas on day 2 of oestrus were exposed to one of the following treatments (n = 9 per group): artificial insemination with 1 ml 0.9% sterile saline (control group), insemination with 1 ml koala semen, stimulation of the urogenital sinus with a purpose built glass rod (designed to mimic the action of the penis during natural mating) and urogenital stimulation with the glass rod followed by insemination of 1 ml koala semen. Confirmation of a luteal phase was based on evidence of a prolonged return to oestrus, parturition and/or elevated progesterone concentrations. Insemination of saline (0/9) and urogenital stimulation (0/9) failed to induce a luteal phase. Insemination of semen without glass rod stimulation resulted in a luteal phase in 4/9 koalas, three of which gave birth. Insemination of semen in combination with urogenital stimulation produced a luteal phase in 7/9 koalas, four of which gave birth. Semen had a significant effect on induction of the koala luteal phase (P < 0.001) but glass rod stimulation had no such effect (P = 0.335). It was concluded that semen must be involved in the induction of a luteal phase in the koala. The results presented in this study will serve to improve optimal timing and induction of ovulation for artificial insemination in the koala.



Johnston, S. D., Mcgowan, M. R., O'Callaghan, P., Cox, R. and Nicholson, V. (2000) Natural and artificial methods for inducing the luteal phase in the koala (Phascolarctos cinereus). Journal of Reproduction and Fertility, 120 1: 59-64.

Abstract: An experiment was conducted in which female koalas were mated for different durations of intromission and ejaculation to confirm that the luteal phase of the oestrous cycle in koalas is induced by the physical act of mating. Results showed that induction of a luteal phase in the koala usually required a complete duration of penile thrusting behaviour from the male. It is proposed that induction of a luteal phase in koalas may involve a copuloceptive reflex, triggered by the thrusting of the male's penis into the female's urogenital sinus. Although interrupted mating in koalas may be used to induce a luteal phase in preparation for an artificial insemination programme, this study showed that there is a 12.5% probability that pregnancy will result from semen prematurely emitted by the teaser male. A dose of 250 iu hCG was administered intramuscularly to eight oestrous females to determine whether it was possible to induce a luteal phase artificially. In contrast to control females, which received sterile saline injections, all females injected with hCG showed a significant increase in progestogen concentration, above that of basal values, indicating that a luteal phase had been induced successfully.



Johnston, S. D., Mcgowan, M. R., O'Callaghan, P., Cox, R. and Nicholson, V. (2000) Studies of the oestrous cycle, oestrus and pregnancy in the koala (Phascolarctos cinereus). Journal of Reproduction and Fertility, 120 1: 49-57.

Abstract: 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.



Wardrop, S., Fowler, A., O'Callaghan, P., Giffard, P., and Timms, T. (1999) Characterization of the koala biovar of Chlamydia pneumoniae at four gene loci--ompAVD4, ompB, 16S rRNA, groESL spacer region. Systematic and Applied Microbiology, 22 1: 22-27.

Abstract: Koalas are infected with two species of Chlamydia, C. pecorum and C. pneumoniae. While it is known that significant genetic diversity occurs in the C. pecorum strains infecting koalas, very little is known about the C. pneumoniae strains that infect this host. In the current study, 10 isolates of koala C. pneu­moniae were analysed at four gene loci and found to be different to both the human and horse C. pneu­moniae strains at all loci (biovar differences ranging from 0.3% at groESL up to 9.0% at ompAVD4). All koala biovar isolates studied were found to be 100% identical at ompAVD4 (all 10 isolates) and at ompB (all three isolates) genes. This lack of allelic polymorphisms at ompAVD4 has now been observed for koala C. pneumoniae, human C. pneumoniae, guinea pig inclusion conjunctivitis C. psittaci and feline conjunctivitis C. psittaci and may be correlated to a lack of antibody response to the chlamydial major outer membrane protein (MOMP) in these same strain/host combinations. This study also provides the first documented case of natural C. pneumoniae infection causing a severe and extended respiratory episode in a captive koala population. This captive episode is in contrast to most free-range observations in which koala C. pneumoniae is rarely documented as causing respiratory, ocular or urogenital tract disease.



Memoirs of the Queensland Museum 33. (1993) A Record of Communal Egg Laying in the Skink Carlia Tetradactyla (Reptilia: Scincidae).

 


Porter, R. (1993) An observation of insectivorous feeding habits in the green tree snake (Dendrelaphis punctulata), Herptofauna, 23 1:36.

Abstract: During a recent visit to Carnarvon Gorge, central Queensland, a brief observation was made of an unusual feeding behaviour in the colubrid snake Dendrelaphis punctulata. At around 1100 hours on a warm sunny day (temperature approximately 28°C) in early March 1993, a green tree snake was seen amongst a clump of sedge grass approximately 1 metre from the river's edge. The snake appeared active and quite agitated, moving nervously in a short "start stop" motion while holding an object in its jaws. On closer inspection the item was seen to be some type of flying insect as a pair of large, elliptical, transparent wings were projecting out of each side of the mouth. The wings were consistent with a dragon -or damsel-fly, a lace wing or a similar type of insect. The wings were seen to move up and down slightly, though this appeared to be a consequence of the snake making a chewing motion rather than movement from the insect. When approached closer the snake rapidly fled into the clump of sedge and disappeared. Many early authors listed insects as prey items for Australian snakes (e.g. Kinghorn, 1929, Worrell, 1963, Gow, 1976). However, Shine (1991 a) states that although insect fragments are often found in snake digestive tracts, in most cases these were secondarily ingested from a vertebrate prey item. He does indicate that occasional crickets have been found in some elapids and in his examination of Australian colubrids (Shine, 1991 b) he lists 76 prey items from 58 D. punctulata, including one insect item, also a cricket. Considering the huge number of Australian snake stomach contents examined by Shine over the last 15 years the occurrence of insects as prey items in Australian snakes (apart from typhlopids) is evidently a very rare event. It is possible that the insect observed being eaten by the green tree snake had been captured by a frog or lizard which was then taken by the snake, both items being swallowed consecutively. It seems likely however, that the frog or lizard would have dropped the prey item when the snake struck. Even if this did occur, the insect may have become contaminated by the lizard or frog's scent (in the same fashion as keepers scent mice for lizard eating snakes) and the insect then eaten independently by the snake. The most parsimonious explanation, however, has to be that the snake captured and ate the insect itself, despite the rarity of such events in Australian snakes. Though the importance of invertebrates in most native snake diets is apparently negligible, this observation still raises some interesting questions regarding the causes of such behaviour.



Osawa, R. and Walsh, T. P. (1993) Effects of Acidic and Alkaline Treatments on Tannic Acid and its Binding Property to Protein. Journal of Agricultural and Food Chemistry, 41 5: 704-707.

Abstract: Effects of acidic and alkaline treatments on tannic acid (T A), one of the hydrolyzable tannins, and its binding property to protein [bovine serum albumin (BSA)] were investigated. Dissociation of TA-BSA complexes was apparent in both highly acidic (pH <3) and alkaline (pH >7) treatments. T A dissociated from the complexes and pure T A was not significantly hydrolyzed in highly acidic (pH 1.0-3.0) media and-retained its protein-binding property. TA did not seem to be hydrolyzed even with a more acidic treatment (in 1 M HCI, pH <0.5), although its protein-binding property was much reduced. The higher pH treatment (pH >6.-5) facilitated the hydrolysis of T A, as judged by the release of its constituent Gallic acid.



Osawa, R., Walsh, T.P., Cork, S. J. (1993) Metabolism of tannin-protein complex by facultatively anaerobic bacteria isolated from koala faeces. Biodegradation, 4 2: 91-99.

Abstract: The metabolic pathways involved in degradation of tannin-protein complex (T-PC) were investigated in vari­ous facultatively anaerobic bacteria, with specific reference to faecal isolates from the koala including T -PC­degrading enterobacteria (T -PCDE), Streptococcus bovis, Klebsiellapneumoniae, and K. oxytoca. It was dem­onstrated that ~-PCDE and S. bovis biotype I were capable of degrading protein complexed with Gallo tannin (a hydrolyzable tannin), but not that complexed with quebracho (a condensed tannin). Subsequent studies showed that these strains metabolized Gallic acid to pyrogallol. Strains of Klebsiella pneumoniae and K. ox­ytoca, which did not degrade T -PC, also metabolized Gallic acid into pyrogallol. Pyrogallol was not degraded by any strains studied, but it was not detected in fresh faeces of the koalas. The majority of strains isolated from faeces could degrade phloroglucinol. Based on these findings, we propose that members of the gut microflora of the koala cooperate in the degradation of T -PC.



Osawa, R., Blanshard, W. H., O'Callaghan, P. G. (1993) Microbiological Studies of the Intestinal Microflora of the Koala, Phascolarctos cinereus. II. Pap, a Special Maternal Faeces Consumed by Juvenile Koalas. Australian Journal of Zoology, 41 6: 611-620.

Abstract: Quantitative and qualitative studies on the micro flora in the faeces of 10 female koalas, Phascolarctos cinereus, were conducted with specific reference to 'pap', a special maternal faeces consumed by the juvenile at about the time of first emergence from the pouch. This specific coprophagy, called 'pap feeding', occurred multiple times in all of the females examined. Pap was higher in water content (81.8%) and pH (7.0) than normal faeces produced by the females before and after pap feeding (54.6-56.4% and 5.5, respectively), suggesting that it is derived directly from the contents of the caecum. Pap had higher (23-41-fold) viable counts of tannin-protein-complex-degradi enterobacteria (T -PCDE) than the normal faeces; in four of the females examined, viable T -PCDE were found in pap but never in the normal faeces. The evidence indicates that pap feeding is an essential physiological activity for the juvenile koala to prepare it for an imminent dietary transition from maternal milk to tannin-rich eucalypt leaves.



Osawa, R. and Walsh, T. P. (1993) Visual Reading Method for Detection of Bacterial Tannase. Applied and Environmental Microbiology, 59 4: 1251-1252.

Abstract: Tannase activity of bacteria capable of degrading tannin-protein complexes was determined by a newly developed visual reading method. The method is based on two phenomena: (i) the ability of tannase to hydrolyze methyl gallate to release free Gallic acid and (ii) the green to brown coloration of Gallic acid after prolonged exposure to oxygen in an alkaline condition. The method has been successfully used to detect the presence of tannase in the cultures of bacteria capable of degrading tannin-protein complexes.



Osawa, R. and Woodall, P. F. (1992) A Comparative Study of Macroscopic and Microscopic Dimensions of the Intestine in Five Macropods (Marsupialia: Macropodidae). I. Allometric Relationships. Australian Journal of Zoology,  40 1: 91-98.

Abstract: Macroscopic and microscopic dimensions of the intestines in five macropod species (the red kangaroo, Macropus rufus; the eastern grey kangaroo, M. giganteus; the agile wallaby, M. agilis; the swamp wallaby, Wallabia bicolor; and the red-necked pademelon, Thylogale thetis) were investigated allo­metrically in relation to body mass. In general, the length of the small intestine changed in an area: volume (A: V) compensating manner, but the circumference showed negative allometry such that the overall change in surface areas, both ground and mucosal (including the contribution of villi), were isometric but also included the coefficient derived from 'Kleiber's Law' (0' 75) in their 95 and 99% confidence limits, respectively. Villous height and width generally showed no significant correlations with body size, but villous density was lower in large individuals. The allometry coefficient for the length of the large intestine was generally near the A: V com­pensating value (0' 5) in most intraspecific analyses but much higher in the interspecific analysis, suggesting that some factor other than body size might be important (possibly dietary fibre). Caecal length was significantly correlated with body size only in two largest species (M. rufus and M. giganteus) and the interspecific analysis gave a value near A: V compensation).



Osawa, R. O., Fujisa, T. and Mitsuoka, T. (1992) Characterization of Gram-negative anaerobic strains, isolated from koala feces, which exhibit satellite growth and pleomorphism. Systematic and Applied Microbiology, 15 4: 628-635.

Abstract: Three strains, which showed satellite growth around colonies of concomitant isolates of Escherichia coli, were isolated from koala feces. Experimental inoculation of these strains and E. coli strains on a plate medium anaerobic, gram-negative, and highly pleomorphic rods. They were bile resistant and asac-charolytic, and produced only a trace amount of propionic acid from peptone yeast-extract Fildes-solution with glucose broth. Their growth was stimulated by succinic acid but completely inhibited by fumaric acid. Subsequent biochemical analysis revealed that the strains produced a large amount of propionic acid from peptone yeast-extract (PY) broth supplemented with sodium succinate. A PY broth agar plate, which contained 30% E. coli-metabolized PY broth, enhanced growth of the strains, and it was found that this spent broth contained succinic acid produced by the E. coli. The evidence suggests that the observed satellite growth was due to succinic acid produced by E. coli strains.



Osawa, R., Blanshard, W. H. and O'Callaghan, P. G. (1992) Microflora of the Pouch of the Koala (Phascolarctos cinereus). Journal of Wildlife Diseases, 28 2: 276-280.

Abstract: 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 micro-organisms 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.



Osawa, R. (1992) Tannin-Protein Complex-Degrading Enterobacteria Isolated from the Alimentary Tracts of Koalas and a Selective Medium for Their Enumeration. Applied Environmental Microbiology, 58 5: 1754-1759.

Abstract: Tannin-protein complex (T -PC)-degrading enterobacteria (T -PCDE) were isolated from the feces and from a layer of bacteria attached to the cecal wall of koalas. The T -PCDE were facultatively anaerobic, gram-negative, pleomorphic, nonmotile bacilli. The bacteria were also oxidase and catalase negative and resistant to vancomycin, reduced nitrates to nitrites, and grew on MacConkey agar. Growth on tannin-treated agar media showed a distinctive clear zone around the colony. From these observations, a selective agar plate medium (vancomycin- and tannin-treated Wilkins-Chalgren anaerobe. agar) was developed to enumerate T-PCDE isolated from the feces of koalas. This medium was highly selective in the enumeration of the fecal T -PCDE and inhibited the growth of concomitant T -PC-degrading Streptococcus bovis. The T -PCDE were isolated from 10 of the 12 captive koalas studied; in 8 of these 10 koalas, the facultatively anaerobic bacterial flora was dominated (more than 60%) by T-PCDE. Viable numbers of T-PCDE were, in most of the animals, much larger (more than 100 times) than the numbers of T -PC-degrading S. bovis, suggesting that T -PCDE played a more active role in digesting T -PC in the alimentary tracts of koalas.


Porter, R. The Current Status of the Knob-tailed Geckos (Nephrurus) in captivity.


Osawa, R. and Sly, L. I. (1991) Phenotypic Characterization of CO2-Requiring Strains of Streptococcus bovis from koalas. Applied Environmental Microbiology, 57 10: 3037-3039.


Ueno, H., Mizuno, S., Osawa, R., Blanshard, W., Timms, P., White, N. and Hashimoto, N. (1991) Serological assessment of chlamydial infection in the koala by a slide EIA technique. Australian Veterinary Journal, 68 12: 393-396.


Porter, R. (1991) Unusual Basking Behaviour in captive bearded dragons (Pongona barbata). Herpetofauna, 21 2:31.

Osawa, R., Carrick, F.N., Hashimoto, N., Takashima, I. and Takahasi, T. (1991) Application of a Blood Sampling Paper Method for Complement Fixation Test Detection of Anti- Chlamydial Antibody in Koalas, Phascolarctos cinereus. In Biology of the Koala. (Eds. Lee, A.K, Handasyde, K.A. and Sanson, G.D. ) pp. 97-108.


Blanshard, W. H. (1991) Growth and development of the koala from birth to weaning. In Biology of the Koala. (Eds. Lee, A.K., Handasyde, K.A. and Sanson, G.D.) pp. 193-202.

 

Osawa, R. and Woodall, P. F. (1990) Feeding Strategies of the Swamp Wallaby, Wallabia-Bicolor, on North Stradbroke Island, Queensland .2. Effects of Seasonal-Changes in Diet Quality on Intestinal Morphology. Wildlife Research Management and Conservation, 17 6: 623-632.

 

Osawa, R. (1990) Feeding Strategies of the Swamp Wallaby, Wallabia bicolor, on North Stradbroke Island, Queensland. I: Composition of Diets. Wildlife Research Management and Conservation, 17 6: 615-621.


Osawa, R. (1990) Formation of a Clear Zone on Tannin-Treated Brain Heart Infusion Agar by a Streptococcus sp. Isolated from Feces of Koalas. Applied Environmental Microbiology,  56 3: 829-831.


Osawa, R. and Mitsuoka, T. (1990) Selective Medium for Enumeration of Tannin-Protein Complex­ Degrading-Streptococcus spp. in Feces of Koalas. Applied Environmental Microbiology,  56 11: 3609-3611.


Osawa, R. and Carrick, F. N. (1990) Use of a dietary supplement in koalas during systemic antibiotic treatment of chlamydial infection. Australian Veterinary Journal, 67 8: 305-307.


Osawa, R. and Mitsuoka, T. (1990) Faecal microflora of captive koalas, Phascolarctos cinereus (Marsupialia: Phascolarctidae). Australian Mammalogy 13: 141-147.

 

Osawa, R. (1989) Road-kills of the Swamp Wallaby, Wallabia bicolor, on North Stradbroke Island, South-east Queensland. Wildlife Research Management and Conservation, 16 1: 95-104.

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