Lone Pine's Research

Lone Pine continues to research Australia's wildlife alone and in conjunction with scientists from Universities and the Government.  Here are some of our published papers with their abstracts.  For more information on this or other research, e-mail us at research@koala.net

Semen-induced luteal phase and identification of a LH surge in the koala (Phascolarctos cinereus)

D Johnston, P O'Callaghan, K Nilsson, G Tzipori and J D Curlewis

School of Animal Studies, The University of Queensland, 4072, Lone Pine Koala Sanctuary, Jesmond Road, Fig Tree Pocket, 4069 and 2 School of Biomedical Sciences, The University of Queensland, 4072, Australia
Correspondence should be addressed to 5 0 Johnston; Email: s.johnston@uq.edu.au

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. Reproduction (2004) 128 629-634

Natural and artificial methods for inducing the luteal phase in the koala (Phascolarctos cinereus)

S. D. Johnston, M. R. McGowan, P. O'Callaghan, R. Cox and V. Nicolson

School of Veterinary Science and Animal Production, The University of Queensland, 4072, Australia; 2Lone Pine Koala Sanctuary,­ Jesmond Road, Fig Tree Pocket, 4069, Australia; and 3Bioquest Ltd, North Ryde, 2113, Australia
.
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.

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

S. D. Johnston, M. R. McGowan, P. O'Callaghan, R. Cox and V. Nicolson

School of Veterinary Science and Animal Production, University of Queensland, 4072, Australia; Lone Pine Koala Sanctuary, Jesmond Road, Fig Tree Pocket, 4069, Australia; and 3Bioquest Ltd, North Ryde, 2113, Australia.

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.

MEMOIRS OF THE QUEENSLAND MUSEUM

A RECORD OF COMMUNAL EGG LAYING IN THE SKINK CARLIA TETRADACTYLA (REPTILIA: SCINCIDAE). Memoirs of the Queensland Museum 33: 000-000. 1993:- During a visit to the Oakey area, southeast Queensland on 25 November 1992, a communal nest of 25 skink eggs was located. The site -a disused railway line ­was approximately)' 8km NW of Oakey township and consisted of a relatively continuous row of wooden sleepers. The nest was located beneath a single sleeper, which was lying on an approximate north-south axis, at its northern end. The whole sleeper was also angled slightly to the west. The eggs varied little in size. All were partially buried in the soil. The eggs were not measured at the time, but their size sug­gested they could belong to Carlia tetradactyla, a very com­mon species at the site. Considering the paucity of records of communal nesting for Carlia, one egg was retained by one of us (RP) for artificial incubation to confirm its identification.

AN OBSERVATION OF INSECTIVOROUS FEEDING HABITS IN THE GREEN TREE SNAKE (DENDRELAPHIS PUNCTULATA)

Robert Porter, Lone Pine Koala Sanctuary, Jesmond Rd, Fig Tree Pocket QLD 4069

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.

Effects of Acidic and Alkaline Treatments on Tannic Acid and its Binding Property to Protein

Ro Osawa and Terence P. Walsh

Veterinary Service and Research, Lone Pine Koala Sanctuary, Fig Tree Pocket, Brisbane, Queensland 4069, Australia, and Centre for Molecular Biotechnology, School of Life Science, Garden Point Campus, Queensland University of Technology, Brisbane, Queensland 4001, Australia

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.

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

Ro Osawa, Terry P. Walsh & Steven J. Cork
Veterinary Service & Research, Lone Pine Koala Sanctuary, Fig Tree Pocket, Brisbane, Queensland 4069, Australia,. Centre for Molecular Biotechnology, School of Life Science, Queensland University of Technology, Faculty of Science, Gardens Point Campus, Brisbane Queensland 4001, Australia, CSIRO Division of Wildlife and Ecology, Lyneham, ACT2602, Australia (* Present address: Department of Food Microbiology, School of Medicine, University of Tokushima, 18-15, 3-chome, Kuramotocho, Tokushima 770, Japan)

Received 10 June 1992; accepted in revised form 30 November 1992

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.

Microbiological Studies of the Intestinal Microflora of the Koala, Phascolarctos cinereus. II. Pap, a Special Maternal Faeces Consumed by Juvenile Koalas

R. Osawa, W. H. Blanshard and P. G. O'Callaghan

Veterinary Service and Research, Lone Pine Koala Sanctuary, Fig Tree Pocket, Qld 4069, Australia.

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.

Visual Reading Method for Detection of Bacterial Tannase

R. OSAWA AND T. P. WALSH

Veterinary Service and Research" Lone Pine Koala Sanctuary" Fig Tree Pocket" Brisbane" Queensland 4069" and Centre for Molecular Biotechnology" School of Life Science" Queensland University of Technology"
Garden Point Campus" Brisbane" Queensland 4001, Australia
Received 19 October 1992/ Accepted 26 January 1993

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.

A Comparative Study of Macroscopic and Microscopic Dimensions of the Intestine in Five Macropods (Marsupialia: Macropodidae). I. Allometric Relationships

R. Osawa and P. F. Woodall

Department of Anatomy, University of Queensland, Brisbane, Qld 4072, Australia.

A Present address: Veterinary Service and Research, Lone Pine Koala Sanctuary,
Jesmond Road, Fig Tree Pocket, Qld 4069, Australia.

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).

Characterization of Gram-negative Anaerobic Strains, Isolated from Koala Feces, which Exhibit Satellite Growth and Pleomorphism

RO OSAWA, TOMOHIKO FUJISA WA, and TOMOT ARI MITSUOKA

Veterinary Service and Research, Lone Pine Koala Sanctuary, Fig Tree Pocket, Brisbane, Australia 4069, and Laboratory for Intestinal Flora, Frontier Research Program, Institute of Physical and 'Chemical Research (RIKEN), Wako-shi, Saitama 351-01, Japan

Received May 4, 1992

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.

Microflora of the Pouch of the Koala (Phascolarctos cinereus)

Tannin-Protein Complex-Degrading Enterobacteria Isolated from the Alimentary Tracts of Koalas and a Selective Medium for Their Enumeration

RO OSAWA
Veterinary Service and Research" Lone Pine Koala Sanctuary" Fig Tree Pocket" Brisbane" Australia 4069
Received 23 October 1991/Accepted 7 March 1992

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.

THE CURRENT STATUS OF THE KNOB-TAILED GECKOS (NEPHRURUS) IN CAPTIVITY

Robert Porter, Lone Pine Koala Sanctuary, Jesmond Road, Fig Tree Pocket, Brisbane, QLD 4069.

There are currently seven described species of Nephrurus, namely N. levis, N. laevissimus, N. asper, N. vertebralis, N. wheeleri, N. stellatus and N. deleani (Wilson and Knowles, 1988). They are all medium to large geckos with a very distinctive appearance, having a relatively large head with a short tail terminating in a small round knob. They are widespread over most of arid Australia, where they are endemic. At least one species, N. deleani, is very restricted in range (Wilson and Knowles, 1988). A detailed review of the biology and ecology of the genus can be found in Greer (1989).

This article was stimulated by discussions in meetings of the Reptile Taxon Advisory Group, the body responsible for the overall management of reptiles in Australasian zoos. Geckos of the genus Nephrurus were chosen for particular attention, among other taxa, due to the low level of knowledge of their reproductive biology and captive requirements, as well as their desirability for captive collections and their unique morphology. The group acknowledges that some of the animals on which this article is based are probably held illegally, but, while it in no way supports illegal take or trade, it places the wide dispersal of knowledge of our native herpetofauna as one of its highest priorities.

Phenotypic Characterization of CO2-Requiring Strains of Streptococcus bovis from koalas

R. OSAWA AND L. I. SLY
Veterinary Service and Research, Lone Pine Koala Sanctuary, Fig Tree Pocket,1 and Department of Microbiology, University of Queensland, St. Lucia,2 Brisbane, Queensland, Australia

Received 16 April 1991/ Accepted 29 July 1991

We examined phenotypic characteristics of six mannitol-fermenting strains of Streptococcus bovis, including two unusual CO2-requiring strains isolated from koala feces. These strains did not grow in air, but grew in air supplemented with CO2 and under reduced oxygen conditions. All six strains had the same biochemical characteristics, except that the CO2-requiring strains did not produce p-N -acetylglucosaminidase.

Streptococcus bovis is commonly found in the normal gastrointestinal floras of many animals (2, 3, 8, 16) and also is often isolated in clinical situations, such as cases of human bacteremia (9, 10, 13), endocarditis (7, 17), and bovine mastitis and udder infections (5, 12). Although S. bovis is usually considered to be a facultative anaerobe (6), Latham et al. (11) reported that this species includes strains which are strictly anaerobic when they are first isolated. Recently, Osawa (14) and Osawa and Mitsuoka (15) investigated the phenotypic characteristics of facultatively anaerobic strains of S. bovis obtained from various sources. We isolated apparently anaerobic strains from the feces of two healthy koalas (Phascolarctos cine reus (Goldfuss), and in this study we characterized these strains.

Six strains of S. bovis, including two that are not able to grow in air, were obtained from our collections at the Lone Pine Koala Sanctuary (strains LPKS 1, LPKS 6, LPKS 31, and LPKS 32) and the Department of Microbiology, University of Queensland, Queensland, Brisbane, Australia (strains UQM 3549 and UQM 3552). The sources of the strains are indicated in Table 1. Cultures of these strains, which were stored as lyophiles, were revived and subcultured anaerobi­cally at least three times on plates containing Columbia blood agar (Oxoid, Ltd., Basingstoke, Hampshire, United Kingdom) in an atmosphere enriched with 8 to 10% CO2 by using Bio-bags (Becton Dickinson and Co., Cockeysville, Md.) before use.

Serological assessment of chlamydial infection in the koala by a slide EIA technique

H UENO, S MIZUNO, I TAKASHIMA, R OSAWA, W BLANSHARD, P TIMMS, N WHITE, N HASHIMOTO

Summary: A rapid and simplified slide enzyme immunosorbent assay (EIA) was developed for the diagnosis of chlamydial infection In the koala. HeLa 229 cells Infected with koala strain Chlamydia psittaci were fixed on the surface of multiwell slides and used as the antigen. The assay consisted of first reacting koala antiserum with the fixed C psittaci antigen, followed by reaction with biotinylated rabbit anti-koala IgG, ABC reagent and substrate. The chlamydial EIA antibody titres obtained were compared with those of a complement fixation (CF) test using koala strain C psittaci as antigen. Of 35 koala sera tested, 16 CF positive sera (~1: 8) also had a positive titre (~1: 200) In the slide EIA test (sensitivity 93.8%, 15/16). Nineteen CF negative sera were also negative in the slide ElA (specificity 100%, 19/19). Sixty-eight samples of koala blood were collected by ear-prick using a sampling paper method and were assayed by both tests. Sensitivity of the slide EIA was 100% (15/15) and specificity of the test was 96.2% (51/53). To simplify the slide EIA for use as a practical screening test, a 3-polnt serum dilution series (1:100, 1: 200, 1: 400) was used. This 3-polnt slide EIA was compared with the CF test using sheep strain chlamydial antigen. Thirty-nine sera were assayed by both tests. The sensitivity of the 3-polnt method was 85.7% (6n) and the specificity was 71.9% (23/32) as compared with the sheep antigen CF test. Of the 10 koalas with discrepant results, 8 (slide EIA positive but sheep antigen CF negative) were either chlamydia positive in cell culture or had evidence of clinical symptoms. This suggests a significant superiority of the slide EIA for detecting Infected animals as compared with the sheep antigen CF test. The 3-polnt slide EIA was also applied to cephalic vein blood collected on sampling paper and good agreement was obtained In 25 of 26 (96.1 %) samples when compared with those of serum samples. These results show that the slide EIA Is at least equal to the koala antigen CF test, and significantly more sensitive than the sheep antigen CF test for the serological assessment of chlamydial infections in koalas. Aust Vet J 68: 393 �E96.

UNUSUAL BASKING BEHAVIOUR IN CAPTIVE BEARDED DRAGONS (POGONA BARBATA)

Robert Porter Lone Pine Koala Sanctuary, Jesmond Road, Fig Tree Pocket, QLD, 4069.

Two fourteen month old, captive hatched Bearded Dragons (Pogona barbata) are currently maintained in an inside enclosure measuring 2400mm x 900mm x 600m high with two adult Shingleback skinks (Tiliqua rugosa). The substrate consists of a layer of dry mulched eucalyptus branches and the inhabitants are supplied with raised basking sites in the form of rocks and branches. Light and heat are provided by two 150 watt white spotlights plus a single 4-foot fluorescent fitting containing an F40T9BL "black light" tube. All are suspended 500mm above the substrate, although the lizards can move closer by climbing onto the basking sites. The enclosure is housed in an air conditioned room which produces a heat gradient of 36° ­37°C directly beneath the spotlights to around 23° -24°C in the coolest corner. The lights are controlled automatically by an electronic timer to simulate the local natural photoperiod. These lizards have been maintained inside with no access to direct sunlight for at least ten months with no apparent health problems.

For a period of three days in mid January 1991 all four lizards were removed to a 900mm x 300mm x 400mm glass tank while their permanent enclosure was thoroughly cleaned. A wooden lid covered with insect mesh and 12mm weld mesh for ventilation was fitted to the top. An incandescent light fitting was situated in the middle of this lid and contained a 75 watt white bulb. Rocks were placed directly beneath the bulb allowing the lizards to bask within 200mm of the light. Unfortunately no temperatures were taken in the enclosure but an estimation would be about 25° -28°C.

Application of a blood sampling paper method for complement fixation test detection of anti-chlamydial antibody in koalas (Phascolarctos cinereus)

R. OSAWA, F. N. CARRICK, N. HASHIMOT0, I. TAKASHIMA and T. TAKAHASI.

Blood samples obtained from 23 koalas were examined for detection of anti-chlamydia CF antibody by two different methods: (1) a conventional CF test on serum; (2) a new CF test on whole blood absorbed in a Blood Sampling Paper. The results obtained from these methods showed good correlation, with consistency in 22 out of 23 samples. This new CF test does not require a large amount of blood, and this may be obtained by pricking the ear vein rather than the more invasive cephalic vein puncture conventionally used. Thus this method greatly facilitates and simplifies the procedure of assessing anti-chlamydia CF antibody status of koalas, which are easily "stressed'' by handling, and seems to provide a more robust technique for monitoring koala populations in the field.

Introduction
INFECTIONS with bacteria belonging to the genus Chlamydia are widespread in the animal kingdom (Storz 1971). One of the most common methods used to screen for infection by these organisms is an assay which detects complement fixing (CF) antibodies to Chlamydia sp. in the blood. Conventional CF tests require 0.2 ml' of serum which would normally be derived from 1 ml of whole blood drawn by venipuncture. This technique is frequently used to screen for Chlamydia psittaci infections in captive colonies of koalas.
The Nobuto Blood Sampling Paper (Toyo Roshi Kaisha Ltd, Tokyo) has been developed as an alternative procedure for the collection, transportation and storage of blood for serological examination, and has potential for use by personnel with no experience in collecting blood by venipuncture. This technique has been used successfully for a wide variety of serological examinations in the microbiological field and is particularly convenient for continuous testing of newborn and small animals, and in surveys. Examples of prior application of the device include studies of toxoplasmosis (Nobuto 1965a), Japanese encephalitis, Newcastle disease, mycoplasmosis and canine distemper (Nobuto 1965b).

The objectives of this study were to evaluate whether samples of whole blood collected from koalas by this method are suitable for use in a CF test, and to compare the perform­ance of the CF test on these samples with the conventional method which utilizes blood serum.

Feeding Strategies of the Swamp Wallaby, Wallabia bicolor, on North Stradbroke Island, Queensland. I: Composition of Diets

Ro Osawa

Department of Anatomy, University of Queensland, St Lucia, Qld 4072; present address: Division of Veterinary Service and Research, Lone Pine Koala Sanctuary, Jesmond Road, Fig Tree Pocket, Qld 4069, Australia.

Abstract

Species composition of plants present in the stomach contents of 34 road-killed swamp wallabies, W. bicolor, on North Stradbroke Island, south-east Queensland, was investigated, and related to seasonal variations in nitrogen and fibre contents of plants in the study area. W. bicolor consumed a wide range of plant categories including shrubs, forbs, grasses, ferns and fungi. Among these, grasses, followed by shrubs, were the major component of the stomach contents. These dietary plants had higher nitrogen contents than those non-dietary plants of the corresponding plant category, which were present in the study site but never found in the stomach contents. Individual grass species of high occurrence in the stomach contents were molasses grass, Melinus minutiflora, green couch, Cynodon dactylon and sedges, Cyperus spp. Although more fibrous, these species had similar or relatively high nitrogen levels compared to native shrubs, except during summer. This suggests that W. bicolor selected dietary plants of high nitrogen content rather than of low fibre content.

Feeding Strategies of the Swamp Wallaby, Wallabia bicolor, on North Stradbroke Island, Queensland.
II: Effects of Seasonal Changes in Diet Quality
on Intestinal Morphology

Ro Osawa and Peter F. WoodaII

Department of Anatomy, University of Queensland, St Lucia, Qld 4072, Australia. A Present address: Division of Veterinary Research, Lone Pine Koala Sanctuary,
Jesmond Road, Fig Tree Pocket, Qld 4068, Australia.

Abstract
The effect of seasonal changes in diet quality on the alimentary tract of the swamp wallaby, W.bicolor, on North Stradbroke Island was investigated by examining 25 road-killed animals. To allow for variation in body weight, allometric analyses of both macroscopic and microscopic dimensions of the intestine were used and differences were tested by analysis of covariance. W. bicolor on a high fibre diet in spring and autumn had significantly shorter villi compared to those on a low fibre diet in winter and summer, suggesting that the intestinal villi of W. bicolor are highly labile and respond rapidly to changes in diet quality.

Formation of a Clear Zone on Tannin-Treated Brain Heart Infusion Agar by a Streptococcus sp. Isolated from Feces of Koalas RO OSAWA

Veterinary Service and Research, Lone Pine Koala Sanctuary, Fig Tree Pocket, Brisbane, Queensland 4069, Australia

Received 5 October 1989/ Accepted 7 December 1989

Gram-positive cocci, isolated from the feces of koalas and identified as Streptococcus bovis biotype I, formed a distinct clear zone on tannin-treated brain heart infusion agar, suggesting that this isolate has the unique characteristic of degrading the tannin-protein complex.

The koala, Phascolarctos cinereus (Goldfuss), is an arbo­real marsupial inhabiting the forests of eastern Australia. It is entirely folivorous, feeding almost exclusively on the foliage of Eucalyptus spp. (5, 6), which are known to have high concentration of tannins (3, 11). Tannins are a diverse group of soluble phenolic compounds that form chemical complexes with proteins. These complexes are considered to be resistant to degradation (i.e., by protease) within the guts of mammals, thereby interfering with digestion and the utilization of dietary protein (8, 13, 16, 18). Cork et al. (2) found a low tannin level in koala feces relative to that in the diet and- suggested that the tannin fraction of the total phenolic compounds was degraded to a considerable extent in the alimentary tracts of koalas. Investigation of the functions of the ceca of the koala (12) and the ring tail possum, Pseudocherius peregrinus, another marsupial feed­ing on eucalyptus diets (A. Lomdahl, Hons. thesis, Monash University, Clayton, 1983), led to the claim that bacterial degradation of tannin-protein complexes in the cecum may make protein available to these animals.

Fresh fecal pellets were collected directly from the ano­genital orifice of 12 captive koalas (nine adult males and three adult females) kept at the Lone Pine Koala Sanctuary. The animals were provided daily with fresh eucalyptus leaves ad libitum. The major species of eucalyptus leaves fed to the animals included forest red gum (Eucalyptus tereticor­nis), tallow wood (E. microcorys), and. grey gum (E. punc­tata). Fecal pellets collected from each animal were weighed aseptically, and approximately 1 to 2 g (wet weight) was taken into a tube containing 20 ml of sterile 0.25-strength Ringer solution (Oxoid Ltd., Basingstoke, Hampshire, United Kingdom) and thoroughly mixed with a homogenizer and a Vortex test-tube mixer.

Growth and development of the koala from birth to weaning

WENDY H. BLANSHARD

Changes in the appearance, dentition and behaviour of juvenile koalas during the first year of life are described. A number of these changes are discussed in relation to the normal timetable of physical and behavioural development of koalas reared naturally by their mothers in zoos and sanctuaries.

INTRODUCTION
IN common with other marsupials, koalas (Phascolarctos cinereus) are born in a very undeveloped state, weighing less than one gram. Although the respiratory, digestive and urinary tracts are functional and the forelimbs are already equipped with claws, the hind limbs are little more than buds (Cork 1987a; Lee and Martin 1988). During the first year of life most of the physical attributes of an adult koala develop, and the young koala becomes physically and physiologically independent of the mother.

Little published information is available on the growth and development of juvenile koalas. Eberhard (1972) examined a number of pouch ­young of the Victorian race (P. cinereus victor) occurring on Kangaroo Island, South Australia, and produced a schedule of first appearance of some external characteristics. As the ages of these animals were unknown, his descriptions were tabulated against body weight and various anatomical measurements. Lee and Martin (1988) gave an outline of the development of P. cinereus victor at intervals between birth and independence, including general information on body weight, head length, physical appear­ance, dentition and behaviour.

Only Smith (1975) and Thompson (1987) have documented the exact ages of individual koalas when describing aspects of their develop­ment. Both authors studied captive-bred animals of the Queensland race (P. cinereus adustus), and recorded stages in their behavioural and dietary transition from life inside to life outside the pouch.

In a search for body traits which may be useful in predicting the age of koalas, Eberhard (1972) found that head length, elbow-wrist length and ankle-knee length were linearly related to each other over a wide range of ages. Both Eberhard (1972) and Martin (1983) plotted the relationships between head length and estimated age for different populations of Victorian koalas. The youngest age represented on any of these plots was 200 days (Martin 1983).
Over the past three years, the author has accepted 21 juvenile Queensland koalas (P. cinereus adustus) for hand-raising. These comprised nine unfurred and four partially furred pouch-young, and eight fully furred back-young. A quantitative method of estimat­ing the ages of the hand-raised koalas was devised, so that their growth and development relative to one another could be meaningfully assessed. This paper documents some of the physical and behavioural changes which occur during the first 12 months of life of the koala, encompassing the period from birth to weaning.

Selective Medium for Enumeration of Tannin-Protein Complex­ Degrading-Streptococcus spp. in Feces of Koalas

RO OSAWA AND TOMOTARI MITSUOKA

Received 15 June 1990/ Accepted 6 September 1990

A selective agar plate medium (tannin-treated brain heart infusion agar supplemented with colistin-oxolinic acid) was developed to enumerate tannin-protein complex-degrading Streptococcus bovis in the feces of koalas. This medium was successfully used to enumerate strains from fecal samples but failed to enumerate those from pure cultures.

Streptococcus bovis is a common isolate from the alimen­tary tracts of ruminants (4, 6, 12, 17) and nonruminants (1, 15). Strains of bovine origin are frequently found to belong to biotype II (mannitol nonfermenting), whereas strains of human origin, which are associated with human systemic infections, have more often been found to belong to biotype I (mannitol fermenting) (5, 7, 11, 16). Recently, Osawa (9) reported that S. bovis biotype I isolated from koala (Phas­colarctos cinereus) feces had the unique characteristic of growing on tannin-treated brain heart infusion agar (T­TBHIA) and forming a clear zone around the colony. In the same work, a strain of S. bovis biotype II used for compar­ison grew on T-TBHIA plates but did not form a clear zone. Strains of S. faecalis (Enterococcusfaecalis) and S. faecium (Enterococcus faecium) did not grow on this medium. On the basis of these findings, Osawa (9) suggested that S. bovis biotype I is capable of degrading the tannin-protein complex, thereby helping koalas obtain dietary protein from tannin­rich eucalypts.

This report describes a selective medium used to enumer­ate the strains degrading the tannin-protein complexes. Such a medium would allow us to monitor the tannin-protein complex -degrading activity of the gastrointestinal microflora in koalas, whose diet consists almost exclusively of tannin­rich eucalyptus leaves (3).

The strains of S. bovis and other streptococcal and enter­obacterial strains used for comparisons in this study are listed in Table 1. Cultures of the strains were maintained on BHIA (Oxoid Ltd., Basingstoke, Hampshire, United King­dom) supplemented with 0.5% yeast extract (Oxoid). Phys­iological characteristics of the streptococci were determined by the API 20 Strep system (API System, Montalieu, Ver­cieu, France) to ensure their identity immediately before inoculation onto test plates. The identities of the enterobac­terial strains were checked by standard methods.

Use of a dietary supplement in koalas during systemic antibiotic treatment of chlamydial infection

Department of Zoology, University of Queensland, St Lucia, Queensland 4067

R OSAWA FN CARRICK

Infection with Chlamydia psittaci in the koala can cause blindness, pneumonia, infertility and urinary tract diseases and has been described by many workers (Cockram and Jackson 1974; Dickens 1976; Cockram 1978; Brown eta11980; Brown and Grice 1984). A survey of both captive and wild koalas throughout eastern Australia from 1979 to 1985 indicated that a large proportion of koalas sampled was affected by C psittaci (Brown and Carrick 1985).

Ad hoc attempts to treat chlamydial infections in koalas with antibiotics usually failed, although Carrick and Wood (1986) found that administration of dietary supplements enabled the majority of koalas treated with oxytetracycline by injection to survive.

Nevertheless, Brown (1987) reported that systemic antibiotic therapy for chlamydial infections in koalas, using either erythromycin or oxytetracylcine, was invariably fatal. The koalas showed rapid loss of body weight and died, although there was a marked reduction in signs of chlamydiosis during the course of the treatments.

As suggested by Carrick and Wood (1986), supplementary feeding using soya-based infant feeding preparation has become a common management practice for captive koalas in many zoos, including Lone Pine Koala Sanctuary in Australia, and Saitama Children's Zoo and Awaji Farm Park in Japan. Handasysde et al (1988) have also since recommended such supplementary feeding for captive koalas showing consistent loss of weight. We have conducted a controlled experiment to evaluate the efficacy of such dietary supplementation during systemic antibiotic' therapy in koalas.

Road-kills of the Swamp Wallaby, Wallabia bicolor, on North Stradbroke Island, South-east Queensland

Ro Osawa

Department of Anatomy, University of Queensland, St Lucia, Qld 4067., present address: Division of Veterinary Service and Research, Lone Pine Koala Sanctuary, Jesmond Road, Fig Tree Pocket, Qld 4069.

Abstract

A total of 127 road-kills of the swamp wallaby, Wallabia bicolor, on North Stradbroke Island in south-east Queensland was recorded between May 1981 and April 1982. The majority of kills were of adults. Road-kills occurred mainly at night, suggesting that W. bicolor is nocturnal, and were significantly correlated with the number of vehicles brought onto the island by ferries. Faecal pellet counts were made in classified habitats along the road in order to determine the relationship between the population density and road-kills of W. bicolor. Road-kills were significantly correlated with the faecal pellet counts in the Disturbed Ares (narrow, artificially cleared areas adjacent to the road) but not with the counts in the Native Area (i.e. native forest). This indicates that the wallabies were attracted to the disturbed area and became more susceptible to being killed by vehicles. There was no significant variation in the number of road-kills between lunar phases.

FAECAL MICRO FLORA OF CAPTIVE KOALAS, PHASCOLARCTOS CINEREUS (MARSUPIALIA: PHASCOLARCTIDAE)

Ro OSAWA AND TOMOTARI MITSUOKA

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

The faecal microflora of four Phascolarctos cinereus kept at the Saitama Children's Zoo in Japan was investigated using 10 selective and 4 nonselective agar media. Total viable counts (in log form) of faecal bacteria (8.2 g of wet faeces) were significantly lower than the direct microscopic counts (11.2 g of wet faeces). Four groups of obligate anaerobes (bacteroidaceae, peptococcaceae, bifidobacteria, and clostridia) and three groups of facultative anaerobes (enterobacteria, streptococci and lactobacilli) were isolated. Dominant components of the flora were obligate anaerobes, in particular bacteroidaceae and peptococcaceae.

R. Osawa, Department of Zoology, University of Queensland, St Lucia, Queensland 4067 Australia. Present address: Veterinary Service and Research, Lone Pine Koala Sanctuary, Jesmond Road, Fig Tree Pocket, Queensland 4069 Australia. 7: Mitsuoka, International Frontiel; Research System, RIKEN, Wako, Saitama 351. Department of Biomedical Science, Faculty of Agriculture, The University of Tokyo, Bunkyo, Tokyo, 113 Japan. Manuscript received 21 September 1989.

KOALAS (Phascolarctos cinereus) are arboreal marsupials living mainly in the forests of eastern Australia. The animal feeds entirely on a folivorous diet, consisting almost exclusively of foliage of trees of the genus Eucalyptus (Eberhard, McNamara, Pearse and Southwell 1975, Eberhard 1978).
McKenzie (1978) observed that the luminal surface of the caecum was completely covered by a tangled mat of bacteria including gram-positive and gram-negative rods, cocci and actinomycete-like organisms. He also suggested that the caecum of P. cinereus was a major site of microbial fermentation of ingesta. However, Cork, Hume and Dawson (1983) reported that when P. cinereus were fed on a diet of the grey gum (Eucalyptus punctata) only about 9 070 of total energy intake was derived from volatile fatty acid production; thus suggesting that the major role of the hindgut micro flora of P. cinereus may not be fermentation of plant cell wall cellulose.

In the present study, the composition of faecal microflora of captive P. cinereus kept at a Japanese Zoo was investigated in order to provide information on the ecology of the gut microflora. Since information on the composition of faecal flora of other animals was provided by Mitsuoka and Kaneuchi (1977), the methods of culturing, isolating, identification, and enumeration used by these workers were used in the present study for quantitative comparison between P. cinereus and those animals.

New Zealand Geckos
Their Natural History and Captive Maintenance

Robert Porter
c/o Lone Pine Koala Sanctuary
Brisbane, Queensland 4069 –Australia
Jesmond Road, Fig Tree Pocket

Isolated from other landmasses for millions of years, New Zealand's ter­restrial fauna is relatively depauperate, particularly when compared to Austra­lia, its closest neighbour. This comparison is especially evident where reptiles are concerned, New Zealand laying claim to around 50 species, while Australia has over 650. Yet New Zealand can boast some of the most unique reptiles, the most famous being the two species of tuatara (Sphenodon sp.), relict surviving members of an extremely ancient group. The country's lizard fauna includes. some of the most beautiful geckos in the world, rivalling even P helsuma in their stunning colors and markings. These geckos are divided into two distinct groups, the green geckos (Naultinus) and the predominantly brown or grey species (Hoplodactylus).

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