PNEUMONIA ASSOCIATED WITH BORDETELLA BRONCHISEPTICA IN CAPTIVE KOALAS

Bordetella bronchiseptica was isolated from 12 cases of purulent bronchopneumonia from a captive koala colony near Brisbane, Queensland. An initial epizootic in March 1967 causing 13% mortality was followed by annual outbreaks causing 2% mortality usually in newly weaned and aged koalas in late winter to early spring. High population density and a low plane of nutrition in winter were thought to predispose to the occurrence of the disease.     

Histological survey for oxalate nephrosis in Victorian koalas (Phascolarctos cinereus)

The Mount Lofty Ranges koala (Phascolarctos cinereus) population in South Australia has a high prevalence of the renal disease oxalate nephrosis, for which an underlying genetic cause is suspected. South Australian koalas primarily originate from those in French Island, Victoria; however, oxalate nephrosis has not previously been reported in Victorian koalas. Examination of kidney tissue sections from 63 koalas across Victoria found that nine koalas were affected by oxalate nephrosis (14.3%). These included 2/5 koalas from French Island (40%), 4/14 koalas from the western regions (29%), 2/11 Raymond Island koalas (18%), and 1/13 Cape Otway koalas (8%). There were no cases of oxalate nephrosis identified in the Strzelecki koalas (n = 12). These findings suggest that oxalate nephrosis occurs in koalas from French Island and populations that have received significant influx of koalas from French Island, but not in the Strzelecki region, which has little to no French Island input. This lends support to the theory that an inherited abnormality of oxalate metabolism could underlie the high prevalence of oxalate nephrosis in the Mount Lofty Ranges koala population, and molecular investigations are currently underway to investigate a genetic cause.     

Lymphoid neoplasia in the koala

Objective Correlation of immunophenotype with history, anatomical and morphological features of lymphoid neoplasia in the koala.
Methods Routine necropsies were performed on 51 koalas with suspected lymphoid neoplasia between 1986 and 1997 in New South Wales and Queensland. Immuno-phenotyping was by an immunoperoxidase method utilising species cross-reactive antibodies raised against human lymphocytes and an antibody raised against koala IgG. Cases were classified according to organs and tissues affected and the morphological features of neoplastic cells.
Results Twenty-six (51%) of the cases were of the T cell immunophenotype, 12 (24%) were of B cell immunopheno-type and 13 (25%) did not stain. The age and sex of koalas did not correlate with immunophenotype (P = 0.686 and P = 1.000, respectively). Thirty-two cases were leukaemic and 36 had multiple organ involvement, probably reflecting presenta tion of koalas at advanced stages of disease. Abdominal tissue involvement was most common (44 cases), followed by nodal (32), atypical (21) and cervicomediastinal (14). The T cell immunophenotype was over-represented among the leukaemic cases (P = 0.013). Generally, the T cell immunophenotype predominated except for many affected atypical tissues. Neoplastic cells were mostly of medium nuclear size with round to oval nuclei. No correlations were found for cell morphology, mitotic index and immunopheno-type.
Conclusion The prognostic value of an immunopheno-typic, anatomical and morphological basis for the classifica tion of lymphoid neoplasia in the koala currently is limited by the need to detect these neoplasms at an early age, the requirement for freshly fixed tissues and the restricted range of available cross-reacting antibodies.     

Expression and in vitro upregulation of MHCII in koala lymphocytes

Understanding and measuring immune activity of the koala (Phascolarctos cinereus), is important to studies of the epidemiology and impact of the widespread chlamydial and koala retroviral (KoRV) infections that occur in this iconic but increasingly threatened species. To explore the interaction of disease and immunity, and to assess the potential for use of class II major histocompatibility complex (MHCII) upregulation as an indicator of lymphocyte activation in in vitro immune assays, we have investigated the expression of MHCII in koala lymphocytes by flow cytometry. MHCII expression was upregulated in mitogen stimulated B lymphocytes in vitro but no such increase was detected in vivo in free-living koalas with active inflammation. In assessing phenotypic baseline data of captive koalas, we have identified that MHCII is expressed predominantly on circulating B lymphocytes (85.7 ± 2.4%) but on very few T lymphocytes (3.4 ± 1.9%), even following activation, and suggest that the latter finding might be compensated by the greater absolute numbers of peripheral blood B lymphocytes in this species relative to many eutherian species.     

Epizootiology of Chlamydia infections in two free-range koala populations

The prevalence of Chlamydia pecorum and Chlamydia pneumoniae infections in two free-range koala populations was assessed using genus-specific PCR combined with species-specific DNA probe hybridisation. Population A had a very high overall level of chlamydial infection (85%) with significantly more of these infections being due to C. pecorum (73%) compared to C. pneumoniae (24%). The second population had a much lower prevalence of infection (10%) with equal levels of both species. An important finding of this study was that. while five of 24 C. pecorum-infected koalas had clinical signs of the disease (both ocular and urogenital sites), none out of seven C. pneumoniae-infected koalas had signs of clinical disease. This suggests that C. pecorum may be the more pathogenic of the two chlamydial species infecting this host. The level of infection (assessed by intensity of the specific hybridisation signal) also differed between chlamydial species, with C. pecorum infections ranging from low to high grade whereas C. pneumoniae infections were always low grade. When the age of infected koalas was examined, 58% of young, sexually immature koalas were found to have C. pecorum infections, increasing to 100% of koalas in the older age groups. This suggests that, in this population at least, young koalas are readily infected with C. pecorum from their mothers. While the infection levels with C. pneumoniae were too low to be statistically significant, again, sexually immature koalas were found to be infected. The recent separation of chlamydial infections in koalas into two species is beginning to indicate different epizootiologies for koala C. pecorum compared to koala C. pneumoniae.     

Season- and Age-related Reproductive Changes Based on Fecal Androgen Concentrations in Male Koalas,Phascolarctos cinereus

The purposes of the present study were to clarify age- and season- related androgen patterns, and to compare the reproductive physiology between Japanese captive koala populations and Australian populations. To measure fecal androgens, feces were collected from male koalas (4.2 to 13.8 years of age) kept in Japanese zoos. Fecal androgens were extracted with methanol from the lyophilized samples and determined by enzyme immunoassay using 4-androstene-3,17-dione antibody. Fecal androgen concentration in male koalas increased after sexual maturation and remained relatively high until old age. In the survey with the Japanese zoo studbook of koalas, copulation (conception) month showed a pyramid shape with a peak in March to June (60.7%) in koalas born and reared in Japanese zoos and from July to April with the highest concentration in September to January (69.7%) in Australian institutes. Japanese zoo koala populations have a characteristic physiological cycle adapted to Japan''s seasonal changes. The suitable month of year for copulation or conception in Japan is diametrically opposed to that in Australia. Mean fecal androgen concentrations by month in the males born and reared in Japan indicated annual changes with the highest concentration in May and the lowest value in November. Fecal androgen analysis may be a noninvasive alternative tool to monitor circulating testosterone and may be helpful in understanding reproductive activity and physiology in male koalas.     

A survey of urinary tract disease in New South Wales koalas

From 1980 to 1988 235 koalas were necropsied and 67 were found to have urinary tract disease. Six affected koalas out of 48 were derived from wildlife parks around Sydney while 61 of 187 were derived from free living populations on the central and north coasts of New South Wales. Sixteen had cystitis alone, 5 had cystitis and associated renal disease only, 16 females had cystitis with genital disease, 23 had urinary disease in combination with other systemic disease and 7 had renal disease only. Overall 49 animals had cystitis (30 females and 19 males; 47 being free living) with 12 of these having renal extension (all free living). Cystitis tended to be active but chronic while associated renal disease was mainly designated as hydronephrosis and pyelonephritis. Other forms of renal disease included lymphosarcoma, oxalate nephrosis, acute and chronic nephritis, and microabscessation related to septicaemia. Female genital disease associated with cystitis was commonly vaginitis and metritis. Paraovarian cysts were detected with and without metritis. Other diseases occurring with urinary tract disease included conjunctivitis, dermatitis/stomatitis, pneumonia and hepatic disease. The higher prevalence of urinary tract disease in free living koalas, especially cystitis, is in contrast to captive koalas and may reflect the interaction between disease cause and habitat.     

Dissociative anaesthesia in free-ranging male koalas and selected marsupials in captivity

Forty seven free-ranging, adult, male koalas were captured and administered an intramuscular injection of the dissociative anaesthetic, Telazol (tiletamine HCl plus zolazepam HCl), at dose rates of 5.0 to 7.7 mg/kg body weight. Anaesthesia induction was rapid and smooth and resulted in a surgical plane of anaesthesia lasting 30 to 45 min. There was no depression of heart rate or respiration. Mild salivation occurred in most animals, but was not a problem because the swallowing reflex was retained. There was no mortality or morbidity and the anaesthesia level was sufficient to allow electroejaculation and multiple blood sampling with no apparent animal discomfort. For 10 of 19 males in which anaesthesia was required for a 90 min protocol, a supplementary Telazol injection (average, 2.5 mg/kg) was necessary. All koalas recovered completely within 3 to 4 h of the initial injection. The results suggest that the optimal Telazol dosage for the adult male koala is 7.0 mg/kg body weight. The retrospective analysis of 259 anaesthesia records involving 14 species indicated that Telazol was equally effective and safe in other captive marsupials.     

Diabetes mellitus in a koala (Phascolarctos cinereus)

Objective To describe a case of diabetes mellitus in a koala (Phascolarctos cinereus).
Design A case report with controls.
Procedures We describe clinical and laboratory findings in a 6-year-old, free-living, female koala presented with traumatic injury and subsequently found to have polydipsia, hyperglycaemia and glucosuria. Over a 5 week period, serum biochemical analyses, haematological examinations, urinal-yses, measurement of serum insulin and fructosamine concentrations, necropsy, histopathological examination of a range of tissues and immunohistochemical examination of the pancreas for insulin-containing cells were done. For reference purposes, serum insulin and fructosamine concentrations were determined in four and two healthy koalas, respectively, and three healthy koalas pancreases were examined histo-logically and immunohistochemically.
Results The koala had persistent hyperglycaemia, hyperlipidaemia, hyponatraemia, hypochloraemia and glucosuria. Serum insulin concentration of the diabetic koala was only marginally smaller than that of healthy koalas, but all concentrations were smaller than reference concentrations in dogs and people. Fructosamine concentration did not allow the diabetic koala to be distinguished from healthy koalas and concentrations of all koala analytes were greater than expected for healthy dogs and people. Histopathological examination revealed extensive degeneration of pancreatic islet cells and fatty infiltration of hepatocytes. Immunoperoxidase staining revealed decreased or absent insulin in the b cells of the affected koala.
Conclusion Clinical signs, clinicopathological results and histopathological changes were consistent with diabetes mellitus. The pathogenesis of the condition could not be determined but may have been related to the administration of a parenteral corticosteroid preparation, the stress of capture or tissue damage and inflammation.     

INTRAVENOUS UROGRAPHY IN THE KOALA (Phascolarctos cinereus)

Urograms were performed on 16 adult koalas to evaluate this procedure for use in the koala. Because there is only small amount of intraabdominal fat, radiographs of the abdomen of koalas lack detail. Contrast medium was excreted more slowly in the koala than in the dog and there was considerable variation in the rate of excretion between koalas. The results of this study indicated that urography was valuable technique for assessing renal anatomy, but there was considerable variation in the time for excretion of the contrast medium.     

Using quantitative polymerase chain reaction to correlate Chlamydia pecorum infectious load with ocular, urinary and reproductive tract disease in the koala (Phascolarctos cinereus)

Complex interactions between Chlamydia pecorum infection, the immune response and disease exist in the koala. We used quantitative polymerase chain reaction to investigate the relationship between C. pecorum infectious load and ocular and urogenital tract disease. Chlamydia pecorum shedding was generally higher in animals with chronic, active disease than in animals with inactive disease. The absence of ocular disease was generally associated with low levels of shedding, but relatively high levels of shedding in the urogenital tract were detected in some koalas without clinical disease signs. These results suggest a complex disease pathogenesis and clinical course in C. pecorum-infected koalas.     

Chlamydial infection in a colony of captive koalas

Forty three koalas in a captive colony were investigated for the presence of Chlamydia psittaci infection and associated disease. Swabs were taken from conjunctivae and urogenital sites for cell culture isolation of C psittaci and for cytological examination (direct smears) for chlamydial inclusions and evidence of inflammation. On the basis of cell culture isolation, 28 samples from 25 koalas were positive for C psittaci (that is, infected). Three koalas were positive from both sites, 5 from conjunctivae alone and 17 from urogenital sites alone. Seven of the 8 koalas with positive conjunctival swabs had overt signs of conjunctivitis, but only 3 of the 20 koalas with positive urogenital swabs had overt signs of 'wet bottom' (continual urine soiling due to cystitis) or purulent discharge. However, 5 of the 20 with positive urogenital swabs had past episodes of 'wet bottom'. Moreover, examination of direct cytological smears showed evidence of inflammation (neutrophils) in 7 of 8 koalas with positive conjunctival swabs and 17 of 20 with positive urogenital swabs. Chlamydial inclusions were rarely identified with surety on direct cytological smears. In the 18 koalas without chlamydia, one had overt conjunctivitis while 2 had past episodes of conjunctivitis. The koala with conjunctivitis at the time of sampling had a prior history of 'wet bottom'. Examination of direct cytological smears revealed 2 of the chlamydial negative koalas had high numbers of neutrophils in urogenital smears. It was concluded that C psittaci infection may cause overt or sub-clinical disease, with the former developing when the koalas were stressed through management procedures or concomitant disease.     

Schirmer tear tests and intraocular pressures in conscious and anesthetized koalas (Phascolarctus cinereus)

Objective To estimate mean Schirmer tear test (STT) and intraocular pressure (IOP) values in healthy koalas both conscious and anesthetized. Methods Data were gathered from koalas in Victoria, Australia. Conscious examinations were performed on captive koalas. Free‐ranging (wild) koalas were examined under anesthesia. Anesthesia was induced using alfaxalone, and animals were maintained on oxygen and isoflurane if required. All animals were healthy and had no surface ocular pathology detectable during slit lamp biomicroscopy. STT I tests were performed using commercial STT test strips placed in the lower fornix for 1 min. IOP was measured using an applanation tonometer after topical anesthesia. The higher value of the two eyes for both STT and IOP was analyzed. STT was measured in 53 koalas (34 conscious, 19 anesthetized) and IOP was measured in 43 koalas (30 conscious, 13 anesthetized). A two‐sample t‐test was used to compare means. A P‐value <0.05 was regarded as significant. Mean ± SD is presented. Results The mean higher STT in conscious koalas was 10.3 ± 3.6 mm wetting/min and in anesthetized koalas it decreased to 3.8 ± 4.0 mm wetting/min (P < 0.0001). The mean higher IOP in conscious koalas was 15.3 ± 5.1 mmHg, and in anesthetized koalas it was 13.8 ± 3.4 mmHg (P = 0.32). There was no effect of sex on either STT or IOP. Conclusions The mean and SD of STT and IOP values for koalas both conscious and anesthetized were reported. The mean STT was significantly reduced by alfaxalone anesthesia.     

Predation by carpet pythons (Morelia spilota) is an important cause of mortality in a free‐living koala (Phascolarctos cinereus) population in South East Queensland

Koalas (Phascolarctos cinereus) are experiencing significant declines across the northern part of their range. However, unbiased, population‐level estimates of mortality are rarely reported, as it's difficult to quantify causes of mortality robustly in this cryptic species. We aimed to determine the relative importance of carpet python (Morelia spilota) predation in a free‐living koala population and describe the characteristic pathological findings during necropsy. In total, 503 koalas were captured, underwent veterinary examination and telemetric tagging, and were monitored after release over a four‐year period. Mortalities were detected when activity data reported by K‐Tracker® biotelemetry collars indicated low or zero activity, or during routine field monitoring events. Experienced koala veterinarians performed thorough, standardised necropsy examinations on retrieved carcasses to determine causes of death. The three, sometimes subtle, cardinal signs used to definitively diagnose carpet python‐caused deaths of koalas were a U‐shaped primary bite site, slicking of the fur by python saliva (particularly around the face), and diffuse, uniform pulmonary congestion. We found that carpet pythons were important predators of koalas, second only to wild dogs (dingoes and dingo hybrids (Canis familiaris dingo)), accounting for 11.6% of predation deaths and 7.2% of total deaths. Less than half (38%) of the koalas killed by carpet pythons were ingested. Though carpet pythons are known predators of koalas, their relative importance as a cause of mortality hasn't previously been recognised. Population viability analyses and conservation management plans benefit from robust cause‐of‐death data collected during longitudinal monitoring studies, requiring telemetry methods that facilitate rapid detection of mortalities.     

Koala lymphoid cells: analysis of antigen-specific responses.

Bovine serum albumin (BSA) and ovine immunoglobulin (OvIgG) were used to induce humoral immune responses in two koalas which were also painted with 2-4-dinitrofluorobenzene (DNFB) and subsequently tested for local delayed-type hypersensitivity (DTH) reactions. The responses observed support the suggestion that the koala is 'immunologically lazy'. Antibody responses to BSA and OvIgG were not detected until 12 weeks after the initial antigen injection and antigen-specific in vitro proliferative responses by the mononuclear cells (PMC) of immunised animals could not be induced, although these cells did respond to concanavalin A and phytohaemagglutinin. Similarly, DTH responses to DNFB could be elicited in vivo, but took a relatively long time to develop and the PMC of the sensitised koalas were unresponsive to the sensitising antigen in vitro. The absence of proliferation when mixed suspensions of PMC from different koalas were cultured in vitro is consistent with these results.     

Purification and initial characterisation of koala immunoglobulins

The production of koala immunoglobulins (Ig) was elicited by the immunisation of a koala (Phascolarctos cinereus) with bovine serum albumin. This Ig was then purified using the highly specific techniques of affinity chromatography. The purified protein was compared with a "potential" koala Ig protein which was subsequently purified by Protein G chromatography and both proteins were further characterised by agarose/SDS-PAGE and immunoelectrophoresis. Results indicate that koalas do produce Ig in response to antigen challenge, koala Ig has a higher net negative charge than that seen in most other mammals and possibly two subclasses of IgG and IgM are present in normal koala serum.     

Resorptive tooth root lesions in the Malayan tapir (Tapirus indicus)

Facial abscessation and osteomyelitis due to dental disease is commonly seen in the Malayan tapir (Tapirus indicus), but little is known about the prevalence or etiology of these lesions. To determine the prevalence of dental ailments, 56 skulls and mandibles of deceased Malayan tapirs were visually and radiographically evaluated. Dental lesions were scored according to severity, and individuals were classified according to their age (juvenile/ young adult/adult) and origin (captive/free ranging). All of the lesions identified were of a resorptive nature. seemingly originating at the cementoenamel junction and burrowing towards the center of the tooth. Overall, 27% of the investigated skulls presented radiolucent dental lesions. The prevalence among captive animals was 52% (13/25), while only 6% (2/31) of the free-ranging tapirs had dental lesions. The second, third, and fourth premolars and first molar were the teeth most commonly affected, and the mandibular teeth were more often involved than the maxillary dentition. This study demonstrates a high prevalence of resorptive dental lesions in captive Malayan tapirs and provides a strong indication that age and captivity are significant risk factors in the development of these lesions. Dental disease, Malayan tapir, radiology, resorptive lesions, Tapirus indicus.     

Further characterisation of the immune response of the koala

Sensitive enzyme immunoassays (EIA) were developed to monitor antibody (Ab) production in the koala, in response to both soluble and particulate antigens (Ag). When compared with a eutherian mammal, the rabbit, both the dynamics and kinetics of Ab production in the koala were found to be severely retarded. In vitro, Ag specific lymphocyte proliferative responses were demonstrated for the first time in this animal by sensitising koalas in vivo with Bacillus Calmet-Guerin (BCG), with the level and timing of this cell mediated immune (CMI) response comparable with those seen in non-metatherian mammals. Levels of circulating B lymphocytes were examined in an attempt to clarify the retarded humoral responses to foreign Ags. In addition, peripheral mononuclear cells (PMC) from koalas, were examined for their reactivity to a range of monoclonal Abs and lectins in an attempt to characterise these cells further. The lectins examined, demonstrated an all or none reactivity with koala lymphocytes and were therefore considered unsuitable as markers for identifying lymphoid subsets in this animal. A monoclonal Ab directed at class II MHC Ags in the mouse, demonstrated cross reactivity with a high percentage of all koala monocytes tested. Using this Ab to probe CMI responses in vitro, it is concluded that immune interactions required for such responses in the koala parallel those seen in other mammals.     

OBSERVATIONS ON DISEASES OF FREE-LIVING AND CAPTIVE KOALAS (PHASCOLARCTOS CINEREUS)

SUMMARY During 1975–1978, pathological examination of 15 free-living and 11 captive koalas from Queensland revealed the following conditions: Bordetella bronchiseptica-associated pneumonia (6 cases), Pseudomonas aeruginosa-associated pneumonia (1), idiopathic chronic interstitial pneumonia (1), severe alveolar emphysema (3), ulcerative stomatitis (1), intestinal blockage (3), urinogenital lesions (3), Ixodes holocyclus paralysis (1), chlamydial keratoconjunctivitis (2), neoplasia (3), trauma/shock (2).     

Review of some pharmacokinetic and pharmacodynamic properties of anti‐infective medicines administered to the koala (Phascolarctos cinereus)

Although koalas are iconic Australian animals, no pharmacokinetic studies of any first‐line medicines used to treat diseased or injured koalas had been published prior to 2010. Traditionally, medicine dosages suggested for this species underwent linear extrapolation from those recommended for domesticated species. The koala, a specialist folivore whose natural diet consists of almost exclusively Eucalyptus spp. foliage has anatomical and physiological adaptations for detoxifying their diet which also affect medicine pharmacokinetic profiles. This review addresses aspects of medicine absorption, clearance, and other indices (such as medicine binding to plasma proteins) of enrofloxacin/marbofloxacin and chloramphenicol used for the systemic treatment of chlamydiosis, and fluconazole ± amphotericin, and posaconazole for the treatment of cryptococcosis. Based on observations from published studies, this review includes suggestions to improve therapeutic outcomes when administering medicines to diseased koalas.