Symmetric dimethylarginine values in koalas (Phascolarctos cinereus) based on oxalate nephrosis status

Oxalate nephrosis is a prevalent renal disease in koalas (Phascolarctos cinereus) of the Mount Lofty Ranges population in South Australia. The symmetric dimethylarginine (SDMA) assay is widely used in companion animals to diagnose renal disease, particularly in the early stages. This study aimed to determine: (1) reference intervals for SDMA in koalas and (2) SDMA values of koalas with oxalate nephrosis. Blood samples were collected from 41 Mount Lofty Ranges koalas euthanased on welfare grounds. Koalas were necropsied and, based on renal histopathology, were classified as unaffected (n = 22) or affected (n = 19) by oxalate nephrosis. Serum or plasma samples were analysed for creatinine, urea and SDMA and urine samples for urine specific gravity (USG). The reference interval for SDMA in unaffected koalas was 2.4–22.9 μg/dL. In koalas with oxalate nephrosis, SDMA was elevated in 74% of cases above the upper limit of the confidence interval. SDMA was elevated in three affected koalas with normal creatinine values. A positive correlation was found between SDMA and creatinine (R = 0.775, P < 0.001) and SDMA and urea (R = 0.580, P < 0.001) and a negative correlation between SDMA and USG (R = −0.495, P = 0.027). In conclusion, SDMA correlates well with other commonly used tests of renal function in koalas and should be included as part of the standard diagnostic process to increase the accuracy of oxalate nephrosis diagnosis in koalas.     

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.     

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.     

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.     

Haematological and biochemical investigations of diseased koalas (Phascolarctos cinereus)

Haematological and biochemical investigations were performed on 14 koalas with uncomplicated cystitis, 8 with complicated cystitis, 8 with conjunctivitis, 8 with lymphosarcoma, and 14 with miscellaneous diseases. Changes were limited and inconsistent in individual koalas with uncomplicated cystitis and conjunctivitis. In contrast, individual koalas with complicated cystitis were more likely to have anaemia, leukocytosis due to neutrophilia, hypoproteinaemia due to hypoalbuminaemia, and azotaemia due to elevated urea concentration. Although these changes were non-specific they did allow assessment of prognosis for survival and response to treatment. Koalas with lymphosarcoma were invariably anaemic, leukaemic, azotaemic and hypoalbuminaemic. Elevated enzymes (aspartate transaminase [AST]. lactate dehydrogenase [LD] and gamma glutamyl transferase [GGT]) were more common in koalas with lymphosarcoma. Koalas affected by miscellaneous conditions showed variable changes but once again anaemia, leukocytosis, azotaemia, elevated AST and LD, and hypoalbuminaemia were not uncommon. On the basis of these findings a minimal profile is suggested for the investigation of sick koalas and would include haematocrit, total and differential leukocyte counts, urea, total protein and albumin concentrations and AST, GGT and LD activities.     

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.     

Plasma concentrations of chloramphenicol after subcutaneous administration to koalas (Phascolarctos cinereus) with chlamydiosis

Nine mature koalas with chlamydiosis, typically keratoconjunctivitis and/or urogenital tract infection, were treated with daily subcutaneous injections of chloramphenicol at 60 mg/kg for 45 days (five koalas), or for a shorter duration (four koalas). All koalas were initially positive for Chlamydia pecorum as determined by real-time polymerase chain reaction (qPCR). Plasma chloramphenicol concentrations were determined at t = 0, 1, 2, 4, 8, and 24 h after the day 1 injection (nine koalas) and after the day 15 injection (seven koalas). Chloramphenicol reached a median (and range) maximum plasma concentration of 3.03 (1.32-5.03 μg/mL) at 4 (1-8 h) after the day 1 injection and 4.82 (1.97-27.55 μg/mL) at 1 (1-2 h) after day 15. The median (and range) of AUC(0-24) on day 1 and day 15 were 48.14 (22.37-81.14 μg·h/mL) and 50.83 (28.43-123.99 μg·h/mL), respectively. The area under the moment curve (AUMC)(0-24) median (and range) for day 1 and day 15 were 530.03 (233.05-798.97 h) and 458.15 (291.72-1093.58 h), respectively. Swabs were positive for chlamydial DNA pretreatment, and all koalas except one, produced swabs negative for chlamydial DNA during treatment and which remained so, for 2-63 days after treatment, however whether chloramphenicol treatment prevented long-term recrudescence of infection was not established. At this dose and dosing frequency, chloramphenicol appeared to control mild chlamydial infection and prevent shedding, but severe urogenital disease did not appear to respond to chloramphenicol at this dosage regime. For koalas affected by severe chlamydiosis, antibiotics alone are not sufficient to effect a cure, possibly because of structural or metabolic changes associated with chronic disease and inflammation.     

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.     

A mortality survey of free range koalas from the north coast of New South Wales

In the period 1980 to 1986, 127 free range koalas from the north coast of New South Wales were presented for necropsy. Thirty-three koalas had urogenital disease alone, 8 had respiratory disease alone, 2 had digestive tract disease alone, 12 had multiorgan disease, 48 had traumatic injuries, 6 had neoplasia, 3 had miscellaneous conditions, while 15 had no significant lesions. Common naturally occurring disease entities included cystitis, conjunctivitis, paraovarian cysts, metritis and pneumonia. Lymphosarcoma was the common neoplasm.     

Within-population diversity of koala Chlamydophila pecorum at ompA VD1-VD3 and the ORF663 hypothetical gene

Infection of koalas by Chlamydophila pecorum is very common and causes significant morbidity, infertility and mortality. Fundamental to management of the disease is an understanding of the importance of multi-serotype infection or pathogen virulence in pathogenesis; these may need consideration in plans involving koala movement, vaccination, or disease risk assessment. Here we describe diversity of ompA VD1-3, and ORF663 hypothetical gene tandem repeat regions, in a single population of koalas with diverse disease outcomes. We PCR amplified and sequenced 72 partial ompA segments and amplified 25 tandem repeat segments (ORF663 hypothetical gene) from C. pecorum obtained from 62 koalas. Although several ompA genotypes were identified nationally, only one ompA genotype existed within the population studied, indicating that severe chlamydial disease occurs commonly in free-ranging koalas in the absence of infection by multiple MOMP serotypes of C. pecorum. In contrast, variation in tandem repeats within the ORF663 hypothetical gene was very high, approaching the entire range reported for pathogenic and non-pathogenic C. pecorum of European ruminants; providing an impetus for further investigation of this as a potential virulence trait.     

Malocclusions in the koala (Phascolarctos cinereus)

Malocclusions are a misalignment or incorrect positioning of the teeth when the upper and lower jaws close. These are poorly described in the koala and can result in irregular mastication which can have lifelong effects on body condition and oral health. A total of 370 koalas from two populations in Queensland (295) and one in South Australia (75) were examined for malocclusions. The prevalence of malocclusions in South Australian free‐ranging koalas, captive Queensland koalas and Queensland free‐ranging koalas was 39% (44), 30% (29) and 22% (29) respectively. Four types of malocclusion were identified based on severity of misalignment of the incisor/canine region, types 1, 2, 3 and 4. Maxillary overbite measurements of the molariform teeth were determined and these anisognathic values were then used to describe malocclusions within familial relationships in captive colonies. Captive koalas with a malocclusion had narrower mandibular width that ranged between 0.5 and 1% less than the normal measurements. The specific malocclusions reported in this study affected individuals by leading to tooth rotation, mobility and erosion with inefficient mastication of food and vegetation compaction. These changes increased the oral cavity pathology, by placing animals at risk of periodontal disease. There was evidence of familial links to malocclusion types in captive animals. Therefore captive breeding recommendations should consider known koala malocclusion traits to minimise their effect on future generations.     

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.     

Hip dysplasia in koalas (Phascolarctos cinereus) at the San Diego Zoo.

A retrospective/prospective radiographic study documented 55 cases of moderate to severe hip dysplasia, with varying degrees of shallowing of the acetabulum, flattening or loss of the femoral head, widening or loss of the femoral neck, and femoral diaphyseal abnormalities in northern koalas (Phascolarctos cinereus) in the San Diego Zoo (San Diego, California, USA) collection. For the retrospective study, historic radiographs were examined when availble. For the prospective study, three standard views (ventrodorsal extended leg, ventrodorsal frog leg, and lateral extended leg) were used. A scoring system was developed using four areas (acetabulum, femoral head, femoral neck, and femur) and ranges of 0 to 5 (0 = not affected to 5 = severely affected) for each area, creating a total score out of 40. Scores were graded as follows: 0-2 = normal dysplasia; 3-6 = mild dysplasia; 10-19 = moderate dysplasia; and 20-40 = severe dysplasia. Thirty koalas were graded as severe, 25 koalas as moderate, and 38 koalas as excellent or mild. Affected koalas may or may not demonstrate gait abnormalities. Mild to severe degenerative joint disease may develop and symptoms may be alleviated with glucosamine/chondroitin sulfate and nonsteroidal anti-inflammatory drugs. The etiology of hip dysplasia in koalas is not currently understood.     

Pyometra and pyovagina in koalas

SUMMARY Four female koalas were found to have pyometra and pyovagina at autopsy. All had soiling of fur around the cloaca and were in poor condition. One koala had cystic ovaries in addition to the pyometra while another had extensive peritonitis due to rupture of one of the uterine horns. Chronic cystitis was present in all koalas while one also had pyelonephritis. Microbiological examination revealed a mixed flora of both aerobes and anaerobes.     

Concentrations of thyroid hormones and iodothyronine binding proteins in serum of the koala (Phascolarctos cinereus)

Objective To examine circulating total and free thyroid hormone (T₃ and T₄) concentrations, determine serum iodothyronine binding characteristics and estimate thyroid stimulating hormone (TSH) activity in sera of coastal and inland koalas.
Design A prospective study.
Procedure Koala serum T₃ and T₄ were measured by radioimmunoassay. T₄ binding parameters were determined by radioligand binding and electrophoresis. Koala TSH values were determined by bioassay.
Results Mean total T₄ concentrations were 3.2 ± 2.1 nM although values were significantly higher in inland-dwelling females in comparison to coastal-dwelling males. Free T₄ was 3.3 ± 2.1 pM. Total and free T₃ were 0.4 ± 0.2 nM and 1.4 ± 0.9 pM respectively, although these values were at the lower end of the assay detection limit and should be viewed with reservation. Electrophoresis of [¹²⁵I]-T₄-labelled serum revealed only two proteins of electrophoretic mobility similar to human transthyretin (TTR) and albumin. Scatchard analysis of T₄ binding to serum gave a curvilinear plot, which could be resolved into two binding sites with affinities identical to that of TTR and albumin but both of low concentration. The bioactivity of the TSH present in the sera was measured using a cell line (JP09) transfected with the human TSH receptor. The mean level of stimulation found in the sera corresponded to a bovine TSH activity of less than 10 mU/L.
Conclusion These results suggest that the serum concentrations of free and total thyroid hormones in koalas are low compared to other marsupials and very low compared to eutherian mammals. The mechanism of maintenance of euthyroidism in this species remains to be determined.     

Screening semen from koalas (Phascolarctos cinereus) for Chlamydia species by PCR

Artificial insemination is a valuable method for facilitating genetic exchange between captive colonies of koalas (Phascolarctos cinereus) and for the maintenance of genetically important remnant populations. However, to reduce potential disease transmission, their semen needs to be screened for venereal diseases caused by organisms such as Chlamydia species. Semen samples from 11 koalas, eight of them with clinical signs of cystitis, were examined for the presence of Chlamydia by an optimised PCR assay. Chlamydia was detected in semen from seven of the 11 animals.     

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.     

Chronic vascular catheterisation of the koala and the rabbit

SUMMARY: Vascular access ports were surgically placed, first in rabbits maintained under laboratory conditions, and then in koalas maintained in a wildlife park. The ports remained patent for 9 months in rabbits and for up to 13 months in the koalas and were removed successfully. They allowed collection of blood samples without assistance or disturbance in koalas, and without stress as reflected by plasma cortisol concentration. The use of retention rings on the cannula tubing is recommended.     

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.