Pharmacokinetic profile of oral and subcutaneous administration of paracetamol in the koala (Phascolarctos cinereus) and prediction of its analgesic efficacy.

The pharmacokinetic profile of paracetamol in koalas is described when administered orally at 15 mg/kg; followed by the same dose, administered every 12 hours (hrs), repeated five times. After the initial oral administration, the median (range) maximal plasma concentration (Cmax), the time Cmax was reached (Tmax) and elimination half-life (t1/2) were 16.93 µg/mL (13.66 to 20.25 µg/mL); 4 hrs (4 to 8 hrs) and 5.54 hrs (4.66 to 7.67 hrs), respectively. When paracetamol was administered orally at 15 mg/mL every 12 hrs, the trough total plasma concentration range remained comparable to the therapeutic range in humans i.e. 4 to 20 µg/mL that is known to provide some analgesia. However, there is a smaller proportion of free drug (i.e. not bound to plasma proteins; and the active form) available in koala plasma (approximately 40% unbound) compared to human plasma (approximately 80% unbound). Consequently, even when there are similar total drug plasma concentrations in both koala and human plasma, the therapeutic efficacy may be reduced in koalas compared to humans. The initial oral dose and subsequent twice daily doses resulted in no obvious adverse effects in any koala. Haematology, plasma electrolyte and biochemical analyte values remained within their reference ranges eight hrs after the last dose but there was a significant change in alanine transaminase (ALT) levels (an increase), and in total protein (a decrease) (both p = 0.03). A dose of 15 mg/kg was also administered as a subcutaneous injection, diluted 50:50 with saline, to two koalas. As the oral formulation and the subcutaneous administration resulted in comparable absorption, the study focused on the oral profile. Based on these results there is an argument to recommend a slight increase in the oral paracetamol dose for the koala, however further investigation is required to confirm whether repeated administration of a slightly higher dose may be associated with more severe or additional significant changes in haematology, electrolytes or biochemical analytes. However, a preferable recommendation would be to administer this dosage of paracetamol in combination with another analgesic such as tramadol, as a subcutaneous injection, to improve efficacy.

Evaluation of a biosecurity survey approach for contamination by Chlamydia pecorum in koala rehabilitation, field capture, and captive settings.

Transmission of Chlamydia pecorum between koalas is a potential risk in field capture or rehabilitation settings, where koalas are held in proximity to each other, or equipment is shared between animals. Given the impact of C. pecorum on koala welfare and population viability it is surprising that quarantine and disinfection protocols in a koala rehabilitation facility or capture settings have not previously been evaluated. This study aimed to evaluate an approach, based on the detection of chlamydial DNA and cell viability, to determine the degree of environmental contamination within a koala care facility. Various fomite sites associated with koala care at a koala rehabilitation facility in New South Wales, Australia were identified as potential sources of chlamydial contamination, following exposure to koalas known to be infected with C. pecorum. Fomite sites were swabbed following exposure, and again after decontamination procedures were carried out. Samples were tested for the presence of chlamydial DNA using qPCR and viability using both RT-qPCR and cell culture. From a total of 239 sampling events, 30 tested qPCR positive for chlamydial DNA, with 19 and 11 samples corresponding to pre-decontamination and post-decontamination events respectively. Detection of chlamydial DNA appeared to be most common in the examination room, especially on fomite sites in direct contact with koalas. Physical removal of chlamydial DNA, or its degradation by the elements, appeared to be more common on outdoor enclosures, clothing, and hands. Based on the cell culture assay, of the pre-decontamination samples with chlamydial DNA, eight had viable chlamydial cells, two of these at low levels. Of the post-decontamination samples with chlamydial DNA, one had a moderate number, and one had a very low number of viable chlamydial cells. RT-qPCR was unsuccessful in determining cell viability due to low yields of RNA and high levels of contaminants from the environmental samples. The outcomes of this study provide a knowledge base for the design of future biosecurity evaluation guidelines in captive and koala rehabilitation facilities. The higher incidence of chlamydial DNA detection by qPCR than viable organism highlights the need to use viability assays in similar studies. However, further investment is still needed to optimise these methods and improve sensitivity for complex environmental samples.

Comparing immunochromatography with latex antigen agglutination testing for the diagnosis of cryptococcosis in cats, dogs and koalas.

Although the point-of-care cryptococcal antigen lateral flow assay (LFA) has revolutionized the diagnosis of cryptococcosis in human patients, to date there has been no large-scale examination of this test in animals. We therefore assessed the LFA in cats, dogs and koalas suspected of having cryptococcosis. In sum, 528 serum specimens (129 from cats, 108 from dogs, 291 from koalas) were tested using the LFA and one of two commercially available latex cryptococcal antigen agglutination test (LCAT) kits. The LCAT is a proven and well-accepted method in veterinary patients and therefore taken as the "gold standard" against which the LFA was compared. The LFA achieved a sensitivity of 92%, 100%, and 98% in cats, dogs, and koalas, respectively, with corresponding negative predictive values of 94%, 100%, and 98%. The specificity of the LFA was 81%, 84%, and 62% for cats, dogs, and koalas, respectively, with corresponding positive predictive values of 76%, 48%, and 69%. These findings suggest the most appropriate role for the LFA is as a screening test to rule out a diagnosis of cryptococcosis in cats, dogs, and koalas. Point-of-care accessibility makes it equally suited for use in the field and as a cage-side test in veterinary hospitals. The suboptimal specificity of the LFA makes it less suited to definitive confirmation of cryptococcosis in animals; therefore, all LFA-positive test results should be confirmed by LCAT testing. The discrepancy between these observations and the high specificity of the LFA in humans may reflect differences in the host-pathogen interactions amongst the species.

Effects of Eucalypt Plant Monoterpenes on Koala (Phascolarctos Cinereus) Cytokine Expression In Vitro.

Protective immunity is crucial for survival of any species, though the koala as a specialist feeder adapted to an exclusive diet of eucalypts that contain plant secondary metabolites of varying toxicity and of immunomodulatory property. Being constantly exposed to such dietary chemicals it raises the question of their immune effects in a specialist eucalypt feeder. This study demonstrates that natural levels of circulating eucalypt plant secondary metabolites have dose dependent in vitro effects on cytokine expression of koala peripheral blood mononuclear cells, suggesting a potential trade-off of reduced function in multiple arms of the immune system associated with koala's use of its specialized dietary niche.

Ingestion and Absorption of Eucalypt Monoterpenes in the Specialist Feeder, the Koala (Phascolarctos cinereus).

The koala is a specialist feeder with a diet consisting almost exclusively of potentially toxic eucalypt leaves. Monoterpenes, an abundant class of plant secondary metabolites in eucalypts, are highly lipophilic. Chronic absorption and systemic exposure can be anticipated for the koala, causing health effects in various ways when consumed in high amounts, but particularly causing alterations in immune function in this species. Therefore, careful leaf selection, efficient detoxification pathways, and other specialist adaptations are required to protect animals from acute intoxication. This is the first paper providing insight into the systemic exposure of koalas to these compounds. Profiles of six selected major monoterpenes were investigated in the ingesta of deceased koalas from four different regions of NSW and South-East Queensland. Concentrations of the same compounds were measured in lymphoid tissues of deceased koalas and in the blood of live koalas from other regions of NSW. Analytical methods included liquid extraction and solid-phase micro-extraction, followed by gas-chromatography/ mass-spectrometry. Concentrations in the ingesta of individual animals vary remarkably, though the average proportions of individual monoterpenes in the ingesta of animals from the four different regions are highly comparable. Blood concentrations of the selected monoterpenes also varied considerably. The highest blood concentrations were found for 1,8-cineole, up to 971 ng/ml. There was similarity between circulating monoterpene profiles and ingesta profiles. Based on the observed lack of similarity between blood and lymph tissue concentrations, individual monoterpenes either exhibit different affinities for lymphatic tissue compared to blood or their accumulation in blood and lymph tissue differs temporally. In general, blood monoterpene concentrations found in koalas were low compared to those reported in other marsupial eucalypt feeders, but significant concentrations of monoterpenes were detected in all samples analysed. This data on blood and lymphatic tissue monoterpene concentrations builds the fundamental groundwork for future research into the effects of dietary monoterpenes on various biological processes of specialist herbivores and into the significance of these animals' metabolic and behavioural strategies for coping with these compounds. We have shown that the systemic exposure of koalas to potentially anti-inflammatory eucalypt monoterpenes is continuous, and we provide data on physiological concentrations which will allow realistic future studies of the effects of monoterpenes on immune cell function.

A Survey of Pesticide Accumulation in a Specialist Feeder, the Koala (Phascolarctos cinereus).

To maintain profitability in Australia's agricultural and urban landscapes pesticides are used throughout the range of koala habitats. The koala is a specialist feeder, reliant on metabolic enzyme capacities to utilise a toxic diet of eucalypt leaves and is potentially prone to adverse effects when xenobiotic interactions between dietary and anthropogenic xenobiotics occur. The aim of this study was to investigate accumulation of frequently used pesticides in wild koalas in 4 areas of New South Wales and Queensland. Liver samples of 57 deceased koalas were collected from care facilities and analysed using a modified QuEChERS extraction method followed by GCMSMS, HRLCMS and LCMSMS. No accumulation of any of the 166 investigated pesticides was found. Data indicate hepatic accumulation of pesticides in this species is uncommon even with close interactions with intensive land use. Despite the lack of hepatic bioaccumulation, this study cannot exclude a direct effect on hepatocellular metabolic pathways.