Validation of an Enzyme Immunoassay to Measure Faecal Glucocorticoid Metabolites in Common Brushtail Possums (Trichosurus vulpecula) to Evaluate Responses to Rehabilitation

Abstract

Volunteer wildlife rehabilitators rescue and rehabilitate thousands of native animals every year in Australia. However, there is little known about how exposure to novel stimuli during rehabilitation could affect the physiology of wildlife. We investigated this question in a species that commonly enters rehabilitation, the common brushtail possum (Trichosurus vulpecula). We evaluated five enzyme immunoassays (EIA) to determine the most suitable for measuring faecal glucocorticoid metabolites (FGM) as a proxy for evaluating the response of brushtail possums to potential stressors during rehabilitation. An adrenocorticotrophin hormone (ACTH) challenge was conducted on wild-caught possums to determine the best-performing EIA based on the successful detection of FGM peaks in at least two of three possums. While a number of assays met these criteria, the 11-oxoaetiocholanolone (abbreviation: 72a) EIA was selected as it had the largest amplitude of change in response to the ACTH challenge. This assay was then used to measure FGM concentrations in 20 possums during rehabilitation. There was high variation in baseline FGM concentrations and response to captivity between possums. Significant changes in FGM levels were detected in most possums during captivity, but were not reliably associated with potentially stressful events that were identified by rehabilitators. The probability of an FGM peak occurring within five days of a potentially stressful event was about 50%, regardless of the type of event. Our study has demonstrated that injured and orphaned possums show changes in FGMs during captivity and rehabilitation and has identified events that can induce a physiological response in some individuals. We recommend that research now focus on the relationship between these responses during rehabilitation and pre- and post-release survival.

Keywords: 11-oxoaetiocholanolone; EIA; captivity; corticosterone; cortisol; dermatitis; enzyme immunoassay; wildlife rehabilitation; wildlife rescue.

Figure 1

Boxplot of faecal glucocorticoid metabolite (FGM) concentrations (ng/g) as detected by the enzyme immunoassays 72a, AA-corticosterone, AA-cortisol, CJM006 and R4866 for all four brushtail possums across 9 days in captivity following the ACTH challenge (including the pre-challenge scat on day zero; boxplot depicts the median (dark line), two hinges (first and third quartiles), two whiskers (extending from the hinges to the smallest and largest values no further than 1.5 × inter-quartile range) and dots (outlying values)).

Figure 2

Faecal glucocorticoid metabolite (FGM) concentrations (ng/g) over 9 days (between 16th and 26th April 2018) in captivity for brushtail possums (Females 1, 2, 3 and Male) as detected by the enzyme immunoassays 72a, AA-corticosterone, AA-cortisol, CJM006 and R4866 (note: ACTH was administered on the night of capture as indicated by an arrow within 24 h of the first faecal sample collection).

Figure 3

Boxplots of baseline faecal glucocorticoid metabolite (FGM) concentration thresholds (ng/g; mean plus 1.5 SD of baseline concentrations) by sex (female n = 14, male n = 5, unknown n = 1), length of stay (short-term n = 2, long-term n = 9, orphan n = 9) and age group (adult n = 8, juvenile n = 9, sub-adult n = 3) for possums in rehabilitation. Boxplots depict the median (dark line), two hinges (first and third quartiles), two whiskers (extending from the hinges to the smallest and largest values no further than 1.5 × inter-quartile range) and dots (outlying values).

Figure 4

Faecal glucocorticoid metabolite (FGM) concentrations (ng/g) for 20 possums during rehabilitation. Potential stress events are represented as arrows, indicating when they occurred relative to sample collection (E = Enter rehabilitation; C = No cover; NC = New cage; NP = New possum; BR = Back rider; H = Handling; S = Separated; I = Injection; OM = Oral medication; EB = excited behaviour and movements; Es = escaped cage; U = a lot of urine noticed in cage; possum 8295 received daily oral medication in addition to labelled events). The grey dashed line represents the baseline threshold above which values are classified as peaks.