One size does not fit all : monitoring faecal glucocorticoid metabolites in marsupials

Marsupial research, conservation, and management can benefit greatly from knowledge about glucocorticoid (GC) secretion patterns because GCs influence numerous aspects of physiology and play a crucial role in regulating an animal's response to stressors. Faecal glucocorticoid metabolites (FGM) offer a non-invasive tool for tracking changes in GCs over time. To date, there are relatively few validated assays for marsupials compared with other taxa, and those that have been published generally test only one assay. However, different assays can yield very different signals of adrenal activity. The goal of this study was to compare the performance of five different enzyme immunoassays (EIAs) for monitoring adrenocortical activity via FGM in 13 marsupial species. We monitored FGM response to two types of events: biological stressors (e.g., transport, novel environment) and pharmacological stimulation (ACTH injection). For each individual animal and assay, FGM peaks were identified using the iterative baseline approach. Performance of the EIAs for each species was evaluated by determining (1) the percent of individuals with a detectable peak 0.125–4.5 days post-event, and (2) the biological sensitivity of the assay as measured by strength of the post-event response relative to baseline variability (Z-score). Assays were defined as successful if they detected a peak in at least 50% of the individuals and the mean species response had a Z ⩾ 2. By this criterion, at least one assay was successful in 10 of the 13 species, but the best-performing assay varied among species, even those species that were closely related. Furthermore, the ability to confidently assess assay performance was influenced by the experimental protocols used. We discuss the implications of our findings for biological validation studies

Univariable analysis assessing select epidemiological variables as predictors for the presence of active herpesvirus infection in common wombats ^a.

<p>^a Reference levels are indicated by odds ratio of 1.0. Results highlighted in bold (log likelihood p ≤0.25) represent variables included in the initial multivariable model, with the exception of pouch young which was excluded due to zero prevalence of herpesvirus infection in females lactating/with pouch young. In the final model (n = 33) only age (adult/aged) and body condition score (≤ 2) were identified as significant factors (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133807#pone.0133807.t008" target="_blank">Table 8</a>). n/a = not applicable.</p><p>Univariable analysis assessing select epidemiological variables as predictors for the presence of active herpesvirus infection in common wombats <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133807#t007fn001" target="_blank">^a</a>.</p

Predicted amino acid alignment and phylogenetic tree of the novel marsupial herpesviruses.

<p>A: Alignment of the predicted amino acid sequence of a portion of the DNA polymerase gene of the novel marsupial herpesviruses, along with other herpesviruses from the three herpesvirus sub-families. B: Maximum likelihood tree generated from the alignment. Bootstrap values of 100 replicates are displayed on the tree branches. Novel herpesvirus species are underlined. Key: PaHV-2 = papiine herpesvirus 2 (AAN87165.1); SaHV-1 = saimiriine herpesvirus 1 (YP_003933809.1); HHV-1 = human herpesvirus 1 (NP_044632.1); MaHV-1 = macropodid herpesvirus 1 ([<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133807#pone.0133807.ref022" target="_blank">22</a>]); <u>VoHV-3 = vombatid herpesvirus 3 (novel sequence)</u>; PCMV = porcine cytomegalovirus (AF268042.1); HHV-6 = human herpesvirus 6A (NP_042931.1); HHV-4 = human herpesvirus 4 (YP_401712.1); HHV-8 = human herpesvirus 8 (ACY00400.1); SaHV-2 = saimiriine herpesvirus 2 (NP_040211.1); BHV-4 = bovine herpesvirus 4 (NP_076501.1); DaHV-1 = dasyurid herpesvirus 1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133807#pone.0133807.ref013" target="_blank">13</a>]; <u>DaHV-2 = dasyurid herpesvirus 2</u> (novel sequence); MaHV-3 = macropodid herpesvirus 3 (ABO61861.1); <u>MaHV-5 = macropodid herpesvirus 5 (novel sequence). PeHV-1 = peramelid herpesvirus 1 (novel sequence</u>); PotHV-1 = potoroid herpesvirus 1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133807#pone.0133807.ref010" target="_blank">10</a>]; <u>VoHV-1 = vombatid herpesvirus 1 (novel sequence)</u>; <u>VoHV-2 = vombatid herpesvirus 2 (novel sequence);</u> PhaHV-1 = phascolarctid herpesvirus 1 (AEX15649.1); PhaHV-2 = phascolarctid herpesvirus 2 (AFN66528.1); EHV-2 = equine herpesvirus 2 (NP_042605.1); CpHV-2 = caprine herpesvirus 2 (ADV92276.1).</p

Univariable analysis assessing select epidemiological variables as predictors for the presence of herpesvirus DNA in koalas ^a.

<p>^a Reference levels are indicated by odds ratio of 1.0. Results highlighted in bold (log likelihood p ≤ 0.25) represent variables included in the initial multivariable model, with the exception of season as the timing of sampling correlated with the location at which it occurred and was thus excluded. In the final model (n = 68) only the presence of <i>Chlamydia pecorum</i> was identified as a significant factor (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133807#pone.0133807.t008" target="_blank">Table 8</a>). n/a = not applicable.</p><p>Univariable analysis assessing select epidemiological variables as predictors for the presence of herpesvirus DNA in koalas <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133807#t005fn001" target="_blank">^a</a>.</p

Anatomical sites of herpesvirus DNA detection in swab samples collected from Australian marsupials in 2010 and 2011.

<p>^a Only species that were sampled in relatively large numbers, from multiple anatomical sites, are included.</p><p>^b Herpesvirus DNA was sometimes detected in more than one swab from the same animal, swabs were not collected from every anatomical site from every animal.</p><p>Anatomical sites of herpesvirus DNA detection in swab samples collected from Australian marsupials in 2010 and 2011.</p

Univariable analysis assessing select epidemiological variables as predictors for the presence of herpesvirus DNA in eastern grey kangaroos ^a.

<p>^a Reference levels are indicated by odds ratio of 1.0. Results highlighted in bold (log likelihood p ≤ 0.25) represent variables included in the initial multivariable model, with the exception of presence of pouch young/lactation as it is correlated with sex and thus excluded. Backward elimination of non-significant variables yielded no significant variables. Multivariable analysis was repeated including presence of pouch young/lactation as a variable instead of sex. Age was excluded from the model due to collinearity. In the final model (n = 42) only the absence of pouch young/lactation was identified as a significant factor (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133807#pone.0133807.t008" target="_blank">Table 8</a>). n/a = not applicable.</p><p>Univariable analysis assessing select epidemiological variables as predictors for the presence of herpesvirus DNA in eastern grey kangaroos <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133807#t004fn001" target="_blank">^a</a>.</p

Univariable analysis assessing select epidemiological variables as predictors for the presence of herpesvirus DNA in Tasmanian devils ^a.

<p>^a Reference levels are indicated by odds ratio of 1.0. Results highlighted in bold (log likelihood p ≤ 0.25) represent variables included in the initial multivariable model, with the exception of season as it was directly influenced by timing of management procedures, and therefore correlated with captive status and was thus excluded. In the final model (n = 50) only captivity was identified as a significant factor (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133807#pone.0133807.t008" target="_blank">Table 8</a>). n/a = not applicable.</p><p>Univariable analysis assessing select epidemiological variables as predictors for the presence of herpesvirus DNA in Tasmanian devils <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133807#t006fn001" target="_blank">^a</a>.</p

Electron micrographs of novel herpesviruses.

<p>Transmission electron microscopy was used to visualise herpesviruses in cultures of primary wombat kidney cells. Herpesvirus capsids (arrowheads) of VoHV-1 (A) and VoHV-2 (B) are shown. Bar = 100 nm.</p