HM Medical Clinic

Proceedings of the 13th australasian vertebrate pest conference, wellington, new zealand, 2005

13th Australasian Vertebrate
Keven Drew
Wellington, New Zealand
2-6 May 2005
Hosted by Manaaki Whenua – Landcare Research
PO Box 69, Lincoln 8152, New Zealand. www.landcareresearch.co.nz
A PRACTICAL BAIT MARKER FOR BRUSHTAIL POSSUMS – WHISKER
MARKING BY RHODAMINE B
Penny Fisher and Louis Tremblay
Landcare Research, PO Box 69, Lincoln, Canterbury, New Zealand
ABSTRACT: Practical field measurement of bait uptake by brushtail possums in New Zealand
will become more important as new bait types, deployment methods, and active control agents
reach field-testing stage. Potential markers of bait uptake were identified, fed to captive possums
and efforts made to detect subsequent physiological marking. Clenbuterol (in hair) and
amiodarone (in blood) were not suitable, but rhodamine B (RB) marked possum whiskers
reliably. Further investigation with captive possums dosed with 10 mg/kg RB found no
significant differences in the mean proportions of marked whiskers at 2, 6, 10, 20 and 40 weeks
after dosing, indicating that marking persistence could be at least 40 weeks in field conditions.
There were also no effects of the position of marked whiskers in the mystacial array on
persistence of marking, and no significant differences in the mean proportions of marked
whiskers in male and female possums. The probability that a randomly selected whisker was
marked was 0.6, indicating that six whiskers should be used as the minimum sample size in field
applications of RB. Fluorescence microscopy thus provides a reliable method for detecting
marking in possum whiskers in field applications of RB as a bait marker.
INTRODUCTION

Bait delivery of active agents (e.g. toxicants, biocontrol agents, vaccines) to populations of
brushtail possums (Trichosurus vulpecula) in New Zealand is currently expected to retain a
significant role in broad-scale management of their impacts. The efficacy of such control
strategies relies on a high proportion of a field population encountering and consuming bait.
Accurate quantification of field bait uptake will become more important as new bait types,
deployment methods, and active agents reach field-testing stage. Techniques for measurement of
bait uptake by possum populations require reliable identification of individuals that have
consumed baits in the field, and are currently limited by practical constraints. Ideal
characteristics of a compound that acts as a biological marker of bait uptake include ease of
application to bait material, no effect on bait acceptance, and easily detected, relatively persistent
marking. Markers with increased persistence are important for field study logistics in order to
allow sufficient time for the recovery of animals to be examined for marking. Three compounds
(clenbuterol, amiodarone and rhodamine B) were assessed against these criteria as bait markers
in captive possums. Clenbuterol is a cardiovascular drug that can be detected in hair following
oral administration and has been used successfully as a bait marker in dogs (Gleixner et al.
1998). Amiodarone is another cardiovascular drug, with a long half-life in humans and rats (e.g.
Gill et al. 1992).
Rhodamine B (RB) is a non-toxic dye that fluoresces orange under ultraviolet light, and
following ingestion can produce systemic marking in hair, detectable through fluorescence
microscopy (Fisher 1999). This property of RB has been utilised in bait marker techniques for
European badgers (Meles meles) (Southey et al. 2002), stoats (Mustela erminea) (Spurr 2002),
house mice (Mus domesticus) (Jacobs et al. 2002) and Australian native mammals, including
brushtail possums (Fairbridge et al. 2003). In possums, contact marking by RB of the mouth and
gastrointestinal tract was found to persist for approximately 1 week (Morgan 1981). Greater
persistence was expected if RB could be shown to reliably mark possum hair (whiskers), and in
following trials we sought to more accurately define the persistence and incidence of systemic
rhodamine B marking in possums using fluorescence microscopy.

METHODS
1. Marking of captive possums by rhodamine B, clenbuterol and amiodarone
Thirty wild-caught brushtail possums were acclimatised to indoor individual housing at the
Landcare Research animal facility, Lincoln. They were dosed with three potential bait markers in
a ration of palatable food (non-toxic cereal pellet bait), and subsequently sampled as shown in
Table 1. Treatments were prepared by soaking individual rations (25-g pellets) in 1.5 mL of a
solution of the appropriate marker in water (37.14 mg/mL RB, 0.33 mg/mL clenbuterol, or 2.0
mg/mL amiodarone), then drying at 40°C for 2 h. Possums were offered treated pellets (Table 1)
for a maximum of three consecutive days, until the required dose (either 50 mg RB, 0.5 mg
clenbuterol, or 3 mg amiodarone) was consumed.
For RB sampling, possums were lightly anesthetised with CO2:O2 (2:1) and three mystacial
vibrissae (whiskers) were sampled from each by plucking, ensuring that the bulb of the whisker was removed intact. Whiskers were sampled at specified intervals after the first and a second dose of RB (Table 1) and washed and mounted for fluorescence microscopy as described by Fisher et al. (1999). Microscopy was carried out using a Zeiss Photomicroscope III, with a high-performance filter set for rhodamine B 200, comprising band pass interference exciter filter BP546/12, barrier filter LP590 and chromatic beam splitter FT570, a halogen UV light source and ×2.5 objective. Individual whiskers were recorded as ‘marked' or ‘not marked' and the position and appearance of any marking described. For clenbuterol marking, sufficient fur to yield approximately 50 mg of hair powder was plucked, soaked in water for 1 h, rinsed twice with 30 mL water, twice with 30 mL 0.1% bovine serum albumin (BSA) solution, and three times with 30 mL 0.2% Tween 80, then dried at 60°C. Samples were pulverised to powder with a Mikro-Dismembrator II then stored at −25°C. Clenbuterol was extracted (1 mL of 50 mM aqueous 1,4-dithiothreitol, 50 µl of 5 M sodium hydroxide and 2.5 mL of tertiary butylmethyl ether (TBE)) and shaken overnight at room temperature. The ether was separated by centrifugation and the extraction repeated for 1 h using only TBE. The ether phases of each sample were combined and evaporated at 60°C. The residues were dissolved in 300 µL assay buffer (7.12 g/L Na2HPO4, 2H2O, 8.5 g/L NaCl, 1 g/L BSA, pH 7.2) and appropriate dilutions assayed using an enzyme immunoassay kit (RIDASCREEN® Clenbuterol Fast, R-Biopharm GmbH, Germany). Recovery efficiency was evaluated using powdered hair samples spiked with clenbuterol at 10, 30 and 50 ng/g. For amiodarone marking, possums were anaesthetised with fluothane and blood samples (1 mL) taken from the tail vein into heparinised tubes and centrifuged to obtain serum, which was frozen at −25°C until analysed. Amiodarone serum levels were measured by HPLC as described by Manfredi et al. (1995), with a least detectable level (LDL) of 0.4 µg/mL. Table 1 Treatment of thirty captive possums with three different chemical markers and collection of
samples for analysis of marking
Day Procedure
0
First treatment RB (50 mg per possum) offered (n = 30) Ten whiskers from each possum sampled for RB detection Clenbuterol (0.5 mg per possum) offered (n = 30) Whiskers (28 days after first RB) and fur (13 days after clenbuterol) sampled Second treatment RB (50 mg per possum) offered (n = 30) Whiskers (56 and 28 days following first and second RB treatments, respectively) and fur (41 days following clenbuterol treatment) sampled Fur (69 days following clenbuterol treatment) sampled Amiodarone (3 mg per possum) offered (n = 12, 6 male and 6 female) Blood sampled for amiodarone (14, 29, 42 and 56 days after treatment)
2. Persistence and reliability of detection of rhodamine B marking in possums
Twenty-four wild-caught possums (12 M, 12 F) were acclimatised to captivity as before, briefly
anaesthetised with fluothane, weighed and gavage dosed with 10 mg/kg RB. This dose was
selected to simulate a field situation where a 3-kg possum fed on 5 g of bait containing 0.5% RB
(w/w). Groups with equal sex ratios were killed for sampling of all whiskers after 2 weeks (n =
6), 6 weeks (n = 6), 10 weeks (n = 4), 20 weeks (n = 4), and 40 weeks (n = 4). Whiskers were
plucked and washed as previously described, identified according to their location on the possum
(Fig. 1), and stored in individual bags (rather than mounted on slides). Strong bilateral symmetry
exists in possum vibrissae (Lyne et al. 1974), and it was assumed that marking on the left-hand
side would be representative of that on the right-hand side. All left-side whiskers were examined
for markings using fluorescence microscopy as described. Individual whiskers were recorded as
‘marked' or ‘not marked' and the position and appearance of any marking described. To
investigate the effects of sex, time since dosing, and location of whiskers (row) on the proportion
of marked whiskers, a generalised linear mixed model was fitted to the data, using GenStat®
version 6.1 (GenStat Committee 2002).
Fig. 1 Location of mystacial, genal
and supraorbital vibrissae follicles
of the brushtail possum (adapted
from Lyne et al. 1974).

RESULTS
1. Marking of captive possums by rhodamine B, clenbuterol and amiodarone
Possums consumed an average of 47.4 mg RB each on the first treatment, and 31.3 mg each on
the second treatment, and may have recognised RB baits as less palatable on a second exposure.
Marking of possum whiskers by RB appeared as a fluorescent-orange band that could be
distinguished from the natural background fluorescence often present at the hair root. Single RB
bands were found on most whiskers. After the second RB treatment, fewer whiskers showed
double bands (Table 2). Possums consumed an average of 0.44 mg clenbuterol over 3 d, but
clenbuterol was not detected in hair extracts. It appeared something in the hair extract, possibly
increased pH, was interfering with the assay so that high background levels were present and a
standard curve could not be produced. The 12 possums offered amiodarone consumed an average
of 2.5 mg each, but amiodarone was not detected in their serum.
Table 2 Number of possum whiskers marked with rhodamine B.
No. whiskers marked Day 14
Day 56 (after first RB dose) &
per sample
Day 28 (after second RB dose)
1 band
66.7 76.7 65.5 12.6 28/29 28/30 26/29 10/29 *30 possums were used but two slides were lost

2. Persistence and reliability of detection of rhodamine B marking in possums
There were no significant differences in the mean proportions of marked whiskers at 2, 6 10, 20
and 40 weeks after dosing (Fig. 2). There was no significant effect of position on the marking of
whiskers (Fig. 3), and no significant differences in the mean proportions of marked whiskers in
male (0.65 ± 95% CI 0.53 – 0.76) and female (0.53 ± 95% CI 0.40 – 0.65) possums.
Fig. 2 Proportion of left-hand-
side possum whiskers (y-axis) ±
SE marked at different sample times after dosing with 10 mg/kg rhodamine B.
Fig. 3 Proportion of left-hand-side possum whiskers (y-axis) ± SE, marked after dosing with 10
mg/kg RB.
The overall mean proportion of left-hand-side whiskers with RB marking was 0.597 (upper 95%
CI = 0.6801, lower 95% CI = 0.5071), suggesting that approximately 60% of vibrissae were
actively growing at the time of dosing with RB. The probability that a random whisker is marked
was approximated as 0.6, with the complementary probability that a random whisker is not
marked being 0.4, so that the probability that at least one whisker will be marked in a random
sample of four was 0.9744 (above 95% chance).
DISCUSSION

Neither amiodarone nor clenbuterol appeared to be suitable bait markers for possums. It was not
possible to fully evaluate clenbuterol as a bait marker because of the limited flexibility of the
relatively expensive commercial kit; regardless, the preparation of hair samples for clenbuterol
extraction is labour intensive and requires specialised equipment, which reduces its practicality
as a bait marker. However, RB marking can be reliably determined in whisker samples taken
from possums up to 40 weeks after the last intake of RB. The practical implications of these
results are that male and female possums are equally likely to be marked by an RB intake of 10
mg/kg or more. Whiskers sampled to examine for marking can be taken from any position in the
mystacial, supraorbital or genal array with an equal probability of detecting marking if it is
present. Allowing for sampling of club hairs (i.e. inactive whiskers not likely to be marked) and
breakage of whiskers during sampling in field conditions, it is recommended that six whiskers
should be used as the minimum sample size in field applications of RB as a bait marker in
possums. For practical application in field conditions, when possums need to be released after
sampling, it is suggested that the genal and supraorbital whiskers of one side are sampled.
Successive intakes of RB can produce a series of fluorescent bands in whiskers (e.g. Fisher 1999;
Purdey et al. 2003), as demonstrated in this trial with possums. The apparently lower incidence
of ‘double marking' in comparison to ‘single marking' may be due to the differences in
individual whisker growth at the times of ingestion of RB, e.g. the probability that a whisker is
actively growing and therefore marked by RB at multiple times of RB ingestion may be lower
than for a single instance.
ACKNOWLEDGEMENTS

This was a contracted research project for the Animal Health Board. Experimental procedures
involving the use of animals were approved by the Landcare Research Animal Ethics Committee
(Project No. 02/10/01). We thank Julie Turner, Karen Washbourne, Vicki Bunt and Michael
Werner for maintenance and husbandry of the possums; Guy Forrester for statistical advice; and
Christine Bezar for editing.
REFERENCES

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