hal- canica isolates. (Robinson et al., 1982). The possum is considered to be the ... Ashby,. 1995). Leptospira interrogans serovar balcani- ca infection is found.
JuurmaI
of
\tll(IIt[(
I)i>ees>->.
:132 l)>sas,’
T’ \\‘ihllife
LEPTOSPIROSIS
IN BRUSHTAIL
INTERROGANS
SEROVAR
POSSUMS:
BALCANICA
1997. PP 254_215) Ass>>ciat>>>,m 19Y7
IS LEPTOSPIRA
ENVIRONMENTALLY
TRANSMITTED? T. D. Day,1-2
J. R. Waas,
‘Department 2
of Biological
Animal
Behaviour
and
Welfare
In New
ABSTRACT:
C. E. O’Connor,2
Sciences,
University
Research
Centre,
the
biological
Zealand,
P. W. Carey,2
of Waikato,
L. R. Matthews,2
Hamilton,
AgResearch,
New
Ruakura
control
and
Research
of introduced
Centre,
Hamilton,
brushtail
New
possunms
Zealand
(Trichosurus
may
be the only affordable option for achieving a significant long term pest numbers on a national scale. Leptospira interrogans serovar balcanica is among biocontrol agents and vectors currently being investigated for this purpose. As thie pathways of L. interrogans serovar balcanica between possums are poorly understood, tive of the study was to determine whether infection could result from exposure to vulpecula)
Pearson2
A. J.
Zealand
reduction in the potential
transmission tine objeccontaminated environments. Sixteen individually housed, uninfected possums, in three groups, were reginlarly exposed over a period of 32 days to contaminated cages or grass enclosures of 16 other experimentally infected possums all shedding leptospires in their urine. None of the 16 challenged possums developed serological evidence of L. interrogans serovar balcanica infection. These results suggest that this organism is unlikely to be transmitted environmentally, supporting previoums circumstantial evidence that social contact may be required for transmission of L. interrogans
balcanica
serovar
ica,
possums.
between
words: biological
Trichosurus
Brushtail possum, control, environmental
Key
vulpecula,
INTRODUCTION
Brushtail cula,
possums,
are
New
trade
mid
19th
(Pracy,
to occupy
century
1974), more
Control
most
through
(Parliamentary Environment, from Australia to
with the population be between 60 and
selective
browsing competition
young,
adversely
establish
(Nugent,
1995)
and
predation
nes, 1995). Possums vectors, maintaining tuberculosis ingstone,
native
adequate
for
long
term and
to
logical control is considered affordable option for achieving major
reduction
in
possum
(Jolly
et
sums
and
presently
under
and
possum 1993).
Bio-
for
serovars
L.
to
be
hardjo
1976;
serovar
first
isolates
serovar
(Haththe
sero-
in
L.
1975;
later
hardjo
of
lepwere
interrogans de
Lisle
identified
balcan et
New
possums of
(Brockie,
Hathaway
which
survival
isolations
Zealand
were
parasites
serovars
The
but
of
lepto-
organisms
present
interrogans
serovar
1975)
of
from
are
thought
al., 254
Two
New
interrogans
numbers
obligate
species
in
al.,
to be the only a long-term
pos-
investigation.
balcanica.
tospires
strain
potential
Hebdomadis
Zealand:
and in-
to suronly
i nterrogans
are
1978).
group
Such
mode
One
among
a host
away,
impair
unable infect
a possum-specific
Leptospires
require
or
humane,
(Leptospira
in
en-
biology.
possums,
1993).
not
agent
reproduction
be
(Jolly,
is
either
populations
of possum
have
balcanica)
(In-
are
al.,
a disease
an infectious
control
aspect from
spirosis
of
poisons and
away
achieved
possum
by
should
vive
action
and,
fauna
national Wright,
ba/can-
of
in
or
other
an agent
(Tb) to cattle and deer (Liv1991). Present methods of con-
(Atkinson
be
introduction
to
some
are also important and spreading bovine
trolling possums based on traps involve ongoing expense control
might
Zealand
gineered
a fur
recently estimated 70 million (Batchelor
affect
Zealand
present
New
and Cowan, 1988). Possums cause severe damage to New Zealand’s native forests by through
New
the
currently
possums have spread 90% of New Zealand,
than
serovar
1992).
vulpe-
Zealand’s
serious vertebrate pest Commissioner for the 1994). Initially introduced in the
interrogans
throughout
Trichosurus
considered
Leptos-pira
transmission.
ica al.,
(Marshall 1978).
et
as L. et
Lepto-
DAY ET AL.-LEPTOSPIROSIS
spira thought
interrogans to have
Zealand
by
and
infected
New
dente, of
New
has
from
European
canica
isolates
The
maintenance balcanica (Hathaway
onstrate
biochemical
al.,
of renal it
al.,
son
and
tious
(Pearson
and is found
sums
over
18
lence
of infection Ashby,
ies
between in
et
larger
the
al.,
for
in the
et
many
the 1991;
Pearvar-
New a
Zealand
higher
preva-
than
in
Several
forests
aspects
of
habitats, nest
such
sharing
contact
the
ground
et
al.,
1991)
in
prevalence.
species
prevato differ
prevalence
in
on
in pos-
al.,
of increased Cowan
balcani-
and
(e.g.,
and with
(see may
cattle),
in-
referaccount contact
with infected urine by an uninfected animal is the principal transmission pathway of leptospires; leptospires shed in urine may
survive
and
pasture
and
under
favorable
They
after
remain
they
However,
remain
damp
lose other
soil
infective for
up
conditions
(Fame,
infectious
and
motility
(Hathaway,
species
in water, to
(e.g.,
the
possibility
has
as
not
a biocomtrol
in
(Day,
yet
been
sys-
present
study
whether
balcanica step
other
of environ-
L.
could
transmitted a first
infect
contact
The
to determine
MATERIALS
appear
The
ranges,
increase
In
not
in pastoral
material
ences
age
1991).
home
possibility
fected
of
habitats
behavior
tial
as
maex-
between assessing
in-
be
enpos-
its
poten-
vector.
infec-
serovar
with
pastoral
sums,
captive
highly
predominantly
habitats
vironmentally
Pear-
to
social
serovar
(Hor-
1991;
shown
investigated.
designed
balcanica
Sexually have been
in
transmission
terrogans
1995).
does
However,
mental was
been
tramspreva-
with L. interrogans intraperitomeal inoc-
by
when
tematically
Island
among
Ashby,
1978),
(Cowan
as
and
1995).
(Hathaway,
possum
et al.,
(Cowan
sexes
and
have
possums.
seropositive
appears
mo
ulatiom possums
social
serovar
infected
signs
of
puberty
age-specific
mature possums. possums that
dem-
sug-
than
the
interrogans
in sexually ture captive
1996).
255
leptospires around
likely
on
balcanica
all
North
less
L.
of
transmit
behavior
serovar
nature
to
interrogans
son
mild
many
1995)
disease
between
to
lence
IN POSSUMS
1981c). Hathaway (1978) sugenvironmental transmission of serovar balcanica between
perimemtally
characteris-
the
are
Leptospira ca infection
lence
failure
Cowan
the
seroZealand
1981a)
of
the
of clinical
pathogenicity
Ashby,
possums
be
interrogans in New
and
1996;
1982). to
(Hathaway,
populations
et
L.
hal-
serovar
changes
low
Victoria
et al.,
the
percentage
ner
based
A lack
damage
has
possum
was
mission,
1991),
lesions
gest
possums
considered
1978). et
A high
serovar to differ
host for infection
(Cowan
kidney
interrogans its genotype
L. interrogans
var
tic
(Presi-
(Robinson is
to
sexual
analysis
L.
possum
thought
through
enzyme
Wales,
shown
are
Australia
from
Restriction
Zealand
is New
(Hathaway, gested that L. interrogans
possums
South
1984).
balcanica
serovar balcanica introduced to
been
TRANSMISSION
74
days 1994).
viable,
even
1981a). hedgehogs)
AND
METHODS
Possums used in this study were trapped in October 1994 and January 1995 using box traps from the Huiarau Range in the Urewera National Park (38#{176}37’S, 177#{176}04’E) and Kawau Island (36#{176}26’S, 174#{176}50’E), New Zealand. Possums in both areas are reportedly free of L. interrogans serovar balcanica infection (Homer et al., 1996; Pearson and Ashby, 1995). Animals were transported in individual sacks to the Animal Behaviour and Welfare Research Centre (AgResearch Ruakura, New Zealand) where
they X
were placed in individual wire 560 X 1050 mm high) suspended
cages (550 from the
ceiling, containing a feed tray, nest sack, shelf (300 mm from top of cage) and water nipple. Each cage was separated by an opaque PVC divider, providing a physical barrier between possums. Possums were fed on a daily diet of wet mash (200 g of cereal-based possum pellets (Northern Rolling Mill, Auckland, New Zealand) and water at a 1:1.5 ratio) and a single apple. Animals were kept on a fixed 12:12 hr day/night light regime and the temperature in the room was maintained above 10 C. All possums were aged by tooth wear (Winter, 1980) during routine veterinary checks. Serological testing for evidence of L. interrogans serovar balcanica infection was examined using a modified version of the Microscopic Agglutination Test (MAT) by the Central Animal Health Laboratory (AgResearch Wallaceville, New Zea-
256
JOURNAL
OF
WILDLIFE
DISEASES,
VOL.
33,
NO.
land). Cross absorption procedures (Fame, 1982) were used to discriminate between L. interrogans serovar balcanica and other serovars. Positive tests were reported as the greatest seruni dilution at which serum showed a reaction. If no reaction was seen in a 1:50 dilution, results were reported as negative. All procedures used in the present study were approved by the AgResearch Ruakura (Hamilton, New Zealand) and University of Waikato (Hamilton, New
Zealand)
Animal
Ethics
Committees
prior
to
experimentation.
infected ± SD
Experimentally
of 16 animals 7 fennales,
(mean 9
aesthetised with 0.5
males),
in
age
three
3.8 groups,
consisting ± 1.6 yr; were
an-
intraperitoneally ml L. interrogans serovar balcanica inoculum (about 1 X 10 organisms). The inoculum was cultured from an infected New Zealand possum at the Department of Veterinary Pathology and Public Health at Massey University
and
possums
(New
inoculated
Zealand)
and
confirmed
as
New
Zealand possum L. interrogans serovar balcanica isolate, using restriction endonticlease analysis and DNA verification. The first two groups (group 1 and 2), each of four possums, were confirmed to be shedding leptospires 28 days post inoculation (pi), by dark field (DF) microscopy (400X) of mid-stream urine samples. The third group of eight possums (group 3) were shown to be infected with L. interrogans serovar balcanica using MAT serology at 24 days pi. MAT serology was found to be more reliable for detecting establishment of infection than DF microscopy, hence it was used in group 3. The infected possums were used as a source of environmental L. interrogans serovar balcanica contamination in cages and in grass enclosures, to which uninfected possums were exposed. The three groups of infected and three groups of uninfected possums used in this study
were
balanced
for
sex
ratio,
origin
and
mean age. Four recently infected possums (group 1) (3.8 ± 2.2 yr; 2 females, 2 males) were used to contaminate cages with L. interrogans serovar balcanica. For this experiment, sacks were laid on the cage floor, allowing urine, faeces, food scraps and shed leptospires to accumulate within the cages. Nest and floor sacks were not changed for the duration of the experiment. Fluid trapped by the sacks was examined under DF microscopy (400X) for leptospires 14 days after swapping began. Four uninfected possums (4.3 ± 1.9 yr; 2 females, 2 males) were regularly exposed to the cages of the infected possums. Over 32 days, each uninfected animal was interchanged with each infected possum’s cage eight times (each of 24 hr duration). The
2, APRIL
1997
swapping was designed to correspond with the maximum period of leptospire shedding after infection (27 to 60 days pi: Hathaway 1981a). Swapping began on day 29 pi and ended on day 62 pi. The entire procedure was then replicated with another group (groinp 2) of four infected (4.8 ± 1.5 yr 2 fennales, 2 males) and cage
four
uninfected
(3.9
±
1.8
yr
2
females,
2
males) possums in another set of cages. Urine samples were collected froni inninfected possums throughout the cage swapping period and were examined by DF microscopy (400x ) for leptospires. A urine sample was taken 18 day’s after swapping began and subsequently
at
four
day
intervals
until
exposure
ended. Blood samples were taken from uninfected possums between 14 and 28 days after the end of the experiment and tested by MAT serology for L. interrogans serovar balcanica infection.
Urine from the four infected possums in group 1 was also sampled on day 62 pi to ascertain if leptospiruria was still present. Urine from the four infected possums in group 2 was examined by DF microscopy every 7 days throughout
the
swapping
period.
Blood
for
rology was collected from the six surviving infected possums at 152 and 186 day’s pi. Eight individimally fenced, outdoor grass enclosures (ranging from 51 ni2 to 130 ni2), containing a wooden nest box and one or two climbing logs, were used in this ex-perimilent. Also
tnsed,
were
eight
indoor
concrete
pens
(3
to 5 inn2), again with pen. Eight recently
a wooden nest box in each infected possimms (group 3) (3.4 ± 1.3 yr 3 females, 5 males) were transferred (on day 22 pi) fronu individual cages in the quarantine facility’ to individual enclosures, seven days prior to the beginning of the experiment. Each enclosure was subject to natural weather conditions and the grass (approximately 300 mm long) was not nuown during the experimental period. Possums were able to contact each other through the wires of neighbouring grass enclosures, but not between indoor pens or between indoor pens and enclosures. Each infected possum alternated daily between the grass enclosure and the indoor pen. In a similar procedure to that used with cages, each of eight uninfected possums (3.9
±
2.0
yr;
4 females,
ed with eight infected indoor pens and outside eight quence
infected
uninfected to
both possums
possums
4 males)
was
possums,
enclosures were
alternat-
between
the
daily. exposed
of the environments over an 18 day’
All in
se-
of all eight period. The
experiment was designed so that all animals were in the outside enclosures day and all uninfected possums were
infected on one outside
DAY
on the
following
day.
This
ensured
that
ET AL.-LEPTOSPIROSIS
no so-
TRANSMISSION
1.
TABLE
Serological
I)F MAT Possum
tutre
(day
pi)
24
257
Microscopic Aggluntinationn pattern of experinnnentallv
Test titres and leptospirmnria infected possunnnis.
cial contact could be made between infected and uninfected possums. Swapping continued for 32 days (day 29 pi to day 60 pm) so that each uninfected possum was exposed to each infected environment two to three times. Posstnms were fed daily with mash and an apple in their
IN POSSUMS
uuriunu .unnalvsis (day . pm) -
28
43
71
1
1:8(5)
M’
N/M1’
2
1:16(5)
M
N/M
N/M
indoor pens and the base of climbing logs in the grass enclosrnres (collected by pipette into sterile jars) were examined under DF micros-
3
1:16(5)
M
N/M
N/M
4
1:16(5)
NI
N/NI
N/NI
5
1:32(5)
NI
N/NI
N/NI
copy
for
6
1:16(5)
NI
X
N/NI
days
after
7
1:1600
NI
N/NI
N/NI
8
1:8(5)
NI
X
N/NI
indoor ( urine
pen and
taken
or outside enclosure. Fluid water) from the concrete
leptospires
expostnre
from
at days
the
28,
Two
once
began.
eight
43
urine
and
71
L.
14
and
21
Urine
samples were group 3 possums
infected pi.
samples
the eight uninfected ping period (days med tinder DF were taken from day’s after swapping
to
between
samples floor of
Xlotilu
were
taken
possums 52
from
during
pm and
62
each the
pi)
of
swap-
and
‘
were present in any possums. Temperature and rainfall data were collected at the Ruakura Meteorological Station (Hamilton, New Zealand) located approximinately 1 km from the enclosure conniplex. Dewfall and
frosts corded
on the daily.
grass
of the
enclosures
were
re-
RESULTS
Using taken
DF
from
(14
days
cages
post
on
non-motile
fluid
sampled
door pens and between 14 and gan
contained During the
20
of the
32
bottom indicated
exposure)
contained
Similarly,
fluid
the
leptospires. period rain
experimental
imum period Temperatures
without ranged
days.
enclosures with cage environment
perature
between possums
ranged captured
be free infection None
were ronments positive balcanica
of L. interrogans before the of
the
exposed
16 to
containing titres to in
MAT
on
max-
rainfall was 2 days. from -2 C to 17 C
in the outdoor 3 nights. In the All
fell
The
L.
over tem-
10 and 19 C. were confirmed serovar experiments
uminfected cages
frosts the
possums or
enclosure
leptospires interrogans
serology
tests.
to
balcanica began.
exposure
In were urine
group shedding at day
1, all four infected motile leptospires 28 pm. Three of the
fected
possums
motile
leptospires)
No
62
from
showed
day
28
pi showed
of the every
in
further
at
in
the
sam-
leptospithroughout was seen
day
62
pi,
al-
showed lepurine sample. the
motile were
urine
samples.
six group
on
urine
that
possums weekly
detected
weekly for
possums in the four in(non-
weekly
2 possums
none
cages
sampling
intermittently period. Shedding
group 2 possums were non-motile leptospires ogy
to the
next
2,
group
pos-
leptospiruria
at the group
continued swapping all
uninfected
In
Leptospires
1 and
urine
from
at day seen
28 pi; in all
MAT
serol-
2 possums
test-
ed at 152 and 186 days pi showed positive titrations to L. interrogans serovar balcanica ranging from 1:200 to 1:400. Group 3 infected possums, used in enclosure L.
that envi-
eight
lep-
pi.
though tospiruria
day were
showed serovar
microscopy
or after
the
enclosures exposure be-
DF
sums during or enclosures.
in
urine
under from
ruria
in-
seen samples
ples
eight
were
in any
samples
cages all eight
all
eight outdoor 21 days after non-motile exposure
tospires
of
leptospires. from
h’tt’ct’d.
dt’tectt’d.
leptospirts
day
microscopy,
sacks
No
dh’tt’cted.
li’ptospires
exam-
microscopy. Blood samples the uninfected possums 38 ended to determine if titres serovar balcanica infection
interrogans
leptospirt’s
Nomm-tumotilt’
X
swaps,
interrogans
showed
positive
serovar
balcanica
titres
Motile samples
leptospires from
24 pi (Table detected in individuals
motile
leptospires
28 pi, 1).
although
1). urine at
day
were not
in
28
serological
pi.
detected all
samples
Only
at all non-
after
day
(Table
258
JOURNAL
OF WILDLIFE
DISEASES,
VOL.
33, NO. 2, APRIL
DISCUSSION
This
study
serovar
showed
possums
by
containing
was
predictions contact,
rogans
that
serovar
(Hathaway,
other
routes for
balcanica
1978;
presuch L.
as
inter-
and
this
experimentally
Presidente,
study
very
showed
similar
by
Hathaway
in
urine over
that
the
nical
the
time.
a
of
be
by
related
DF
Day
(1996)
that when pairs of possums infected with L. interrogans canica in each (12 to 69 days sures
used
mission
occurred
in
pairs. lowing
This showed intraperitoneal same
culture when
L.
two
periment, The
to loss
affect
were
best
survival
a lack
of direct
concentrations 1978).
factors are suitable vars may survive
(see When and
74
mod-
C)
and
cage
con-
to lepto-
through-
not
in
density, more
that
fa-
are imendemic
species
1978).
(see
Other
population host
have
re-
factors
species
of
(e.g.,
rate, social in maintaining
host
structure) infection
as
references in Hathavav, 1978). Hath(1978) attempted to assess which facenvironmental conditions or possum
population portant
characteristics, maintaining
in
ovar
balcanica
that
the
three
different
ciated different
with
were more intcrrogans
L.
in possums.
prevalence
of
He this
environmental ecosystems,
concluded in
not
was
and
imser-
infection
populations
variables that
asso-
in tine possum
factors (such as possum contact important in the maintenance
organism
could
however, not
with contaminated The data obtained
shown
that
alone
is not
ba/can
serovar
not,
did
be
in
would therefore L. interrogans
sufficient
Social
sion. However, not present in
contact appear serovar
Hath-
ica.
show
spread
by
environments. in this study’
environmental
possums.
some leptospire seroremain infective in
but to
survival.
host
Hathaway,
contact important
or
environmental
recorded likely
suggested
in
maintenance
this
tem-
salt
leptospiral have
describe
of lepto-
conditions
were been
infection
references
of
survival
environmental conditions for the maintenance of
leptospiral
tact
the environmental
affected
(1978)
by
to ensure lepthree of tine 32
reduced
workers
interrogans
references
to
some
and rainfall, Frosts on
or pH have
detrimentally
away
sunlight
up
environment
each
Neither
.
of L.
moderate
in
have
population rate) were
Leptospires
alkaline
19
ensured
dew fall survival.
viability
is influenced factors.
moisture,
and
both
does or
in slightly
adequate
ex-
leptospires.
of leptospires
excretion
survive
salt
in this
infectiousness
environmental
possums
used
The
folfrom
balcanica
other
to
of each
concentration neither would
(see away tors,
male/female
enclosures,
of motility
following
Hathaway,
of
an infection inoculation
non-motile
several
high
trans-
to
1981a).
peratures
study,
serovar
and
contain
(Hathaway,
with
this
environments
their
spires
enclo-
50% that
to the tospire
the
together grass
interrogans
cages
shown
housed
for
moisture,
(10
present
searchers
found
same
in
was infectious in social contact.
The
in
(one possum serovar hal-
were in the
were
the
that
pair) pi)
to tech-
leptospires
microscopy.
study,
were
vorable portant
suggests
due
sufficient
possums
Several
all individ-
(1981b) may
In
leptospires
for
in detecting
urine
infected
inoculation inoculum.
of
Hathaway
inconsistency
possum
also
consistent
difficulties
In
who
infected
days may leptospires
previously
detection
not
in
characteristics
described
(1981a),
was
uals
those
intraperitoneal quantities
studies,
possums
infection
to
possums by with similar both
infected
cages,
re-exposure
spires
soil
out the trial period. Based on weather data, each enclosure should also have contamed sufficient moisture in tine grass, due
1979). The
damp
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temperatures
tinual
transmission
Durfee
the
erate
supports
required
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In
to
environments
This
are
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and
pasture
days
transmitted
with
leptospires.
social
L.
not
contact
vious
water,
that
balcanica
1997
that con-
have
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infection
in
possums
to be required for ba/can ica transmis-
in the wild, other factors this study’ (foot pad cuts,
DAY
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may
enhance
vironmental
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with
the transmission
transmission
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Although
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likely
to
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This research was funded by the New Zealand Ministry of Agriculture and Fisheries. Thanks are due to Tony Painting, Matthew Fawkes and Lynette Hartley for assistance in animal manipulations, to Murray Ashby for blood sampling and to Tony Day for veterinary assistance. We are indebted to Roger Marshall and Julie Collins-Emerson at the Department of Veterinary Pathology and Public Health, Massey University, New Zealand for providing L. interrogans serovar balcanica inoculum and advice.
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