5I1I(Iltf’
of
34(3). (t) \\iI(IIik
TRANSMISSION
OF LEPTOSPIRA
BALCANICA
INFECTION
POSSUMS
IN NEW
T. D. Day,123
INTERROGANS
AMONG
SOCIALLY
HOUSED
J. R. Waas,2
A. J. Pearson1
and
1995
BRUSHTAIL
L R. Matthews1
2
3
Corresponding
(e-mail:
571i-5S1
ZEALAND
C. E. O’Connor,1
Author
PP
Ass(Xitmi(
SEROVAR
Animal Behaviour and Welfare Research Centre, AgResearch, Ruakura Research Department of Biological Sciences, University of Waikato, Hamilton, New Zealand
,
199S.
I)isast’
Centre,
Hamilton,
New
Zealand
[email protected])
Leptospira interrogans serovar balcanica is a potential vector being investigated for spreading a biological control agent among introduced brushtail possums (Trichosurus vulpecula) in New Zealand. As previous studies have shown that possums are unlikely to contract leptospirosis through a contaminated environment alone, the objective was to determine whether L. interrogans serovar balcanica could be transmitted between sexually mature, socially housed possums. Possums were infected experimentally with L. interrogans serovar balcanica and housed in pairs or groups with uninfected possums for either 70 or 140 days, during the breeding or non-breeding seasons. No transmission occurred between any infected and uninfected possums during the non-breeding season. However, transmission occurred between females that had been socially housed in pairs or groups in the breeding season. Mixed sex transmission also occurred in pairs and groups, both from males to females and from females to males. Mixed sex transmission usually occurred rapidly (18 mo of age Ashby,
in
host
that
possums.
a
is
infection
or kidney
interrogans
and
tospirosis
al.,
indicate
in
of
possum
1978).
et
damage
Transmission
its specificity
dictate
(Hathaway,
appears to be predominantly
control
The balcanica
1981) L.
a vector
maintenance
of renal
possums,
presently
as
the
signs
biological interrogans
serovar
Zealand
pathogenicity
infect and surhave a possum(Jolly,
clinical
use
agent.
to be
(Hathaway,
(Living-
biological
New
of
for
organisms for
interrogans
evidence
stone, 1991). Biological control may be the only affordable option for achieving a long-term major reduction in possum numbers throughout New Zealand (Jolly et
potential
control
in
a vector
Trans-
to occur that L. in-
Leptospira
investigated
considered L.
in
transmission,
biological
browsing
through
and (Tb)
(Tn-
severe
selective
and predation, adversely na (Innes, 1995). Possums in
possums
seasons.
but is unlikely study suggest
desired
is among
being
Zealand, vulpecula)
behaviour, in this
brushtail possum, disease Trichosurus vulpecula.
INTRODUCTION
In New chosurus
or the non-breeding
sexual determined
types host
when pass
body directly
sexual
Indirect
occur
occasionally
hosts species,
or
of
lep-
species
usu-
fluids
from
to
contact
another or
suck-
transmission between
from maintenance as a result of contact
mainhosts with
DAY
environments containing is not known whether sion of L. interrogans occurs New
from Zealand.
trials
this
possums to However,
leptospire
al., 199Th). transmission canica has
terrogans
possums gests be
the
associated
away,
1978;
appear
to be
by
contact (Day et
and
and was
L.
Presidente, designed
transmitted mature
behaviours
MATERIALS
sugmay
possums associated
to with
AND METHODS
in this
study were box trapped 1995 and January 1997 from (36#{176}26’S, 174#{176}50’E) and Nelson (41#{176}20’S, 172#{176}43’E), New Zealand. Possums from both areas are reportedly free of L. interrogans serovar balcanica infection (Homer et al., 1996). Animals were transported to the
December Kawau Island
Animal
Behaviour
and
Welfare
Research
Cen-
tre (AgResearch, Ruakura, Hamilton, North Island, New Zealand). All possums were aged by tooth wear (Winter, 1980) and sexual maturity was determined during routine veterinary checks. All procedures used in the present study were approved by the AgResearch Ruak-
ura (Hamilton, New Waikato (Hamilton, Ethics Committees.
Zealand) New
for
the
presence
of lepto-
Public
Health
(Massey
University,
Palmer-
ston
1979).
and
(400x)
spires. None of the captured possums used in this study had serological or urinary evidence of infection; therefore they were considered to be uninfected with L. interrogans serovar balcanica prior to the commencement of the experiments. Twenty three sexually mature experimentally infected possums (mean age ± SE = 4.2 ± 0.3 yr; 12 female, 1 1 males) were used in this study. Possums were anaesthetised and inoculated intraperitoneally with 0.5 ml L. interrogans serovar balcanica (about 1 X 10 organisms). The inoculum was cultured from an infected possum at the Department of Veterinary Pathology
and
(Hath-
to deterserovar haldirectly be-
interrogans
be
sexually
used
1995)
maturity
sexual
identify likely transmission.
Possums between
Ashby,
with study
present whether canica could
(Hathaway, of L. inin immature
of infection
Durfee
The mine
tween
microscopy
not
acquisition
577
in
balcanica
Furthermore, no “vertical” of L. interrogans serovar halbeen seen between infected
(Pearson that
IN POSSUMS
If no reaction was seen in a 1:50 dilution, resuits were reported as negative. Mid-stream urine also was collected from possums before the experiments and examined under dark-fiehd
possums environments
and their offspring A very low seroprevalence serovar balcanica
TRANSMISSION
It
urine. transmis-
other species in experimental
does
transmitted between with contaminated
mothers 1978).
infected indirect serovar
ET AL.-LEPTOSPIROSIS
and University of Zealand) Animal
Serological testing for any evidence of L. interrogans serovar balcanica infection was conducted prior to the beginning of the experiments using a modified version of the Microscopic Agglutination Test (MAT) by the Central
Animal Health Laboratory (AgResearch Wallaceville, Wellington, North Island, New Zealand). Cross absorption procedures (Fame, 1982) were used to discriminate between L. interrogans serovar balcanica and other serovars. Positive tests were reported as the greatest serum dilution at which serum showed a reaction.
North, New Zealand) and confirmed as New Zealand possum L. interrogans serovar balcanica isolate, using restriction endonuclease analysis and DNA verification. The characteristics of experimental and free-living L. interrogans serovar balcanica infection in possums have been thoroughly examined (Hathaway, 1981), and do not appear to differ significantly. Using MAT serology, all expenmentally infected possums were shown to be infected with L. interrogans serovar balcanica at 12 days post inoculation (pi).
Each
experimentally
infected
possum
was
housed with other animals in a grass enclosure from day 12 pi, either with one uninfected possum (in pairs) or with 3 uninfected animals (in groups). Each of the eight enclosures used was individually fenced (ranging in size from 51m2 to 130m2), contained at least two wooden nest
boxes
and
natural bitum
weather conditions. access to fresh pellets
Mill,
two
Auckland,
climbing
logs and
Zealand),
New
was
Possums (Northern
subject had
apples
to
ad iiRolling
and
wa-
ter
in their enclosure. Seventeen pairs of sexually mature possums (four female/female, eight male/female, five male/male pairs), each containing one of the infected animals, were housed in the enclosures
for
a 70
day
period,
ing” or “non-breeding” were housed together
during
the
“breed-
seasons. Thirteen in
the
breeding
pairs season
female/female, six male/female, four male/male) and four pairs in the non-breeding season (one female/female, two male/female, one male/male). In four of the mixed sex pairs a male was infected and in the other four pairs a female was infected. Serum was sampled and tested for evidence of L. interrogans serovar balcanica infection from each infected possum on day 0 (12 pi) and day 70 (82 pm) of social (three
578
JOURNAL
OF WILDLIFE
DISEASES,
VOL.
34, NO. 3, JULY
housing, and from uninfected possums on days 30, 44 and 70. Mid-stream urine was sampled from infected possums at day 30 (42 pm) and examined under dark field microscopy (400x) for the presence of leptospires. All females also
were
examined
(pouch
Four
for
evidence
young)
at days
30,
groups
of four
sexually
44
of and
1997)
season
and
the
70
and
day
groups
(82
pm)
0 (12
for
the
breeding
pi)
and
day
housed
together
to
seasons.
from
infected
Mid-stream
Paired
t-tests
were
titers tween
over time. Differences the breeding and
were tests
used
L. interrogans
serological
analyzed in the
(Release thamsted
using
to
was
sampled
analyze
changes
Fisher’s
exact
Genstat 5 statistical analysis package 3.1; Lawes Agricultural Trust, RoExperimental Station Hertfordshire,
of
the
23
had serovar
experimentally
L. by
interday
12
pi (mean ± SE, 1:420 ± 70; range, 1:100 to 1:1,600). Leptospires were detected in the urine of 19 of the infected possums at either day 42, 82 or 152 pi (4 with motile and
15 with non-motile leptospires). At day 82 pi, the mean L. interrogans serovar balcanica titre had decreased (1:200 ± 40; range, 1:50 to 1:800; t(17) 5.31, P < 0.001),
but
was
still
present
female possum. However, in non-motile urinary leptospires
Non-breeding
1’
33
6
31)
50
Male
4
0
0
Female
1
0
0
2
0
0
1
0
0
sex
Mixed
sex
Male .)
Tramusmuuissioml
I,
Tsvo
after
days
1t)
tramusniissiomus
frommu
s(xial
mimaIc
Imousimug.
to femmuale amud 0mw
from
ft._
imuale to imuale.
in all but this
day 82 pi. At day 152 pi, L. serovar balcanica titers were still positive (1:180 ± 90; range, 1:50 to 1: 400) in the four infected possums. Transmission occurred between possums in three of six male/female pairs during the breeding season, but in neither of
one
female, were still
on
the two breeding passed from sion One two
male/female season both
from
pairs (Table males
1). to
outside Infections
the
females
and
females to males, with all transmisoccurring by day 44 of association. transmission also occurred between females after 160 days during the
breeding season. corded between no transmission breeding season, cally rate
infected
positive serological balcanica titers
of
trans‘ mmmis- Traumssinim nuissiolu
3
Mixed
interrogans
RESULTS All
type
Female
detected
UK).
possums rogans
Breeding
col84, in
serovar balcanica in transmission benon-breeding season
one-tailed
Pair
Seasomu
Un-
possums between days 77 and 82 pi or days 142 and 152 pi and examined for the presence of leptospires. Females from all social groups were examined for pouch young when each senmm sample was collected. in
ci-
the
seasons.
urine
(luring
groups,
pm) for
infected possums in all groups had serum lected on days 21, 30, 44 and 70, plus days 98, 1 12, 126 and 140 for the groups housed
both
of socially
seasons.
her
March March
(152
both
interrogans
tYpes period
Nuimmu-
season
in
timer the breeding
of L
possums
(mid
140
housed
transmissions
between different pairs over a 70 day or mion-breeding
Oh-
May 1997). Two additional groups of four possums (one all female, one mixed sex), also contaming an infected possum, were housed in the enclosures for a 70 day period during the breeding season only. Serum was collected from each infected possum on day 0 (12 pi) and day
balcanica possum
70.
to early
season
1.
serovar
SerV(’(I
mature
(January
breeding
Observed
TABLE
offspring
(one all female, two mixed sex, one all male group), each containing one of the infected animals, were also housed in separate enclosures for a 140 day period that encompassed both the
non-breeding
1998
significant between
No transmission was remales (Table 1). Although was observed in the nonthere was not a statistidifference seasons (P
in transmission 0.30), due
to
the relatively low rate of transmission overall in the breeding season (31%). The initial serological L. interrogans serovar balcanica titres of possums that became naturally infected during social housing were similar to those seen after recent experimental infections (mean ± SE, 1:550 ± 170; range, 1:200 to 1:800; t(3) = -0.78, P > 0.05). All possums that became infected
in the
pairs
continued
to
have
de-
tectable L. interrogans serovar balcanica titres until the end of the experiment (70
DAY
2.
TABLE as
Serological
a result
of
at which
social
serum
contact
showed
reactions of the six with experimentally a reaction.
ET AL.-LEPTOSPIROSIS
possums naturally infected infected animals. Titres
Da Possum
0
of s(x’ial
44
with L. interrogans serovar given are the greatest serum
luousing 70
98
126
l)
-
1:200
1:100
1:50
Neg
1:800
1:400
1:100
-
-
3
Neg
Neg
1:400
1:200
-
-
-
4
Neg
Neg
Neg
Neg
-
-
1:800
-
-
5
Neg
1:1,600
1:200
Neg
Neg
6
Neg
1:12,800
1:200
1:50
1:50
with
no
not
reaction
at
1:50
serum
Table
by the females housed transmission occurred
No
during
Neg
2).
the
dilution.
No
offspring
were
pro-
in pairs. in the groups
non-breeding
season.
tospires
were
these
ter
During
detected
animals
the
first
balcanica
positive
tween males during the breeding season. Again, there was no statistically significant difference in transmission between sea-
mating would have male with a pouch fected throughout
sons came
(P = 0.35). infected
od.
high
litres
and
(1:1,600
which
one
The two possums that the groups initially
of
quickly
these
balcanica from 40 days other, a titre was
detected in the after
90
days
3.
serovar
balcanica
possum
groups and
Non-motile
transmissions
in different over 70 day
non-breeding
types periods
Breeding
seasons.
Transmission type Female
Mixed Male Non-breeding
lep-
of L. interrogans of socially housed during the breed-
sex
Female
Mixed Male
sex
young from
This serovar
tial transmnission
Ohserved tramus-
urine
of
days
af-
51
L. interrogans
the
were
found
mixed
sex
season, during
serovar in
three
groups
fe-
during
two of which had the period in which occurred. The other feyoung remained uninthe experimental peri-
study showed balcanica can
tween when
sexually housed
season. with
%
mnis-
Trans-
sion
mnissiomu
work
sums
(Day
some
aspect
for
interrogans
had
17
4
0
0
sums
4
0
0
mals
4
0
0
4
0
0
one
gans
and
no
infected
therefore be
im-
longer
of
the had
balcanica after the
possums of
those
1981).
titres, described
However, infected
socially detectable
in
infection
balcanica to
experimentally two
serovar 90 days
transpos-
must
characteristics
serovar
the
and
and
contact
(Hathaway,
of
con-
indirect between
199Th),
direct
similar
previously
13
that
shown
environments
experimentally
(L.
1
has
transmission.
leptospiruria)
1
al.,
of
study
6
possums breeding
to allow leptospire
et
this
8
brushtail during the
contaminated
alone is insufficient mission of this
The
that L. interrogans be transmitted be-
mature together
Previous
tact
portant Poten-
Season
2).
interro-
titre was no longer after infection and no longer detected
of infection.
Observed
TABLE
L.
the and
DISCUSSION
(Table
the
Pouch males
respec-
decreased
possums,
serovar
gans
1:12,800
behad
46
titre.
the breeding positive titres
in
in
between
the breeding season, transmission occurred between an infected female and uninfected male and between two females (Table 3). No transmission occurred be-
lively)
-
tested.
duced
ing
halcanica dihution
Nega
days;
In
579
1
mn(hcates
-
30
IN POSSUMS
2
a Negative
h
TRANSMISSION
pos-
infected
L.
ani-
interro-
titres between establishment
30 of in-
JOURNAL
580
fection. tected between
OF WILDLIFE
DISEASES,
VOL.
34, NO. 3, JULY
Non-motile leptospires were dein the urine of all of these animals 46 and 82 days after infection
(between 8 days before the negative titre was transmission
and 16 days after detected), but no
occurred
the
in
group
con-
taming the experimentally infected female that ceased to have a detectable titre. This typ e of transient serological response to L. interrogans serovar balcanica has been described previously in a possum after experimental days, was
infection
L. recovered but
(titer
lasting
interrogans serovar from kidneys 70
infection; tenance
Hathaway, hosts for
negative
animals
1981). L.eptospira
that
are
only
30
balcanica days after
spire. in
Therefore,
the
transmission
balcanica
of
must
have
role
L.
remains
potential for non-breeding Transmission
of
mating
serovar
uncertain,
resulted
itself
interrogans as
transmission season. between
to
from
than mating, also However, female
does
occur
the in the
females,
which
behaviour
other
occurred to female
in this study. transmission
also only occurred during the breeding season, suggesting that transmission is associated with particular behaviours occurring during this period. In female to female and mixed sex contacts between cap-
In many spp.,
mainsero-
tive den
infected
with
are
still
1998
possums, sharing, common
affiliative allogrooming,
behaviours food
(e.g., sharing)
during
breeding
season
the
leptospirosis are found. This infers that leptospires, which are generally localized in the renal tubules, exert only minor antigenic stimulus on maintenance hosts (Hathaway, 1978). Therefore, the animals in this study that no longer had L. inter-
(Day, 1996), but occur less frequently in the non-breeding season. These behaviours may be important for the transmission of L. interrogans serovar balcanica
rogans
serovar
to still presence
be
iours towards no transmission
urine were
around observed.
Based rogans primarily
balcanica
titres
were
infected, as is suggested of non-motile leptospires the
time
that
likely by the in the
negative
titres
son and Ashby, 1995), transmission during the son in this study, direct with sexual maturity
no successful presence/absence
and the lack of non-breeding seacontact associated and possibly the
appears to of infection. mating of
(as pouch
be
important In the pairs,
measured young)
by oc-
curred, so it is unknown whether the mixed sex transmission occurred as a result of unsuccessful mating or through some other behaviour. Successful curred in the groups between uninfected possums, although evitably rogans
result serovar
behaviour (such stage of infection critical for the
in this
study.
mission
of
gans
on the observation that L. interserovar balcanica infection occurs in sexually mature animals (Pear-
breeding season in the acquisition
between possums. sums very rarely
in transmission balcanica. The
mating ocinfected and did not inof L. timing
interof a
as mating) relative to the in the possum, may be transmission of the lepto-
serovar
sexual mission
Furthermore, show affiliative
each
other occurred
mice,
the
mice
and
trans-
leptospire (L. interroalso is dependant on
maturity, but not on mating. can occur between mature
female
posbehav-
(Day, 1996) and between males
In laboratory another ballum)
male
mature
Transpairs of
mixed-sex
pairs,
but ture
does not occur between sexually mamale mice (Hathaway, 1978). The rate of transmission of L. interrogans serovar balcanica in this study was lower in the groups than in the pairs. While the reasons for this are unknown, it is possible
that
fected possum contact with the would have had fore, transmission contacts such iours (as our
in
the
groups,
each
as affiliative data suggest),
or sexual behavmay occur rel-
atively slowly in a group situation. ever, the transmission rate observed groups is comparable to the rate mission tive
found
unin-
had less opportunity for infected animal than they if housed in pairs. Therethat is reliant on close
previously
possums (Pearson Social transmission
in groups and of
Howin the of transof cap-
Ashby, 1995). L. interrogans
DAY
serovar sirable
balcanica from the
control,
as it would
species-specific
agent
more
that
et
is transmitted that
al.,
actively season
FAINE,
may
appears during
be
to the
associated
behaviour.
with
affiliative
L.
Therefore,
ovar
balcanica
appears
sion
properties
that
biological
suited
1994.
HATHAWAY.
S.
animals
use
Health,
serovar
tion.
New
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This research was funded by the New Zealand Ministry of Agriculture and Fisheries. Thanks are due to T Painting and L. Hartley for assistance in animal manipulations and to T. Day for veterinary assistance. We are indebted to R. Marshall and J. Collins-Emerson for providing L. interrogans serovar balcanica inoculum and advice.
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of
Science
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the
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,
Possums breeding
Medical
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ic(, in
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IN POSSUMS
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control. ing the
to
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by sexual
primarily
rogans
biological
likely
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be
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between
perspective
ET AL.-LEPTOSPIROSIS
19.97.
\‘ihdhife
in
Tnic/aoRe-
