Glasgow,. G61 1QH, Scotland. Scottish. Agricultural. Colleges. Veterinary. Investigation. Centre, Drummondhill,. Stratherick. Road,. Inverness, 1V2 4JZ, Scotland.
Journal
SEROLOGIC
STUDY
HARBOR
SEALS
OF PHOCINE
DISTEMPER
of
Wildlife
Diseases, © Wildlife
1992, pp. 21-27 1992
28(1), Disease
Association
IN A POPULATION
OF
IN SCOTLAND
P. M. Thompson, H. J. C. Comwell,2 H. M. Ross,3 Lighthouse Field Station, Department of Zoology, University Cromarty, IV1 1 8YJ, Scotland Department of Veterinary Pathology,
Veterinary
2
Bearsden, Glasgow, G61 1QH, Scotland Scottish Agricultural Colleges Veterinary Inverness, 1V2 4JZ, Scotland A serologic
Investigation
of
harbor
of the
University Centre,
(Phoca
seals
prevalence
of Glasgow,
Drummondhill,
Stratherick
vitulina)
from
of morbillivirus northeastern
Road,
was conducted in a where mortality was comparatively low during the 1988 phocine distemper virus outbreak. None of the 12 seals sampled before the epizootic were seropositive. Thirty-five (52%) of 68 seals sampled after the beginning of the epizootic were seropositive, although there were significant age-related differences in both the number of seropositive individuals and in antibody levels. Marking studies showed that most seropositive seals caught during the peak of the epizootic survived for several months. Thus, the low mortality observed in this population did not appear to result from a lack of contact with the ABSTRACT:
population
survey
School,
and D. Miller of Aberdeen,
antibodies Scotland,
virus.
Key
words:
serologic
Phoca
seal, study.
Harbor
field
survey,
vitulina,
Phocine
INTRODUCTION During among
1988,
a major
harbor
populations
seal in
North
to
20%
in
some
and
an
differed varying
considerably from only
northeastern Miller,
Scandinavian
on
Scotland 1991)
waters
to
in al.,
Although ondary
seals
generally
infections
pneumonia,
such
et
the
died as
primary
acute
of
of the
zootic was a previously undescribed billivirus, phocine distemper virus Kennedy
et
der, 1988). veloped for temper harbor pus)
1988; Osterhaus Virus-neutralization the closely related al.,
from
to PDV
the during
North 1988
Sea
had
(Osterhaus
been
morbillivirus taken from
serum
found different
antimor-
from
seals
samples
antibodies gray seals
in serum samples in the United King-
dom
between 1977 and 1987. If the epidemiology of the 1988 epizootic is to be understood in detail, data are required on the proportion of the population which came into contact with the virus and on the fate of those individuals
both gry-
exposed and
antibefore Nether-
in to a rehabilitation center be1984 and 1987. On the other hand, only samples from UK waters, Haret al. (1989) found no evidence of
epi-
and Vedtests decanine dis-
virus (CDV) confirmed that and gray seals (Halichoerus
morbillivirus
(1989)
Osterhaus et al. to an antigenically
wood
mor(PDV)
of
lands, bodies
sec-
bacterial
cause
prevalence
in
groenhispida)
(Dietz et al., 1989a). limited data are available populations In the
brought tween in the
1989b).
epizootic,
bodies in North Sea seal and during the epizootic.
billivirus
60%
(Dietz
the
morbillivirus,
and harp seals (Phoca ringed seals (Phoca
from Greenland However, only
L.)
killing
individuals (Dietz et al., et al., 1989). Harbor seals affected to some extent,
but mortality rates between populations, 10
vitulina Sea,
virus,
et al., 1989), landica) and
occurred
(Phoca
the
estimated 17,000 1989b; Harwood in all areas were
(Thompson
epizootic
distemper
Ved-
der, 1988; Harwood et al., 1989). In addition, morbillivirus antibodies were detected from other regions, in samples from Baikal seals (Phoca sibirica) (Likhoshway
exposed to assess mortality
to it. Such whether resulted
of contact
with
information can be used observed differences in from variations in rate the
virus,
in resistance to the virus. This paper presents data logic survey of a population 21
or of
variations
from a seroof harbor seals
22
JOURNAL
OF WILDLIFE
DISEASES,
VOL.
28, NO.
1992
1, JANUARY
dom) and diazepam (Valium, Roche Products Ltd., Wellwyn Garden City, AL7 3AY, United Kingdom) (Baker et al., 1988) or tiletamine hydrochloride and zolazepam (Zoletil, Reading, Z.A.C., 17 rue des Marronniers, 94240 L’Hayles-roses, France) (Stirling and Sjare, 1988). Standard length and girth measurements were taken while seals were on the restraining board. All seals less than 1.1 m standard length were assumed to be juveniles in their first or second
LJ
year
(Harwood
et al.,
to determine seals
the than
longer
1989).
It was
precise age 1.1 m were
not
of older therefore
possible
animals; classed
as adult. Blood
Invenss
FIGURE
1.
Location
of
harbor
seal
haul
in
the
of the out
study
area
and
position
(#{149}).
sites
Firth,
Moray
Flrth
northeastern
(57#{176}35’N; 4#{176}O’W).Mortality
Scotland in
this
popu-
lation was low (10 to 20%), with most deaths occurring during August, September and October 1988 (Thompson and Miller, 1991). A capture and marking program was started early in 1988 and blood was collected Sera
routinely from analyzed to of morbillivirus
were
prevalence seals
caught
before,
epizootic. In made on seals to assess antibody
during,
addition, marked
the fate levels.
all captured seals. assess changes in antibodies in and
observations during the
of individuals
after
the
were epizootic
with
known
METHODS
Study area and capture The
Moray
least 1,100
Firth
harbor
techniques
contains
a population
seals which
haul-out
of at regularly
on intertidal part of the
sand and mudbanks in the inner firth (Fig. 1). Seals were captured by rushing at haul out groups with an inflatable boat or fourwheel drive vehicle and catching animals individually using hoop nets. They were then transfered to a restraining board and, where
lightly
with
necessary,
sedated
hydrochloride Lambert,
(Vetalar, Parke Pontypool NP4 8YA,
either
ketamine
Davis/Warner United King-
sampling
and marking
techniques
Blood samples were taken from the epidural vein of restrained seals using either plain or heparinized (heparin hydrochloride) blood vacuum tubes (Vacutainers, Becton Dickinson, UK Ltd., Between Towns Road, Cowley, Oxford, 0X4 3LY, United Kingdom). Serum or plasma was removed within 24 hr and frozen at -20 C for 12 mo. Between April 1988 and June 1989, blood samples were obtained from 79 individual harbor seals, with samples being taken before, during and after the period when most deaths occurred (Table 1). Repeat blood samples were also obtained from two individuals in different seasons. During August and September 1988, 28 harbor seals (14 adults, 14 juveniles) were caught at haul
out
sites
in the
Inverness
and
Dornoch
Firths. All seals were marked with a numbered tag in each hind flipper and twenty four individuals also were marked with a large colored number or letter (approximately 20 cm high) on their back (Thompson, 1989). Very high frequency (VHF) radio-transmitters were glued (Fedak et al., 1983) to the hair on the heads of six adult seals from the Inverness Firth. Throughout
1988
and
1989,
observations
were
made at known haul out sites at least twice each month. Haul out groups were counted using a 30 x 70 telescope and the presence of any colormarked individuals was noted. Flipper tags could not be read using a telescope but were used for the identification of carcasses or individuals that were recaptured after the color-marks had been lost in the moult. Radio-tagged individuals were located daily by triangulation during November 1988 and January 1989, and their presence and activity around haul out sites was monitored using automatic recording equipment (Thompson and Miller, 1990). Virus
neutralization
test
Neutralization tests were carried out in microtitration plates by a method based on that described by Appel and Robson (1973). Four-
THOMPSON
TABLE
after
1. Age a phocine
and sex distemper
of
harbor
seals
caught
and
AL.-PHOCINE
E
bled
in the
Moray
DISTEMPER
Firth
IN HARBOR
1988-1989,
before,
Pre-epizootic
April
Male
1988
epizootic
Aug-Sept
Post-epizootic
Feb-May
1989
Female
1
9
4
10
15
21
20
fold dilutions of test serum, from 1:16 to 1:16,384, were made up with graduated pipettes. The Onderstepoort-Bussell Strain (A. E. Churchhill,
of
UK
Intervet,
Ltd.,
Science
Park,
Cambridge CB4 4FP, United Kingdom) of canine distemper virus (CDV) was diluted to 80320 tissue culture infectious doses 50% (TCID5O) per ml of growth medium with 5% fetal bovine serum (FBS) and 1 ml of this added to an equal volume of each serum dilution. The serum-virus mixtures were left in the dark at room temperature (18-20 C) for 1 hr and at 4 C for a further 1.25 hr to allow neutralization to proceed. Each mixture (0.2 ml/well) was then inoculated into four wells of Vero cell monolayers in a 96 well microtiter plate (Nunclon, Gibco Ltd., P.O. Box 35, Trident House, Paisley, PA3 4EF, Scotland). These cultures had been prepared the previous day by seeding the wells with a suspension containing 200,000 cells per ml, 0.2 ml per well. Growth medium consisted of medium 199 with 25 mM Hepes buffer and L-Glutamine and 10%
FBS. incubated atmosphere containing 3% CO2. Each well was then examined microscopically and the proportion of wells showing the characteristic cytopathogenic effect was recorded for each serum dilution. Titers were expressed as the reciprocal of the serum dilution which reduced the proportion of wells infected from 100% to 50%. All samples with titers 90 were assumed to be seropositive. Serum and virus controls were included on each occasion that the test was carried out. Following
at
inoculation,
37 C for
plates
were
in a humidified
3 days
Male
Unknown
2 1988
Total
formerly
during
and
Adult
Juvenile
During
23
epizootic.
period
Sampling
SEALS
5
-
1
1
Thirty-five
(52%)
10
23
16
of
= 5.25, 1988 and Furthermore,
seropositive
higher
(
adults
than
adult
titer
2.69,
=
those
33
significant
samples
spring
1989
taken
2.41,
=
were
log10
in autumn P
samtiters
juveniles titer
There in
0.05)
significantly
juvenile
(t-test,
af-
P < 1989 log10
seropositive
differences
tween
1 df, spring the
P
Survival of marked seals with known antibody titers Eleven
color-marked
negative.
Three
seals
of these
were
sero-
individuals
were
found dead 4 to 6 wk after capture and another three were seen alive and apparently healthy after 21 to 30 wk (Table 2).
Log
Antibody
Titer
3.
2
12
2
21
2
RESULTS 12
Changes
in the prevalence
41
and
3
1
2
level of antibodies Antibody
all low, ples first
titers
ranging
taken deaths
quently body
after due had
(Fig.
before from 8
much 2).
August to PDV higher
the
epizootic to 32. Blood 1988, were levels
when seen, of
MAMJJ
were samthe freanti-
A
SOND
JFMA
1988
FIGURE
free-living
2.
temper
epizootic
(August
to September
(February
1989
Serum
harbor
to May
M
morbillivirus seals
(April
from
before
1988), 1988),
1989).
antibody the
during and
after
titers
phocine the
epizootic
the
epizootic
in
dis-
24
JOURNAL
OF
DISEASES,
WiLDLIFE
VOL. 28, NO. 1, JANUARY
% Positive Ad
Ad
71%
1992
One
juvenile
after signs
3 wk. It displayed of PDV infection
63%
1989b)
36%
L
tive
been died after was
0 Outbreak
3.
seropositive epizootic.
Poet-Outbreak
88
Aug-Sept
FIGURE
Age
Feb-May
differences
seals caught sizes are
Sample
89
in the proportion and
during presented
after
of
the
in Table
its titer
was none (see
remained
found of the Dietz at
Two of thirteen color-marked seals were recovered dead,
after
During
(32038)
8.
sick classic et al., After
treatment with antibiotics it recovered and was released to the wild. The remaining seals were either not subsequently seen or were seen alive only soon after marking.
Juv
50
and
seal
1988 1.
marking.
An adult
seroposi1 and 3 wk
female
which
had
radio-tagged (32041) probably also because her radio-signal disappeared less than 1 wk. This disappearance preceded by several days of unusual
behavior when she spent almost all her time either on the shore or at the surface. A further six individuals were observed alive between 13 and 44 wk, well beyond the period when deaths due to PDV were
TABLE 2. 1988 when
Fate most
of seals with and without morbillivirus seal deaths were seen in the Moray
antibodies
Antibody
Seal Seals
number
with
no
antibodies
against
M
32
8.29.88
32032
A
M
32
9.2.88
32035
J
32036
16
