Jan 23, 1992 - suis biovar. 4-challenged bison. Brucella abortus was isolated from several tissues in all control ..... fall at hunting season. Although the. B.
Journal
Wildlife
of
32(4).
l)(ses
© Wildlife
THE
PATHOGENICITY
Julia S. Bevins,1 John Donald S. Davis2
OF BRUCELLA E. Blake,1
L. Garry
SUIS
BIOVAR
Jamie
Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, Alaska, 99775, Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M College Station, Texas, 77843-4467, USA Green Valley Veterinary Services, 1483 Green Valley Road, Watsonville, California,
The pathogenicity of 2.1 X 10’ colony cows. Six pregnant
inoculation six pregnant strain
2308
calving
as
or
tological internal
a
positive
abortion
control.
examinations
Bison
they conducted
which
after
organs.
of Brucella suis biovar forming units (CFU) bison were inoculated
were Terminal
serum
on
from
Assuxiation
1996
BISON
K. Morton3
and
95076,
USA
bison (Bison bison) was evaluated by nil saline into the conjunctival sac of 1.27 X 10’ CFU of Brucella abortus on 23 January 1992, and observed until and necropsied. Bacteriological and hisreproductive tract, mammary gland, and
euthanized, lymph nodes,
samples
551-.555
4 for in 0.1 with
inoculated
were were
PP
USA University,
2
ABSTRACT:
Disease
4 FOR
J. W. Templeton,2
Adams,2
I.
calves
and
cows
were
evaluated
by
card,
rivanol
standard tube agglutination, cold complement fixation tube, indirect bison conjugated enzyme linked immunosorbent assay (EUISA), competitive ELISA, and particle-concentration fluorescence immunoassay. No clinical signs of brucellosis were seen in bison inoculated with B. suis biovar 4, and infection was found only in lymph nodes of two animals. There was no evidence of metastasis of this organism to the mammary gland or the reproductive tract. There were no detectable levels of antibodies to Brucella spp. in terminal blood samples taken from B. suis biovar 4-challenged bison. Brucella abortus was isolated from several tissues in all control bison. All B. abortus-challenged animals developed uterine infection and five developed mammarv gland infection. Reproductive disease resulted in abortions in five B. abortu.s’-challenged bison and neonatal death in the remaining calf. Brucella .suis hiovar 4 does not appear to he pathogenic for bison. Key words: Brncella suis biovar 4, brucellosis, bison, Bison bison, pathogenesis. precipitation,
INTRODUCTION
Brucellosis (Ran
4;
causes
late-term birth
rate
symptoms
Bruce!losis of reindeer
have
1961).
Alaskan
arctic
(Meyer, range
1966). from less
in some
arthritis
moschatus) 1984).
with One
mentally infection biovar to
4 can cattle
(Alces
developed et al., be via
and tesZabrodin,
been
reindeer. of
Alaska
(USA),
(Gates alces)
in
in
usually
or the
infects
herds
of
cattle
of weak has
bison
to specan
abortus
birth
organism
calves
been
main-
independent
infected
with
area. the
Brucella
The
a free
susceptible
abortus,
1990).
abortion
in that are
which
a!.,
bison.
tamed
cattle
of
(Tessaro,
1986).
isolated
In
response
to the
the perceived Delta Junction
al.,
experi-
ka,
a generalized 1991). Brucella with
et
contact
et
Brucella
of Brucella
cause
areas
with
about
4 into
present
bison)
Junction,
concerns
biovar
B.
catgame
Delta
raised
of B. sins herd
than
in intensive near
(Bison
Litof
other
Interest has
1993).
pathogenicity
ruminants
reindeer
bison
(Davis
Tessaro,
the
4 for
farming
cies
and
about
tle and
moschatus
experimentally contact
biovar
infection
in most located in
(Ovibos
bursitis
moose
infected (Dieterich
4 has
suis
Bison
1981).
biovar muskox
wild
is known
ranging
and
coastal
(Forbes
tle
introduction
of bruce!losis to 30% or more
(Dieterich,
suis
a
and
Prevalences than 1%
and a poor
Additional
are
enzootic caribou
regions
herds
Brucella
from
is and
disease
females,
with associated lameness infection (Golosov and
1959). herds
mitted
in
seen
suis
the
that
(Davidov,
caribou
Brucella
calves
commonly
bursitis ticular
and
abortion
of weak
survival
suis
reindeer
spp.) is caused by in these animals,
gfer
biovar the
in
reindeer
sins
we
evaluated
biovar
jective
trans-
could
infected 581
threat bison the
4 infection
was be
public
to
infected
concern
over
of brucel!osis herd in Interior pathogenicity
in
determine with
to
of
bison.
Our
whether B.
suis
the Alas-
4, and
B.
obbison if so,
582
JOURNAL
if
they
would
OF WILDLIFE
would
be
transmit
the
DISEASES,
adversely bacteria
VOL.
32, NO. 4, OCTOBER
affected to other
or bison.
1996
samples
of
lymph
nodes;
froni
MATERIALS Twelve
second
pies
AND METHODS tri mester
pregnant
bison
were
ol)tained frolli a privately owned bnicelherd located near Gillette, Wyoming (USA) (43#{176}40’to 43#{176}41N, 105#{176}28’to 105#{176}29’W). All bison were seronegative for brucellosis by card test (Alton et al. , 1975) and had not l)een vaccinated with B. a/)Ortu.s strain 19. These hison were shipped via truck to the Brucella spp. research facility at the Texas Veterinary Medical Park, Texas A&M University (College Station, Texas, USA). All bison were given hay and water ad lihitum and fed a balanced ration at 2 to 3% of body weight (TAMU Mix Number losis-free
1; Producer’s
Cooperative
Association,
College
Station, Texas). Experimental protocols for the studies were approved by an independent campus-wide animal welfare committee. Six bison were moved into an animal facility that met published Bio Safety Level 3 requireinents (Richardson and Barkle 1988). Bison were housed in individual pens within this facility. These animals were challenged on 23 January 1992 with 2.1 X 10’ colony forming units (CFU) B. suis biovar 4 in a 0.1 nil physiological saline suspension placed in the conjunctival sac. The B. suis biovar 4 culture was originally isolated from the carpus of a reindeer shot near Nome, Alaska in 1977 and had been passed once th rough lemmings (Lenunu.s sibiricus) to assure virulence. This culture and challenge protocol had been employed successfully in similar challenge experiments in reindeer at the University of Alaska, Fairbanks, Alaska (Dieterich et al., 1981). Two lemmings and a guinea iig (Cavia porcellu.s) were inoculated with the challenge suspension, as an additional virulence check. The remaining six pregnant bison were challenged with 1.27 X 10’ CFU of B. abortus strain 2308 as positive controls. These positive control bison remained in outdoor biocontainment pens at the Brucella spp. research facility. All bison were observed until the time of abortion or calving, after which they were thanized with an overdose of 19.5 g pentobarbital sodium and 2.5 g phenvtoin sodium (Beuthanasia-D, Shering-Plough Animal Health Corp. Phoenix, Arizona, USA) and necropsied from 21 February to 14 May 1992. Samples taken at the time of necropsy included blood for serology and 49 tissues for bacterial culture. These tissues included: right an(l left atlantal, axillary,
internal
liteal, prefelnoral, malnmary, and
iliac,
mandibular, prescapular,
suprapharangeal
parotid,
each
pop-
of
hepatic,
niediastinal
tissue mammary
kidney,
and
samples gland
liver,
mesenteric
an(l
milk
quarter;
lungs,
both
swabs
and
sam-
adrenal
glands,
spleen, tonsil, both uterine horns, vagina, and cervix. Tissues from B. suis biovar 4-challenged bison were frozen at - 70 C and later thawed and inoculated onto trypticase soy agar ( Baltimore Biological Laboratories, Cockeysville, Maryland, USA) and a more selective media containing per liter: 25g tryptose broth (Difco Laboratories, Detroit, Michigan, USA), 20 g agar (Bacto agar, Difco Laboratories), 0.15 ml Tergitol 7 (Sigma Chemical Co., St. Louis, Missouri, USA), 25 ml Tween 40 (Sigma Chemical Co.), 1.4 ing ethyl violet (Sigma Chemical Co.), 1.44
g
Co.),
sodium 1
vial
of
laulryl
sulfate
CNV
(Cholistin
(Sigma
Chemical sulfate,
7,500
mcg, nystatin, 12,5(X) units, vancomycin, 3,000 mcg, Difco Laboratories) and 500 rug of cycloheximide (Actidione, Sigma Chemical Co.) Tissues from B. abortus-cha!!enged bison were frozen at -70 C and later thawed and plated on Farrell’s restrictive media (Farrell, 1974). Fetuses were necropsied within 12 hr of abortion. Healthy calves were bled, euthanized, and necropsied at the time of adult necropsy. Tissues taken for bacterial culture frolu calves and fetuses included liver, kidney, spleen, lung, abomasal and rectal swabs, and mediastinal lymph nodes. Serum salnples were riot taken from bison l)etween the time of challenge and necropsy because of the risk of inducing stress-related abortions. Serum samples from calves and cows were evaluated by card (Aiton et al., 1975), rivanol precipitation (Alton et a!., 1975), standard tube agglutination (Alton et a!., 1975), cold complement fixation tube (Alton et al., 1975), indirect bison conjugated enzyme linked i bent assay (ELISA) (Davis et al., 1990), competitive ELISA (Adams and Mia, 1992), and particle concentration fluorescence immunoassay (PCFIA) (Reynolds, 1987). Minimum criteria for diagnostically positive reactions for bison on these tests are listed in Animal an(l Plant Health Inspection Service (1992). RESULTS Two biovar this
of
3 wk
renal,
suipra-
mo
lymph
nodes;
tamed
from the
pathogen.
One no
the
fetus. from bison
All Both
B.
SUiS
case
brucellosis.
isolated second
post-challenge.
this
with neither
in
post-challenge;
was of
infected but
to
isolated
pyogene.s
uterus
bison
attributable
aborted were
the
4 aborted,
was bison
pathogens
Actinoinyces
the that
fetal
fetus
and
aborted,
2
tissues bison
cows
also
BEVINS
TABLE
and
Pre-inoculation
1.
I)iovar
ET AL.-PATHOGENICITY
terminal
serology
OF BRUCELLA
in bison
challenged
SUIS
with
BIOVAR
Brucella
4 FOR
and
ahortus
583
BISON
Brncella
suis
4.
Indirect
ELISA
B. ahortu.s (n = 6) Pre-inoculation
0.194
B.
±
0.068I Terminal
6)
0.280
±
±
0.217
±
0.2.33
0.167
0.428e
0.125
B. abortus
B.
0.601
0.604
±
0.000
0.123
0.975
Calf
=
Competitiv
PCFIAh
4
sni.s
(ii
0.152
±
results
1) Particle
concentration
are
> 1 (XX)
ELISA.
Cornpetitive
Positive
± standard
Mean
optical
((3D)
±
4.98
7.22
±
1.76
±
0.601
0.046
0.000
0.64
0.601
±
71.65
±
23.02
6.24
±
2.30
4.14
±
0.98
2.72
±
units.
iinmunoa.ssay. results
4.58
o.ooo
density
fluorescence
±
B. .‘iui.s 4
0.008
o.iii a Positive
e ELISAC
B. abortus
4
suis
are
>70%
Positive
results
inhibition.
Bison
are
40%
inhibition.
deviation.
1.
ii
to
=
4.
were
infected
with
B.
was
isolated
from
a
node
of
the
dibular
first
cow
and
a
second Brucella
tracts
or
and
both
was
node no
lymph
from
of
the
the of
reproductive
glands.
The
four
re-
maining bison challenged with B. suis biovar 4 bore healthy calves at term, approximately 4 mo post-challenge. These four cows and for brucellosis brucellosis son
their when
were
observed
challenged
A!!
six
calves cultured.
with
bison
were No
suis
vano! nation Results
for
Similarly,
all
negative (Table By
surviving
by all tests 1). comparison,
five
lenged with B. ahortus 50 days post-challenge, weak Four 24
calf
that
swabs
taken
calving
or
from abortion
of
six
card,
animal
yielded
ri-
the
2).
sero-
bison
cha!-
after
to a
birth.
placentas Vaginal shortly
cultures
after of B.
also
six of
calf
and
obtained
cows
five
Many tissues contained
from
the
were
all
of
glands
(Table 2). challenged
obtained in
Cultures
uterus
mammary
serologic
test
and
from
of these
ani-
in each
of the (Ta-
B. abortus
(Table
1).
DISCUSSION Based
on a sample size of six animals, biovar 4, at a dose of 2 X i07 CFU, not pathogenic for bison. Although bi-
suis
son may become infected biovar 4, these preliminary idence that with infection
of necropsy
had retained parturition. each
at
33 days one bore
shortly
sampled
ble 2). All bison infected with B. ahortus developed antibodies to the disease and were strongly positive on more than one
was
suis
tests
were
aborted and
died
of these bison hr following
a!!
PCFIA and in Table 1.
time
were
tissues
(Table
mals bison
B.
tube agglutifixation tests.
calves at the
bi-
B.
ELISA, are given
ELISA
of the
with
standard complement
Indirect
competitive
of
4.
challenged
precipitation, and cold
signs
biovar
biovar 4 were seronegative by the time of necropsy, including
Cultures
negative
in any
B.
eight
fetuses
evidence
in the
mammary
all
man-
parotid
lymph
There infection
abortus.
lymph
from
a
popliteal
cow. spp.
4, which
mandibular
nodes,
lymph
node
biovar
suis
the
ripheral which
lymph aborted,
pathogens idence
that
ed.
There
B.
suis
reproductive mentally is unlikely
infection localized nodes. lack
with results
B.
are
In the of any
was no evidence 4 to the mammary
that
B.
ev-
was sub-clinical, in regional or peone bison significant
in the fetus and uterus was this abortion was stress-relat-
tract infected
suis
of metastasis gland or
evof the
in any of the experibison, and therefore, it suis
biovar
4 would
shed by these animals. The experimental dose of B. suis biovar 4 should have been a sufficient challenge dose. A dose of 1.4 X i03 CFU B. abortus
be
584
JOURNAL
TABLE
2.
OF WILDLIFE
Tissues
DISEASES,
VOL.
32, NO. 4, OCTOBER
Brucella
spp.
was
which
from
isolated
1996
in bison
challenged
with
B.
ahortus.
Animal
and
Tissues
Head
ivniph
organs
nodesa
Organ
lymph
Other
peripheral
nodesb lymph
nodese
lymph
Supramammarv
Mammary
gland
Reproductive Other
808
nodes
and
swahsd
Calf
glands or
fetal
Right
and
left
I) Bronchial.
Right
tjssuesg
and
Tissue
left
from
Right
and
Liver,
long,
g Calf
atlantal.
hepatic.
mammary
each
uterine
spleen.
cervix,
and
819
6/8
5/8
418
5/8
3/6
3/6
4/6
2/6
0/6
4/10
9/10
SIlo
SIlo
4110
4110
2/2
2/2
2/2
212
1/2
2/2
2/8
5/8
2/8
0/8
5/8
1/8
1/4
2/4
1/4
1/4
1/4
1/4
0/4
3/4
1/4
0/4
0/4
0/4
1/1
1/1
0/1
0/1
1/1
0/1
0/2
0/2
0/2
0/2
0/2
0/2
4/8
8/8
8/8
2/8
7/8
8/8
and
suprapharvngeal
and
right
and
lymph
nodes.
left adrenal
popliteal.
and
corresponding
with
817
8/8
prefemoral.
quarter
horns, and
fetal
or
parotid. nuesenteric.
prescapular.
axillarv.
left
(808)
mandibular. mediastinal,
816
0/6
Tonsil Adrenal
812
3/8
organse
organst
SI()
lymph
internal
nodes.
lymph
iliac
nodes.
swabs.
vagina.
kidney.
(all others)
liver,
lung.
kid,ie
spleen.
aboniasal
aboma.sum.
rectal
SWOI).
swab,
and
mediastinal
lymph
node.
is
sufficient
to
infect
1939).
Tests
of
al.,
species
a cow
(McEwen
vaccine
et
efficacy
have used 106 or i07 CFU of Bruspp. as challenge doses (Plommet,
cella
1990). If bison rally-occurring
B.
were
the been
of i07 CFU should to produce infection.
challenge sufficient
suis
If
these biovar
the
susceptibility
susceptible
suis
to
biovar
4
the
species,
natu-
if B.
biovar 4 was not shed by infected imals, then it is possible to extrapolate state that B. suis biovar 4 is unlikely maintained
in
a bison
source
unlike
B. abortus
infection both
ease
and
causes mammary
son, and the in populations
disease of
has wild
fected
cattle
been bison
of
(Tessaro,
1986;
in
able
levels
bodies biovar
in-
Meagher,
bison
for Brucella tion between
in this
study
spp.-specific the
of
necropsy.
challenged
with
time
were antibody
biovar
tested produc-
of challenge
It is possible B. .cuis
not
and that
bison
4 developed
The
lack
are
infec-
of
detect-
anti-
necropsy in B. bison is evidence to this
E!evated
wild
or more spp.
spp.-specific
of
bison
in yearly
organism re-
unlikely
samplings, in
of
antibody are
sampled
suis
to
be
particularly
the
fall
at
if
hunting
season.
did we
Although the B. SiLZS biovar 4 organism not appear to be pathogenic for bison, are recommending that precautions be
taken when reindeer are shipped from the Seward Peninsula to Alaska’s interior. Such precautions include adequate fencing for of bison
antine
and
In
summary,
results
from
causes
and
testing
reindeer
of
and
shipped
quar-
reindeer
1991).
(Stahmann, the
months
Brucella
are
in detect-
Bnzcella
immunoreactivity
separation
The
several
at the time 4 -challenged
animals
dis-
with
of
they significance.
produced
species.
detected
maintained in North
produced,
active
other
of
1973).
time
tions
in
in bi-
contact
to
sponses
Bra-
infections
for
response
were
protocol.
diagnostic
usually
quantities
in
be
is clearly
in bison. reproductive
gland
independent
America
This
were
are
which
experimental
little
species.
and to
these of
reduced in this
an-
independent
of infection.
abortus
cella
herd
indeed
able
responses
by our
Antibodies
have
suis
another
If
infection,
and
antibody
detected
probably
results of an experimenta! B. 4 infection in six bison reflected of
temporary
not
other
only
based
six sub-clinical
bison,
on
the B.
experimental suis
infections
biovar in bison.
4
BEVINS
Imported
reindeer
cellosis
are
which
not
may
expected
cellosis disease of Alaska.
risk
ET AL-PATHOGENICITY
to interior
tus
bru-
carry
to present
from
sion nal
This
ence
study
was
and
supported
L.
B.,
S. T. TESSARO.
brucellosis
Alaskan
Technology Foundation, Medical Center,
A&M Veterinary stitute of Arctic Fairbanks.
Biology,
the Texas and the In-
University
of
C.,
box
,noschatus
nal
of Wildlife
of
AND
D-Tec
A.
S.
A.
Brucella
petitive
A”,
a monoclonal-based
com-
im munosorbent
assay
enzyme-linked
(cELISA) tle.
for
serodiagnosis
Proceedings
of
States
Animal
United
Field evaluation
of brucellosis
the
Annual Health
Association
peutics National
of
the
tional
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