Experimental infection of Bordetella bronchiseptica to rabbits


Yoda, H.; Nakayama, K.; Nakagawa, M.

Jikken Dobutsu. Experimental Animals 31(2): 113-118

1982


Infectivity and pathogenicity of Bordetella bronchiseptica to rabbits were investigated by intranasal inoculation of the organism to suckling and young animals. Results are summarized as follows. 1. By inoculation of 5 × 104 and 106 organisms, the infection developed in 60% and 100% of young rabbits, respectively. 2. In young rabbits, the growth of the organism was evident in the trachea within 5 days after inoculation and in the lung on the 10th day. The organism began to disappear from the lung and trachea of some animals from the 40th day after inoculation, but remained in the external nares and nasal cavity of all animals even on the 90th day. Neither clinical sign nor pneumonic lesion was observed in any stages of the infection. 3. Pneumonic lesions and serous nasal secretion were found in all suckling rabbits by inoculation of 5 × 106 organisms, but no fatal case was detected.

Exp.
Anim.
31(2),
113-118,
1982
Experimental
Infection
of
Bordetella
bronchiseptica
to
Rabbits
Hachiharu
YODA,
Kazue
NAKAYAMA
and
Masaro
NAKAGAWA
Department
of
Veterinary
Science,
National
Institute
of
Health,
10-35,
Kamiosaki
2-Chome,
Shinagawa-ku,
Tokyo
141,
Japan
(Received
for
publication
:
November
10,
1981)
Infectivity
and
pathogenicity
of
Bordetella
bronchiseptica
to
rabbits
were
investigated
by
intranasal
inoculation
of
the
organism
to
suckling
and
young
animals.
Results
are
summarized
as
follows.
1.
By
inoculation
of
5
x
10
4
and
10
6
organisms,
the
infection
developed
in
60%
and
100%
of
young
rabbits,
respectively.
2.
In
young
rabbits,
the
growth
of
the
organism
was
evident
in
the
trachea
within
5
days
after
inoculation
and
in
the
lung
on
the
10th
day.
The
organism
began
to
disappear
from
the
lung
and
trachea
of
some
animals
from
the
40th
day
after
inoculation,
but
remained
in
the
external
nares
and
nasal
cavity
of
all
animals
even
on
the
90th
day.
Neither
clinical
sign
nor
pneumonic
lesion
was
observed
in
any
stages
of
the
infection.
3.
Pneumonic
lesions
and
serous
nasal
secretion
were
found
in
all
suckling
rabbits
by
inoculation
of
5
x
10
6
organisms,
but
no
fatal
case
was
detected.
Bordetella
bronchiseptica
is
generally
regarded
as
one
of
the
causal
agents
of
snuffles
and
pneumonia
in
rabbits
[6,9].
However,
only
a
few
investigators
have
reported
natural
outbreaks
of
these
diseases
due
to
B.
bronchiseptica
in
rabbits
[1,5]
and
others
considered
the
organism
a
common
inhabitant
of
the
normal
respira-
tory
tracts
[2,
3].
Thus
the
pathogenicity
of
B.
bronchiseptica
to
rabbits
has
not
yet
fully
evaluated.
In
the
present
study,
infectivity
and
pathogenicity
of
B.
bronchiseptica
are
investigated
in
experimentally
infected
suckling
and
young
rabbits.
Materials
and
Methods
Rabbits
:
Three
-day
-old
sucklings
and
2
to
3
-month
-old
young
of
Japanese
White
rabbits
were
used
in
this
experiment,
regardless
of
their
sexes.
They
were
obtained
from
a
rabbit
colony
free
from
Pasteurella
multocida,
Eimeria
spp.
as
well
as
B.
bronchiseptica.
The
rabbit
colony
had
been
maintained
in
a
non-barriered
facility
but
carefully
isolated
from
possible
contamination
sources
of
the
organism
such
as
other
laboratory,
domestic
and
wild
animals
and
pets
by
use
of
sterilized
bedding,
cages
and
other
equipments.
B.
bronchiseptica
strain
:
A
fresh
isolate
from
the
lung
of
a
naturally
infected
rabbit,
which
was
named
as
R-36
strain,
was
used
throughout
the
experiment.
The
strain
was
Gram-negative
and
motile
rod,
and
showed
such
biochemical
properties
as
nitate
reduction
+,
Simmons
citrate
me-
dium
+,
H
2
S
production
—,
indol
production
—,
urease
+
and
glucose
fermentation
—.
The
strain
also
showed
a
strong
reaction
in
slide
agglutination
test
by
use
of
a
hyperimmune
rabbit
serum
against
B.
bronchiseptica
strain
64L
[8].
It
had
been
maintained
on
nutrient
agar
by
subcultu-
ring
twice
before
use.
Experimental
infection
:
A
loopful
(2
mm
diameter)
amount
of
dilutions
of
48
114
hour
nutrient
broth
(1
%
polypeptone
and
0.5%
NaCI
in
fresh
beef
infusion
of
pH
7.2)
culture
containing
5x
10
2
,
10'
and
10
6
viable
count
was
smeared
into
the
external
nares
of
a
unanesthetized
rabbit.
The
rabbit
was
housed
in
a
40x50
x
40cm
metal
cage
with
wire
mesh
floor
and
given
100g
commercial
pellets
(Funabashi
Farm)
a
day
and
tap
water
ad
libitum.
In
the
case
of
sucklings,
they
were
housed
in
the
above
mentioned
metal
cage
with
a
30x
50
x40cm
nest
box,
together
with
their
dam.
The
cages
were
placed
separately
each
other
by
putting
a
partition
between
the
cages,
and
the
rabbits
were
carefully
handled
to
prevent
the
transmission
of
the
organism
from
one
to
another.
At
the
termination
of
the
experiment,
the
rabbits
were
sacrificed
by
inhalation
of
chloroform.
After
taking
blood
by
heart
puncture,
swab
samples
were
obtained
from
the
mucous
membrane
of
the
external
nares,
nasal
cavity
and
upper
(cervix)
and
lower
(chest)
parts
of
the
trachea
of
rabbits,
cultivating
on
DHL
agar
plate
(Eiken
Co.).
A
piece
of
lung
material
was
also
obtained
from
each
lobe,
minced
with
scissors
and
cultivated
on
a
5
%
horse
blood
agar
plate.
Agglutination
test
:
The
tube
aggluti-
nation
test
was
carried
out
by
use
of
serum
samples
inactivated
at
56°C
for
30
minutes
and
48
hour
nutrient
broth
culture
of
strain
R-36
as
antigen,
which
had
been
killed
in
0.6%
formaline
saline
for
3
days
at
37°C,
washed
3
times
with
saline
and
then
adjusted
the
turbidity
to
tube
no.
3
of
MacFarland's
nephelometer
series
with
saline.
After
making
a
2
-fold
serial
dilu-
tion
of
the
serum
sample,
0.5m1
of
each
serum
dilution
and
0.5m1
antigen
were
mixed
and
left
at
37°C
for
2
hours
and
then
4°C
overnight.
Antibody
titer
was
recorded
by
the
reciprocal
of
serum
dilu-
tion
showing
positive
agglutination.
Results
1.
Infectivity
of
B.
bronchiseptica
to
young
rabbits
In
order
to
examine
the
relationship
between
inoculum
sizes
of
the
organism
and
development
of
the
infection
in
2
to
3
-month
-old
rabbits,
5
each
of
the
rabbits
were
inoculated
intranasally
with
5
x
10
2
,
5
x
10'
and
5x
10
6
organisms.
They
were
sacrificed
20
days
after
inoculation
for
post-mortem
examinations
such
as
cultiva-
tion
of
the
organism
from
various
parts
of
the
respiratory
tract,
observation
of
pneu-
monic
lesions
and
checking
of
serum
agglutination
titers.
Results
are
shown
in
Table
1.
None
of
the
rabbits
received
5x
10
2
viable
cells
harbored
the
organism
in
any
parts
of
the
respiratory
tract
and
was
suffered
from
the
pneumonic
lesions.
Their
serum
agglutination
titers
were
1
:
10
or
lower,
except
one
which
showed
1
:
20
titer
in
spite
of
negative
isolation
of
the
orga-
nism.
Three
of
5
rabbits
inoculated
with
5
x
10'
viable
cells
were
suffered
from
the
infection,
yielding
a
large
number
of
the
organism
from
all
parts
of
the
respiratory
tract
examined.
However,
neither
pneumo-
nic
lesion
nor
significant
rise
of
agglutina-
tion
titer
was
observed
in
these
infected
animals.
By
administration
of
5x
10
6
cells,
all
of
5
rabbits
harbored
the
organism
throughout
the
external
nares,
nasal
cavity,
trachea
and
lung,
while
macroscopic
lung
lesions
failed
to
appear
in
any
of
them.
Agglutination
titers
rose
to
some
degrees
as
1:
20
to
1:
40.
It
was
apparent
from
these
results
that
10
6
organisms
were
enough
numbers
for
development
of
the
infection
of
B.
bronchiseptica
in
rabbits.
Thus
an
inoculum
size
of
10
6
organisms
was
used
in
all
the
following
experiments.
2.
Periodical
observations
on
the
course
of
infection
Thirty-five
young
rabbits
of
2
to
3
-
month
-old
of
age
were
inoculated
with
5x
10
6
cells
of
B.
bronchiseptica
and
five
each
of
them
were
periodically
sacrificed
up
to
90
days
after
inoculation
for
isolation
of
the
organism,
observation
on
lung
lesions
115
Table
1.
Development
of
B.
bronchiseptica
infection
in
young
rabbits
according
to
inoculum
sizes
of
the
organism
Isolation
of
the
organism
Inoculum
Animal
size
no.
External
Nasal
nares
cavity
Trachea
upper
lower
Pneumonic
Aggl.
Lung
lesion
titer
1
2
5x10
2
3
4
5
1
:
10
1
:
10
<
1
:
10
1
:
20
<
1
:
10
6
7
5X10
4
8
9
10
ilk
1W
1W
1W
1
:
10
1
:
10
1
:
20
1
:
10
1
:
10
11
12
5
X
10
6
13
14
15
1W
1W
1W
iff
1W
+I+
1W
1W
1W
1W
1W
1W
1W
1W
1W
Animals
were
sacrificed
20
days
after
inoculation
of
*
Colony
numbers
developed
on
DHL
or
horse
blood
agar
plate
on
isolation
;
*
:
more
than
100,
-H-
:
21-100,
:
1-20,
:
no
colony
1W
1W
1W
1W
the
organism.
and
titration
of
serum
agglutinin.
Results
are
summarized
in
Table
2.
The
organism
was
isolated
from
the
upper
respiratory
tract
but
not
from
the
lung
5
days
after
inoculation,
when
neither
lung
lesion
nor
rise
of
serum
agglutinin
titer
was
observed.
After
10
days,
the
organism
became
detectable
in
the
lung
as
well
as
in
the
upper
respiratory
tract
of
many
rabbits,
but
no
macroscopic
lung
lesion
was
observed
even
in
animals
sacri-
ficed
thereafter.
Serum
agglutination
titers
began
to
rise
in
many
animals
after
10
days
and
distributed
between
1
:
20
and
1
:
160
thereafter.
From
the
40th
day,
the
orga-
nism
began
to
disappear
from
the
lung
and
trachea
in
some
animals,
thus
the
organism
was
not
isolated
from
lung
sam-
ples
of
all
animals
sacrificed
on
the
90th
day
when
the
experiment
was
terminated.
Such
a
clearance
phenomenon
of
the
orga-
nism
was
also
observed
in
the
trachea
of
3
of
5
animals
on
the
90th
day
post-inocula-
1
:
40
1
:
40
1
:
40
1
:
40
1
:
20
tion,
but
all
the
animals
still
harbored
the
organism
in
their
nares
and
nasal
cavities.
Apparent
symptoms
of
snuffles
were
not
observed
in
any
animals
during
the
experimental
period.
3.
Experimental
infection
in
suckling
rabbits
In
order
to
study
the
susceptibility
of
suckling
rabbits
to
B.
bronchiseptica,
thir-
teen
3
-day
-old
sucklings
from
2
litters
were
intranasally
inoculated
with
5
x
10
6
organisms,
and
3
to
4
each
were
sacrificed
weekly
or
biweekly
for
post-mortem
observations.
Results
are
shown
in
Table
3.
A
large
amount
of
the
organism
was
isolated
from
all
parts
of
the
respiratory
tract
including
the
lung
of
all
3
animals
sacrificed
2
weeks
after
inoculation,
but
no
pneumonic
lesion
was
found
at
this
stage
of
the
infection.
Thereafter,
the
organism
was
also
isolated
in
a
large
amount
from
entire
parts
of
the
respiratory
tract,
and
pneumonic
lesions
were
detected
macros-
116
Table
2.
Periodical
observation
on
young
rabbits
infected
with
B.
bronchiseptica
Days
after
inoculation
of
the
organism
Animal
no.
Isolation
of
the
organism
External
Nasal
Trachea
nares
cavity
upper
lower
Pneumonic
Aggl.
Lung
lesion
titer
1
2
**
Off
+I+
Off
Off
1
:
10
1
:
10
5
3
iff
fff
1
:
10
4
Off
<
1
:
10
5
Off
fff
1
:
10
6
*
*
+I+
44
+
1
:
10
7
* * #
+I+
d+
1
:
80
10
8
*
* * *
+
1
:
20
9
*
II+
*
+H-
4+
1
:
20
10
* *
*
+I+
1
:
20
11
+I+
* *
Off
+F
1
:
10
12
* * *
+I+
+H
1
:
20
20
13
* * * *
+
1
:
40
14
+I+
* *
1-14
4H-
1
:
10
15
* * *
* *
1
:
80
16
* *
* * *
1
:
40
17
* * * *
*
1
:
10
30
18
*
+I+
41+
+I+
+F
1
:
80
19
*
4ff
* * *
1
:
80
20
* * * * *
1
:
10
21
*
+1-
+I-
1
:
160
22
41+
Off
*
+I-
-1+
1
:
40
40
23
+I+
--H-
+
+
1
:
160
24
1
:
80
25
Off
Off
1
:
80
26
+F
+F
1
:
10
27
+F
1+
1
:
80
70
28
+F +F
1
:
80
29
4+
1
:
40
30
+I+
+F
1
:
160
31
+I+
-1+
1
:
80
32
1
:
80
90
33
4+
Off
+1-
1
:
40
34
4+
1
:
80
35
41+
-
FF
1
:
40
Animals
were
intranasally
inoculated
with
5x10
6
organisms.
*
See
the
foot
-note
of
Table
1.
copically
in
all
animals
examined
during
the
3rd
to
6th
weeks,
usually
appearing
in
one
lobe
(left
or
right
upper
lobe)
of
the
lung.
Although
no
fatal
case
was
observed,
they
from
the
nose.
serum
agglutinin
all
animals,
even
showed
serous
secretion
Nevertheless
a
rise
of
titer
was
not
observed
in
in
those
sacrificed
at
the
117
Table
3.
Experimental
infection
of
B.
bronchiseptica
to
suckling
rabbits
Weeks
after
inoculation
of
the
organism
Animal
no.
Isolation
of
the
organism
External
nares
2
1-1
1-2
2-1
1W
3
1-3
2-2
2-3
1W
4W
Nasal
Trachea
cavity
upper
lower
4W
1W
Pneumonic
Aggl.
Lung
lesion
titer
44
4-1-4
CIF
44
<
1
:
10
<
1
:
10
1
:
10
11-
411
off
4W
4W
1-4
1-5
2-4
4W
4W
if
f
4W
4W
:
10
1
:
10
1
:
10
<
1
:
10
<
1
:
10
1
:
10
6
1-6
1-7
1-8
2-5
-Hf
fly
4*
fff
Hi
CIF
4*
414
Animals
were
intranasally
inoculated
with
5x10
6
organisms.
*
See
the
foot
-note
of
Table
1.
6th
week,
showing
1
:
10
or
lower.
Discussion
Bordetella
bronchiseptica
infection
has
been
mainly
studied
in
guinea
pigs
among
laboratory
animals,
indicating
that
10
4
or
more
organisms were
enough
inoculum
sizes
for
development
of
the
infection
[8],
the
bronchopneumonia
appeared
in
about
80
per
cent
of
infected
animals
[8]
and
the
infected
guinea
pigs
usually
recovered
to
be
in
non
-carrier
state
of
the
organism
within
20
weeks
after
infection
[10].
Com-
paring
with
these
results,
the
rabbit
seemed
to
have
the
same
susceptibility
to
the
organism
as
the
guinea
pig,
because
of
development
of
the
infection
in
3
of
5
animals
received
5x
10
4
organisms.
However,
development
of
the
pneumonic
lesions
was
dependent
on
age
of
rabbits,
being
positive
in
sucklings
but
negative
in
young
in
spite
of
the
presence
of
the
organism
in
the
lung.
Maeda
and
Shimizu
[4]
reported
a
high
incidence
of
the
pneu-
monia
in
70
hour
newborn
rabbits
intra-
1
:
10
1
:
10
<
1
:
10
<
1
:
10
nasally
inoculated
with
B.
bronchiseptica
strains
of
swine
and
rabbit
origins,
but
no
young
or
adult
rabbit
was
used
in
their
experiment.
Likewise,
Mayer
[5]
reported
natural
outbreaks
of
enzootic
pneumonia
due
to
B.
bronchiseptica
infection
in
young
rabbits
in
two
large
rabbit
farms,
and
some
fatal
cases
were
included
in
these
outbreaks.
In
our
experiment,
however,
such
a
severely
affected
case
was
not
observed
even
in
sucklings.
As
regards
the
fate
of
the
infection,
the
organism
was
cleared
from
the
lung
and
trachea
of
2
of
5
young
rabbits
sacri-
ficed
90
days
after
infection,
but
all
the
5
rabbits
still
harbored
the
organism
in
their
external
nares
and
nasal
cavities.
In
a
long-term
observation
on
30
rabbits
natu-
rally
infected
with
B.
bronchiseptica,
it
was
found
that
24
animals
shed
the
organism
from
their
noses
during
longer
than
one
year
after
infection
(unpublished
data).
Thus
the
complete
recovery
of
the
infection
seemed
to
be
rare
in
rabbits.
Serum
agglutinin
titers
of
infected
rabbits
were
generally
lower
than
those
of
118
guinea
pigs
[7],
usually
ranging
1
:
20
to
80,
and
some
of
them
showed
as
a
low
titer
as
1
:
10
even
in
the
middle
and
late
stages
of
the
infection.
None
of
the
infected
suckling
rabbits
also
showed
significant
agglutinin
titers
exceeding
1
:
10
which
was
common
in
non
-infected
ones.
This
phenomenon
must
be
taken
into
consideration
in
the
serological
diagnosis
of
B.
bronchiseptica
infection
in
rabbits.
References
Ferry,
N.
S.
(1913-14).
Bacteriology
and
control
of
acute
infections
in
laboratory
animals.
J.
Path.
Bact.
18,
445-455.
Flatt,
R.,
and
Dungworth,
D.
L.
(1971).
Enzootic
pneumonia
in
rabbits:
Microbiology
and
comparison
with
lesions
experimentally
produced
by
Pasteurella
multocida
and
a
chlamydial
organism.
Amer.
J.
Vet.
Res.
32,
627-637.
Griffin,
C.
A.
(1952).
Respiratory
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among
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Control
and
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Proc.
Anim.
Care
Panel,
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Meeting,
3-13.
Maeda,
M.,
and
Shimizu,
T.
(1975).
Nasal
infec-
tion
of
Alcaligenes
bronchiseptica
(Bordetella
C5]
[
6
]
C7]
[8]
C9]
[10]
Bordetella
bronchiseptica
bronchiseptica)
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Nat.
Inst.
Anim.
Hlth
Quart.
15,
29-37.
Mayer,
von
H.
(1971).
Bordetellainfektionen,
ein
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der
Massentierhaltung
bei
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Berl.
Muench.
Tieraerztl.
Wochenschr.
Nr.
14,
273-
274.
M'Gowan,
J.
P.
(1911).
Some
observations
on
a
laboratory
epidemic,
principally
among
dogs
and
cats,
in
which
the
animals
affected
presented
the
symptoms
of
the
disease
called
"distemper".
J.
Path.
Bact.
15,
372-426.
Nakagawa,
M.,
Muto,
T.,
Nakano,
T.,
Yoda,
H.,
Ando,
K.,
Isobe,
Y.,
and
Imaizumi,
K.
(1969)•
Some
observations
on
diagnosis
of
Bordetella
bronchiseptica
infection
in
guinea
pigs.
Exp.
Anim.
18,
105-116.
Nakagawa,
M.,
Muto,
T.,
Yoda,
H.,
Nakano,
T.,
and
Imaizumi,
K.
(1971).
Experimental
Bordetella
bronchiseptica
infection
in
guinea
pigs.
Jpn.
J.
Vet.
Sci.
33,
53-60.
Weisbroth,
S.
H.,
and
Scher,
S.
(1969).
The
establishment
of
a
specific
-pathogen
-free
rabbit
breeding
colony.
II.
Monitoring
for
disease
and
health
statistics.
Lab.
Anim.
Care,
19,
795-799.
Yoda,
H.,
Nakagawa,
M.,
Muto,
T.,
and
Imaizumi,
K.
(1972).
Development
of
resistance
to
reinfection
of
Bordetella
bronchiseptica
in
guinea
pigs
reco-
vered
from
natural
infection.
I
pn.
J.
Vet.
Sci.
34,
191-196.
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