Simultaneous subcutaneous and conjunctival administration of the influenza viral vector based Brucella abortus vaccine to pregnant heifers provides better protection against B. abortus 544 infection than the commercial B. abortus S19 vaccine


Tabynov, K.; Orynbayev, M.; Renukaradhya, G.J.; Sansyzbay, A.

Vaccine 34(42): 5049-5052

2016


In this study, we explored possibility of increasing the protective efficacy of our novel influenza viral vector based B. abortus vaccine (Flu-BA) in pregnant heifers by adapting an innovative method of vaccine delivery. We administered the vaccine concurrently via the conjunctival and subcutaneous routes to pregnant heifers, and these routes were previously tested individually. The Flu-BA vaccination of pregnant heifers (n=9) against a challenge B. abortus 544 infection provided protection from abortion, infection of heifers and fetuses/calves by 88.8%, 100% and 100%, respectively (alpha=0.004-0.0007 vs. negative control; n=7). Our candidate vaccine using this delivery method provided slightly better protection than the commercial B. abortus S19 vaccine in pregnant heifers (n=8), which provided protection from abortion, infection of heifers and fetuses/calves by 87.5%, 75% and 87.5%, respectively. This improved method of the Flu-BA vaccine administration is highly recommended for the recovery of farms which has high prevalence of brucellosis.

Vaccine
xxx
(2016)
xxx-xxx
ELSEVIER
Contents
lists
available
at
ScienceDirect
Vaccine
journal
homepage:
www.elsevier.com/locate/vaccine
Vaccine
Short
communication
Simultaneous
subcutaneous
and
conjunctival
administration
of
the
influenza
viral
vector
based
Brucella
abortus
vaccine
to
pregnant
heifers
provides
better
protection
against
B.
abortus
544
infection
than
the
commercial
B.
abortus
S19
vaccine
Kaissar
Tabynov
Mukhit
Orynbayev
a
,
Gourapura
J.
Renukaradhya
b
,
Abylai
Sansyzbay
a
'The
Research
Institute
for
Biological
Safety
Problems,
Zhambulskaya
oblast,
Kordaiskiy
rayon,
080409
Gvardeisky,
Kazakhstan
b
Food
Animal
Health
Research
Program,
Ohio
Agricultural
Research
and
Development
Center,
Department
of
Veterinary
Preventive
Medicine,
The
Ohio
State
University
(OSU),
Wooster,
OH
44691,
USA
ARTICLE INFO
Article
history:
Received
8
April
2016
Received
in
revised
form
19
August
2016
Accepted
23
August
2016
Available
online
xxxx
Keywords:
Brucella
abortus
Vaccine
Influenza
viral
vector
Protection
Abortion
Infection
Pregnant
heifer
Vaccination
method
ABSTRACT
In
this
study,
we
explored
possibility
of
increasing
the
protective
efficacy
of
our
novel
influenza
viral
vec-
tor
based
B.
abortus
vaccine
(Flu
-BA)
in
pregnant
heifers
by
adapting
an
innovative
method
of
vaccine
delivery.
We
administered
the
vaccine
concurrently
via
the
conjunctival
and
subcutaneous
routes
to
pregnant
heifers,
and
these
routes
were
previously
tested
individually.
The
Flu
-BA
vaccination
of
preg-
nant
heifers
(n
=
9)
against
a
challenge
B.
abortus
544
infection
provided
protection
from
abortion,
infec-
tion
of
heifers
and
fetuses/calves
by
88.8%,
100%
and
100%,
respectively
(alpha
=
0.004-0.0007
vs.
negative
control;
n
=
7).
Our
candidate
vaccine
using
this
delivery
method
provided
slightly
better
pro-
tection
than
the
commercial
B.
abortus
S19
vaccine
in
pregnant
heifers
(n
=
8),
which
provided
protection
from
abortion,
infection
of
heifers
and
fetuses/calves
by
87.5%,
75%
and
87.5%,
respectively.
This
improved
method
of
the
Flu
-BA
vaccine
administration
is
highly
recommended
for
the
recovery
of
farms
which
has
high
prevalence
of
brucellosis.
©
2016
Elsevier
Ltd.
All
rights
reserved.
1.
Introduction
Bovine
brucellosis
is
an
economically
devastating
disease
caused
by
the
gram
-negative
bacterium
Brucella
abortus,
through
induction
of
abortion
and
decreased
fertility
in
infected
cattle,
and
chronic
zoonotic
infection
in
humans
[1].
For
prophylaxis
of
this
dangerous
disease,
we
previously
developed
a
novel
vector
based
vaccine,
Flu
-BA,
a
recombinant
influenza
virus
subtypes
H5N1
and
H1N1
vaccine
expressing
the
Brucella
Ompl6
and
L7/
L12
proteins.
The
Flu
-BA
vaccine
was
shown
to
be
safe
[2]
and
effective
[3]
in
cattle,
including
in
pregnant
animals
[4,5].
Our
ear-
lier
study
using
Flu
-BA
vaccination
demonstrated
protection
against
B.
abortus
544
infection
by
70-80%
and
abortion
by
80-
90%
in
heifers.
Pregnant
heifers
are
more
sensitive
to
brucellosis
than
adult
cows
[6].
The
protective
response
induced
by
the
Flu
-
BA
vaccine
was
equivalent
to
the
current
commercial
vaccine
B.
abortus
S19
[5].
These
encouraging
results
suggested
that
the
Flu
-
BA
vaccination
combined
with
other
proper
control
measures
may
*
Corresponding
author.
E-mail
addresses:
tabynov
81@mail.ru,
tabynov
81@biosafety.kz
(IC
Tabynov).
http://dx.doLorg/10.1016/j.vaccine.2016.08.072
0264-410X/©
2016
Elsevier
Ltd.
All
rights
reserved.
be
effective
in
eradication
of
brucellosis
from
endemic
countries.
In
Kazakhstan,
brucellosis
is
endemic
with
a
high
disease
prevalence
[7],
so
we
need
to
develop
a
highly
effective
vaccine
which
pro-
vides
sterilizing
immunity
(100%)
to
eradicate
this
malady
in
cat-
tle.
In
this
study
our
main
goal
was
to
further
enhance
the
effectiveness
of
the
Flu
-BA
vaccine
in
pregnant
heifers
by
innova-
tive
strategies.
Therefore,
we
employed
an
innovative
method
of
concurrent
administration
of
the
Flu
-BA
vaccine
by
the
conjuncti-
val
and
subcutaneous
routes
to
animals.
2.
Materials
and
methods
2.1.
Vaccination
Twenty-five
15-19
months
-old
Kazakh
white
breed
pregnant
heifers
at
2-3
months
of
pregnancy
from
brucellosis
-free
herds
were
obtained.
The
heifers
were
artificially
inseminated
and
the
pregnancy
was
confirmed
at
an
early
stage
(up
to
60
days
of
preg-
nancy)
using
the
hormonal
method
(progesterone
concentration)
and
by
rectal
palpation.
These
animals
at
3-4
months
gestation
2
K.
Tabynov
et
al./Vaccine
xxx
(2016)
xxx—xxx
were
randomly
divided
into
three
groups:
experimental,
positive
and
negative
controls.
Pregnant
heifers
in
the
experimental
group
(Flu
-BA
vaccine;
n
=
9)
were
immunized
twice
concurrently
via
subcutaneous
(1
ml)
and
conjunctival
(0.5
ml
to
each
eye)
routes
at
an
interval
of
28
days
with
vaccines
generated
from
the
influ-
enza
viral
vectors
(IVV)
subtypes
H5N1
(prime
vaccination;
6.2-
6.5
log
10
EID
50
/animal)
and
H1N1
(booster
vaccination;
6.1-6.3
log
10
EID
50
/animal).
Animals
in
the
positive
control
groups
(B.
abor-
tus
S19;
n
=
9)
were
immunized
once
subcutaneously
in
the
neck
region
(right
side)
with
the
commercial
vaccine
B.
abortus
S19
(Shchelkovsky
Biokombinat,
Moscow
oblast,
Russia;
8.0
x
10
10
CFU/animal)
according
to
the
manufacturer's
instruc-
tions.
Pregnant
heifers
in
the
negative
control
group
(n
=
7)
were
administered
with
10%
Montanide
Ge101
adjuvant
in
PBS
in
the
same
manner
as
the
Flu
-BA
vaccine.
2.2.
Assessment
of
vaccine
protectiveness
On
the
day
56
post
-vaccination,
i.e.,
when
pregnant
heifers
at
5-6
months
gestation
belong
to
the
experimental,
negative
and
positive
control
groups
were
subcutaneously
challenged
with
a
vir-
ulent
strain
of
B.
abortus
544
(5.0
x
10
8
CFU/animal).
Clinical
observation
of
the
challenged
animals
was
performed
up
to
calving
or
abortion
for
3-4
months.
Animals
that
gave
birth
to
non
-viable
calves
(stillbirths)
were
considered
under
the
aborted
group.
From
aborted
fetuses
and
newborn
calves
within
12
h
collected
sub
-
mandibular,
retropharyngeal,
right
subscapular,
left
subscapular,
mediastinal,
bronchial,
portal,
para-aortic,
pelvic
and
mesenteric
lymph
nodes,
and
also
liver,
kidney,
spleen
and
bone
marrow
for
bacteriological
analysis.
After
abortion
or
calving
the
heifers
were
euthanized
and
collected
similar
samples
listed
above
in
addition
to
mammary
lymph
nodes
and
placentome.
Euthanasia
of
heifers
and
their
newborn
calves
was
performed
by
intravenous
injection
pentobarbital
sodium
(100
mg/kg)
(Euthasol,
Le
Vet.
Pharma
BV,
Oudewater,
Netherlands).
The
tissue
slices
and
homogenates
were
plated
on
Brucella
agar plates
and
incubated
at
37
°C
for
4
weeks,
and
bacterial
colonies
were
counted
periodically
during
this
time.
The
concentration
of
bacteria
(CFU/g
of
tissue)
in
the
tissue
sam-
ples
were
determined
by
performing
standard
plate
counts.
An
ani-
mal
was
considered
to
be
infected
if
a
Brucella
colony
was
detected
from
the
culture
of
one
or
more
organs.
The
bacteriological
exam-
ination
was
assessed
by
determining
the
number
of
animals
from
which
no
colonies
were
isolated
(effectiveness
of
vaccination)
and
index
of
infection
(the
number
of
organs
and
lymph
nodes
from
the
animals
and
their
fetuses
or
calves
from
which
Brucella
organisms
were
isolated).
All
isolates
were
identified
using
routine
methods
described
previously
[8].
2.3.
Statistical
analysis
Difference
in
infection
or
abortion
rates
between
vaccinated
and
control
groups
were
compared
by
one-sided
Fisher's
exact
test
for
two
proportions
at
a
significance
level
of
alpha
<
0.5.
Signifi-
cance
of
the
differences
in
index
of
infection
and
colonization
of
Brucella
in
tissues
between
groups
was
analyzed
using
one-way
ANOVA
followed
by
Tukey's
multiple
comparisons
test.
P
val-
ues
<
0.05
were
considered
significant.
Means
are
reported
with
standard
errors
(SEM).
Statistical
analysis
of
all
the
experimental
data
was
performed
using
Graphpad
Prism
Software,
version
6.0
(Graphpad
Software
Inc.,
CA,
USA).
2.4.
Other
materials
and
methods
Information
about
the
generation
of
IVV
and
vaccine
prepara-
tion,
as
well
as
aspects
of
bioethics
are
included
under
Supplemen-
tary
Materials.
3.
Results
Our
results
indicated
that
both
the
Flu
-BA
and
B.
abortus
S19
vaccines
provided
significant
protection
against
abortion
(alpha
=
0.03-0.004)
and
B.
abortus
544
infection
(including
fetuses
or
calves;
alpha
=
0.008-0.0007)
in
pregnant
heifers
(Table
1).
In
negative
control
group,
the
rates
of
abortion
and
infection
(includ-
ing
fetuses
or
calves)
in
pregnant
heifers
were
71.4%
abortion
(5/7),
and
100%
and
85.7%
infection
in
the
pregnant
heifers
and
their
fetuses
or
calves
(vaccine
effectiveness),
respectively.
While
the
Flu
-BA
vaccine
provided
the
maximum
protection
(100%)
with
0%
abortion
(0/9),
and
11.2%
and
0%
infection
in
pregnant
heifers
and
their
fetuses
or
calves
(vaccine
effectiveness),
respectively.
In
B.
abortus
544
challenged
positive
control
pregnant
heifers
vacci-
nated
with
B.
abortus
S19,
12.5%
abortion
(1/8),
and
25%
and
15.5%
infection
in
the
pregnant
heifers
and
their
fetuses
or
calves
(vaccine
effectiveness),
respectively,
were
observed.
The
abortions
in
Brucella
challenged
heifers
were
observed
20-35
days
before
the
predicted
calving
period.
With
respect
to
other
indicators
of
protection,
such
as
the
index
of
infection
(Fig.
1)
and
Brucella
colonization
in
tissues
of
pregnant
heifers
and
their
fetuses
or
calves
(Table
2),
both
the
vaccines
pro-
vided
significant
protection
(P
=
0.04
to
<0.0001)
compared
to
the
negative
control
group.
Further,
these
parameters
were
not
signif-
icantly
different
(P
=
0.3
to
>0.99)
between
these
two
vaccines
received
animals.
It
should
be noted
that
in
heifers
vaccinated
with
the
Flu
-BA
vaccine,
Brucella
colonization
(5
CFU/g
of
tissue)
was
only
found
in
one
animal
at
one
of
the
regional
lymph
nodes
(right
subscapular)
around
the
injection
site
of
the
B.
abortus
544
strain
challenge.
While
in
the
Brucella
positive
pregnant
heifers
belong
to
positive
and
negative
control
groups,
Brucella
was
mainly
iso-
lated
from
70%
to
90%
of
animals
from
the
right
subscapular,
mam-
mary
and
mesenteric
lymph
nodes
and
placentome.
In
Brucella
positive
fetuses
and
newborn
calves,
the
highest
frequency
(87.5%)
of
Brucella
colonization
was
observed
in
the
spleen
and
bone
marrow.
4.
Discussion
The
commercial
B.
abortus
S19
and
B.
abortus
RB51
vaccines
are
widely
used
to
mitigate
Brucella
outbreaks
in
cattle.
But
both
the
vaccines
have
significant
shortcomings,
including
they
cause
abor-
tion
in
some
vaccinated
cattle
and
importantly
pathogenic
in
humans
[9,10],
which
limit
their
global
widespread
use.
This
work
is
a
continuation
of
our
series
of
studies
aimed
at
developing
a
novel
and
safe
vaccine
against
B.
abortus
infection
with
comparable
efficacy
of
the
commercial
vaccines.
Our
studies
with
Flu
-BA
vac-
cine
not
only
achieved
that
goal
[2-5,11-13],
it
also
accomplished
the
most
important
criteria
of
an
"ideal
Brucella
vaccine"
described
earlier
by
Schurig
et
al.
[14]
and
Ko
and
Splitter
[15].
In
addition
to
its
ability
to
protect
against
B.
abortus
infection
in
cattle
[12],
our
vaccine
was
shown
to
provide
cross
-protection
against
B.
melitensis
infection
in
cattle
[13].
In
fact,
for
the
fi
rst
time
after
20
years
since
the
introduction
of
the
B.
abortus
RB51
vaccine,
our
Flu
-BA
vaccine
has
shown
huge
promise
in
repeated
experimental
studies
in
cat-
tle,
and
now
it
is
entering
fi
eld
trials
in
2016-2017
in
Kazakhstan.
As
previously
mentioned,
brucellosis
is
endemic
with
a
high
prevalence
in
Kazakhstan
[7].
Therefore,
for
total
eradication
of
Brucella
infection
from
Kazakhstan,
the
vaccine
efficacy
should
be
as
high
as
100%
along
with
other
exhaustive
control
measures.
Within
the
framework
of
our
planned
fi
eld
trials
in
Kazakhstan,
we
will
attempt
to
test
in
a
large
Brucella-infected
cattle
farm.
Therefore,
this
study
was
conducted
to
further
improve
its
effi-
ciency
from
our
earlier
study
from
70%
to
80%
protection,
which
was
akin
to
the
commercial
B.
abortus
S19
vaccine
[5].
K.
Tabynov
et
al./
Vaccine
xxx
(2016)
xxx-xxx
3
Table
I
Rates
of
abortion,
parturition
and
infection
among
pregnant
Kazakh
white
breed
heifers
after
challenge
with
the
virulent
strain
B.
abortus
544.
Group
Abortion,
/7'
(%)
Parturition,
n
b
(%)
Isolation
of
B.
abortus
from
heifers
Isolation
of
B.
abortus
from
fetuses
and
calves
Total
Positive
(%)
Negative
(%)
Positive
(%)
Negative
(%)
Flu
-BA
vaccine`
0
(0)
*
9
(100)
1
(11.2)
*
8
(88.8)
0
(0)
*
9
(100)
9
B.
abortus
S19
1
(12.5)
*
7
(87.5)
2
(25)
*
6
(75)
1
(12.5)
*
7
(87.5)
8
d
Control
5
(71.4)
2
(28.6)
7
(100)
0
(0)
6
(85.7)
1
(14.3)
7
Number
of
aborted
heifers,
also
includes
animals
that
delivered
stillbirths.
b
Number
of
heifers
that
delivered
a
viable
calf.
`
Influenza
viral
vector
based
B.
abortus
vaccine.
d
One
heifer
aborted
at
day
51
post
-vaccination
with
B.
abortus
S19.
Bacteriological
and
serological
studies
confirmed
that
abortion
of
this
animal
was
caused
by
the
vaccine
(smooth
colonies
of
Brucella
were
isolated
from
the
organs
of
the
aborted
fetus);
this
heifer
was
excluded
from
the
study.
alpha
=
0.03-0.0007
vs.
control
group,
one-sided
Fisher's
exact
test.
A
15
12
0
C,
9
4
.
X
CD
C
O
A
A
A
0
Flu
-BA
vaccine
B.
abortus
S19
A
Control
A
o
h
o
Heifers
Fetuses
and
calves
B
100-
80-
60
ae•
40
20
0
Heifers
Fetuses
and
calves
s.
+,
v
+,\\,+
\
4,
,7\,?
\
>
-
\,+ce\,•+,e
A
*
\
\,
bS
ci
V,04
0
Ab
c
'
xi•
c
'
e
\
c
V
cle
c
:e
t
sc
c'
e
ZP.\
t9
2R
IP
e
eZs
.
N
t'
0
*
of"
et
f
e
(
'
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„,\*
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e
e
4.
0
l
c,
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of
owe
8`
.
•c•
Fig.
1.
Brucella
infection
index
in
pregnant
heifers,
aborted
fetuses
and
newborn
calves.
(A)
The
frequency
of
Brucella
infection
index
from
Brucella
positive
heifers
and
their
fetuses
or
calves;
and
(B)
the
percent
of
different
tissues
of
Brucella
positive
heifers
and
their
fetuses
or
calves
positive
for
Brucella
organisms
challenged
with
the
virulent
strain
B.
abortus
544.
Vaccination
of
pregnant
heifers
was
carried
out
twice
at
an
interval
of
28
days
with
the
influenza
viral
vector
B.
abortus
(Flu
-BA)
vaccine
by
concurrent
conjunctival
and
subcutaneous
routes
of
administration.
The
positive
control
group
was
vaccinated
subcutaneously
once
with
the
commercial
vaccine,
B.
abortus
S19,
and
the
negative
group
was
administered
with
Montanide
Ge101
adjuvant
in
PBS.
All
the
heifers
were
challenged
with
the
virulent
B.
abortus
544
via
the
subcutaneous
route.
The
index
of
infection
is
the
number
of
organs
and
lymph
nodes
from
the
animals
and
their
fetuses
or
calves
Brucella
organisms
were
isolated;
*
from
P=
0.002
to
<0.0001,
two-
way
ANOVA
followed
by
Tukey's
multiple
comparisons
test.
Among
the
possible
methods
of
improving
vaccine
protective-
ness,
we
selected
an
approach
which
was
shown
previously
to
be
effective.
In
our
earlier
study
with
the
Flu
-BA
vaccine
in
cattle
[5],
prime
-boost
vaccination
was
followed
by
either
conjunctival
or
subcutaneous
routes,
and
both
the
routes
provided
comparable
high
protection
against
B.
abortus
544
challenge
infection.
Activa-
tion
of
the
protective
immune
response
following
the
conjunctival
immunization
in
cattle
was
shown
to
be
induced
by
conjunctiva
-
associated
lymphoid
tissue
(CALT)
and
eye
-associated
lymphoid
tissue
(EALT).
CALT
can
sample
antigens
on
the
ocular
surface
and
present
to
naïve
T
cells
to
generate
protective
effector
lympho-
cyte
activity
[16,17].
However,
as
the
conjunctival
method
led
to
difficulties
associated
with
low
productivity
and
the
complexity
of
controlling
the
dosage,
we
selected
subcutaneous
immunization
as
the
primary
administration
mode
in
further
studies.
Subcuta-
neous
immunization
of
the
Flu
-BA
vaccine
resulted
in
cross
-
protection
against
B.
melitensis
infection
[13],
as
well
as
the
pro-
longed
protective
immune
response
against
B.
abortus
544
infec-
tion
was
maintained
for
at
up
to
12
months
after
booster
vaccination
[12].
Therefore,
in
this
study
to
further
improve
the
Flu
-BA
vaccine
efficacy,
we
concurrently
used
subcutaneous
and
conjunctival
routes
of
administration.
To
note
in
our
earlier
stud-
ies,
increasing
the
Flu
-BA
vaccine
dose
(6.0-7.0
log
10
EID
50
/animal)
or
concentration
of
the
adjuvant
Montanide
Ge101
(10-20%)
did
not
have
a
significant
impact
on
its
effectiveness
in
cattle
[5,12].
Our
results
in
this
study
met
our
expectation
by
concurrent
administration
of
Flu
-BA
vaccine
to
pregnant
heifers
by
the
con-
junctival
and
subcutaneous
routes,
which
provided
100%
protec-
tion
from
abortion
to
a
B.
abortus
544
challenge,
with
88.8%
of
the
pregnant
heifers
and
100%
of
their
fetuses
or
calves
protected
against
infection.
Moreover,
Flu
-BA
vaccine
provided
slightly
bet-
ter
protection
than
the
commercial
B.
abortus
S19
vaccine.
As
expected
B.
abortus
S19
vaccine
caused
abortion
in
1
out
of
9
vac-
cinated
pregnant
heifers.
In
Brucella
challenged
Flu
-BA
vaccinated
heifers,
the
Brucella
colonization
was
found
in
one
animal
in
a
regional
lymph
node
around
the
site
of
injection
of
the
B.
abortus
544
strain.
Therefore,
a
generalized
infectious
process
was
not
noted
in
this
Brucella
positive
heifer.
Consequently,
the
effective-
ness
of
the
Flu
-BA
vaccine
in
pregnant
heifers
was
approximately
100%.
This
result
with
the
Flu
-BA
vaccine
in
pregnant
heifers
is
superior
to
our
previous
results
with
the
conjunctival
(mucosal)
or
subcutaneous
(parenteral)
routes
of
administration
[5,12].
Over-
all,
the
improved
Flu
-BA
vaccine
efficacy
is
a
consequence
of
the
mode
administration
in
combination
with
other
factors
such
as
cross
-immunization
scheme,
wide
tropism
of
IVV,
mucoadhesive-
ness
of
the
Montanide
Ge101
adjuvant,
and
Brucella
Ompl
6
protein
adjuvant
properties
[3].
Thus,
we
conclude
that
concurrent
subcutaneous
and
conjunc-
tival
administration
of
our
novel
Flu
-BA
vaccine
provides
height-
ened
protection
against
B.
abortus
544
infection
in
pregnant
4
K.
Tabynov
et
al./
Vaccine
xxx
(2016)
xxx-xxx
Table
2
Colonization
and
recovery
of
B.
abortus
in
tissues
after
challenge
with
B.
abortus
544
in
vaccinated
heifers
and
their
fetuses
and
calves.
Sample
type
Mean
±
SEM
CFU/g
of
tissue
(number
recovered/total
number)
Flu
-BA
vaccine'
B.
abortus
S19
Control
Heifers
Submandibular
LN
b
o
±
0
(0/9)
*
0
±
0
(0/8)
*
0.6
±
0.4
(2/7)
Retropharyngeal
LN
0
±
0
(0/9)
*
0.1
±
0.1
(1/8)
*
1.7
±
0.5
(5/7)
Right
subscapular
0.05
±
0.05
(1/
0.3
±
0.2
(2/8)
*
3.0
±
0.5
(6/7)
LN
9)
*
Left
subscapular
LN
0
±
0
(0/9)
*
0
±
0
(0/8)
*
1.2
±
0.5
(4/7)
Mediastinal
LN
0
±
0
(0/9)
*
0
±
0
(0/8)
*
2.7
±
0.7
(5/7)
Bronchial
LN
0
±
0
(0/9)
*
0
±
0
(0/8)
*
3.1
±
0.5
(6/7)
Portal
LN
0
±
0
(0/9)*
0±0
(0/8)*
3.0
±
0.7
(6/7)
Para
-aortic
LN
0
±
0
(0/9)
*
0
±
0
(0/8)
*
2.1
±
0.7
(5/7)
Pelvic
LN
0
±
0
(0/9)
*
0±0
(0/8)
*
1.4
±
0.3
(6/7)
Mesenteric
LN
0
±
0
(0/9)*
0.1
±
0.07
(2/
2.1
±
0.7
(5/7)
8)
*
Udder
LN
0
±
0
(0/9)
*
0.1
±
0.1
(2/8)
*
3.0
±
0.6
(6/7)
Liver
0
±
0
(0/9)
0±0
(0/8)
0.3
±
0.2
(2/7)
Kidney
0
±
0
(0/9)
0±0
(0/8)
0.1
±
0.1
(1/7)
Spleen
0
±
0
(0/9)
*
0
±
0
(0/8)
*
1.1
±
0.3
(5/7)
Bone
marrow
0
±
0
(0/9)
0±0
(0/8)
0.07
±
0.07
(1/
7)
Placentome
0
±
0
(0/9)
*
0.4
±
0.4
(1/8)
*
5.6
±
0.7
(6/7)
Fetuses
and
calves
Submandibular
LN
0
±
0
(0/9)
0
±
0
(0/8)
0
±
0
(0/7)
Retropharyngeal
LN
0
±
0
(0/9)
0
±
0
(0/8)
0
±
0
(0/7)
Right
subscapular
0
±
0
(0/9)
0
±
0
(0/8)
0
±
0
(0/7)
LN
Left
subscapular
LN
0
±
0
(0/9)
0
±
0
(0/8)
0.2
±
0.1
(2/7)
Mediastinal
LN
0
±
0
(0/9)
*
0
±
0
(0/8)
*
0.3
±
0.2
(2/7)
Bronchial
LN
0
±
0
(0/9)
*
0
±
0
(0/8)
*
0.5
±
0.2
(3/7)
Portal
LN
0
±
0
(0/9)
*
0±0
(0/8)
*
0.4
±
0.2
(3/7)
Para
-aortic
LN
0
±
0
(0/9)
0
±
0
(0/8)
0.2
±
0.1
(2/7)
Pelvic
LN
0
±
0
(0/9)
0±0
(0/8)
0±0
(0/7)
Mesenteric
LN
0
±
0
(0/9)
0±0
(0/8)
0.04
±
0.04
(1/
7)
Liver
0
±
0
(0/9)
0±0
(0/8)
0±0
(0/7)
Kidney
0
±
0
(0/9)
0±0
(0/8)
0±0
(0/7)
Spleen
0
±
0
(0/9)
*
0.2
±
0.2
(1/8)
*
0.9
±
0.3
(5/7)
Bone
marrow
0
±
0
(0/9)
*
0.1
±
0.1
(1/8)
*
1.2
±
0.3
(5/7)
All
isolates
from
tissue
samples
were
identified
as
B.
abortus.
a
Influenza
viral
vector
based
Brucella
abortus
vaccine.
b
LN,
lymph
nodes.
P=0.04
to
<0.0001
vs.
control
group,
two-way
ANOVA
followed
by
Tukey's
multiple
comparisons
test.
heifers
against
abortion;
which
is
better
than
the
commercial
B.
abortus
S19
vaccine.
This
method
of
vaccine
delivery
is
recom-
mended
in
combination
with
other
control
measures
to
aid
in
recovery
of
farms
with
high
prevalence
of
brucellosis;
and
in
other
situations
Flu
-BA
vaccine
could
be
administered
only
by
the
subcu-
taneous
route.
Acknowledgements
We
thank
B.
Yespembetov,
Sh.
Ryskeldinova,
N.
Zinina,
Zh.
Kydyrbayev,
Y.
Kozhamkulov,
D.
Inkarbekov
and
M.
Sarmykova,
and
employees
of
the
Research
Institute
for
Biological
Safety
Prob-
lems
for
their
assistance.
The
work
was
carried
out
under
the
pro-
ject
0402
"Development
of
Products
for
Preventing
Bovine
Brucellosis"
as
part
of
the
research
program
0.0600
"Bovine
Bru-
cellosis:
Monitoring
the
Epizoological
Situation
and
Developing
Means
of
Diagnosis
and
Prevention"
for
2012-2014,
and
the
grant
1296/GF4
for
2015-2017
funded
by
the
Science
Committee
of
the
Ministry
of
Education
and
Science
of
the
Republic
of
Kazakhstan.
Appendix
A.
Supplementary
material
Supplementary
data
associated
with
this
article
can
be
found,
in
the
online
version,
at
http://dx.doi.org/10.1016/j.vaccine.2016.08.
072.
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