Simultaneous intramammary and intranasal inoculation of lactating cows with bovine herpesvirus 4 induce subclinical mastitis


Wellenberg, G.; Bruschke, C.; Wisselink, H.; Barkema, H.; Oirschot, J.-Van

Veterinary Microbiology 86(1-2): 115-129

2002


In this study, we examined whether an experimental bovine herpesvirus 4 (BHV4) infection can induce bovine mastitis, or can enhance bovine mastitis induced by Streptococcus uberis (S. uberis). Four lactating cows were inoculated intramammarily and intranasally with BHV4, and four lactating control cows were mock-inoculated. After 14 days, two of four cows from each group were inoculated intramammarily with S. uberis. No clinical signs were recorded in cows inoculated only with BHV4, and their milk samples showed no abnormal morphology, despite the fact that BHV4 replicated in inoculated quarters. Somatic cell count increased significantly in milk from three of six BHV4-inoculated quarters, compared to the non-inoculated quarters of the same cows (within-cow) and the quarters of mock-inoculated cows (control group) on days 8, 9 and 11 post-inoculation (pi). BHV4 was isolated from nasal swabs between days 2 and 9 pi. Clinical mastitis was observed in all four cows intramammarily inoculated with S. uberis. A preceding BHV4 infection did not exacerbate the clinical mastitis induced by S. uberis. S. uberis infections appeared to trigger BHV4 replication. From one quarter of each of two cows inoculated with BHV4 and S. uberis, BHV4 was isolated, and not from quarters inoculated with BHV4 only. In conclusion, BHV4 did not induce bovine clinical mastitis after simultaneous intranasal and intramammary inoculation. However, the BHV4 infection did induce subclinical mastitis in 50% of the cows and the quarters.

veterinary
microbiology
ELS
EV
IER
Veterinary
Microbiology
86
(2002)
115-129
www.elsevier.com/locate/vetmic
Simultaneous
intramammary
and
intranasal
inoculation
of
lactating
cows
with
bovine
herpesvirus
4
induce
subclinical
mastitis
G.J.
Wellenberg
a
'
*
,
C.J.M.
Bruschke
a
,
H.J.
Wisselink
a
,
H.W.
Barkema
b
,
J.T.
Van
Oirschot
a
'Division
of
Infectious
Diseases
and
Food
Chain
Quality,
Institute
for
Animal
Science
and
Health
(ID-Lelystad),
Food
Chain
Quality,
Edelhertweg
15,
P.O.
Box
65,
8200
AB
Lelystad,
The
Netherlands
b
Animal
Health
Service,
P.O.
Box
361,
9200
AA
Drachten,
The
Netherlands
Abstract
In
this
study,
we
examined
whether
an
experimental
bovine
herpesvirus
4
(BHV4)
infection
can
induce
bovine
mastitis,
or
can
enhance
bovine
mastitis
induced
by
Streptococcus
uberis
(S.
uberis).
Four
lactating
cows
were
inoculated
intramammarily
and
intranasally
with
BHV4,
and
four
lactating
control
cows
were
mock-inoculated.
After
14
days,
two
of
four
cows
from
each
group
were
inoculated
intramammarily
with
S.
uberis.
No
clinical
signs
were
recorded
in
cows
inoculated
only
with
BHV4,
and
their
milk
samples
showed
no
abnormal
morphology,
despite
the
fact
that
BHV4
replicated
in
inoculated
quarters.
Somatic
cell
count
increased
significantly
in
milk
from
three
of
six
BHV4-inoculated
quarters,
compared
to
the
non-inoculated
quarters
of
the
same
cows
(within-cow)
and
the
quarters
of
mock-inoculated
cows
(control
group)
on
days
8,
9
and
11
post-inoculation
(pi).
BHV4
was
isolated
from
nasal
swabs
between
days
2
and
9
pi.
Clinical
mastitis
was
observed
in
all
four
cows
intramammarily
inoculated
with
S.
uberis.
A
preceding
BHV4
infection
did
not
exacerbate
the
clinical
mastitis
induced
by
S.
uberis.
S.
uberis
infections
appeared
to
trigger
BHV4
replication.
From
one
quarter
of
each
of
two
cows
inoculated
with
BHV4
and
S.
uberis,
BHV4
was
isolated,
and
not
from
quarters
inoculated
with
BHV4
only.
In
conclusion,
BHV4
did
not
induce
bovine
clinical
mastitis
after
simultaneous
intranasal
and
intramammary
inoculation.
However,
the
BHV4
infection
did
induce
subclinical
mastitis
in
50%
of
the
cows
and
the
quarters.
©
2002
Elsevier
Science
B.V.
All
rights
reserved.
Keywords:
Bovine
herpesvirus
4;
S.
uberis;
Intramammary
and
intranasal
inoculation;
Mastitis;
Lactating
cows;
Virus
Abbreviations:
BHV4,
bovine
herpesvirus
4;
S.
uberis,
Streptococcus
uberis;
pi,
post-inoculation;
IPMA,
immunopemxidase
monolayer
assay;
SCC,
somatic
cell
count
*
Corresponding
author.
Tel.:
+31-320-238219;
fax:
+31-320-238050.
E-mail
address:
g.j.wellenberg@id.wag-ur.nl
(G.J.
Wellenberg).
0378-1135/02/$
see
front
matter
©
2002
Elsevier
Science
B.V.
All
rights
reserved.
50378-1135(01)00496-5
116
G.J.
Wellenberg
et
al.
/Veterinary
Microbiology
86
(2002)
115-129
1.
Introduction
Mastitis
is
a
disease
with
large
economic
impact
on
dairy
cattle
industry
(Miller
and
Dorn,
1990;
Schakenraad
and
Dijkhuizen,
1991).
Bacteria
and
non-bacterial
pathogens
play
a
role
in
clinical
mastitis
(Radostits
et
al.,
1994;
Watts,
1988).
Despite
intensive
implementation
of
control
programs,
still
around
20-35%
of
clinical
cases
of
bovine
mastitis
have
an
unknown
aetiology
(Miltenburg
et
al.,
1996).
The
percentage
of
bacteriological
culture-negative
milk
samples
of
both
clinical
and
subclinical
mastitis
cases
is
still
approximately
25%
(Barkema
et
al.,
1998).
Recently,
in
a
case-control
study,
bovine
herpesvirus
4
(BHV4)
was
isolated
from
milk
samples
collected
from
cows
with
clinical
mastitis,
whereas
no
virus
was
isolated
from
matched
controls
(Wellenberg
et
al.,
2000).
Concomitant
development
of
BHV4
antibodies
in
cows
supported
the
hypothesis
that
BHV4
may
play
a
role
in
mastitis
in
dairy
cows.
In
the
same
study,
a
positive
association
was
found
between
BHV4
isolation
and
the
isolation
of
Streptococcus
uberis
from
milk
samples.
This
study
was
performed
to
examine
whether:
(a)
mastitis
was
induced
after
a
simultaneous
intramammary
and
intranasal
inoculation
of
lactating
cows
with
BHV4;
(b)
a
preceding
BHV4
infection
exacerbated
clinical
mastitis
induced
by
S.
uberis;
(c)
a
S.
uberis
infection
triggered
the
replication
of
BHV4.
2.
Materials
and
methods
2.1.
Cell
culture,
virus
and
bacteria
A
pestivirus-
and
mycoplasma-free
bovine
umbilical
cord
endothelial
(BUE)
cell
line
was
used
for
virus
isolation
and
multiplication
(Van
de
Wiel
et
al.,
1989).
BUE
cells
were
grown
and
maintained
in
Dulbecco's
minimal
essential
medium
(DMEM)
(Gibco
Labora-
tories,
Life
Technologies,
USA)
supplemented
with
10%
foetal
bovine
serum
(Gibco
Laboratories,
Life
Technologies,
USA)
and
0.5%
antibiotic
mix
(stock
mix
contained:
10,000
IU
penicillin,
11.25
mg
streptomycin,
10
mg
kanamycin
and
5000
IU
nystatin
per
ml).
The
BHV4
strain
Tolakker,
which
has
been
isolated
from
a
cow
with
clinical
mastitis,
was
used
for
inoculation
(Wellenberg
et
al.,
2000).
After
isolation,
this
virus
was
passaged
once
in
BUE
cells
to
obtain
a
virus
stock.
The
stock
virus
batch,
used
for
animal
inoculation,
contained
10
7
'
3
median
tissue
culture
infective
doses
(TCID
50
)/ml
of
BHV4
strain
Tolakker,
and
was
free
of
other
herpesviruses,
pestiviruses,
mycoplasmas
and
bacteria.
S.
uberis
strain
0140J
has
originally
been
isolated
from
a
cow
with
mastitis,
and
was
kindly
provided
by
Milner
et
al.
(1996).
Prior
to
inoculation,
the
S.
uberis
strain
0140J
was
cultivated
in
Todd-Hewitt
broth
for
24
h
at
37
°C,
and
was
found
to
be
pure.
2.2.
Animals
and
experimental
design
Eight
cows
were
selected
from
three
different
farms
2
weeks
before
the
start
of
the
study.
Inclusion
criteria
for
selection
were:
(a)
no
clinical
or
subclinical
mastitis,
(b)
the
same
parity
(first
or
second
parity),
(c)
no
history
of
Streptococcus
(uberis)
infections,
(d)
first
half
of
lactation,
and
(e)
no
treatment
with
antibiotics
during
the
last
month.
G.J.
Wellenberg
et
al./Veterinary
Microbiology
86
(2002)
115-129
117
Table
1
Inoculation
scheme
of
cows
inoculated
with
BHV4,
BUE
cell
culture,
or
S.
uberis
Cow
Intranasally
Intramammarily
into
quarters
RF
and
LH
with
BHV4
(day
0)
BUE
(day
O)
a
BHV4
(day
0)
BUE
(day
0)
S.
uberis
(day
14)
B1
+
b
+
+
B2
+
+
B3
+
+
B4
+
+
+
Cl
+
+
+
C2
+
+
+
C3
+
+
C4
+
+
a
BUE
cell
culture.
b
Not
inoculated.
In
addition,
milk
samples
from
each
udder
quarter
and
blood
samples
were
collected
from
these
eight
cows
to
examine
whether:
(a)
these
cows
contained
no
antibodies
against
BHV4,
(b)
milk
samples
of
the
four
quarters
were
free
of
udder
pathogens,
and
(c)
cows
were
BVDV-free.
The
eight
cows
were
randomly
allotted
to
a
BHV4
and
a
control
group.
The
BHV4
and
the
control
group
were
housed
in
two
separate
isolation
units,
and
cows
were
tied
individually.
Hygienic
rules
were
strictly
followed
to
prevent
transmission
of
viruses
and
bacteria
between
the
two
groups
and
from
one
cow
to
another.
The
four
cows
of
the
BHV4
group
(B1,
B2,
B3
and
B4)
were
inoculated
intramammarily
into
the
right
forequarter
(RF),
and
left
hindquarter
(LH)
with
10
7
'
3
TCID
50
of
the
BHV4
strain
Tolakker
diluted
in
20
ml
of
phosphate
buffered
salt
solution
(PBS)
per
quarter
(Table
1).
The
other
two
quarters
(right
hindquarter
(RH)
and
left
forequarter
(LF))
were
not
inoculated
and
served
as
within-cow
control
quarters.
Prior
to
each
intramammary
inoculation
via
the
teat
channel,
teats
were
disinfected
with
alcohol.
After
each
inoculation,
using
a
syringe
with
a
short
blunt
ended
needle,
the
udder
was
massaged
to
distribute
the
inoculum.
The
four
cows
of
the
BHV4
group
were
also
intranasally
inoculated
with
1
ml
containing
10
7
'
3
TCID
50
of
BHV4
per
nostril,
using
a
nozzle
attached
to
a
syringe
to
produce
a
spray.
Cows
of
the
control
group
(C1,
C2,
C3
and
C4)
were
mock-inoculated
into
the
same
two
quarters
as
described
above
for
the
BHV4
group
with
20
ml
BHV4-free
BUE
cell
culture
medium.
The
cows
of
the
control
group
were
also
intranasally
inoculated
as
described
for
the
BHV4
group
with
BHV4-free
BUE
cell
culture
medium.
Fourteen
days
after
the
BHV4
inoculation,
the
RF
and
LH
quarters
of
cows
B1
and
B4
from
the
BHV4
group,
and
of
cows
C1
and
C2
from
the
control
group,
were
inoculated
intramammarily
with
800
colony-forming-units
(cfu)
of
S.
uberis
strain
0140J
in
20
ml
of
PBS.
Prior
to
inoculation,
teats
were
disinfected
with
alcohol
and
after
inoculation
the
udder
was
massaged
to
distribute
the
bacterium.
All
inoculations
were
performed
after
the
morning
milking
between
10:00
and
12:00
a.m.
The
cows
were
milked
twice
daily
using
a
quarter
milking
device.
During
this
study,
cows
were
not
treated
with
antibiotics,
except
cow
C4
that
was
treated
with
Duoprim
(intramuscularly)
and
Delvomast
(LH)
daily
on
days
9-17
pi.
118
G.J.
Wellenberg
et
al.
/Veterinary
Microbiology
86
(2002)
115-129
2.3.
Collection
of
samples
A
volume
of
50
ml
of
milk
from
each
quarter
of
each
cow
was
collected
daily
just
before
the
a.m.
milking
of
the
cows.
Milk
samples
were
collected
according
to
the
procedure
described
by
the
National
Mastitis
Council
(Harmon
et
al.,
1990),
and
transported
immediately
to
the
laboratory,
homogenised
and
divided
for
storage.
Milk
samples
for
virus
isolation
were
directly
stored
at
—70
°C.
Milk
samples
used
for
the
determination
of
somatic
cell
counts
(SCCs),
bacterial
isolation
and
antibody
detection
were
immediately
stored
at
—20
°C.
Weekly,
blood
samples
were
obtained
from
the
vena
jugularis,
cen-
trifuged
at
2000
x
g
for
10
min,
and
sera
were
stored
at
—20
°C.
Daily,
blood
samples
were
collected
in
tubes
containing
EDTA
starting
on
day
—2
till
day
28
pi
for
white
blood
cell
counts
and
for
blood
cell
differentiation.
Daily,
nasal
swabs
for
BHV4
isolation
and
titration
were
collected,
processed
and
stored
directly
at
—70
°C,
as
described
by
Kaashoek
et
al.
(1994).
All
collected
milk,
nasal
secretions
and
serum
samples
were
stored
in
duplicate.
2.4.
Clinical
signs
and
morphology
of
the
milk
Clinical
signs
and
rectal
temperatures
were
recorded
daily
in
the
morning.
Udders
were
palpated
for
signs
of
infection
(swollen
or
painful).
The
morphology
of
the
milk
samples
was
examined
daily.
Therefore,
the
first
streams
of
milk
secretion
were
discarded,
and
the
foremilk
was
checked
for
colour,
clots
and
morphology.
2.5.
Assays
of
samples
2.5.1.
Somatic
cell
count
Somatic
cells
in
daily
quarter
milk
samples
were
counted
by
means
of
a
Fossomatic
(Foss
Electric,
Hiller4d,
Denmark).
Subclinical
mastitis
was
defined
high
quarter
milk
SCC
of
>250,000
cells/ml
(Dohoo
and
Leslie,
1991;
Smith,
1996),
without
any
visible
abnormality
of
the
milk
or
the
udder
(International
Dairy
Federation,
1987).
2.5.2.
White
blood
cell
counts
and
differentiation
The
white
blood
cell
(WBC)
counts
were
determined
using
an
automated
cell
counter
(Sysmex
F-800,
Charles
Goffin,
the
Netherlands).
Each
day,
thin
blood
films
were
prepared
from
venous
blood
collected
in
tubes
containing
EDTA.
The
slides
were
stained
by
using
the
May—Grunwald—Giemsa
staining
method
(Merck
KGaA,
Darmstadt,
Germany),
and
the
WBC
were
differentiated
by
typing
100
cells.
2.5.3.
BHV4
isolation
In
order
to
isolate
virus,
milk
samples
were
thawed
and
defatted
by
centrifugation
at
1500
x
g
for
10
min.
A
volume
of
0.2
ml
of
the
defatted
milk
was
added
to
0.3
ml
of
DMEM
and
pipetted
on
a
semi-confluent
monolayer
of
BUE
cells
in
24-wells
cell
culture
plates.
Virus
isolation
was
further
performed
as
described
earlier
(Wellenberg
et
al.,
2000).
Cell
cultures
were
observed
daily
for
cytopathic
effect
(cpe).
After
a
freeze/thaw
cycle,
a
second
passage
was
performed
by
inoculating
the
virus/cell
suspensions
on
semi-confluent
mono-
layers
of
BUE
cells.
BUE
cell
cultures
were
incubated
again
for
7
days
at
37
°C
(5%
CO2).
G.J.
Wellenberg
et
al./Veterinary
Microbiology
86
(2002)
115-129
119
In
each
test
run,
five
controls
were
incorporated.
Three
controls,
containing
10,
100
and
1000
TCID
50
BHV4/ml
milk,
served
as
positive
controls.
A
BHV4-free
milk
sample
and
a
non-inoculated
BUE
cell
culture
control,
served
as
negative
controls.
The
BHV4
titre
was
determined
in
milk
samples
from
two
arbitrarily
chosen
cows
B1
(RF)
and
B3
(LH)
on
days
1,
3,
5,
7,
9
and
10
or
11
pi,
and
in
BHV4
positive
milk
samples
obtained
after
S.
uberis
inoculation
(Table
2).
Virus
titration
was
performed
by
preparing
serial
10-fold
dilution
steps
in
DMEM
(10
-1
-10
-8
).
A
volume
of
200
pi
of
each
dilution,
plus
300
pi
of
DMEM,
was
inoculated
in
12-fold
on
BUE
cells
cultivated
in
24-wells
cell
culture
plates.
Plates
were
incubated
as
described
above.
After
a
freeze/thaw
cycle,
a
second
passage
on
BUE
cells
was
performed,
and
cells
were
examined
for
the
appearance
of
cpe
after
an
incubation
period
of
7
days
at
37
°C
(5%
C
O
2).
Isolation
and
titration
of
BHV4
from
nasal
secretions
was
performed
on
BUE
cells
using
96-wells
cell
culture
plates.
For
BHV4
isolation,
BUE
cells
in
suspensions
of
150
pi
were
inoculated
with
50
pi
of
thawed
nasal
swab
suspension
and
cells
were
examined
for
cpe
after
an
incubation
for
7
days
at
37
°C
(5%
CO
2
).
For
virus
titration,
serial
10-fold
dilutions
of
nasal
swab
suspensions
were
prepared
in
DMEM
(10
°
40
-4
).
Each
dilution
was
ino-
culated
in
eightfold
on
BUE
cells
as
described
above
and
incubated
at
37
°C
(5%
CO
2
).
Plates
for
virus
titration
were
examined
after
7
days
for
the
appearance
of
cpe.
In
each
virus
titration
assay,
the
endpoint
titres
were
expressed
as
log
10
TCID
50
/ml
by
using
the
method
of
Reed
and
Muench.
2.5.4.
BHV4
antibodies
Serum
and
pooled
milk
samples
from
all
four
quarters,
in
dilutions
of
1:20
and
1:5,
respectively,
were
screened
for
antibodies
against
BHV4
by
immunoperoxidase
monolayer
assay
(IPMA)
(Wellenberg
et
al.,
1999).
Samples,
containing
antibodies
against
BHV4,
were
titrated
in
serial
twofold
dilutions,
starting
at
a
dilution
of
1:20
(serum)
or
1:5
(milk).
The
titre
of
the
test
sample
was
taken
as
the
reciprocal
of
the
highest dilution
giving
a
positive
reaction.
2.5.5.
Bacterial
isolation
Bacteriological
culturing
of
milk
samples
was
performed
based
on
standards
of
the
International
Dairy
Federation (1984).
Briefly,
milk
samples
(0.025
ml)
were
inoculated
on
Oxiod
Blood
Agar
Base
No.
2,
containing
5%
blood
and
0.1%
aesculin.
Plates
were
incubated
at
37
°C,
and
bacterial
growth
was
evaluated
after
24
and
48
h.
Bacterial
colonies
were
identified
and
bacteria
were
considered
to
be
pathogenic
or
non-pathogenic.
A
quarter
was
considered
to
have
an
intramammary
infection
when
>500
cfu/ml
of
the
same
udder
pathogen
was
cultured
from
two
out
of
three
consecutive
milk
samples,
or
when
>100
cfu/
ml
of
a
bacterial
species
was
cultivated
from
a
quarter
with
clinical
signs
of
mastitis
(Barkema
et
al.,
1999).
If
three
or
more
bacterial
species
were
cultivated
from
a
sample,
the
sample
was
considered
to
be
contaminated.
2.6.
Statistical
analysis
To
approximate
the
normal
distribution,
a
natural
logarithmic
transformation
of
the
SCC
was
used
(Shook,
1982).
Statistical
analysis
was
used
to
compare
the
natural
logarithm
of
120
G.J.
Wellenberg
et
al.
/Veterinary
Microbiology
86
(2002)
115-129
SCC
between
quarters
inoculated
with
BHV4,
control
quarters
of
the
same
cow
of
the
BHV4
group
(within-cow
controls),
quarters
of
the
control
cows
inoculated
with
BUE
cell
culture,
and
quarters
of
the
control
cows
that
were
not
inoculated.
The
following
linear
mixed
model
was
used:
Yi
d,
k(i
d
)
=
CQi
d
tk(i
d
)
eid,k(id)
where
Yid,k(id)
is
the
average
natural
logarithm
of
SCC
of
quarter
j
(j
=
1,
,
4)
of
cow
i
(i
=
1,
. . .
,
8)
under
the
assigned
treatment
k,
m
the
overall
mean,
C,
the
random
effect
of
cow
i,
CQ
id
the
random
effect
of
quarter
j
within-cow
i,
and
t
k(id)
the
fixed
effect
of
the
treatment
k
and
finally
eid,k(id)
the
residual
error.
The
treatment
effect
has
a
factorial
structure,
since
it
consists
of
combinations
of
an
intramammary
inoculation
(+
or
—)
and
inoculum
(BHV4
or
BUE
cell
culture).
Estimation
of
the
parameters
in
the
model
is
done
by
restricted
maximum
likelihood
(REML)
and
the
Wald-test
is
applied
to
assess
significance
of
effects.
All
statistical
calculations
were
performed
with
Genstat
5
Com-
mittee
(1993).
Statistical
significance
was
declared
at
p
<
0.05.
3.
Results
3.1.
Clinical
signs
3.1.1.
Days
0-14
pi
Within
the
period
of
0-14
days
pi,
clinical
signs
of
mastitis
or
rectal
temperatures
>39.5
°C
were
recorded
neither
in
the
four
cows
of
the
BHV4
group
nor
in
cows
Cl
and
C2
of
the
control
group.
No
changes
in
udder
health
or
morphology
of
the
milk,
collected
from
BHV4-inoculated
quarters,
BUE
cell
culture-inoculated
quarters,
and
non-inoculated
quarters
were
recorded
in
these
six
cows.
Cow
C4
of
the
control
group
showed
an
increase
in
rectal
temperatures
(>39.5
°C)
for
6
days,
starting
at
day
9
pi.
Swelling
of
the
udder
and
changes
in
the
morphology
of
the
milk
from
all
four
quarters
from
cow
C4
were
recorded
starting
at
day
9
pi.
This
was
also
observed
for
the
LF
quarter
from
cow
C3
starting
on
day
2
pi.
No
clinical
signs
or
increase
of
rectal
temperature
were
further
recorded
for
cow
C3,
and
no
changes
in
the
morphology
of
the
milk,
collected
from
the
three
remaining
quarters
were
recorded.
Gram
negative
bacteria
(Klebsiella
sp.)
were
isolated
from
the
milk
of
these
clinical
mastitis
cases.
Therefore,
all
data
of
all
four
quarters
from
cow
C4,
that
were
obtained
after
day
8
pi,
and
all
data
of
the
LF
quarter
from
control
cow
C3
were
excluded
from
statistical
analysis.
3.1.2.
Days
15-28
pi
After
the
intramammary
inoculation
of
cows
B1
and
B4
(BHV4
group)
and
Cl
and
C2
(control
group)
with
S.
uberis
on
day
14
pi,
increased
rectal
temperatures
(>39.5
°C)
were
recorded
for
all
four
cows
starting
between
days
16
and
19
pi.
Thereafter,
rectal
temperatures
varied
between
38.2
and
41.6
°C
(Table
2).
The
morphology
of
the
milk
from
S.
uberis-inoculated
quarters
altered
and
clots
were
observed
initially
between
days
15
and
20
pi.
Changes
in
the
morphology
of
the
milk,
in
combination
with
painful
and
swollen
quarters,
were
recorded
for
all
S.
uberis-inoculated
quarters
up
to
day
28
pi.
G.J.
Wellenberg
et
al./Veterinary
Microbiology
86
(2002)
115-129
121
Table
2
Rectal
temperatures,
morphology
of
the
milk,
SCC
and
peripheral
WBC
from
cows
inoculated
intramammarily
with
S.
uberis,
preceded
by
a
BHV4
infection
(day
0),
or
with
S.
uberis
only
(day
14
pi)
Response
of
infected
cows
(BHV4
+)
S.
uberis
S.
uberis
B1
B4
Cl
C2
Rectal
temperature
First
day
temperature
>39.5
°C
5a
5
4
2
No.
of
days
>39.5
°C
(n
=
•)
4
a
4
7
5
Morphology
of
the
milk
First
day
clots
in
the
milk
1
5
3
2
No.
of
days
of
abnormal
milk
(n
=
•)
13
6
12
12
Somatic
cell
counts
First
day
of
>250,000/m1
2 2 2 2
No.
of
days
>250,000/m1
(n
=
•)
12
13 13
13
Peripheral
WBC
First
day
of
changes
in
WBC
count
4
5
3
5
No.
of
days
that
the
amount
of
band
form
neutrophils
>2
(n
=
•)
6
1
6
3
a
Days
after
S.
uberis
inoculation
(S.
uberis
inoculated
on
day
14
pi).
No
changes
in
the
morphology
were
observed
in
milk
from
non-S.
uberis-inoculated
quarters
of
these
four
cows.
There
were
no
marked
differences
in
rectal
temperatures
or
the
morphology
of
the
milk
from
quarters
inoculated
with
BHV4
and
S.
uberis
(cows
B1
and
B4)
compared
to
those
of
the
two
cows
that
were
inoculated
with
S.
uberis
only
(C1
and
C2)
(Table
2).
Between
days
15-28
pi,
cows
B2
and
B3
(cows
from
the
BHV4
group
but
not
inoculated
intramammarily
with
S.
uberis)
and
control
cow
C3
(except
for
quarter
LF)
showed
no
clinical
signs,
increase
of
rectal
temperatures,
painful
or
swollen
udders
or
any
changes
in
the
morphology
of
the
milk.
3.2.
Somatic
cell
count
3.2.1.
Days
0-14
pi
Prior
to
the
inoculation
of
BHV4,
SCC
in
milk
from
two
quarters
(LH
from
cow
B1
and
RF
from
B4)
was
>250,000
cells/ml,
and
therefore,
SCC
of
these
two
quarters
was
not
used
for
statistical
analysis.
In
three
of
the
six
remaining
BHV4-inoculated
quarters,
the
SCC
in
milk
increased
above
250,000
cells/ml
(cows
B3
(RF
and
LH)
and
B4
(LH)),
and
reached
levels
up
to
981,000
cells/ml.
No
increased
SCC
was
recorded
in
the
quarters
of
cows
B1
(RF)
and
B2
(RF
and
LH).
An
increase
in
SCC
was
recorded
between
days
6-14
pi
(Fig.
1).
No
SCC
>250,000
cells/ml
was
recorded
in
milk
samples
collected
from
the
eight
non-
BHV4-inoculated
quarters
of
four
cows
of
the
BHV4
group
(within-cow
controls),
from
the
two
quarters
(RF
and
LH)
from
cows
C1, C2,
C3
and
C4
(up
to
day
9
pi)
of
the
control
group
that
were
inoculated
with
BUE
cell
culture
suspension,
and
from
the
two
non-
inoculated
quarters
of
these
four
cows
of
the
control
group
(except
for
the
LF
quarter
of
SCC
(x
10
00
ce
lls
/m
1)
700
600
500
400
300
200
100
122
G.J.
Wellenberg
et
al.
/Veterinary
Microbiology
86
(2002)
115-129
BHV4
inoculated
—E—
BHV4
non-inoculated
Control
BUE
inoculated
Control
non-inoculated
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Days
post-inoculation
Fig.
1.
Mean
SCC
in
milk
from
BHV4-inoculated
quarters
(n
=
6),
non-BHV4-inoculated
quarters
(within-
cow-controls)
(n
=
8),
BUE
cell
culture-inoculated
quarters
(rt
=
6,
plus
data
from
cow
C4
up
to
day
9
pi), and
non-inoculated
quarters
(n
=
5,
plus
data
from
cow
C4
up
to
day
9
pi).
SCC
in
milk
from
quarters
inoculated
with
BHV4
were
significantly
higher
than
the
SCC
in
milk
from
the
other
quarters
on
days
8,
9
and
11
pi
(*
p
<
0.1;
**
p
<
0.05;
***
p
<
0.01).
cow
C3
as
indicated
in
Section
3.1).
In
only
one
occasion,
day
12
pi,
SCC
in
milk
from
quarter
LH
from
cow
C1
was
>250,000
cells/ml
(360,000
cells/nil).
On
days
8,
9
and
11
pi,
SCC
in
milk
from
quarters
inoculated
with
BHV4
was
significantly
higher
than:
(a)
SCC
in
milk
from
the
non-BHV4-inoculated
quarters
from
the
same
cows
(within-cow
controls),
(b)
SCC
in
milk
from
quarters
inoculated
with
BUE
cell
culture
(control
group),
and
(c)
SCC
in
milk
from
non-inoculated
quarters
of
the
control
group
(Fig.
1).
The
differences
between
the
SCC
in
milk
from:
(a)
the
non-BHV4-inoculated
quarters
from
cows
of
the
BHV4
group,
(b)
the
quarters
inoculated
with
BUE
cell
culture
(control
group),
and
(c)
the
non-inoculated
quarters
of
the
control
group,
were
not
significant.
3.2.2.
Days
15-28
pi
The
SCC
in
milk
from
the
RF
and
LH
quarters
from
cows
Cl,
C2,
B1
(RF),
and
B4
(LH)
that
were
inoculated
with
S.
uberis
on
day
14
pi
(n
=
6),
started
to
increase
above
250,000
cells/ml
around
day
16
pi
(Table
2),
and
reached
levels
above
1,000,000
cells/ml.
The
morphology
of
the
milk
changed
between
days
15
and
20
pi
(Table
2).
The
SCC
in
milk
from
the
two
non-S.
uberis-inoculated
quarters
LF
and
RH
of
the
same
four
cows
(n
=
8)
stayed
below
250,000
cells/ml.
In
only
two
occasions,
the
SCC
increased
above
250,000
cells/ml,
namely
for
the
non-inoculated
LF
quarter
of
cow
B4
on
days
23
and
24
pi
(341,000
and
376,000
cells/ml,
respectively).
However,
no
BHV4,
S.
uberis
or
other
bacteria
was
isolated
from
the
milk
of
this
quarter.
G.J.
Wellenberg
et
al./Veterinary
Microbiology
86
(2002)
115-129
123
3.3.
WBC
counts
and
differentiation
of
WBC
No
effects
on
WBC
counts
were
recorded
in
blood
from
the
four
cows
inoculated
with
BHV4
and
the
control
cows
Cl
and
C2
up
to
day
14
pi,
and
no
shift
was
recorded
in
the
peripheral
differential
WBC
of
these
cows.
WBC
counts
started
to
decrease
between
days
17-19
pi,
3-5
days
after
the
inoculation
with
S.
uberis,
and
WBC
counts
of
1.3-2.4
x
10
9
1
were
recorded
in
all
four
cows
between
days
19-22
pi.
In
blood
smears
of
all
four
cows
that
were
inoculated
with
S.
uberis,
a
shift
in
the
peripheral differential
WBC
was
detectable.
An
increase
of
band
form
neutrophils,
up
to
10%
of
the
WBC,
was
recorded
in
blood
smears.
In
all
four
cows
the
WBC
counts
started
to
increase
again
to
normal
values
(5-10
x
10
9
1)
after
day
22
pi.
No
differences
in
WBC
counts,
or
in
the
number
of
band
form
neutrophils
in
blood
smears
were
recorded
in
cows
B1
and
B4
compared
to
those
in
the
two
cows
that
were
inoculated
with
S.
uberis
only
(C1
and
C2)
(Table
2).
No
effects
on
WBC
counts
were
recorded
in
blood
from
cows
B2
and
B3
of
the
BHV4
group,
which
were
not
inoculated
with
S.
uberis,
and
no
shift
was
recorded
in
the
peripheral
differential
WBC
of
these
cows
up
to
day
28
pi.
3.4.
BHV4
isolation
BHV4
was
isolated
from
all
eight
BHV4-inoculated
quarters
(RF
and
LH)
from
day
1
pi
up
to
days
9-14
pi,
and
the
titres
in
milk
samples
from
cows
B1
and
B3
varied
between
10
1.3
and
10
2.9
TCID
50
/ml
(Table
3).
BHV4
was
isolated
neither
from
milk
samples
from
the
two
non-inoculated
quarters
RH
and
LF
of
these
cows
(within-cow
controls),
nor
from
all
four
quarters
from
the
cows
of
the
control
group.
After
the
intramammary
inoculation
with
S.
uberis,
BHV4
was
isolated
from
milk
collected
from
the
RF
quarter
from
cows
B1
and
B4
on
several
days
between
days
22
and
27
pi;
BHV4
titres
varied
between
10
12
and
10
31
TCID
50
/ml
(Table
3).
BHV4
was
not
isolated
from
milk
samples
from
cows
B2
and
B3
that
were
inoculated
intramammarily
with
BHV4,
but
not
with
S.
uberis.
BHV4
was
isolated
from
nasal
swabs
from
all
four
cows
of
the
BHV4
group
between
days
2
and
9
pi.
The
peak
titres
varied
between
10
2.9
and
10
3.9
TCID
50
/ml
(Fig.
2).
No
BHV4
was
isolated
from
nasal
swabs
after
the
intramammary
S.
uberis
inoculation.
3.5.
BHV4
antibody
responses
In
all
four
cows
of
the
BHV4
group,
serum
antibodies
against
BHV4
were
first
detected
on
day
14
pi
(Table
4),
and
reached
levels
between
320
and
2560
on
day
28
pi.
In
milk
samples
of
the
four
cows
of
the
BHV4
group,
antibodies
against
BHV4
were
detectable
around
day
12
pi,
and
BHV4
antibody
titres
increased
up
to
160.
In
sera
and
milk
samples
of
the
four
cows
of
the
control
group
no
BHV4
antibodies
were
detected.
3.6.
Isolation
of
bacteria
on
days
0-14
pi
No
pathogenic
bacteria
were
isolated
from
milk
samples
collected
from
the
quarters
of
the
four
cows
of
the
BHV4
group,
from
the
control
cows
Cl,
C2,
from
the
remaining
124
G.J.
Wellenberg
et
al.
/Veterinary
Microbiology
86
(2002)
115-129
Table
3
Isolation
of
BHV4
from
milk
and
BHV4
titres
in
milk
from
cows
inoculated
with
BHV4
Days
pi
BHV4
isolation
(log
io
TC1D
50
/in1)
B1
B2
B3
B4
RF
LH
RF
LH
RF
LH
RF
LH
0
__a
1
+(2.1)
"
+
+
+ +
+(1.8)
+
+
2
+ + +
+ + +
+ +
3
+
(1.4)
+
+
+ +
+
(2.4)
+
+
4
+ + +
+ + +
+ +
5
+
(1.4)
+
+
+ +
+
(2.0)
+
+
6
+ + +
+
+ +
+ +
7
+
(1.8)
+
+
+ +
+
(2.9)
+
+
8
+ + +
+
+ +
+ +
9
+
(1.6)
+
+
+ +
+
(1.5)
+
+
10
+
(1.3)
+
+ + +
+
11
+
+ +
+
(1.3)
+
12
+
+ + +
13
+
+ +
14
+
+
15
16
17
18
19
20
21
22
+
(1.2)
23
+
(2.3)
24
+
(3.1)
25
+
(3.0)
26
+
(2.4)
+
(1.3)
27
+
(1.9)
28
a
No
BHV4
isolated.
b
BHV4
titre.
quarters
RF,
RH
and
LH
from
control
cow
C3,
and
from
the
four
quarters
of
control
cow
C4
up
to
day
9
pi.
3.6.1.
Isolation
of
bacteria
on
days
15-28
pi
S.
uberis
was
isolated
from
all
inoculated
quarters
of
cows
C
1
,
C2,
B1
and
B4
(LH;
intermittent)
(Table
2),
whereas
no
S.
uberis
was
isolated
from
the
non-S.
uberis-inoculated
quarters
of
these
cows.
No
pathogenic
bacteria
were
isolated
from
the
quarters
of
cows
B2
and
B3
that
were
inoculated
with
BHV4
only
(day
0),
or
from
the
remaining
quarters
RF,
RH
and
LH
from
control
cow
C3.
G.J.
Wellenberg
et
al./Veterinary
Microbiology
86
(2002)
115-129
125
40—
B1
-
B2
B3
B4
4
3
0
0
—I
2
detection
limit
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
28
Days post-inoculation
Fig.
2.
BHV4
titres
in
nasal
secretions
from
cows
intranasally
inoculated
with
BHV4.
Table
4
BHV4
antibody
titres
in
serum
and
milk
from
cows
inoculated
intramammarily
and
intranasally
with
BHV4
Days
pi
BHV4
antibody
titres
B1
B2
B3
B4
Serum
Milk
Serum
Milk
Serum
Milk
Serum
Milk
0
<20
<5
<20
<5
<20
<5
<20
<5
7
<20
<5
<20
<5
<20
<5
<20
<5
10
_a
<5
<5
<5
<5
12
<5
20
10
10
14
40
5
80
40
40
20
20
20
16
20
80
80
20
18
20
80
80
20
20
20
80
160
20
21
80
320
320
80
22
20
80
160
80
24
40
80
80
160
26
80
80
160
160
28
320
160
1280
80
2560
160
320
160
a
Not
determined.
4.
Discussion
In
this
study,
a
simultaneous
intramammary
and
intranasal
inoculation
of
lactating
cows
with
BHV4
did
not
lead
to
clinical
mastitis.
However,
subclinical
mastitis
was
induced,
as
126
G.J.
Wellenberg
et
al.
/Veterinary
Microbiology
86
(2002)
115-129
evidenced
by
BHV4
replication
in
all
BHV4-inoculated
quarters,
by
the
increased
SCC
of
>250,000
ml
in
two
of
four
cows
and
in
three
of
six
BHV4-inoculated
quarters,
and
by
the
absence
of
such
an
increase
in
SCC
in
controls.
General
phenomena,
such
as
differences
in
susceptibility
or
genetic
background
of
individual
cows,
might
explain
why
the
BHV4
infection
induced
an
increase
of
SCC
in
only
50%
of
the
inoculated
quarters.
The
increase
in
SCC
in
BHV4-inoculated
quarters
was
not
the
result
of
an
intramammary
infection
by
bacteria,
as
evidenced
by
the
absence
of
udder
pathogens
in
milk
during
the
first
14
days
pi.
We
have
chosen
to
use
two
routes
of
inoculation
to
enhance
the
possibility
of
inducing
clinical
mastitis.
However,
a
disadvantage
of
this
approach
is
that
a
possible
interference
of
the
BHV4
infection,
that was
induced
after
the
intranasal
inoculation,
on
the
intramam-
mary
BHV4
infection
cannot
be
excluded.
But,
based
on
the
facts
that:
(1)
BHV4
was
only
isolated
from
the
inoculated
quarters
and
not
from
the
non-inoculated
quarters
of
the
same
cows,
and
(2)
an
increase
in
SCC
was
only
detected
in
milk
from
BHV4-inoculated
quarters,
it
is
likely
that
the
induced
subclinical
mastitis
was
primarily
the
result
of
the
intramammary
BHV4
infection.
The
SCC
is
an
indicator
of
subclinical
mastitis,
and
the
major
factor
affecting
SCC
is
infection
of
the
mammary
gland
(International
Dairy
Federation,
1987;
Dohoo
and
Leslie,
1991;
Harmon,
1994).
Inflammation
of
the
mammary
gland
by
udder pathogens
often
results
in
an
increase
of
SCC
>10
6
ml
within
1
or
2
days
post-infection
(Paape
et
al.,
1981),
as
was
the
case
in
all
four
cows
intramammarily
inoculated
with
S.
uberis.
However,
the
SCC
in
milk
from
the
BHV4-inoculated
quarters
did
not
increase
before
day
6
pi.
The
reason
for
this
difference
in
increase
in
SCC
may
be
that
different
defence
mechanisms
are
involved
in
bacterial
and
viral
infections
of
the
mammary
gland.
In
bacterial
udder
infections,
innate
immunity,
also
known
as
non-specific
responsiveness,
is
the
predominant
defence
during
the
early
stages
of
infection
(Harmon,
1994).
In
the
early
stages
of
infection,
neutrophils,
and
the
migration
of
neutrophils
from
the
blood
into
the
udder,
which
occurs
by
chemotaxis,
play
a
major
role
in
host
defence
against
environmental
(coliform)
mastitis
in
cows
(Kremer
et
al.,
1990).
We
may
speculate
that
the
increase
of
SCC
in
milk
from
BHV4-inoculated
quarters
was
the
result
of
cytotoxic
responses
against
BHV4-infected
cells
mediated
by
other
defence
mechanisms,
i.e.
the
occurrence
of
the
late
cytokine
cascade
(including
the
production
of
interferon-y),
the
development
of
cell-
mediated
immunity
and
the
more
specific
antibody-dependent
cell-mediated
cytotoxicity
(ADCC),
as
reported
for
BHV1
(Rouse
et
al.,
1976;
Campos
et
al.,
1994).
Interferon-y
plays
an
important
role
in
the
generation
of
non-MHC-restricted
cytotoxic
responses
(Campos
et
al.,
1989),
and
it
can
activate
macrophages
to
kill
virus-infected
cells,
as
reported
for
BHV1
(Babiuk
et
al.,
1996).
The
peak
activities
of
these
cell-mediated
immune
responses
occurred
7-10
days
pi
(Campos
et
al.,
1994).
Unlike
BHV4,
experimental
intramammary
inoculation
of
bovine
herpesvirus
1
(Greig
and
Bannister,
1965;
Corner
et
al.,
1967;
Straub
and
Kielwein,
1966),
foot-and-mouth
disease
virus
(Burrows
et
al.,
1971;
Blackwell
and
Yilma,
1981),
and
parainfluenza
3
virus
(Kawakami
et
al.,
1996)
have
been
found
to
induce
clinical
mastitis.
Increased
tempera-
tures,
swollen
udders
and
changes
in
the
morphology
of
the
milk
were
reported
for
all
three
virus
infections.
In
addition,
an
increase
of
SCC
was
recorded
after
the
BHV1
and
PI3
inoculation,
and
an
intramammary
inoculation
with
BHV1
and
foot-and-mouth
disease
virus
also
resulted
in
reduced
milk
yields.
G.J.
Wellenberg
et
al./Veterinary
Microbiology
86
(2002)
115-129
127
For
cases
of
subclinical
mastitis,
bovine
leukaemia
virus
has
been
detected
in
mammary
tissue
of
affected
cows
(Yoshikawa
et
al.,
1996).
But,
there
is
no
clear
evidence
that
this
virus
plays
a
role
in
the
etiology
of
bovine
subclinical
mastitis,
and
no
experimental
studies
have
been
reported
to
examine
whether
this
virus
was
able
to
induce
bovine
subclinical
or
clinical
mastitis.
Consequently,
to
our
knowledge
this
is
the
first
report
on
a
virus
that
induces
bovine
subclinical
mastitis
after
experimental
inoculation.
In
the
preceding
study
(Wellenberg
et
al.,
2000),
BHV4
was
isolated
from
milk
from
cows
with
clinical
mastitis
in
combination
with
udder
pathogens,
especially
S.
uberis,
a
major
udder
pathogen
(Thomas
et
al.,
1994).
To
study
whether
BHV4
might
play
an
indirect
role
in
the
aetiology
of
bovine
clinical
mastitis,
we
examined
whether
a
preceding
BHV4
infection
would
exacerbate
bovine
clinical
mastitis
induced
by
S.
uberis.
Based
on
data
of
clinical
signs,
morphology
of
the
milk,
SCC
and
peripheral
WBCs,
we
conclude
that
a
preceding
BHV4
infection
did
not
exacerbate
bovine
clinical
mastitis
induced
by
S.
uberis.
After
the
S.
uberis
inoculation,
BHV4
was
isolated
from
milk
from
two
of
four
quarters
inoculated
with
BHV4
and
S.
uberis,
whereas
no
BHV4
was
isolated
from
milk
from
four
quarters
that
were
inoculated
with
BHV4
only.
These
data
indicate
that
BHV4
remained
present
in
the
infected
cow,
and
that
the
S.
uberis
infection
appeared
to
trigger
replication
of
BHV4.
Replication
of
latent
BHV4
from
various
tissues
has
been
reported
after
dexamethasone
treatment
(Castrucci
et
al.,
1987;
Dubuisson
et
al.,
1989).
Whether
BHV4
persisted
in
latent
form
in
cells
of
the
mammary
gland
or
in
peripheral
blood
leukocytes
(Osorio
and
Reed,
1983;
Lopez
et
al.,
1996;
Egyed
and
Bartha,
1998),
or
as
infectious
virus
in
milk
cell
fractions
(Donofrio
et
al.,
2000)
was
not
examined.
However,
this
study
demonstrates
for
the
first
time
that
bacteria,
i.e.
S.
uberis,
appear
to
trigger
the
replication
of
BHV4.
Which
process
triggered
BHV4
replication
during
the
S.
uberis
infection
is
unknown.
In
an
experimental
mastitis
model,
an
increase
in
serum
cortisol
level
has
been
detected
shortly
after
Escherichia
coli
infection
(Shuster
et
al.,
1993).
An
increase
of
serum
cortisol
levels
or
comparable
processes
that
activate
BHV4
replication
could
be
induced
in
S.
uberis
mastitis.
More
research
is
warranted
to
define
whether
bacterial
infections
may
trigger
reactivation
of
latent
BHV4
and
which
processes
are
involved.
After
the
S.
uberis
inoculation,
BHV4
was
only
detected
in
milk
and
not
in
nasal
secretions.
This
suggests
a
local
trigger
on
the
replication
of
BHV4
that
did
not
result
in
systemic
spread.
In
conclusion,
this
study shows
that
a
simultaneous
intramammary
and
intranasal
inoculation
of
lactating
cows
with
BHV4
did
not
induce
clinical
mastitis,
but
induced
subclinical
mastitis.
In
addition,
no
exacerbation
of
a
S.
uberis
infection
by
a
preceding
BHV4
infection
was
noted,
and
S.
uberis
infections
may
have
triggered
BHV4
replication.
This
study
indicates
that
it
is
unlikely
that
BHV4
is
a
major
mastitis
pathogen,
but
it
may
play
a
role
in
the
aetiology
of
bovine
subclinical
mastitis.
Acknowledgements
The
authors
gratefully
acknowledge
K.
Weerdmeester,
J.
Maissan
and
B.
Verstraten
for
technical
assistance,
Dr.
A.
Lammers
for
bacterial
analyses,
Dr.
J.
de
Bree
for
statistical
advices,
and
H.
Rutgers
and
co-workers
for
taking
care
of
the
animals.
Streptococcus
uberis
strain
0140J
was
kindly
provided
by
Dr.
P.
Milner
(Compton,
United
Kingdom).
128
G.J.
Wellenberg
et
al.
/Veterinary
Microbiology
86
(2002)
115-129
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