Clinical experience with cross-protective anti-endotoxin antiserum in dogs with parvoviral enteritis


Dimmitt, R.

Canine Practice 16(3): 23-26

1991


Out of 51 puppies testing positive for parvovirus, 30 were divided into three groups of ten each for antiserum treatment. A significant difference (p<0.01) was found between the survival rate of the antiserum treatment group (83%) and control group (52%). Survival rates were 70, 90, and 90%, in the 1, 2, and 4 ml antiserum per lb treatment groups, respectively. Escherichia coli were isolated from 61% of the antiserumtreated puppies, suggesting that gramnegative endotoxins are important contributors to complications of parvoviral infections. No anaphylactic responses were noted.

CANINE
PRACTICE
INFECTIOUS
DISEASE
Out
of
51
puppies
testing
positive
for
parvovirus,
30
were
divided
into
three
groups
of
ten
each
for
antiserum
treatment.
A
significant
difference
(p
<
0.01)
was
found
between
the
survival
rate
of
the
antiserum
treatment
group
(83%)
and
control
group
(52%).
Survival
rates
were
70,
90,
and
90%,
in
the
1,
2,
and
4
ml
antiserum
per
lb
treatment
groups,
respectively.
Escherichia
coli
were
isolated
from
61%
of
the
antiserum-treated
puppies,
suggesting
that
gram-negative
endotoxins
are
important
contributors
to
complications
of
parvoviral
infections.
No
anaphylactic
responses
were
noted.
Clinical
Experience
With
Cross-Protective
Anti-Endotoxin
Antiserum
in
Dogs
With
Parvoviral
Enteritis
Reed
Dimmitt,
DVM
Animal
Emergency
Clinic
1214
Natural
Bridge
Road
Bridgeton,
Missouri
63044
Introduction
Anti-endotoxin
antibodies
have
provided
cross-
protection
to
swine,'
cattle,
24
and
horses
5
'
6
suffer-
ing
from
gram-negative
septicemias.
Escherichia
coli,
Salmonella
sp.,
Shigella
sp.,
Klebsiella
sp.,
Pseudomonas
sp.,
Haemophilus
sp.,
and
other
gram-negative
bacteria
produce
deadly
endo-
toxins
and
are,
therefore,
serious
threats
to
domes-
tic
animals.
A
recent
retrospective
study
yielded
a
90%
isolation
rate
of
E.
coli
from
the
livers
and/or
lungs
of
dogs
with
parvovirus-positive
accessions
to
a
state
diagnostic
laboratory.'
Results
of
that
report
and
the
findings
of
the
investigators
cited
previously
led
to
the
hypothesis
that
the
cross-pro-
tective
antibodies
produced
in
response
to
a
mutant
Salmonella
typhimurium
bacterin-toxoid
could
pro-
vide
an
improvement
in
the
survival
rate
of
puppies
suffering
from
the
gram-negative
endotoxin
com-
plications
of
canine
parvoviral
enteritis.
The
purpose
of
this
study
was
to
determine
if
gram-negative
organisms
were
the
preponderant
secondary
invaders
following
parvoviral
enteritis
and
to
investigate
whether
or
not
cross-protective
antibodies
provided
by
antiserum
(Endoserum
®
:
Immvac
®
,
Inc.,
Columbia,
Mo.)
would
protect
par-
voviral
enteritis
puppies
from
endotoxins.
Materials
and
Methods
A
total
of
51
parvo/enteritis
puppies
were
ad-
ministered
routine
therapies
always
including
10
mg/lb
of
body
weight
of
intravenously
(IV)
admin-
istered
cephalosporin,
isotonic
lactated
Ringer's
solution
to
correct
blood
volume
deficits,
the
iso-
tonic
combination
of
lactated
Ringer's
solution
and
dextrose
to
correct
blood
volume
and
energy
source
deficits
as
needed,
and
1
mg/kg
of
body
weight
of
promazine
hydrochloride
intramuscular-
ly
(IM)
as
needed
to
reduce
intestinal
motility.
Thirty
of
the
puppies
were
randomly
divided
into
three
groups
of
ten
each,
and
were
treated
with
equine-origin
anti-endotoxin
antiserum
as
follows:
Group
1
1
ml/lb
of
body
weight;
Group
2
2
ml/lb
of
body
weight;
and
Group
3
4
ml/lb
of
body
weight.
The
antiserum
was
diluted
at
a
ratio
of
at
least
1:1
with
either
lactated
Ringer's
solution
or
lactated
Ringer's/dextrose
solution.
Responses
of
the
30
antiserum-treated
puppies
were
compared
to
the
Continued
CANINE
PRACTICE
Vol.
16,
No.
3
May/June
1991
PAGE
23
The
Relation
of
Survival
Rate
to
Antiserum
Dosage
......
90%
2MI
amt
80%
70%
m1
1
60%
50%
Rx
40%
Survival
Rate
Rate
=
-0.1
+
exp(0.172
+
1.581
*dose)
30%
I
-
exp(0.172
*
1.581
dose)
-Rx
-
minus
antiserum
1
ml
1
ml
of
antiserum
per
lb
bd
wt
2
ml
-
2
ml
of
antiserum
per
lb
bd
wt
4
ml
-
4
ml
of
antiserum
per
lb
bd
vet
20%
10%
wt.
I
I
1
2
3
Antiserum
Dosage
(ml/lb)
1
Su
rv
iva
l
Ra
te
PARVOVIRAL
ENTERITIS
(Continued)
21
control-group
puppies
in
terms
of
whether
the
puppies
lived
or
died,
and
whether
or
not
the
equine-
derived
antiserum
could
be
safely
administered
to
dogs.
The
safety
of
the
antiserum
was
evaluated
by
development
of
any
clinical
signs
of
anaphylaxis.
Fecal
samples
taken
via
sterile
rectal
swabs
were
tested
to
determine
whether
or
not
parvovirus
was
present
via
enzyme
immunoassay
(CITE
®
Ca-
nine
Parvovirus
Test
Semi-QuantTM
:IDEXX
Corp.,
Portland,
Maine)
and
cultured
to
identify
the
spe-
cies
of
secondary
bacterial
invaders.
All
of
the
28
fecal
samples
retrieved
from
the
antiserum-treated
puppies
were
examined
for
frank
blood,
leukocytes,
and
ovarian
parasites;
and
homogenized
and
plated
on:
MacConkey's
agar
to
differentiate
lactose
fer-
menting
Enterobacteriaceae
from
the
nonlactose
fermenters
Salmonella
sp.
and
Shigella
sp.;
xylose-lysine-deoxycholate
(XLD)
and
Hektoen
(HE)
to
differentiate
coliforms
(especially
E.
coil
which
appear
as
yellow
lactose
fermenting
colonies
on
XLD
and
salmon-yellow
colonies
on
HE);
and
5%
sheep
blood
(BA)
and
phenyl-ethyl-alco-
hol
(PEA)
plates
to
isolate
Streptococci
sp.,
Staph-
ylococci
sp.,
and
yeasts.
All
plates
were
streaked
for
isolation,
incubated
at
37°C
in
6%
CO2,
and
examined
at
24,
48,
and
72
hour's
incubation.
The
specimens
also
were
streaked
for
isolation
on
Campy
blood
agar
(plates
incubated
at
37°C
and
42°C
in
Campy-Pak
jars
for
vibrio-forms;
and
on
chenodeoxycholate
[CDC]
anaerobic
blood
plates
incubated
anaerobically
for
isolation
of
Clostridium
sp.).
Species
identifications
were
determined
by
Gram
staining,
conventional
biochemical
tests,
and
the
automated
API20E
®
and
AN-IDENT"'
sys-
tems
(API
®
Analytab
Products,
Inc.,
Plainview,
N.Y.
A
division
of
Sherwood
Medical
Co.,
St.
Louis).
In
terms
of
statistical
analysis,
the
predeter-
mined,
acceptable
probability
level
was
0.05
or
less.
The
death
rates
(survival
rates)
versus
treat-
ment
levels
were
subjected
to
logistic
regression
analysis"
via
a
JMP
®
software
computer
program
(SAS
Institute,
Inc.,
Cary,
N.C.).
Results
There
was
a
statistically
significant
(p
<
0.01)
relationship
between
the
survival
rate
and
antise-
rum
treatment
levels
(Fig.
1).
Survival
rate
of
the
antiserum-treated
puppies
was
83%
(25
of
30)
compared
to
52%
(11
of
21)
in
the
conventionally
treated
group.
Death
rate
of
the
antiserum-treated
group
was
17%
(5
of
30)
compared
to
48%
(10
of
FIG.1
The
survival
rate
of
puppies
was
compared
to
various
antiserum
dosage
levels
via
logistic
regression
analysis.
A
least
squares
sigmoid
curve
fitted
to
the
data
also
suggested
a
highly
significant
(p
<
0.01)
relationship,
and
thatthe
4
ml/lb
dose
of
antiserum
maximized
its
effect.
21)
in
the
conventionally
treated
group.
There
was
a
70%
survival
rate
in
the
group
of
puppies
treated
with
antiserum
at
1
ml/lb,
a
90%
survival
rate
in
the
2
ml/lb
group,
and
a
90%
survival
rate
in
the
4
ml/lb
group.
There
was
a
30%
(1
ml/lb),
10%
(2
ml/lb),
and
10%
(4
ml/lb)
death
rate
in
the
respective
treat-
ment
groups
(Table
1).
All
puppies
in
the
control
and
treatment
groups
tested
positive
for
the
presence
of
parvovirus
in
their
gastrointestinal
tracts.
E.
colt
was
isolated
from
61%
of
the
puppies
cultured.
Streptococcus
sp.
(43%),
Bacteroides
fragilis
(22%),
Clostridium
sp.
(22%),
Proteus
sp.
(3%),
and
Shigella
sp.
(3%)
were
also
isolated
from
those
puppies
cultured.
Two
species
of
bacteria
were
isolated
from
11
dogs,
and
3
species
of
bacteria
from
2
dogs,
of
the
28
dogs
cultured.
The
1
ml/lb,
2
ml/lb,
and
4
ml/lb
dosage
rates
of
antiserum
did
not
result
in
any
anaphylactic
or
anaphylactoid
reactions,
i.e.,
there
were
no
inci-
dences
of
systemic
responses
or
localized
heat,
swelling,
or
pain
observed
in
the
dogs
following
antiserum
administration.
Discussion
A
significant
correlation
between
survival
rate
and
antiserum
administration
was
demonstrated.
Continued
PAGE
24
May/June
1991
Vol.
16,
No.
3
CANINE
PRACTICE
TABLE
1
Characteristics
of
Parvo/Enteritis
Puppies
Antiserum
Treatment
Case
Number
Sex
Weight
(Ibs)
Age
(wks)
Breed
(X
if
Cross)
EC
a
PR
b
SP
d
SF
°
BF
f
Days
Final
CL
9
(Hosp)
Result
1
F
12.00
9
Basset
7
Died
2
M
9.50
9
Basset
8
Lived
3
M
7.75
7
Mix
3
Died
4
F
7.75
10
Rott
6
Died
5
F
40.00
24
Lab
5
Lived
6
M
17.00
13
Dobe
5
Died
7
F
14.00
10
Shep
3
Lived
8
F
36.20
24
Dalmation
4
Lived
9
M
10.00
10
Cocker
4
Died
10
M
3.00
7
Scottie
6
Livedd
0
ml/lb
11
M
35.00
16
LabX
6
Lived
12
M
6.00
12
Mix
4
Died
13
F
1.00
8
Poodle
3
Died
14
F
28.00
24
LabX
5
Lived
15
F
8.00
9
Pitbull
4
Died
16
F
11.00
8
Pitbull
7
Lived
17
M
14.00
8
Basset
5
Lived
18
F
25.00
12
DobeX
5
Died
19
M
7,75
8
LabX
4
Lived
20
F
58.00
24
Gold
Ret
3
Lived
21
F
15.00
8
ShepX
3
Died
1
F
2.50
7
Min
Schnau
+
5
Died
2
F
33.50
15
Gt
Dane
5
Lived
3
M
32.00
16
LabX
Shep
+
4
Lived
4
F
25.00
16
LabX
Shep
4
Lived
1
ml/lb
5
F
78.00
40
RottX
4
Lived
6
M
5.00
12
Aust
ShepX
+
6
Lived
7
F
43.00
20
Rott
3
Lived
8
F
16.60
11
Boavdefl
au
+
3
Lived
9
M
14.50
13
ShepX
3
Died
10
M
15.50
32
LhasaX
4
Died
1
F
18.00
12
Shep
+
- - - -
+
-
6
Lived
2
M
11.00
8
Dobe
+
- - - -
+
-
6
Lived
3
F
32.00
16
LabX
- - -
+
- - -
3
Lived
4
F
9.00
9
Maltese
- -
- - - - -
4
Lived
2
ml/lb
5
F
4.70
20
Shitzu
- - -
+
- -
+
6
Lived
6
F
2.00
8
Chi
- -
-
+
- - -
3
Died
7
M
25.00
12
RottX
+
- - - - - -
5
Lived
8
F
8.50
9.5
Mix
+ +
- - -
+
-
4
Lived
9
F
2.00
8
Chi
5
Lived
10
F
10.50
16
Mix
5
Lived
1
F
13.00
16
CockerX
- - - - - - -
4
Lived
2
F
35.00
16
Lab
+
-
- - - - -
5
Lived
3
F
30.00
16
Lab
- - -
- -
-
+
6
Lived
4
M
45.00
24
ShepX
Lab
+
-
-
+
- - -
4
Lived
4
ml/lb
5
M
28.00
3.5
Irish
Set
+
- - - - - -
7
Lived
6
M
4.00
8
Poodle
- - -
+
- -
+
7
Lived
7
F
18.00
12
Spitz
+
- -
+
- -
+
3
Lived
8
F
1.50
6
York
- - -
+
-
+
-
3
Died
9
M
12.00
8
BeagleX
- - - - - - -
7
Lived
10
F
15.00
9
Rott
+
- - - -
-
6
Lived
a
EC
=
Escherlchia
coil
b
PR
=
Proteus
sp.
C
SH
=
Shigella
sp.
d
SP
=
Streptococcus
sp.
°
SF
=
Staphylococcus
sp.
fB
F
=
Bactedodes
ha
gills
9
C1.
=
Clostridium
sp.
Continued
CANINE
PRACTICE
Vol.
16,
No.
3
May/June
1991
PAGE
25
PARVOVIRAL
ENTERITIS
(Continued)
Analysis
of
the
dosage
level
data
suggested
that
there
was
not
a
significant
difference
in
the
survival
rate
of
the
group
that
received
2
ml/lb
of
antiserum
and
the
group
that
was
administered
4
ml/lb.
A
dosage
recommendation
of
more
than
2
ml/lb
of
the
antiserum
does
not
appear
to
be
necessary;
however,
the
possible
therapeutic
advantage
of
re-
peating
the
administration
of
the
antiserum
at
any
of
the
dosage
rates
was
not
pursued
in
this
study.
The
fact
that
all
of
the
puppies
tested
in
this
study
were
positive
to
the
enzyme
immunoassay
test
confirmed
that
parvovirus
was
constant
among
the
puppies
studied.
The
61%
recovery
rate
of
E.
coin
from
the
cultured
puppies
confirms
the
findings
of
Turk
and
coworkers'
that
coliform
organisms
are
the
primary
secondary
invaders
of
parvovirus
in-
fection
in
dogs.
Because
gram-negative
organisms
are
the
most
common
secondary
invaders
following
parvovirus
infections,
the
chances
for
developing
gram-nega-
tive
endotoxemia
are
very
high.
Therefore,
the
ad-
ministration
of
antiserum
containing
anti-endo-
toxin
antibodies
was
rational
when
one
considers
that
gram-negative
endotoxins
are
produced
both
during
the
rapid-growth
phase
and
upon
the
kill-off
of
gram-negative
bacteria.
The
mechanisms
responsible
for
the
efficacy
demonstrated
by
the
results
of
this
study
may
not
be
completely
explained
by
the
fact
that
the
an-
tiserum
contains
cross-protective
anti-endotoxin
antibodies.
For
instance,
infusion
of
Tumor
Necro-
sis
Factor
(TNF)
into
dogs,
8
sheep,
9
and
calves
10
has
resulted
in
reproduction
of
the
signs
associ-
ated
with
IV
administration
of
endotoxins.
The
car-
diovascular
depression
characteristic
of
toxic
shock
may
be
induced
by
either
gram-negative
or
gram-positive
toxins
inducing
effector
cells
to
re-
lease
TNF-alpha,
8
and
human
immunoglobulin
has
been
shown
to
suppress
TNF-alpha
production
when
administered
to
rabbits
subsequently
chal-
lenged
with
endotoxin.
11
The
antiserum
used
in
this
study
will
subsequently
be
analyzed
to
determine
the
level
of
anti-TNF-alpha
antibodies
present.
The
1
ml/lb,
2
ml/lb,
and
4
ml/lb
dosage
rates
of
the
antiserum
did
not
result
in
any
anaphylactic
or
anaphylactoid
reactions.
However,
one
should
al-
ways
have
ready
access
to
epinephrine
and
other
appropriate
therapeutic
agents
when
using
equine-
origin
antiserum
or
any
other
biological
product.
The
simultaneous
administration
of
antiserum
and
antibiotics
is
rational
from
the
standpoint
of
treating
the
possible
gram-positive
as
well
as
gram-negative
bacterial
invaders.
When
antibio-
tics
kill
gram-negative
organisms,
however,
endo-
toxin
is
often
released,
thereby
initiating
or
poten-
tiating
endotoxemia.
Although
repeat
administration
might
be
indi-
cated
in
severe
cases
of
endotoxemia,
none
of
the
puppies
in
this
study
received
a
second
dose
of
antiserum.
If
the
administration
interval
of
repeated
doses
of
equine-origin
antiserum
was
10
or
more
days
after
initial
administration,
then
one
could
ex-
pect
recipient
anaphylactic
responses.
Repetition
of
antiserum
administration,
however,
would
prob-
ably
be
safe
if
carried
out
within
5
to
7
days
follow-
ing
the
initial
administration.
13
The
outcome
of
any
parvoenteritis/endotoxemia
emergency
case
has
usually
been
determined
in
7
days
or
less.
Conclusion
The
equine-origin
antiserum,
as
used
in
this
study,
appears
to
be
safe
and
effective
in
the
clinical
man-
agement
of
secondary
gram-negative
endotoxemias
associated
with
parvoviral
enteritis
in
dogs.
The
cross-protective
anti-endotoxin
antibodies
appear
to
account
for
at
least
part
of
the
efficacy
of
the
an-
tiserum
but
other
factors
that
may
contribute
to
its
effectiveness
remain
to
be
determined.
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REPRINTS
of
this
article
may
be
obtained
from
the
author
ONLY
IF
a
self-addressed
return
envelope
is
enclosed.
PAGE
26
May/June
1991
Vol.
16,
No.
3
CANINE
PRACTICE