Canine Parvoviral Disease: Experimental Reproduction of the Enteric Form with a Parvovirus Isolated from a Case of Myocarditis

Abstract

Five 7-week-old pups and four 4-week-old pups, all seronegative to canine parvovirus, were inoculated intravenously with 1000 haemagglutinating units of canine parvovirus originally isolated from the myocardium of a dog with naturally occurring myocarditis. After three days, pups in both litters became pyrexic, anorectic and depressed, with vomiting and diarrhoea. The 4-week-old pups were killed on day 4, and the 7-week-old pups died or were killed on day 5 post-inoculation. Histological examination showed degeneration and necrosis of intestinal crypt epithelial cells and villous atrophy. All pups had thymic atrophy caused by lymphoid depletion. Peyer's patches, mesenteric lymph node and spleen also had lymphoid depletion. Lymphoid necrosis was present occasionally in these tissues. In the bone marrow, granulocytes and granulocyte and erythroid precursors were depleted. Amphophilic intranuclear inclusion bodies were abundant in crypt epithelial nuclei, less so in myocardial nuclei. Canine parvovirus was isolated from intestinal contents, thymus, spleen, mesenteric lymph node and liver in most pups, but not from kidney or myocardium.

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Veterinary Pathology Online
http://vet.sagepub.com/content/17/5/589
The online version of this article can be found at:
DOI: 10.1177/030098588001700508
1980 17: 589Vet Pathol
W. F. Robinson, G. E. Wilcox and R. L. P. Flower
Parvovirus Isolated from a Case of Myocarditis
Canine Parvoviral Disease: Experimental Reproduction of the Enteric Form with a
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Vet.
Pathol.
17:
589-599 (1980)
Canine Parvoviral Disease: Experimental Reproduction
of
the Enteric
Form with a Parvovirus Isolated
from
a
Case
of
Myocarditis
W.
F.
ROBINSON,
G.
E.
WILCOX and
R.
L.
P.
FLOWER
Division of Veterinary Biology, School of Veterinary Studies, Murdoch University,
Murdoch, Western Australia, Australia
Abstract.
Five 7-week-old pups and four 4-week-old pups, all seronegative to canine
parvovirus, were inoculated intravenously with 1000 haemagglutinating units of canine
parvovirus originally isolated from the myocardium of a dog with naturally occurring
myocarditis. After three days, pups in both litters became pyrexic, anorectic and depressed,
with vomiting and diarrhoea. The 4-week-old pups were killed on day 4, and the 7-week-old
pups died or were killed
on
day
5
post-inoculation. Histological examination showed degen-
eration and necrosis of intestinal crypt epithelial cells and villous atrophy.
All
pups had thymic
atrophy caused
by
lymphoid depletion. Peyer’s patches, mesenteric lymph node and spleen
also had lymphoid depletion. Lymphoid necrosis was present occasionally in these tissues.
In
the bone marrow, granulocytes and granulocyte and erythroid precursors were depleted.
Amphophilic intranuclear inclusion bodies were abundant in crypt epithelial nuclei, less
so
in
myocardial nuclei. Canine parvovirus was isolated from intestinal contents, thymus, spleen,
mesenteric lymph node and liver in most
pups,
but not from kidney or myocardium.
Two new viral diseases of dogs, parvoviral enteritis
[5,
9, 12, 171
and parvoviral
myocarditis [7,
8,9,
10,
131
have been described. Clinical signs in dogs with parvoviral
enteritis include anorexia, pyrexia, vomiting and diarrhoea. Panleucopenia often is
present in the early phase. The characteristic lesion is necrosis of rapidly dividing
cells in bone marrow, intestinal crypt epithelium and lymphoid tissues. Intranuclear
inclusion bodies are found in intestinal crypt epithelial cells. Canine parvoviral
enteritis mimics the classical form of feline panleucopenia in many respects
[
151.
Canine parvoviral myocarditis occurs only in pups
3
to
8
weeks old. Clinical signs
are
severe ventricular arrhythmias
[lo,
13,
191,
resulting in sudden death or death
following
a
brief period of restlessness and dyspnoea. Subclinical ventricular arrhyth-
mias may be detected by electrocardiography in affected animals
[
191.
The primary
lesion is multifocal myocardial necrosis, with an inflammatory reaction of variable
intensity. Large intranuclear inclusion bodies are found in myocardial cells and are
pathognomic for the disease.
Although an apparently identical parvovirus has been isolated from naturally
occurring cases
of
both diseases
[5,
6,
11,
17,
181,
clinicopathologic overlap between
589
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590
Robinson,
Wilcox
and Flower
them has not been reported. It has been suggested that the form the disease takes is
a function
of
the age of the puppy at the time of infection
[7,
201,
but pathogenesis
remains to
be
defined.
We report the reproduction of the enteric form of the disease with a parvovirus
isolated from the myocardium of a dog with naturally occurring myocarditis.
Materials and Methods
Virus
Canine parvovirus isolate MV54 isolated from a 4-week-old puppy with histologically
confirmed myocarditis
[
181
was inoculated onto
50%
to
60%
confluent Crandell feline kidney
cells with Eagle’s minimal essential medium containing 10% foetal calf serum with 100
international units/ml penicillin, neomycin
(50
pg/ml) and amphotericin
B
(2.5
pg/ml). The
medium was replaced 72 hours later with Eagle’s minimal essential medium containing
2%
foetal calf serum and antibiotics. After another 72 hours’ incubation, the cultures were frozen
and thawed, sonicated and centrifuged at
1000
X
gravity for
20
minutes at room temperature.
The supernatant was aspirated and tested for haemagglutinating activity as described
[
181. An
uninoculated culture of Crandell feline kidney cells was treated identically.
Experimental animals
A 7-week-old litter of seven crossbred puppies and a 4-week-old litter of
six
were housed in
separate rooms for one week of acclimatization and observation prior to inoculation. Serum
samples were taken and tested for haemagglutinating antibodies against canine parvovirus as
described [MI. Following the period of acclimatization and observation, two puppies from
each litter were removed and housed in a separate building as controls.
Experimental design
Nine puppies, five from the 7-week-old group (litter
1)
and four from the 4-week-old group
(litter
2)
were inoculated intravenously with
5
ml of the supernatant containing canine
parvovirus with a haemagglutination titre of 1000. The two remaining puppies from each litter
were inoculated intravenously with
5
ml of the supernatant from the control culture. All were
observed daily and rectal temperatures were taken. A complete blood count was done on days
3
and 4 post-inoculation. Serum samples were taken on day 4 post-inoculation or at death.
Puppies either died or were killed by an intravenous injection of pentobarbitone sodium.
Necropsy procedure
Complete necropsies were done on all puppies. Samples for histologic examinations were
taken from the following tissues and fixed in 10% neutral buffered formalin: tongue, oesoph-
agus, stomach, duodenum, jejunum, ileum, colon, thymus, retropharyngeal and mesenteric
lymph nodes, tonsil, spleen, adrenal, thyroid and parathyroid, costochondral junction, brain,
pituitary, eyes, heart, lung, pancreas, parotid salivary gland and bone marrow. After routine
processing, tissue samples embedded in paraffin were sectioned at
6
pm and stained with
haematoxylin and eosin (HE). Samples of heart, thymus, spleen, mesenteric lymph node, liver,
kidney and intestinal contents were taken aseptically and stored at -70°C. In addition, l-mm3
pieces of duodenum, jejunum and ileum from all puppies were fixed in
3%
glutaraldehyde in
0.1
mol/liter phosphate buffer pH 7.4. These blocks were then fmed
in
1%
osmium tetroxide
in
the same buffer and processed for epon embedding. Sections for light microscopy were cut
1
pm thick and stained with toluidine blue. Thin sections were cut from selected areas, stained
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Canine
Parvoviral
Disease
T
591
0
CONTROL
(L)
A
LITER1 (5)
"4
37
LmER
2
[LI
0
1
2
3
L
DAYS-POST
INOCULATION
Fig.
1:
Mean
+-
standard deviation of rectal temperature of two inoculated litters and
4
control pups. Rise in rectal temperature in two inoculated groups
on
days
3
and
4
after
infection. Number of pups per group in brackets.
with lead citrate and uranyl acetate and examined in an electron microscope. Bone marrow
smears were made for cytologic examination.
Viral
isolation
Tissues collected at necropsy were minced and homogenized in tissue grinders with Eagle's
minimal essential medium to approximately a
10%
suspension, centrifuged at 1000
X
gravity
for 15 minutes, and the supernatant collected. Approximately 0.1 ml of each tissue supernatant
was inoculated onto
50%
to
60%
confluent Crandell feline kidney cells and allowed
to
adsorb
for two hours at
37°C.
Eagle's minimal essential medium containing
2%
foetal calf serum with
antibiotics was added and changed
24
hours later to Eagle's minimal essential medium
containing
2%
foetal calf serum with antibiotics. Cell cultures were incubated for four days.
The culture media negative for haemagglutination were passaged. This procedure was repeated
for a third passage. Samples were considered negative if haemagglutination was not evident
after three passages.
Results
Clinical findings
Preinoculation serum samples from
all
puppies had haemagglutination inhibition
titres of
-=
10. This indicated lack of previous
exposure
to
canine
parvovirus.
From
day
0
to
day
2
post-inoculation, puppies
in
both
experimental litters remained
clinically
normal.
On
day
3,
they were pyrexic
(fig.
1)
and
depressed. Mild diarrhoea
was present
in
litter
2.
Four puppies
in
litter
1
were
lymphopenic.
On
day
4,
both
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592
Robinson,
Wilcox
and
Flower
Table
I.
Total and differential white-cell counts from dogs with experimental canine
Darvovirus infection (mean
f
standard deviation)
Days Litter
1
Litter 2
post-
inocu- Inocu- Inocu-
lated (4)
lation lated
(5)
Controls (2j Controls (2)
Total white cell 3 15,849 14,350 15,900 16,050
count per pl +4,125
f
1,060 f5,500 f3,606
4 3,699 12,500 14,350 15,605
+3,581 k141 f4,5
11
+4,392
Total neutrophil 3
count per pl 4
14,531
+3,569
2,565
f2.904
Total lymphocyte 3
count per
pl
4
983
f5 17
749
+616
6,573
+270
6,807
f895
6,888
+509
4,879
k762
12,080
+3,332
10,929
+3,775
1,926
+783
1,095
f502
10,146
+3,275
10,737
22,456
3,598
f698
2,765
f1.435
litters were pyrexic, anorexic, depressed and vomiting with litter
1
more severely
affected.
A
pink-white diarrhoea containing red flecks of blood also was present.
Four puppies
in
litter
1
were panleucopenic and three puppies in litter
2
were
lymphopenic (table I). Litter
2
was killed on the morning of day
4.
By
the afternoon
of day
4,
puppies in litter
1
had deteriorated rapidly and were vomiting, anorexic,
markedly depressed and diarrhoeic. The diarrhoea was dark red. On day
5,
three
puppies in litter
2
were found dead and one was moribund and soon died. The
remaining puppy was depressed, dehydrated and vomited frequently, and was killed.
Gross
post-mortem
fmdings
Lesions were similar in all puppies in litter
1.
The puppies were dehydrated and
thin with little subcutaneous fat. Serosal congestion and a moderate number of
petechiae were present from the duodenum to the ileocaecal junction, most severely
in
the jejunum. The small intestinal was dilated and atonic, and contained much
clear yellow fluid. In some cases, the fluid was reddish in the jejunum, becoming
darker brown in the ileum. The colon was usually empty. The intestinal mucosa
usually appeared normal, but in some, thin strands of fibrin adhered to the surface.
The thymus was always very small and consisted of a thin light brown band of tissue.
The bone marrow was macroscopically normal.
The pups in litter
2
were thin and dehydrated, and showed muscle wasting. One
puppy had numerous serosal petechiae of the ileum. The mucosa in this pup was
covered with fibrin strands. The remaining puppies had semi-fluid whitish-pink
intestinal contents. The thymus in all puppies was small.
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Canine Parvoviral Disease
593
Histology
and electron microscopy
Lesions in both litters were similar, consisting of degeneration, necrosis and
hyperplasia of the epithelium of the crypts from the pylorus to the rectum; the
duodenum and jejunum were affected most severely. In many crypts the lesion was
focal. Affected crypts were dilated and contained cellular debris in the lumen. A
varied proportion of surviving crypt epithelial cells were flattened. Many epithelial
cells contained large intranuclear basophilic inclusion bodies, often surrounded by
eosinophilic material (fig.
2).
Villous atrophy and fusion were prominent in many
pups, particularly those with the more severe crypt lesions. Goblet cell metaplasia
occasionally was present in the duodenum and there was involution of Peyer's
patches with
loss
of lymphocytes from germinal centres
(fig.
3).
Coccidia were present
in sections of ileum from litter
2.
Electron microscopic examination of affected crypts showed desquamated cells in
the lumen. The cells lining the crypt had moderately dilated rough endoplasmic
reticulum; some nuclei contained inclusion bodies (fig.
4)
characterized by chromatin
margination and abundant particles
20
nm in diameter, which were interpreted as
parvovirus virions.
There was loss of cortical lymphocytes in the thymus, but small lymphocytes were
still present, although reduced in number, in the medulla. The number of thymic
epithelial cells appeared greater because of this loss. Hassal's corpuscles also were
larger. Some thymuses were diffusely congested. Periarteriolar lymphoid depletion
and lymphocyte necrosis were present in the spleen. The paracortical and medullary
areas of mesenteric and retropharyngeal lymph nodes were depleted of lymphocytes;
there was also an increase in the number of fixed macrophages lining the sinusoids
(fig.
5).
Committed myeloid series and early erythroid series were lost from the bone
marrow (fig.
6,
7).
Megakaryocytes were normal, as were the later stages of the
erythroid series. Large basophilic rectangular intranuclear inclusion bodies were
found in a few myocardial cells.
No
fibre degeneration or inflammatory cells were
present.
Virology
The results of virus isolation are shown in table
11.
Canine parvovirus was isolated
consistently from intestinal contents, thymus, spleen, liver and mesenteric lymph
node from litter
2,
but only from intestinal contents in litter
1.
Serology
All puppies were sero-negative prior to inoculation. Terminal serum samples from
puppies inoculated with canine parvovirus taken on day
4
post-inoculation from
litter
2
were negative, whereas litter
1
had haemagglutination titres from
320
to
1280.
The four control puppies remained negative.
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594
Robinson, Wilcox and
Flower
Fig.
2
Dilated duodenal crypt (bottom). Inclusion bodies correspond to eosinophilic inclu-
sions seen with
HE
(broad arrow). More basophilic inclusion bodies (narrow arrow) associated
with marginated chromatin. One-pm section. Toluidine blue.
Fig.
3
Terminal ileum: crypt dilation, villous atrophy and fusion. Depletion of lymphocytes
in Peyer’s patches (arrow) and lymphatic diluation.
HE.
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Canine Parvoviral Disease
595
Discussion
The original object of this study was to reproduce experimentally canine parvoviral
myocarditis, but the most interesting result was the induction instead of enteric
parvoviral disease. The critical factor in the pathogenesis
of
parvoviral infections is
actively-replicating DNA, and thus a population of rapidly dividing cells [21]. The
vulnerability of any particular tissue could be expected to be different in the prenatal,
neonatal and adult animal, with periods
of
transition occurring at different times for
different tissues. This is well demonstrated by the differing clinicopathologic features
of parvoviral disease in neonatal kittens and adults [3,4, 151. In neonatal kittens, the
bone marrow and external granular layer of the cerebellum are most affected, while
intestinal lesions are minimal [4]. It has been shown that the parvoviral isolate used
in this experiment
(MV54)
has a close serologic relationship to an isolate recovered
from a case of canine parvoviral enteritis [18]. It was suggested that the two isolates
were identical. The findings in our study substantiate those virologic findings and
emphasize the possibility that the clinicopathologic manifestations
of
canine parvo-
viral infection are related to age at infection, myocarditis occurring when pups are
infected at a younger age than that at which enteritis occurs. Further indications are
seen by comparing the difference in severity of the disease between the two litters.
The younger litter
(4
weeks) had neither as marked clinical signs nor as severe gross
pathological changes as the older litter
(7
weeks). Indeed, by the fourth day post-
inoculation, the 4-week-old litter was improving clinically whereas the older pups
(7
weeks old) were deteriorating rapidly. The younger litter was lymphopenic whereas
the older litter was panleucopenic. The implication
is
that the intestinal crypt
epithelium and bone marrow contained a larger number of vulnerable cells in the
older animals. The severity of clinical signs in conventional dogs in this study is
in
contrast to the mild enteric disease in specific pathogen free dogs given canine
parvovirus
[
11.
The reasons for the difference remain undefined. Factors influencing
the expression of the disease may include the route of infection, the
amount
of virus
administered and the composition
of
the microbial flora in the intestinal tract.
Data are not available on the normal replacement time of intestinal crypt epithelial
cells in the dog, but it is known that in the pig the transition from the slower neonatal
replacement time to the faster adult replacement time occurs at about three weeks of
age [14, 161.
Canine parvovims was isolated consistently from the intestinal contents, thymus,
spleen and liver in litter
2,
and cell destruction had occurred in all but the liver.
However, unequivocal inclusion bodies were found only in the nuclei of intestinal
crypt epithelium and myocardium. Post-mortem findings were similar in pups in
litter 1 which died or were killed 24 hours later than litter 2, with virus consistently
isolated only from intestinal contents. These findings, coupled with the extensive
lymphoid depletion with little remaining evidence of necrosis in the thymus, spleen,
mesenteric lymph node and Peyer’s patches and acute crypt epithelial necrosis,
suggest that the virus initially replicated in lymphoid tissue and subsequently invaded,
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596
Robinson,
Wilcox
and
Flower
Fig.
4
Electron micrograph of affected jejunal crypt. Nuclei containing inclusion bodies
(arrows):
electron dense outer nuclear membrane, marginated chromatin and many small
round particles
fill
nucleus.
Fig.
5
Mesenteric lymph node. Extensive reticuloendothelial hyperplasia
in
medullary
sinusoids.
HE.
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Canine Parvoviral Disease
591
Fig.
6
Bone marrow
from
control pup.
Fig.
7:
Bone marrow from inoculated pup: extensive depletion
of
myeloid and erythroid
precursors. Committed erythroid series cells and megakaryocytes still present. Severe conges-
tion.
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Robinson,
Wilcox
and
Flower
Table
11.
Recovery of canine parvovirus from inoculated pups
Litter
1
Litter 2
Inoculated Controls Inoculated Controls
Intestinal contents 3/4 0/2 4/4
0/2
Spleen 0/4 0/2 3/4 0/2
Thymus 2/4
o/
1
3/4 0/2
Mesenteric lymph node 2/4
0/2
2/4 0/2
Liver
1
/4
0/2
4/4
0/2
Kidney 0/4
0/2
0/4 0/2
Mvocardium 0/4
0/2
0/4 0/2
replicated in and destroyed intestinal crypt epithelial cells. This is analogous to
findings in experimental feline panleucopenia
in
which lymphoid, reticulo-endothe-
lial and bone marrow precursor cells are infected initially, followed by a second
phase with invasion and destruction of crypt epithelial cells
[3].
Inclusion bodies in the myocardium have not been reported in natural cases of
canine parvoviral enteritis. However, myocardial inclusion bodies without myocardial
necrosis have been described in experimental panleucopenia in neonatal kittens
[3].
The lack of myocardial cell necrosis and inflammation could be explained by the
pups succumbing to the extracardiac effects of the virus before myocardial damage
could occur. This seems unlikely, however, as extra-cardiac effects of the virus have
not been seen in naturally occurring cases of myocarditis. It is known that myocardial
cell division is maximal in puppies during the first three weeks of life
[2].
It is likely
that in both groups of experimental pups, the period of greatest myocardial vulner-
ability had passed at the time of inoculation.
Acknowledgements
We gratefully acknowledge the excellent technical
assistance
of
Messrs. H. Findlay, P.
Fallon and P. Hinchliffe and Ms.
J.
Robertson.
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