A.M. JONAS Lesions in Puppies Surviving Infection with Canine Herpesvirus
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Vet. Path. 8: 37-53 (1971)
Lesions in Puppies Surviving
Infection with Canine Herpesvirus
D.M. ALBERT,J.M. KING
D.H. PERCY,L.E. CARMICHAEL, and
A.M. JONAS
Sections of Laboratory Animal Sciences and of Ophthalmology,
Yale School of Medicine, Yale University, and Veterinary Virus Research Institute,
and Department of Pathology, New York State Veterinary College, Cornell University
Abstract. Four puppies experimentally infected with canine herpesvirus (CHV)and
one spontaneously infected animal, all of which survived the disease, were examined.
Histologically, there were a focal granulomatous encephalitis, interstitial pneumonitis,
and segmental renal necrosis with dysplasia. In the animal with naturally occurring in-
fection there was segmental cerebellar and retinal dysplasia, and CHV antigen was demon-
strated in the cerebellar cortex by fluorescence microscopy. The cerebellar and retinal
dysplasia indicate that CHV can impair the differentiation of such tissues.
Death of newborn puppies due to canine herpesvirus (CHV) is a well-
recognized entity [7, 10, 181. The naturally occurring acute disease normally
affects only puppies less than 2 weeks of age [7, 181. The characteristic lesions
at necropsy are disseminated foci of necrosis in lungs, liver, and kidneys,
acute hemorrhagic splenitis, focal nonsuppurative meningoencephalo-
myelitis, and ganglioneuritis [7,10,22,27,38]. The susceptible newborn pup
usually is infected with CHV during parturition or shortly after birth [7, 10,
181, but intrauterine infection may occur [34]. Older puppies generally are
resistant to the systemic disease [2]. Although characteristically a disease of
the neonatal pup, there has been considerable controversy regarding the role
of this virus in epizootics of tracheobronchitis in older dogs [23]. Recently
CHV has been isolated from a dog with malignant lymphoma [21].
CARMICHAEL et al. [8] have altered the course of the generalized disease
in neonatal puppies by artificially elevating the environmental temperature.
The newborn puppy has a 'subnormal' body temperature with an incom-
pletely developed homeothermic mechanism, and a rise in environmental
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temperature causes a concurrent elevation of body temperature [I I]. This
phenomenon has resulted in the occasional absence of clinical signs in
inoculated pups or in the deveIopment of mild clinical disease with subse-
quent recovery. Simultaneously inoculated littermates housed at normal
room temperature died with peracute CHV infection [8]. This communi-
cation describes the lesions observed in puppies that did not die after
inoculation with CHV. The puppies were later killed.
Materials and Methods
Four Beagle puppies (No. 2, 5, 6, and B,) inoculated intraperitoneally (i.p.) at 2 days
of age with from 102.5 to 103-5 tissueculture infective doses flcID50) of CHV survived
the infection. They were housed at an environmental temperature of approximately 86°F.
Animal B, was killed at 11 days post inoculation (pi.), and 2, 5, and 6 were killed at 16
days p.i. Also included in the study was a Malamute puppy (39) killed at 31 days of age.
This animal was a field dog raised in the Ithaca area and had ataxia and blindness of
several days duration prior to euthanasia. Four littermates of this animal died a t approxi-
mately 2 weeks of age of CHV infection.
All animals were killed by intraperitoneal or intracardiac administration of peutabar-
bitone.
Tissues collected at necropsy for histopathological examination were from brain, eyes,
lung, heart, liver, kidney, spleen, and intestinal tract. They were fixed in 10% formalin or
Born's fluid, embedded in paraffin, sectioned at 6 F, and stained with haematoxylin and
eosin (HE). In addition, selected tissues were stained with MASON'S trichrome, PTAH,
BROWN and BRENN,PAS, Prussian blue, VONKOSSA,and fibrin stains [3].
Sera from all puppies and from the dam of 39 were collected and tested for CHV by
methods previously described [28]. Portions of spleen, kidney, lung, and liver from animals
Ba and 39 were collected for viral isolative studies. In addition, sections from the cere-
bellum of 39 were collected for viral isolation and immunofluorescent microscopy (FA).
The methods used for the isolation of virus and immunofluorescent microscopy (direct
method) have been described previously [2,71.
Results
Clinical Signs and Macroscopic Findings
Animal B2,killed at I1 days p.i., did not have any sign of illness foIlowing
inoculation of CHV. At necropsy, however, the lungs were edematous with
scattered patchy areas of consolidation. The kidneys had irregular surfaces
and circumscribed pale to hemorrhagic areas in the renal cortices on cut
section.
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Animals 2, 5, and 6 had mild signs of clinical illness characterized by
nasal discharge, dyspnea, and anorexia at 6 to 8 days p.i. with CHV. Animal
5 was lethargicwith a tendency to circle to the right at the time of euthanasia.
Bilateral rales were detected in animal 6 on auscultation. All puppies,
however, had apparently survived the initial infection with CHV and ap-
peared to be in the process of recovery when killed at 16 days p.i. At necropsy
their lungs were congested, with tan to grey mottled areas scattered in the
parenchyma. The bronchial lymph nodes were edematous. Multiple lesions
that varied from hemorrhagic foci to depressed pale grey areas were scattered
in the renal cortices.
Animal 39 had become ataxic and was blind when necropsied at 31 days
of age. At necropsy the cerebellum was markedly reduced in size although
the folia were clearly defined. The kidneys had scattered depressions on the
surface with pale grey areas extending into the cortices.
Histopathological Findings
Central Nervous System
In all animals there were multiple disseminatedfoci of increased cellularity
with obliteration of normal architecture. Lesions were most frequent in the
brain stem and cerebellum, with a few areas scattered throughout the CNS.
Lesions were relatively sparse in animal 5 and were numerous in puppy 6,
both examined at 16 days p.i. In animal B2, killed 11 days pi., there were
foci of increased cellularity consisting of microglial cells and infiltrating
mononuclear cells (fig. 1). These lesions were similar to those in animals
dying with acute CHV encephalitis [27].
In the 3 pups examined at 16 days p.i. there was focal granulomatous
encephalitis characterized by microgliosis, astrogliosis, and proliferation of
neocapillaries (fig. 2, 3). Some lesions were discrete and circumscribed and
consisted of dense collections of mononuclear cells with irregular to elongate
nuclei. Reactive foci were up to 100 to 250 p in diameter. There were other
areas with a more obvious destructive process. Such lesions were most
numerous in the periventricular region of the fourth ventricle, internal
capsule, and thalamus in animal 6. Some foci had a marked punched-out
appearance with a scattering of cells with round, eccentrically placed nuclei,
and densely eosinophilic cytoplasm (gemistocytic astrocytes) (fig. 4, 5).
Occasionally proliferating cells assumed a fibrillar pattern tending to be
arranged in whorls and sheets of longitudinally aligned cells (fig. 2,6). Some-
times multinucleate giant cells were scattered at the periphery of these repa-
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Canine Herpesvirus in Puppies 41
rative lesions (fig. 2, 6). Many of the multinucleate cells appeared to be
composed of proliferating astrocytes, some with peripherally located nuclei
and others with nuclei scattered throughout the cytoplasm. Areas of destruc-
tion of this type were most numerous in the white matter. Frequently, a few
densely basophilic homogeneous bodies were scattered in these regions and
were positive for iron with the Prussian blue technique. In addition to
circumscribed bodies, there was frequently a scattering of finely granular
material which stained with the VON K o s s technique
~ and appeared as densely
basophilic material when stained with HE (fig. 6). Some areas in the
reparative stages consisted almost entirely of proliferating mononuclear cells
forming a fibrillar pattern with relatively uniform nuclei and prominent
cytoplasmic fibrils.
In animal 39, lesions were numerous in the metencephalon and myelence-
phalon. Affected areas in the cerebellum were most numerous in the ventral
folia, both in the vermis and paramedian lobules of the lateral hemispheres.
A few degenerate foci were scattered in other regions of the cerebellar cortex.
Lesions tended to be focal to segmental and usually were bounded by histo-
logically normal areas. Many cerebellar folia were relatively normal histo-
logically. The inner granular layer was poorly delineated in affected segments
and contained reduced numbers of cells (fig. 7,s). PURKINJE cells in degenerate
areas were frequently reduced in number, pyknotic, and sometimes scattered
in the inner granular layer (fig. 7). In the corpus medullaris there were
irregular regions of malacia with a scattering of pleomorphic proliferating
astroglial and histiocytic cells.
One degenerate area in the anterior vermis contained aggregates of densely
basophilic material up to 2 0 p in diameter which reacted as iron with the
Prussian blue stain. Similar deposits were in the adjacent leptomeninges. In
addition, iron was deposited in the walls of blood vessels in the brain and
leptomeninges of this area (fig. 8). Iron deposits were in both the adventitia
and media of affected vessels. There were many gitter cells with ballooned,
vacuolated cytoplasm in the adjacent leptomeninges (fig. 8). Occasionally a
few gitter cells were scattered in the meninges elsewhere in the cerebellum.
Fig.1. Focus of microgliosis in puppy 6 (killed at 16 days p.i. of CHV). The dense
cellular infiltrate with obliteration of normal architectureis typical of acute CHV encepha-
litis. HE, ~ 2 7 5 .
Fig. 2. Focus of granulomatous encephalitis with pleomorphic mononuclear cells and
multinucleate giant cells in thalamus of puppy 6. HE, x 375.
Fig. 3. Areain brain of puppy 6,characterizedby granulomatousinflammationwith prom-
inent proliferatingneocapillariesandreactiveastrocytes at periphery oflesion.PAS, x 185.
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et al.
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Eye
In animal 39 the intact globes measured approximately 14 X 15 x 14 mm.
There were clumping of pigment and scattered white foci on the peripheral
retina. Microscopically, there were areas of retinal dysplasia peripherally.
Foci consisting chiefly of undifferentiatedretinal cells were arranged around
a central lumen (fig. 9). Under higher magnification, a limiting membrane
was discernible with rod-like projections extending into the lumen (fig. 9).
Other areas of the retina and the cornea, anterior chamber, lens, ciliary body,
and choroid were histologically normal. No ocular lesion was observed
microscopically in eyes from the other animals in this study.
Lung
In most animals there was moderate to marked thickening of the interal-
veolar septa. Although many septa1 walls were hypercellular, there were
relatively few pneumocytes in the alveolar spaces. In some areas there was
patchy consolidation of the parenchyma with obliteration of the architecture
by polymorphonuclear and mononuclear cells. The adjacent bronchi and
bronchioles frequently contained cellular exudate. However, the majority of
air passages were free of inflammatory cells. The histological pattern in most
cases was suggestive of viral pneumonitis with superimposed secondary
bacterial infection. No inclusion body was observed.
Heart
Animal 5 had a degenerate focus in the left ventricle. In this area there
were proliferating mononuclear cells and multinuclear giant cells. This was
interpreted to be a reparative lesion subsequent to acute focal myocardial
degeneration.
Liver
In most puppies there was a moderate periportal cellular infiltration of
polymorphonuclear and mononuclear celIs. There was no focal hepatic
necrosis.
Fig.4. Focus of malacia in the periventricular region of the fourth ventricle of puppy 6.
Vacuolation of neuropil and the numerous cells with densely staining cytoplasm and
nuclei, interpreted as gemistocytic astrocytes. HE, x 130.
Fig.5. Higher magnification of figure 4 to illustrate the vacuolation and the gemisto-
cytic astroglia. HE, x 325.
F&. 6. MuItipIe areas of mineralization in the thalamus of animal 6. Enlongated mono-
nuclear cells and multinucleate cells (upper and lower center) are numerous in this region.
HE, x18.5.
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Fig.9. Retina, choroid, and sclera of animal 39. There are foci of retinal dysplasia in the
peripheral retina. x 30. Inset: higher magnification of marked area to illustrate the undif-
ferentiated retinal cells arranged around central lumens. HE, x 185.
Kidney
There were multiple segmental areas in the renal cortices with distortion
of the normal architecture, disruption and obliteration of tubules, pro-
liferation of fibroblasts, and minimal to moderate infiltration of mononuclear
cells (fig. 10,12). Some glomeruli were markedly hypercellular and completely
obliterated BOWMAN’S space. Glomeruli contained an admixture of pleo-
morphic inflammatory cells, cellular debris, and proteinaceous material
(fig. 10). A few glomeruli were distended with densely eosinophilic material,
portions of which stained positively for fibrin. In animal B2, examined at 11
Fig. 7. Cerebellar folium, ansiform lobe of animal 39. Outer germinal layers are at the
upper rigbt and lower left. Segments of the inner germinal and molecular layers are poorly
differentiated and relatively acellular. PURKINJEcells are also reduced in number. HE,
X 160.
Fig.8. Leptomeninges and adjacent cerebellar cortex in puppy 39. Note the densely
stained, mineralized vascular walls and cellular infiltrate, including vacuolated gitter cells
in the leptomeninges. The cerebellar cortex at the right is poorly differentiated. HE, x 170.
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F&. 13. Fluorescent antibody preparation of puppy 39 (cerebellar cortex). There is
viral antigen in both the molecular and inner germinal layers. Note the fluorescent areas
in an adjacent folium (upper right).
days p.i., rend cortical lesions varied from those of some duration with
parenchymal collapse to areas of recent necrosis with eosinophilic homo-
geneous material in remnants of tubules and glomeruli (fig. 10, 11). Affected
convoluted tubules were lined by poorly differentiated epithelial cells of
varying shapes and sizes (fig. 12). Some tubules were dilated and lined by
flattened epithelial cells (fig. 10, 11). Frequently some glomeruli and tubules
appeared vestigial and poorly differentiated (fig. 12).
Fig. 10. Kidney of animal B2,killed 11 days pi. of CHV, with coagulative necrosis of a
few convoluted tubules and marked vatiation in size of other tubules. Many glomeruli are
hypercellular and are obliterated by the destructive inflammatory process. HE, x 130.
Fig.11. Another area of kidney in puppy Bz.Two glomeruli are completely obliterated
by proteinaceousexudate. A few tubules are dilated and lined by flattened epithelial cells.
HE, X140.
Fig.12. Kidney of a n k a l 5. Renal capsule is at the left. Marked distortion of con-
voluted tubules and variation in size of cells lining such tubules. Mononuclear cells, in-
cluding lymphocytes, are scattered in the interstitial regions. A vestigial glomerulus is at
the upper right. HE, x 400.
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et al.
Spleen and Small Intestine
No significant finding was observed in these tissues.
Fluorescent-Antibody Studies
Segments of some cerebellar folia in animal 39 contained multiple fluores-
cent areas with both intranuclear and intracytoplasmicfluorescence. Fluores-
cence was most extensive in the inner granular and molecular layers, some-
times with involvement of displaced PURKINJE cells in the underlying white
matter (fig. 13).
Serological and Viral Studies
Puppy B2 did not have serum neutralizing antibody to CHV at the time
of necropsy; virus was isolated from its lung, liver, spleen, and kidney.
Cytopathic effects (CPE) occurred in less than half of the inoculated cell
cultures at the 10-1 dilution but did not occur at higher dilutions.
Animals 2,5, and 6, killed at 16 days p i , had serum neutralizing antibody
titers to CHV greater than 1 :5. Viral isolation was not attempted in these
PUPS.
Animal 39 had a serum neutralizing antibody titer to CHV of 1:64. The
dam of this pup had an antibody titer to CHV of 1:32. CPE was not observed
in cell cultures inoculated with brain, lung, liver, spleen, or kidney from 39.
As noted above, however, there was abundant viral antigen on FA examina-
tion of the cerebellum from this animal.
Discussion
There are several aspects of this disease syndrome which have not been
recognized previously. Although normally CHV infection in the susceptible
newborn puppy is a peracute fatal disease, occasionally animals may recover
with residual lesions in the CNS, lung, and kidney. Lesions in such animals
could be confused with other disease processes. The presence of cerebellar
and retinal dysplasia in one animal indicates that canine herpesvirus may,
on occasion, impair the normal differentiation of such tissues.
The circumscribed residual lesions in the CNS of the puppies in this study
might be confused with diseases such as toxoplasmosis. K o m m and COLE
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[25] have described lesions in the CNS of dogs with toxoplasmosis. They
observed foci of necrosis with microgliosis, macrogliosis, and vascular pro-
liferation. Such lesions were most common in young dogs, including puppies
which apparently became infected in utero. However, mineralization was not
described in the CNS of these animals with toxoplasmosis. The foci of
mineralization in puppy 39 are probably relatively nonspecific sequels to a
prior destructive process in the CNS. Calcified material has been observed
in CNS lesions of infants that have died with toxoplasmosis [37] and cyto-
megalic inclusion disease [5]. The focal aggregates of iron in the nervous
tissue and blood vessels in one area of the metencephalon are suggestive of
previous hemorrhage, hemolysis, and subsequent deposition of iron. Min-
eralized foci that stained positively for iron but not for calcium have been
described in the CNS of rabbits that recovered from herpetic encephalitis
[12]. Mineralization was confined to areas of the brain where a marked
destructive process had occurred. It was suggested that the deposits were of
hematogenous origin. Ferrocalcareous deposits are frequent in cerebral
vessels of clinically normal horses over 6 years of age (“cerebrovascular side-
rosis”) [3 1 ]. Neither calcification nor siderosis has been previously described
in the brain substance of dogs [19]. Cerebrovascular siderocalcinosis was
described in one dog 12 years of age [14]. Of particular interest was the
segmental cerebellar dysplasiain puppy 39. Active differentiation and cellular
division occur in the canine cerebellar cortex for 2 or more weeks after birth
[30]. Such dividing cells would serve as ideal sites for the replication of certain
viruses such as CHV. KILHAMet al. [24] have described cerebellar hypoplasia
in the offspring of susceptible pregnant cats and ferrets inoculated with the
virus of feline panleukopenia. Cerebellar hypoplasia has also been observed
in the progeny of sows inoculated with the virus of hog cholera [13] and in
the calf from a pregnant cow inoculated with the virus of bovine viral
diarrhea (BVD)[36]. The predilection of CHV for the germinal layers of the
cerebellum in some puppies has been observed by both light and fluorescent
microscopy [29]. However, there were differences between the cerebellar
lesions of puppy 39 and those normally seen in cerebellar atrophy in kittens
due to the virus of panleukopenia. Cerebellar atrophy in kittens tends to
involve entire cerebellar folia with minimal inflammatory response 1241. On
the other hand cerebellar cortical lesions in puppy 39 were focal to segmental
with evidence of a prior acute destructive process with focal mineralization
and gitter cells in the adjacent leptomeninges.
The retinal dysplasia in puppy 39 may be of considerable clinical impor-
tance. Retinal dysplasia may occur ‘spontaneously’ in Beagles [17] and has
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PERCY
been described in the Sealyham Terries as an apparent autosomal recessive
factor [4]. In addition, dysplasia of anterior portions of the retina has been
produced in Beagle pups irradiated at 2 days of age [32]. Retinal dysplasia
has been induced in other species by a variety of factors [15, 351. Recently
retinal dysplasia has been induced experimentally in lambs with bluetongue
virus [33]. Foci of retinal destruction have been described in puppies experi-
mentally infected with CHV [27]. In addition, uveitis, cataracts, and dysplasia
of the optic nerve have been seen in pups infected with CHV [I]. Ocular
lesions in man attributed to the virus of herpes simplex include conjunctivitis
[6], keratitis 1161, and uveitis [26]. The retinal dysplasia observed in one
animal in this series may well be a result of an intraocular infection with
canine herpesvirus. The ocular changes in this disease will be described in
more detail [l].
The pneumonic lesions in our animals with CHV infection were considered
to be relatively non-specific and similar to other viral pneumonias such as
that of canine distemper with secondary bacterial invaders [20]. Of interest
in these animalswas the relative sparing of the bronchial tree, and the absence
of inclusion bodies in the respiratory epithelium.
The renal lesions in these puppies emphasize the susceptibility of the
kidney to invasion by CHV. The distortion of the tubules and marked mor-
phologic variation of epithelial cells lining such tubules were indicative of
both degenerative and regenerative processes. In addition, there was histo-
logical evidence of arrested evolvement of many glomeruli. Impaired and
arrested nephrogenesis have been described in kittens infected in utero with
the virus of feline panleukopenia [24]. The increased cellularity in some
glomeruli probably represent the inflammatory and reparative phase subse-
quent to an initial glomerular damage. A superimposed bacterial infection
could be a factor although it was not demonstrated in such lesions using
appropriate stains. Of particular importance is that these renal lesions could
probably persist for many months in such animals and could therefore be
erroneously attributed to other causes.
Of interest was the failure to isolate CHV from the cerebellum of puppy
39 although FA examination indicated viral antigen to be abundant in some
cerebellar folia. CONNOLY et al. [9] have failed to isolate measles virus in
cases of subacute sclerosing panencephalitis although viral antigen was
demonstrated by fluorescent microscopy. CF and HI antibodies to measles
virus were demonstrated in the CSF and serum of these patients. Similarly
the failure to isolate CHV from the cerebellum of puppy 39 was probably
due to the relatively high neutralizing antibody titer in the serum of this
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animal. The high neutralizing antibody titer to CHV in puppy 39 and his
dam indicates recent exposure to the virus. The death of several littermates
at 2 weeks of age with characteristic lesions of CHV infection is suggestive
of a neonatal rather than an intra-uterine infection with this agent.
Acknowledgements
This work was financed in part by grants USPHS 5-PObRR40393, USPHS 54301-
FR05358, National Institutes of Health grant A 107516-03 and NEI lROl EY 00108-01.
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Author’s address. Dr. D.H. PERCY,
Health Sciences Centre, University of Western Ontario,
London 72, Ont. (Canada)
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