Domestic Animals–Brief Communication

Veterinary Pathology
2019, Vol. 56(4) 604-608
Lymphoplasmacytic Meningoencephalitis ª The Author(s) 2019
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and Neuronal Necrosis Associated With DOI: 10.1177/0300985819837723
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Parvoviral Infection in Cats

Anna Kokosinska1, Grazieli Maboni1, Kathleen M. Kelly2 ,

Alex Molesan2, Susan Sanchez1, Jeremiah T. Saliki1,
and Daniel R. Rissi1

Abstract
Neurologic manifestations other than cerebellar hypoplasia are rarely associated with feline panleukopenia virus (FPV) infection in
cats. Here the authors describe lymphoplasmacytic meningoencephalitis and neuronal necrosis in 2 cats autopsied after exhibiting
ataxia and nystagmus. Gross changes consisted of cerebellar herniation through the foramen magnum, with flattening of cere-
brocortical gyri and narrowing of sulci. Histologically, lymphoplasmacytic meningoencephalitis, extensive neuronal necrosis, and
neuroaxonal degeneration with digestion chambers were present in the telencephalon and brain stem in both cats. Frozen brain
tissue of both cats was positive for parvoviral antigen via fluorescent antibody testing, and formalin-fixed, paraffin-embedded
tissue sections of brain were immunoreactive for parvovirus antigen and positive for parvoviral DNA on in situ hybridization.
Frozen brain tissue from 1 case was positive for parvovirus NS1 and VP2 genes using conventional polymerase chain reaction, and
subsequent DNA sequencing and phylogenetic analysis revealed that the viral strain was a FPV. Reverse transcription quantitative
polymerase chain reaction on formalin-fixed, paraffin-embedded brain tissue revealed high levels of parvovirus in both cases,
supporting an acute and active viral infection. Although rare, FPV infection should be considered in cases of lymphoplasmacytic
meningoencephalitis and neuronal necrosis in cats.

Keywords
feline panleukopenia, neuropathology, cats, brain, parvovirus, encephalitis

Although many parvoviruses can infect cats, infection is most hydranencephaly, and retinal dysplasia.1–3,12,14,23,25 Parvoviral
commonly associated with feline panleukopenia virus (FPV), a infection of rapidly replicating external germinal layer cells
nonenveloped, single-stranded DNA virus of the family Par- and Purkinje cells leads to cerebellar hypoplasia and
voviridae.10,12,25,26 FPV is closely related to other parvoviruses ataxia.1,18,20 Other rare neurologic changes associated with
and infects primarily domesticated cats but also wild felids parvoviral infection in cats include demyelination with or
worldwide.10 Infection typically takes place in tissues with a without neuronal degeneration4,8,16,27 and lymphohistiocytic
high mitotic rate, such as intestine, bone marrow, and lymphoid cerebellar meningoencephalitis.22 Some of these rare manifes-
tissue.12 Transmission occurs via direct contact with contami- tations can affect adult cats8,27 and can occur without the clas-
nated feces, urine, blood, and placenta or by indirect contact sic cerebellar involvement.4,8,16,27
through the environment.15 The high risk for transmission and
the high stability of the virus in the environment create a seri-
ous threat of infection in shelters or other environments with
high population density or multiple animals from different 1
Department of Pathology and Athens Veterinary Diagnostic Laboratory,
sources.15 University of Georgia College of Veterinary Medicine, Athens, GA, USA
2
Clinical disease associated with FPV or other parvoviruses Department of Biomedical Sciences, Cornell University College of Veterinary
in young or adult cats is usually progressive and fatal, leading Medicine, Ithaca, NY, USA
to fever, vomiting, and diarrhea, associated with pancytopenia Supplemental material for this article is available online.
and necrotizing enteritis.12 Fetal infection by FPV causes a
wide range of clinical and pathologic manifestations. Corresponding Author:
Daniel R. Rissi, University of Georgia College of Veterinary Medicine,
Early-term infection most commonly leads to abortion or Department of Pathology and Athens Veterinary Diagnostic Laboratory,
stillbirths, whereas late-term or neonatal infection is more Athens, GA 30602, USA.
often associated with cerebellar hypoplasia, hydrocephalus, Email: rissi@uga.edu
Kokosinska et al 605

cerebral cortex and hippocampus (Fig. 4), as well as the ves-

tibular and abducens nuclei in case 1. Affected neurons had
hypereosinophilic and shrunken cytoplasm, with nuclear
pyknosis or karyorrhexis. Dilation of the periaxonal spaces,
with swollen axonal spheroids or foamy macrophages (Gitter
cells, digestion chambers), were present throughout the white
matter of the distal brain stem, especially within the facial
nerve fibers (Fig. 5). The neuroparenchyma adjacent to the
affected areas throughout the brain was vacuolated because
of edema. Small intestinal crypt necrosis and mucosal collapse,
typical of parvoviral infection, was present in case 2. No other
pathologic changes were present in either cat.
Using fluorescent antibody testing (FAT), frozen brain tis-
sue (cases 1 and 2) was positive for feline parvovirus (Fig. 6)
and negative for rabies, feline infectious peritonitis virus, and
Toxoplasma gondii. Immunohistochemistry (IHC) for feline
parvovirus (mouse monoclonal, 1:500 dilution for 60 minutes;
Bio-Rad, Hercules, CA; MCA2064) was conducted using
Fig. 1. Necrotizing meningoencephalitis, brain, cat, case 1. The cere- formalin-fixed tissue sections of brain and small intestine
bellar vermis is herniated through the foramen magnum. There is also (cases 1 and 2). Control tissues included canine small intestine
flattening of cerebrocortical gyri (arrows) due to cerebral edema. with confirmed parvoviral enteritis. Widespread, robust immu-
nolabeling was detected mainly in cerebrocortical neurons,
Here we characterize the neuropathologic and molecular Purkinje neurons, and Golgi neurons and less commonly in
features of 2 cases of lymphoplasmacytic meningoencephalitis microglial nodules and endothelial cells throughout the brain
and neuronal necrosis associated with parvoviral infection in in both cases (Fig. 7). Immunolabeling was also present in the
cats. Both individuals were part of a group of 3 rescued stray cerebellar external granular layer neurons in case 1. Immuno-
cats that were waiting for adoption and shared the same room in labeling was also detected within necrotic areas in the intestine
a veterinary clinic. All 3 cats had acute onset of neurologic in case 2; no immunolabeling was present in the intestine in
signs. Case 1 was an 8-week-old female domestic shorthaired case 1. In situ hybridization (ISH) was performed on formalin-
cat with a 1-week history of ataxia and nystagmus that was fixed sections of brain stem (cases 1 and 2) according to pre-
euthanized because of a poor prognosis. Case 2 was a 5- viously published work.7,13 A strong parvoviral ISH signal was
month-old male neutered domestic shorthaired cat with a 24- localized within the cytoplasm and nuclei of necrotic and
hour history of acute ataxia that died 1 day after cat 1 was viable neurons across the brain (Fig. 8).
euthanized. A third cat was also ataxic and died 4–5 days A conventional polymerase chain reaction (PCR) was per-
before the other 2 cats exhibited clinical signs; no other infor- formed on frozen brain tissue from case 2. Viral DNA was
mation was provided about that cat. All individuals had tested extracted using a commercial kit (QIAmp cador Pathogen Kit,
negative for feline immunodeficiency virus and feline leuke- Qiagen, Hilden, Germany) according to the manufacturer’s
mia virus. Cases 1 and 2 were submitted for autopsy. instructions. Using published primers,5,6 a 583-bp fragment
Gross anatomic changes in both cats consisted of cerebellar of the capsid protein 2 gene (VP2) and a 2007-bp fragment
herniation through the foramen magnum, with mild widespread of the nonstructural protein 1 gene (NS1) were amplified. A
flattening of cerebrocortical gyri and narrowing of sulci previously sequenced field strain of canine parvovirus (CPV)
(Fig. 1). Representative samples of brain, heart, trachea, lungs, was used as a positive control, as reported elsewhere.9 PCR
liver, spleen, kidneys, urinary bladder, gastrointestinal tract, products from both VP2 and NS1 genes were purified and
lymph nodes, thyroid and parathyroid glands, adrenal glands, submitted for Sanger DNA sequencing, as previously
and bone marrow were fixed in 10% buffered formalin, routi- described.24 Basic Local Alignment Search Tool analysis
nely processed for histology, and stained with hematoxylin and (http://www.ncbi.nlm.nih.gov/BLAST) was used on each
eosin. For the brain, sections of frontal, parietal, temporal, and sequence to identify related viruses. Phylogenetic trees were
occipital telencephalon, basal nuclei, thalamus, hippocampus, built using the NS1 and VP2 sequences from this study and 46
mesencephalon, cerebellum, and pons were examined histolo- VP2 gene nucleotide sequences and 31 NS1 gene sequences
gically.28 Neurohistologic changes were similar in both cases retrieved from GenBank. The retrieved sequences were repre-
and consisted of multiple perivascular accumulations of small sentative strains from FPV, mink enteritis virus, and CPV from
to moderate numbers of lymphocytes and plasma cells within different parts of the world. VP2 and NS1 sequences from this
the neuroparenchyma and leptomeninges (Fig. 2). Endothelial study are available in the public database under accession num-
cells were swollen, and nodular areas of microgliosis were bers MH581488 and MH577049, respectively. Sequence align-
present throughout the neuroparenchyma (Fig. 3). Extensive ments were performed using ClustalW software, and
neuronal necrosis was present within the frontal and parietal phylogenetic trees were inferred using the neighbor-joining
606 Veterinary Pathology 56(4)

Figs. 2–8. Feline parvovirus–associated meningoencephalitis, brain, cat. Fig. 2. Thalamus, case 1. A venule is surrounded by lymphocytes and
plasma cells. Hematoxylin and eosin (HE). Fig. 3. Thalamus, case 2. There is a distinct glial nodule. HE. Fig. 4. Hippocampus, case 1. There are
multiple necrotic neurons. Inset: a necrotic neuron with hypereosinophilic cytoplasm and pyknotic nucleus. HE. Fig. 5. Case 1. There is dilation
of periaxonal spaces (center), which contain accumulations of foamy macrophages (digestion chambers). Inset: a digestion chamber contains
foamy macrophages and cell debris. HE. Fig. 6. Case 1. Strong labeling using fluorescent antibody test for parvovirus. Fig. 7. Obex, case 1.
Strong immunolabeling for feline panleukopenia virus (FPV) in a neuron (right) and a glial nodule (left). Immunohistochemistry for FPV. Fig. 8.
Obex, case 2. Labeling of neuronal cytoplasm and nucleus for parvoviral nucleic acid. In situ hybridization.

method21 available in Molecular Evolutionary Genetics Anal- likelihood method. The percentage of replicate trees in which
ysis version 7 (http://www.megasoftware.net).11 Evolutionary the associated taxa clustered together using bootstrapping
distances were computed using the maximum composite (1000 iterations) is shown next to the branches. The amplified
Kokosinska et al 607

sequences were 99% homologous to NS1 and VP2 gene neurodegenerative changes with minimal or no accompanying
sequences from FPV, CPV, and mink enteritis virus available inflammation.4,8,16,22,27 FPV infection was suspected in cases
in GenBank. Phylogenetic analysis of NS1 placed this of bilateral and symmetric demyelination of the brain stem and
sequence in the FPV clade (Suppl. Fig. S1). Demarcation cri- spinal cord, as well as neuronal degeneration in the gray matter
teria from the International Committee on Taxonomy of of the spinal cord in the 1970s, but infection could not be
Viruses for the parvovirus genus are based only on the NS confirmed, and the role of FPV in the development of those
gene; therefore, the strain investigated in this study was classi- changes remains elusive.4 Mild cerebellar lymphohistiocytic
fied as FPV. The VP2 gene phylogeny (Suppl. Fig. S2) had a meningoencephalitis was reported in a cat with parvoviral
closer association with mink enteritis virus, followed by FPV, infection in which immunolabeling was detected in neurons,
which might be the consequence of recombination events and macrophages and microglia, and ependymal cells; no further
might have an impact on the unusual tropism and virulence of viral characterization was conducted.22 Focal neuronal satelli-
the strain. tosis and neuronophagia were described in a subset of cats that
For reverse transcription quantitative PCR (RT-qPCR), died of necrotizing enteritis due to FPV infection; immunola-
nucleic acid was extracted from two 10-mm sections of beling was detected in neurons of the interthalamic adhesion
formalin-fixed, paraffin-embedded brain tissue (cases 1 and and glial cells.8 Neuronal vacuolation with positive immuno-
2) sectioned into sterile Eppendorf tubes. Samples were depar- labeling for FPV was described in the thoracic spinal cord of a
afinized in lysis buffer for 60 minutes at 70 C and then incu- cat that also had necrotizing enteritis, similar to one of the
bated overnight at 60 C with proteinase K. After incubation the present cases.16 The presence of tissue FPV was confirmed via
tubes were briefly centrifuged and allowed to cool to room PCR and sequencing of viral DNA in only 2 of these previously
temperature; 200 mL of lysate from each tube was transferred reported occasions.8,16 In addition, vacuolation of the neuropil
to a 96-well plate for extraction using a Biomek 4000 auto- and neuronal degeneration in the lateral geniculate nuclei, cer-
mated workstation (Beckman Coulter, Indianapolis, Indiana) ebral cortex, hippocampus, and pons was associated with CPV-
Total Nucleic Acid preset program and Agencourt Formapure 2 in a group of cats; immunolabeling was detected mainly in
Kit (Beckman Coulter) according to the manufacturer’s neurons and less often glial and endothelial cells, and the diag-
instructions. RT-qPCR was performed on these samples using nosis was supported via PCR and DNA sequencing.27
primers and probe to amplify the VP2 gene, according to pre- The successful replication of parvovirus is highly dependent
viously published work.7 Supporting the conventional PCR and on the host cell’s achieving the S phase of its cycle, which
sequencing results, high levels of parvovirus were amplified by makes tissues with high a mitotic rate a preferred target for
RT-qPCR in both cases (1.35  105 and 1.64  105 copies/200 infection in young and adult individuals.17 Intrauterine or neo-
mg of nucleic acid in cases 1 and 2, respectively). natal central nervous system infection occurs mainly within
The diagnosis of parvoviral meningoencephalitis and neu- dividing neuroblasts of the cerebellar external granular
ronal necrosis in the present cases was achieved on the basis of layer.1,10,12,17,25 Purkinje neurons have been shown to be post-
the FAT, IHC, ISH, and PCR results. Subsequent DNA sequen- mitotic after 40 days of gestation,19 but parvoviral infection has
cing and phylogenetic analysis revealed that the NS1 gene been demonstrated within these cells during late pregnancy or
sequence best matched the FPV clade, indicating that the par- neonatal life,1,2,8,18–20,27 which suggests that parvoviruses are
vovirus in these cats was closely related with FPV strains able to replicate in terminally differentiated neurons.25 In the
detected in cats from Italy and Japan; the estimated confidence present cases, viral antigen was detected within neurons, glial
levels of the VP2 gene sequence further support this hypoth- cells, and endothelial cells using IHC, whereas viral nucleic
esis. An acute and active viral infection and a causal relation- acid was present only within neurons. These results differ from
ship were supported by the results of the RT-qPCR.7 a previous study using the same ISH technique for detection of
Gross neuropathologic changes associated with parvoviral CPV-2 in the hearts of dogs, which revealed a more widespread
infection in cats have been limited to cerebellar hypoplasia, parvoviral signal with ISH.7 The reasons for these discrepan-
hydrocephalus, and hydranencephaly.1,12,25 Evidence of cere- cies are elusive and may be related to technical differences in
bral edema, such as cerebellar herniation with flattening of fixation, IHC protocols, or antibodies (mouse monoclonal vs
cerebrocortical gyri and narrowing of sulci, have not been rabbit polyclonal)7 or to differences in the dynamics of viral
described in cases of parvoviral infection in cats and were replication, with active replication within neurons and latent
present in both of the present cases.1,8,12,16,18,20,25,27 The neu- replication within other cells.16
rohistologic changes in the present cases were also distinct In summary, we describe previously undocumented neuro-
from those previously attributed to parvoviral infection in cats, pathologic changes associated with parvoviral infection in 2
which include cerebellar hypoplasia, depletion of the granular cats. Viral antigen was detected within neurons and endothelial
layer, Purkinje cell heterotopia and vacuolation, Purkinje cell cells via FAT and IHC, and parvoviral nucleic acid was
dendrite disarray, and a reduction of myelinated detected within neurons via ISH. Phylogenetic analysis of the
fibers.4,8,16,20,22,27 No gross or histologic evidence of cerebellar NS1 gene classified the strain as a FPV. Abundant viral nucleic
hypoplasia was present in these cases. acid (by RT-qPCR), suggesting an acute and active viral
Other central nervous system manifestations of parvoviral infection, was associated with these neuropathologic changes.
infection in cats are rare and consist mainly of Although uncommon, parvoviral infection should be
608 Veterinary Pathology 56(4)

considered in cases of lymphoplasmacytic meningoencephali- 9. Hong C, Decaro N, Desario C, et al. Occurrence of canine parvovirus type 2c in
tis and neuronal necrosis in cats. the United States. J Vet Diagn Invest. 2007;19(5):535–539.
10. Hueffer K, Parrish CR. Parvovirus host range, cell tropism and evolution. Curr
Acknowledgements Opin Microbiol. 2003;6(4):392–398.
11. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics
We thank Jillian Fishburn (Athens Veterinary Diagnostic Laboratory, Analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33(7):1870–1874.
University of Georgia College of Veterinary Medicine) for support 12. Lamm CG, Rezabek GB. Parvovirus infection in domestic companion animals.
with the FAT and Dr Randall Renshaw (Department of Population Vet Clin North Am Small Anim Pract. 2008;38(4):837–850.
Medicine and Diagnostic Services, Cornell University College of 13. McEndaffer L, Molesan A, Erb H, et al. Feline panleukopenia virus is not
Veterinary Medicine, Ithaca, NY) for the support with RT-qPCR. associated with myocarditis or endomyocardial restrictive cardiomyopathy in
cats. Vet Pathol. 2017;54(4):669–675.
Declaration of Conflicting Interests 14. Percy DH, Scott FW, Albert DM. Retinal dysplasia due to feline panleukopenia
The author(s) declared no potential conflicts of interest with respect to virus infection. J Am Vet Med Assoc. 1975;167(10):935–937.
the research, authorship, and/or publication of this article. 15. Pesavento PA, Murphy BG. Common and emerging infectious diseases in the
animal shelter. Vet Pathol. 2014;51(2):478–491.
Funding 16. Pfankuche VM, Jo WK, van der Vries E, et al. Neuronal vacuolization in feline
panleukopenia virus infection. Vet Pathol. 2018;55(2):294–297.
The author(s) received no financial support for the research, author-
17. Poncelet L, Garigliany M, Ando K, et al. Cell cycle S phase markers are
ship, and/or publication of this article.
expressed in cerebral neuron nuclei of cats infected by the feline panleukopenia
virus. Cell Cycle. 2016;15(24):3482–3489.
ORCID iD
18. Poncelet L, Heraud C, Springinsfeld M, et al. Identification of feline panleuko-
Kathleen Kelly https://orcid.org/0000-0002-5120-6266 penia virus proteins expressed in Purkinje cell nuclei of cats with cerebellar
Dan Rissi https://orcid.org/0000-0003-4574-2836 hypoplasia. Vet J. 2013;196(3):381–387.
19. Poncelet L, Springinsfeld M, Ando K, et al. Expression of transferrin receptor 1,
References proliferating cell nuclear antigen, p27(Kip1) and calbindin in the fetal and
1. Aeffner F, Ulrich R, Schulze-Ruckamp L, et al. Cerebellar hypoplasia in three neonatal feline cerebellar cortex. Vet J. 2013;196(3):388–393.
sibling cats after intrauterine or early postnatal parvovirus infection. Dtsch 20. Resibois A, Coppens A, Poncelet L. Naturally occurring parvovirus-associated
Tierarztl Wochenschr. 2006;113(11):403–406. feline hypogranular cerebellar hypoplasia—a comparison to experimentally-
2. Csiza CK, De Lahunta A, Scott FW, et al. Pathogenesis of feline panleukopenia induced lesions using immunohistology. Vet Pathol. 2007;44(6):831–841.
virus in susceptible newborn kittens II. Pathology and immunofluorescence. 21. Saitou N, Nei M. The neighbor-joining method: a new method for reconstruct-
Infect Immun. 1971;3(6):838–846. ing phylogenetic trees. Mol Biol Evol. 1987;4(4):406–425.
3. Csiza CK, Scott FW, De Lahunta A, et al. Pathogenesis of feline panleukopenia 22. Schwab S, Herden C, Seeliger F, et al. Non-suppurative meningoencephalitis of
virus in susceptible newborn kittens I. Clinical signs, hematology, serology, and unknown origin in cats and dogs: an immunohistochemical study. J Comp
virology. Infect Immun. 1971;3(6):833–837. Pathol. 2007;136(2–3):96–110.
4. Csiza CK, Scott FW, De Lahunta A, et al. Respiratory signs and central nervous 23. Sharp NJ, Davis BJ, Guy JS, et al. Hydranencephaly and cerebellar hypoplasia
system lesions in cats infected with panleukopenia virus. A case report. Cornell in two kittens attributed to intrauterine parvovirus infection. J Comp Pathol.
Vet. 1972;62(2):192–195. 1999;121(1):39–53.
5. Decaro N, Elia G, Campolo M, et al. New approaches for the molecular 24. Stimmelmayr R, Rotstein DS, Maboni G, et al. Morbillivirus-associated lipid
characterization of canine parvovirus type 2 strains. J Vet Med B Infect Dis Vet pneumonia in Arctic foxes. J Vet Diagn Invest. 2018;30(6):933–936.
Public Health. 2005;52(7–8):316–319. 25. Stuetzer B, Hartmann K. Feline parvovirus infection and associated diseases.
6. Fei-Fei D, Yong-Feng Z, Jian-Li W, et al. Molecular characterization of feline Vet J. 2014;201(2):150–155.
panleukopenia virus isolated from mink and its pathogenesis in mink. Vet 26. Truyen U, Parrish CR. Feline panleukopenia virus: its interesting evolution and
Microbiol. 2017;205:92–98. current problems in immunoprophylaxis against a serious pathogen. Vet Micro-
7. Ford J, McEndaffer L, Renshaw R, et al. Parvovirus infection is associated with biol. 2013;165(1–2):29–32.
myocarditis and myocardial fibrosis in young dogs. Vet Pathol. 2017;54(6): 27. Url A, Truyen U, Rebel-Bauder B, et al. Evidence of parvovirus replication in
964–971. cerebral neurons of cats. J Clin Microbiol. 2003;41(8):3801–3805.
8. Garigliany M, Gilliaux G, Jolly S, et al. Feline panleukopenia virus in cerebral 28. Vandevelde M, Higgins R, Oevermann A. Veterinary Neuropathology: Essen-
neurons of young and adult cats. BMC Vet Res. 2016;12:28. tials of Theory and Practice. Hoboken, NJ: John Wiley; 2012.