J Vet Diagn Invest 22:628–632 (2010)
CASE REPORTS
Polyomavirus infection in a free-ranging California sea lion (Zalophus californianus) with
intestinal T-cell lymphoma
Kathleen M. Colegrove,1 James F. X. Wellehan Jr., Rebecca Rivera, Peter F. Moore,
Frances M. D. Gulland, Linda J. Lowenstine, Robert W. Nordhausen, Hendrik H. Nollens
Abstract. An adult female California sea lion (Zalophus californianus) that stranded in central California
was found to have a small glossal polypoid mass on gross necropsy. Histologically, the mass was consistent
with a fibropapilloma, and intranuclear inclusions were found within endothelial cells lining small arterioles
within the mass. Electron microscopy revealed 40-nm virions within endothelial intranuclear inclusions.
Rolling circle amplification was used to obtain a partial viral genomic sequence. Sequence analysis identified
the virus as a novel polyomavirus, tentatively named California sea lion polyomavirus 1. In addition, the sea
lion had a severely thickened small intestine and swollen pale kidneys on gross examination. Severe renal
amyloidosis with chronic interstitial nephritis was diagnosed histologically as well as T-cell intestinal
lymphoma, which was confirmed via immunophenotyping and molecular clonality. The relationship, if any,
between polyomavirus infection and the other disease processes in this sea lion is not known, but it is
considered unlikely that the polyomavirus induced the lymphoma.
Key words:
California sea lion; fibropapilloma; molecular clonality; polyomavirus.
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Members of the Polyomaviridae family are small DNA
viruses that infect birds and mammals. Among mammals,
infection has been identified in humans, nonhuman primates,
rodents, bats, and cattle.7,9,14,21 In non–immune-compromised
endemic mammalian hosts, natural infections have not
typically been linked with severe disease but may lead to
subclinical, persistent, ubiquitous infections.9 In humans,
reactivation can occur in immune-suppressed individuals,
leading to severe and sometimes fatal disease including
hemorrhagic cystitis, progressive multifocal leukoencephalopathy, and Merkel cell carcinoma.5,9,10,20 Some human
polyomaviruses, including Simian virus 40 (SV40), BK polyomavirus, and JC polyomavirus, can cause cancer in animal
models or neoplastic transformation of cell cultures.15 Hamster
polyomavirus is associated with skin tumors.6,7 In contrast,
polyomavirus infection in avian species can cause acute and
chronic diseases such as budgerigar fledgling disease caused by
Psittacine polyomavirus and hemorrhagic nephritis and
enteritis of geese caused by Goose hemorrhagic polyomaFrom the Zoological Pathology Program, University of Illinois at
Urbana-Champaign, College of Veterinary Medicine, Maywood, IL
(Colegrove); University of Florida, College of Veterinary Medicine,
Gainesville, FL (Welleham, Nollens); Hubbs-SeaWorld Research
Institute, San Diego, CA (Rivera); Department of Pathology,
Microbiology, and Immunology, School of Veterinary Medicine,
University of California, Davis, CA (Moore, Lowenstine); The
Marine Mammal Center, Sausalito, CA (Gulland); and California
Animal Health & Food Safety Laboratory, Davis, CA (Nordhausen).
1
Corresponding Author: Kathleen M. Colegrove, Zoological
Pathology Program, University of Illinois, Loyola University Medical
Center, Building 101, Room 0745, 2160 South First Avenue,
Maywood, IL 60153. [email protected]
virus.10 The purpose of the current report is to describe the
histopathologic and molecular features of polyomavirus
infection and intestinal T-cell lymphoma in a free-ranging
California sea lion (Zalophus californianus), both of which have
not been previously reported in any marine mammal species.
An adult female sea lion was found stranded along the
central California coast and was taken to The Marine
Mammal Center (Sausalito, California). On initial examination, the sea lion was lethargic and was unable to raise itself
from sternal recumbency. Blood work abnormalities included elevated blood urea nitrogen, creatinine, sodium, and
phosphorus levels, consistent with renal disease and
hypoalbuminemia. Ultrasound examination revealed bilaterally enlarged kidneys, with multifocal cysts of varying size.
Differential diagnoses included acute nephritis, amyloidosis,
leptospirosis, and chronic renal disease.8 Urogenital carcinoma was also considered as a differential diagnosis due to
the sonographic finding of renal cysts, as ureter obstruction
and hydronephrosis are common sequelae of metastatic
urogenital carcinoma in sea lions.4 Because of poor
prognosis, the sea lion was humanely euthanized.
On gross examination, the dorsal surface of the tongue had
a 0.7 cm in diameter, light tan papillary, nodular mass
(Fig. 1). In addition, the proximal one third to one half of the
small intestine, including the duodenum, was markedly
thickened and plicated. On cut section, the intestinal mucosa
was thickened and rough. There was no evidence of ureter
obstruction or hydronephrosis. However, the kidneys were
enlarged, swollen, and diffusely pale tan with loss of
corticomedullary and renule differentiation. Multiple cysts
were scattered throughout the cortex. Representative tissue
sections were placed in 10% neutral buffered formalin and
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Case Reports
629
Figure 1. Tongue from a California sea lion (Zalophus californianus) with a small focal polypoid mass (arrow) on the dorsal
mucosal surface.
Figure 2. Tongue mass; California sea lion (Zalophus californianus). The hyperplastic epithelium overlies a thick stalk of dense
fibrovascular connective tissue. There is mild superficial erosion and segmental ballooning degeneration in the epithelium. Hematoxylin
and eosin. Bar 5 1 mm.
Figure 3. Tongue mass; California sea lion (Zalophus californianus). Blood vessels within the fibrous connective tissue contain
amyloid deposits and occasionally are lined by endothelial cells with enlarged nuclei, peripheral chromatin, and pale basophilic
inclusions (arrow). Hematoxylin and eosin. Bar 5 50 mm.
Figure 4. Tongue mass; California sea lion (Zalophus californianus). Transmission electron microscopy of a blood vessel with large
endothelial nuclei and intranuclear inclusions containing numerous 40-nm virions (inset, higher power). Bar 5 100 nm.
Figure 5. Small intestine, California sea lion (Zalophus californianus). The lamina propria contains a monomorphic population of
densely packed neoplastic lymphocytes. Inset, Higher magnification of neoplastic lymphocytes. There is occasional epitheliotropism.
Hematoxylin and eosin. Bar 5 100 mm.
Figure 6. Small intestine, California sea lion (Zalophus californianus). Immunohistochemical staining for the T-cell marker cluster
of differentiation (CD)3 illustrating diffuse immunoreactivity of the neoplastic lymphocytes. Bar 5 200 mm.
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Case Reports
submitted to the Pathology Service, William R. Pritchard
Veterinary Medical Teaching Hospital, University of California (Davis, California) for evaluation. Tissues were
embedded in paraffin, processed routinely, sectioned at
5 mm, and stained with hematoxylin and eosin for routine
light microscopy. Representative sections of tongue and
kidney were stained with Congo red. Immunohistochemistry
was completed on sections of tongue and small intestine.
Sections of tongue were incubated with polyclonal antibodies
to Bovine papillomavirus-1 (rabbit polyclonal antibody).a
Sections of intestine were incubated with monoclonal
antibodies to clusters of differentiation (CD)3 (CD3–12),b
CD79a (HM57),c and CD20 (rabbit polyclonal antibody)d as
previously described.16
For transmission electron microscopy (TEM), formalinfixed sections of tongue were processed and embedded in
epoxy resine as previously described.11,24 Cut thick sections
were mounted on glass slides, stained with Toluidine blue
O, and examined by light microscopy. Thin sections were
mounted on 150-mesh copper grids, stained as previously
described, and examined under a transmission electron
microscopef at 60-kV accelerating voltage.19
DNA was extracted from the fresh tongue lesion material
and paraffin-embedded intestine tissue using a commercial
kit,g and rolling circle amplification was performed with the
DNA amplification kith following the manufacturer’s
instructions. Products were digested using EcoRI enzyme,i
cloned into pUC18,j expanded in One Shot Max Efficiency
DH5alpha-T1R Competent Cells,k and sequenced using
M13 primers. The resultant sequences were compared with
those in GenBank (http://www.ncbi.nlm.nih.gov/Genbank/),
European Molecular Biology Laboratory (http://www.embl.
org/ ), and Data Bank of Japan (http://www.ddbj.nig.ac.jp/
index-e.html) databases using the BLASTX algorithm.2
Histopathologically, the mass on the tongue was composed
of hyperplastic stratified squamous epithelium supported by a
thick base of dense fibrovascular connective tissue (Fig. 2).
There was segmental superficial erosion of the epithelium
accompanied by mild neutrophilic inflammation. Segmentally, epithelial cells in the superficial layer exhibited mild
ballooning degeneration; however, no inclusions were noted in
the epithelium. In the underlying fibrous connective tissue,
many small arterioles had thickened walls containing smudgy
pale eosinophilic, acellular, congophilic material, consistent
with amyloid.3 Several of these vessels were lined by
endothelial cells with markedly enlarged nuclei containing
pale basophilic smudgy inclusions and peripheral chromatin
(Fig. 3). Immunohistochemistry for papillomavirus was
negative. Using TEM, numerous nonencapsulated virions
averaging 40 nm in diameter were noted within endothelial cell
nuclei (Fig. 4). The size and morphology of the virions were
consistent with viruses of the families Papillomaviridae or
Polyomaviridae. Rolling circle amplification of the tongue
DNA resulted in a 904–base pair (bp) product. BLASTX
results of the 904-bp product showed the highest identity score
with Myotis polyomavirus major capsid protein (VP1;
GenBank accession no. FJ188392.1; 68% amino acid identity)
and Murine pneumotropic virus major capsid protein
(GenBank accession no. EF186666.1; 67% amino acid
identity), confirming the identity of the virus as a polyoma-
virus. The virus was tentatively named California sea lion
virus 1, strain CSL6994. The sequence was submitted to
GenBank (accession no. GQ331138). Sections of intestine
were negative for polyomavirus DNA using the rolling circle
amplification polymerase chain reaction (PCR) method and
negative using the papillomavirus immunohistochemical stain.
In sections of the small intestine, the mid and superficial
lamina propria was expanded and contained a monomorphic
population of medium-sized immature lymphocytes (Fig. 5).
Villous tips were often fused and disrupted, with the
epithelium artifactually lifted away from the villi. The
immature lymphocytes were densely packed and occasionally
formed small aggregates within the mucosal epithelium. Cells
had scant amounts of basophilic cytoplasm and round to
indented nuclei with clumped chromatin (Fig. 5, inset). The
immature lymphocytes were mixed with small to moderate
numbers of small mature lymphocytes, plasma cells, globular
leukocytes, and eosinophils, especially in the lower portion of
the lamina propria. Scattered immature lymphocytes and
plasma cells were noted in the submucosa and muscularis,
often surrounding blood vessels. No inclusions were noted
within the affected intestine or any other tissue examined.
The dense monomorphic, occasionally epitheliotrophic
population of immature lymphocytes in the lamina propria
were diffusely immunoreactive to CD3 (Fig. 6). Lymphocytes in the more heterogeneous population in the lower
lamina propria occasionally expressed CD20. CD79a, a
monoclonal antibody specific for B cells, did not cross-react
with sea lion B cells. Based on these results, T-cell
lymphoma was diagnosed.
Further confirmation of T-cell intestinal lymphoma was
accomplished via molecular clonality analysis for T-cell
receptor gamma (TCRG) gene rearrangements using PCR.
DNA was extracted from 25-mm sections cut from paraffin
blocks containing small intestine as previously described.16
Amplification of the CDR3 region of TCRG by PCR was
used to assess TCRG gene rearrangement. Consensus primers
derived from the feline TCRG V and TCRG J segment were
used as previously described for feline samples.16 The positive
clonal control was DNA extracted from formalin-fixed,
paraffin-embedded sections of feline intestinal T-cell lymphoma, and the polyclonal control was DNA extracted from
feline lymph node. Template DNA was omitted from the
negative control reaction. The PCR reactions were run in
triplicate to distinguish true clonal samples from pseudoclonal
samples. The PCR analysis using feline-specific primers
amplified DNA bands of 108 and 124 bp, consistent with a
clonal T-cell population with biallelic TCRG rearrangement.
Sequencing of amplified sea lion DNA confirmed that the
products were 80–90% homologous to the feline TCRG.
In addition, the liver contained several small nodular
aggregates of monomorphic lymphocytes similar to those
noted in the affected small intestine. These cells were diffusely
immunoreactive to CD3, suggestive of early metastasis of the
intestinal T-cell lymphoma to the liver. Renal lesions
included severe interstitial and glomerular amyloidosis with
interstitial fibrosis and lymphoplasmacytic nephritis, similar
to lesions previously described in sea lions with amyloidosis.3
In addition, sections of vagina contained segmental areas of
Case Reports
intraepithelial neoplasia consistent with sea lion urogenital
carcinoma.4
Although advanced renal disease was the primary cause of
this sea lion’s clinical signs, hematological abnormalities,
and biochemical abnormalities, the intestinal T-cell lymphoma may have contributed to debilitation. The glossal
mass was small and unlikely to have caused significant
adverse effects. Although polyomavirus infection has been
associated with skin tumors in hamsters and with Merkel cell
carcinoma in humans, the relationship between polyomavirus infection and the glossal fibropapilloma in this sea lion
is not clear.6,20 In Merkel cell carcinomas, viral DNA can be
detected within tumor cells but not in nonneoplastic cells.20
As viral particles were noted within only endothelial cells
and not within epithelial or stromal cells within the glossal
mass, it is possible that neoplasia and severe renal disease
caused reactivation of latent viral infection.
The relationship, if any, between the polyomavirus
infection and the T-cell lymphoma is unknown. Viral
inclusions were noted only in the tongue lesion, and
polyomaviral DNA was not detected in the intestine. Tcell lymphoma may cause immunosuppression, resulting in
reactivation of latent virus. Polyomaviral disease has been
reported in human T-cell lymphoma patients.5 On the other
hand, some polyomaviral T-antigens have been found to
interfere with tumor suppressor genes including p53 and
Rb, promoting oncogenesis.1 Although DNA from polyomaviral-induced tumors has been shown to induce
lymphomas when injected into hamsters, these lymphoid
tumors did not contain detectable polyomaviral particles.7
There is conflicting evidence regarding an association
between SV40 and lymphomas, and even those studies
finding an association have regarded B-cell lymphomas
rather than T-cell lymphomas.17
Lymphoma has rarely been reported in pinniped species,
and there is a single documented case of lymphoma in a
California sea lion.12,13,22,23 Immunophenotyping has
rarely been performed in reported cases.12 The morphologic characteristics of the lymphoid infiltrate in the sea lion
in the present case were very similar to those noted in early
T-cell, mucosal-associated intestinal lymphomas in cats. In
cats, mucosal T-cell lymphoma is characterized by dense
lymphoid aggregates that often form a band-like pattern in
the mid and superficial lamina propria. There is often
epitheliotropism and only mild extension into the submucosa and muscularis.16 In cats, it is thought that these
lymphomas arise from the diffuse mucosal-associated
lymphoid tissue (MALT) in the small intestine. This tissue
consists of both an intraepithelial and lamina propria
component and is composed predominantly of CD3+ T
cells.18 The MALT of sea lions has not been characterized;
however, a similar pathogenesis may exist. In feline
inflammatory bowel disease (IBD), there is an expansion
of the MALT T-cell populations similar to what occurs in
feline intestinal T-cell lymphoma. Molecular clonality
analysis can help to distinguish lymphoma from IBD. In
cases of IBD, a smear or ladder of bands covering a range
of PCR product sizes would be expected, indicating a
polyclonal, reactive lymphoid population within the
sample.16 In the sea lion in the current study, cross-
631
reactivity of the feline TCRG primers with the TCRG
region of the California sea lion enabled confirmation of
clonality within the intestinal lymphoid population.
Neither polyomavirus infection nor primary intestinal
lymphoma has previously been described in marine
mammals. Further information is needed to determine the
pathogenicity and tissue tropism of the newly discovered
sea lion polyomavirus. In addition, the present case
illustrates the utility of molecular phenotyping in diagnostic
pathology of nondomestic species.
Sources and manufacturers
a.
b.
c.
d.
e.
f.
g.
h.
i.
j.
k.
Dako North America Inc., Carpinteria, CA.
Serotec Inc., Oxford, UK.
Dako North America Inc., Carpinteria, CA.
Lab Vision Corp., Fremont, CA.
Eponate-12 epoxy resin, Ted Pella Inc., Redding, CA.
Zeiss 906E, Zeiss Electron Microscopy, Thornwood, NY.
DNeasy, Qiagen Inc., Valencia, CA.
TempliPhi, Amersham Biosciences, Piscataway, NJ.
Invitrogen Corp., Carlsbad, CA.
Clontech Laboratories Inc., Mountain View, CA.
Clontech Laboratories Inc., Mountain View, CA.
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