Brief Communication

Veterinary Pathology
1-6
Systemic Necrotizing Vasculitis in Sheep ª The Author(s) 2018
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Is Associated With Ovine Herpesvirus 2 DOI: 10.1177/0300985818795166
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Patricia A. Pesavento1 , Rahul B. Dange2, M. Carmen Ferreras3,

Akbar Dasjerdi4, Valentin Pérez3, Anna LaRoca4,
Julio Benavides Silván3, Santiago Diab5, Kenneth Jackson1,
Ida L. Phillips6, Hong Li7, Cristina W. Cunha7,
and Mark Wessels8

Abstract
Ovine herpesvirus 2 (OvHV-2) is one of the gammaherpesviruses in the genus Macavirus that can cause malignant catarrhal fever
(MCF) in ungulates. Sheep are the adapted host for OvHV-2 and it is generally assumed that infection is not associated with
disease in this species. However, cases of “polyarteritis nodosa” or idiopathic systemic necrotizing vasculitis reported in sheep are
similar to vascular lesions in clinically susceptible species with MCF. Using a recently developed in situ hybridization (ISH) method,
we were able to identify OvHV-2 nucleic acids within lesions and correlate the viral distribution with systemic necrotizing
vasculitis in 9 sheep, including both naturally and experimentally OvHV-2–infected animals. ISH, combined with polymerase chain
reaction and histology, identify OvHV-2 as the likely agent responsible for sporadic, MCF-like vascular disease in sheep.

Keywords
arteritis, gammaherpesvirus, malignant catarrhal fever, ovine, ovine herpesvirus 2, polyarteritis nodosa, sheep, vasculitis

Systemic necrotizing vasculitis is a rare, sporadic disease
recognized in individuals or clusters of sheep. 9,11,20,22,26
The histologic lesions have been considered typical of
“polyarteritis nodosa” (PAN), which also has been reported
in pigs,15 cats,5 dogs,23 rats,21 and humans.7,10 Many veter-
inary reports use a definition of PAN derived from human
studies, which is a necrotizing arteritis of medium or small
arteries without vasculitis in arterioles, capillaries, or
venules.10 In rats, PAN is recognized in naturally occurring
or drug-induced hypertension and is used as a model for
investigating the correlative human disease.21 Etiologies for
PAN in veterinary species are likely to be multifactorial, as is
the case in humans, in whom a distinction between “primary”
(idiopathic) and “secondary” PAN is made when an etiologic
agent, most commonly a persistent viral infection such as
hepatitis B virus, human immunodeficiency virus, or cytome-
galovirus, is found within the lesions.7,10 By extrapolation,
this raises the possibility that a persistent viral infection may
be the cause of systemic necrotizing vasculitis in sheep.
Important differentials for viral-associated vasculitis in sheep
include Maedi-Visna, which has been reported to produce a
vasculitis but only in association with overt lesions typical of
the disease;6 border disease, which is associated with only
mild periarteritis;27 and bluetongue, in which edema and
hemorrhage occur without histologic evidence of vascular
inflammation.16
Natural infections with ovine herpesvirus 2 (OvHV-2) occur
in a wide range of animals, including sheep, cattle, bison, deer,
moose, and swine.18 Sheep are considered the adapted host for
OvHV-2 and are the natural reservoir source for infection of
clinically susceptible species such as cattle and bison.
1
Department of Pathology, Microbiology and Immunology, School of Veter-
inary Medicine, University of California, Davis, CA, USA
2
California Animal Health and Food Safety Laboratory System, UC Davis,
Davis, CA, USA
3
Dpto. Sanidad Animal, Instituto de Ganaderı́a de Montaña (Uninversidad de
León-CSIC) Facultad de Veterinaria, Universidad de León Campus de Vega-
zana, León, Spain
4
Animal and Plant Health Agency-Weybridge, Addlestone, Surrey, UK
5
Santa Rosa, Argentina
6
Department of Biomedical Sciences, Oregon State University, Corvallis, OR,
USA
7
Animal Disease Research Unit USDA-ARS and Department of Veterinary
Microbiology and Pathology/Paul G Allen School for Global Animal Health,
Washington State University, Pullman, WA, USA
8
Finn Pathologists, One Eyed Lane, Weybread, Diss, Norfolk, UK
Supplemental material for this article is available online.
Corresponding Author:
Patricia A. Pesavento, Department of Pathology, Microbiology and Immunol-
ogy, School of Veterinary Medicine, University of California, 4206 VM3A: PMI,
School of Veterinary Medicine, UCD, Davis, CA 95616, USA.
Email: papesavento@ucdavis.edu
2 Veterinary Pathology XX(X)

Table 1. Description of Cases Evaluated in This Study, Including OvHV-2 PCR and ISH Results.

Case ISH

No. Signalment Presenting Illness References PCRa Tissues Resultb

1 2.5 mo, Dorper Diarrhea, ataxia This study (103 to 105) Liver, intestine, rumen, soft þ
palate, tongue
2 4 mo, cross-bred Respiratory, lymphadenopathy 20 (103 to 106) Adrenal, lung, esophagus, þ
thymus, ileum
3 7 mo, Texel Diarrhea, lymphadenopathy 26 þ Lymph node, intestine þ
4 10 mo, Beltex Hemoabdomen 26 þ Kidney, mesentery, lymph þ
node, intestine
5 1 to 2 y, Assaf Swollen joints 8 þ Uterus, kidney þ
6 3 y, Assaf Weakness, recumbency 8 þ Uterus, kidney þ
7 2 to 3 y, Assaf Swollen joints 8 NA Uterus, ovary, liver þ
8 9 mo, Assaf 8 NA Kidney, skin þ/–
9 9 mo, cross-bred, OvHV-2 Respiratory 14 (104 to 105) Spleen, liver, lung þ
experimental infection
10 9 mo, cross-bred Clinically normal This study (2  102 to 6  102) Spleen, lung, nasal turbinate –
11 9 mo, cross-bred, OvHV-2 Clinically normal This study – Lung –
uninfected control
Abbreviations: ISH, in situ hybridization; OvHV-2, ovine herpesvirus 2; PCR, polymerase chain reaction.
a
þ and – indicate positive and negative, respectively, by OvHV-2 nested PCR. Values in parentheses refer to the range of OvHV-2 genome copies detected per 50
ng tissue DNA, as tested by OvHV-2 quantitative PCR in select cases where fresh tissue was available. NA signifies that no DNA for PCR was available.
b
þ and – indicate positive and negative, respectively, detection of OvHV-2 nucleic acids by ISH. In case No. 8, kidney was positive and skin was negative.

Clinically susceptible species are those that develop the sys-
temic vasocentric disease termed malignant catarrhal fever
(MCF). OvHV-2 infection in sheep is usually subclinical, and
comparatively little is known about the pathogenesis of
OvHV-2 infection in its adapted host species. Hüssy et al12
quantitatively analyzed tissue distribution of OvHV-2 in nor-
mal sheep, and infection of circulating lymphocytes and/or
epithelium has been demonstrated in field infections17 and
under experimental conditions.24 Natural disease in OvHV-
2–infected sheep has been proposed,9,11,20 and experimen-
tally, lambs exposed to a high dose of OvHV-2 can develop
an MCF-like syndrome.3,13 But because most sheep are sub-
clinically infected with OvHV-2,1,12,14,18 detection of viral
DNA or antibodies that confirms infection and/or exposure
cannot uncover disease association. Diagnosticians have long
lacked any method able to recognize the distribution of
OvHV-2 within lesions in formalin-fixed, paraffin-
embedded tissues. In this study, we use in situ hybridization
(ISH), along with polymerase chain reaction (PCR) and his-
topathology, to test the hypothesis that OvHV-2 is the agent
responsible for sporadic cases of systemic necrotizing vascu-
litis in sheep.
The ovine vasculitis cases investigated in this study are
summarized in Table 1 and Supplemental Table S1. Case
Nos. 1 to 8 are naturally occurring cases in 5 independent
flocks from 3 countries (Spain, United Kingdom, and
United States). All of these cases have been previously
described as idiopathic polyarteritis 8 ,2 6 or OvHV-2-
associated MCF,20 and they were selected based on the histolo-
gic criteria of a systemic (or multiorgan) necrotizing vasculitis.
Case No. 9 was a lamb with vasculitis following experimental
inoculation of a high dose of OvHV-2 by intranasal
nebulization.13
Age, breed, and presenting illnesses in these cases were
variable (Table 1), but histologic detection of vasculitis (which
was a criterion for case selection) was present in all cases. The
tissues examined in each case are listed in Table 1. Each case
included a subset of the following tissues: gastrointestinal tract,
liver, lungs, adrenal glands, heart, pancreas, spleen, meninges,
urinary bladder, gallbladder, kidneys, uterus, and/or mammary
glands. Regardless of tissue type, affected tissues shared a
primarily lymphocytic vasculitis that targeted small- to
medium-sized arteries (Figs. 1a, 2a, 3a, 3c). Two clinically
normal sheep were also included in this study; 1 was an
OvHV-2 naturally infected but asymptomatic lamb (case No.
10), and the other was OvHV-2 uninfected (case No. 11).
OvHV-2 DNA was detected by PCR in all clinically affected
animals (case Nos. 1–6 and 9).2,25 Extracted DNA was not
available from case Nos. 7 and 8, and thus no PCR data were
obtained. Quantitation of OvHV-2 DNA was estimated by real-
time PCR on case Nos. 1, 2, 9, and 10, which were the cases
that had fresh tissue available, and results from individual tis-
sues are reported in Supplemental Table S1. Viral quantity
ranged from 103 to 106 viral genome copies per 50 ng total
DNA (Table 1 and Suppl. Table S1) in clinical animals (case
Nos. 1, 2, and 9). The infected but clinically normal animal
(case No. 10) was positive by real-time PCR but had a low
OvHV-2 genome copy number, ranging from 200 to 600 per
50 ng total DNA, which was quantified independently in 3
tissues (Suppl. Table S1). The uninfected control animal (case
No. 11) was OvHV-2 negative by PCR. During this study, a
reevaluation of the whole-genome sequencing (Illumina
Pesavento et al 3

Figure 1. Vasculitis, mesenteric artery, lamb, case No. 4. (a) Lymphocytic inflammation of the artery is transmural and circumferential, most
densely affecting the adventitia and outer muscular wall. Hematoxylin and eosin (HE). (b) The nuclei of scattered cells contain ovine herpesvirus
2 (OvHV-2). In situ hybridization (ISH). The boxed (blue) region is ISH performed with a negative control (unrelated, DapB) probe. (c) Boxed
(black) region of (a). The nuclear morphology of the infiltrative lymphocytes is variable, some round with compact chromatin and others
oblong and open. HE. (d) Boxed (black) region of (b). The ISH-positive nuclei are round to polygonal, many with open, loose chromatin.
Figure 2. Vasculitis, arcuate artery, ewe, case No. 6. (a) Scattered lymphocytes are present within the inner muscular wall, the subintimal space,
and in circulation. HE. (b) Cells within the subintimal space (arrows) and lumen as well as marginated leukocytes (arrowheads) contain OvHV-2
nucleic acid. ISH.

MiSeq, San Diego, CA, USA) that had been previously reference sequence (accession number NC_007646.1) with
obtained from splenic tissue of one of the clinically affected 6.77% average coverage of the virus genome. No other viral
lambs (case No. 4)26 indicated a match to the OvHV-2 BJ1035 sequences were detected through this mapping analysis.
4 Veterinary Pathology XX(X)

Figure 3. Vasculitis, lymph node, lamb, case No. 3. (a) A medium-sized artery is the focus of an inflammatory reaction that disrupts the
arterial wall. Hematoxylin and eosin (HE). (b) Ovine herpesvirus 2 (OvHV-2)–positive cells are present within the tunica muscularis and
scattered within the sinusoids and rarely within the cords of the lymph node. (c) Boxed region of (a). Dense lymphocytic inflammation in the
tunica adventitia and perivascular space. (d) Boxed region of (b). The nuclei of many of the inflammatory cells are positive by in situ
hybridization (ISH). ISH, OvHV-2 probe.

To investigate the distribution of OvHV-2 within tissues, a
recently developed ISH was used to detect viral nucleic acids in
formalin-fixed, paraffin-embedded sections.19 Tissues were
selected based on the presence of vasculitis, with consideration
for the quality of the section and time of tissue fixation (all
samples were in 10% formalin for <7 days). Colorimetric ISH
was performed as previously described.19 Briefly, a RNAscope
2.5 Red assay kit (cat. 322360; ACD, Newark, CA, USA) and
an OvHV-2–specific probe, V-OvHV2-orf25-orf50 (cat.
501091; ACD), were used per manufacturer instruction. Neg-
ative controls included an unrelated (DapB, bacterial gene)
probe and tissue sections from an OvHV-2–negative animal
(case No. 11). Slides were counterstained with hematoxylin
and examined on a 40 objective with bright-field illumination
and digitalized using an Olympus VS120 scanner (Center Val-
ley, PA, USA). All clinically affected animals, including nat-
ural infections (case Nos. 1–8) and the experimental infection
(case No. 9), had ISH-detectable OvHV-2 nucleic acid in all
tissues tested (Figs. 1–3 and Suppl. Figs. S1–S3), excepting a
single ulcerated section of haired skin in case No. 8 (Table 1).
Tissues tested included the mesentery (Fig. 1 and Suppl.
Fig. S1), kidney (Fig. 2), lymph node (Fig. 3), liver, spleen,
Pesavento et al
lung (Suppl. Fig. S2), uterus (Suppl. Fig. S3), and intestines,
and in all cases, the nuclei of a subset of lymphocytes were
hybridization targets for the OvHV-2 probe. Lymphocytes con-
taining viral nucleic acid (ISH positive) transmigrated the arter-
ial walls, expanded the adventitia, and surrounded multifocal
arteries (Figs. 1b, 1d, 2b, 3b, 3d). The histologic character of
the lymphocytes was mixed, with some small and some having
open, large nuclei (Fig. 1c,d). OvHV-2 nucleic acid was also
detectable in circulating lymphocytes within the lumen of
arteries (Fig. 2a,b). As with other MCF-susceptible animals,
the inflammation was predominantly CD3þ lymphocytes
(clone CD3-12, IgG1rat; generous gift from the Moore labora-
tory; Suppl. Figs. S1b, S2b and De Virgilio et al7 and Zakarian
et al27). In addition to their arteriocentric distribution, ISH-
positive cells were also scattered in sinusoids and rarely cords
of lymph nodes (case Nos. 3–4; Fig. 3b,d), the lamina propria
of the intestine (case Nos. 1, 3, and 4), hepatic portal areas (case
Nos. 1, 7, and 9), within the interstitium around individual renal
glomeruli (case Nos. 4–6 and 8), and multifocally expanding
the uterine interstitium and intercalating within glandular cells
(case Nos. 5–7; Suppl. Fig. S3a,b). Sequential sections of all
OvHV-2–positive tissues (eg, Fig. 1b, blue boxed insert) were
negative when probed with an unrelated probe (all tissues; eg,
Fig. 1b, blue boxed inset). The lung, liver, and spleen of the
uninfected animal (case No. 11) probed with the OvHV-2
probe were negative. No ISH-positive signal was detected in
any tissues from the OvHV-2–infected, clinically normal sheep
(case No. 10), although these same tissues were quantitatively
PCR positive with a lower genome copy number than affected
animals (Table 1 and Suppl. Table S1).
It is generally assumed that sheep are well-adapted hosts
for OvHV-2 and that infection is quite common in clinically
normal sheep.1,14 This creates a challenge with suggesting a
diagnosis of MCF in sheep since PCR detection of OvHV-2
DNA by PCR alone in sheep is insufficient evidence of caus-
ality. Using OvHV-2–specific ISH, we demonstrate that vas-
culitis is directly associated with the presence of OvHV-2
nucleic acid. Colocalization of viral nucleic acids within lym-
phocytes in lesioned tissues supports the hypothesis that
OvHV-2 causes an MCF-like disease in sheep. It is notable
that higher levels (by quantitative PCR) of OvHV-2 DNA
were present in tissues of sheep with vasculitis compared to
the levels detected in unaffected persistent carriers (Suppl.
Table S1), suggesting that ISH-positive results associated
with vasculitis and high levels of OvHV-2 DNA can be used
to confirm cases of MCF in sheep.
Two historic reports that did not invoke OvHV-2 specifi-
cally as a cause but described an “idiopathic” polyarteritis or
polyarteritis nodosa (PAN) in sheep were included in this
study,8,26 and all 6 cases tested were positive by OvHV-2–
specific ISH. Immunohistochemical assessment, whether this
study (case No. 1; Suppl. Fig. S1b) or previously performed
(case Nos. 3–8),8,26 demonstrated that transmural arterial infil-
trates were predominantly lymphocytic (Suppl. Fig. S1b), with
fewer macrophages and plasma cells. The character of the
inflammation correlates well with PAN lesions in humans
5
where T-cell–mediated immune mechanisms are important in
the development and perpetuation of the lesions.4 Our data
indicate that OvHV-2 plays a role in the development of vas-
culitis in sheep and can be an etiological agent in certain cases
of PAN, but we cannot rule out the possibility of a comicrobial
pathogen. Our data broaden the potential range of MCF disease
to include the adapted host species.
Acknowledgements
We thank Shirley Elias (Washington State University) for technical
assistance.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
Funding
The author(s) received the following financial support for the
research, authorship, and/or publication of this article: Support fund-
ing to the Li laboratory for this project is from USDA-ARS CWU
2029-3200-037-00D. In the Pesavento laboratory, the study was sup-
ported by Boehringer Ingelheim Vetmedica and the Bernice Barbour
Foundation for research in naturally occurring infectious disease.
ORCID iD
Patricia Pesavento http://orcid.org/0000-0001-6593-9607
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