Veterinary Immunology and Immunopathology 127 (2009) 1–18

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Veterinary Immunology and Immunopathology

journal homepage: www.elsevier.com/locate/vetimm

Review paper

Pathogenesis and immunopathology of systemic and nervous canine

distemper
A. Beineke, C. Puff, F. Seehusen, W. Baumgärtner *
Department of Pathology, University of Veterinary Medicine, Bünteweg 17, 30559 Hannover, Germany

A R T I C L E I N F O A B S T R A C T

Article history: Canine distemper is a worldwide occurring infectious disease of dogs, caused by a
Received 9 May 2008
morbillivirus, closely related to measles and rinderpest virus. The natural host range
Received in revised form 17 September 2008
comprises predominantly carnivores. Canine distemper virus (CDV), an enveloped,
Accepted 18 September 2008
negative-sense RNA virus, infects different cell types, including epithelial, mesenchymal,
neuroendocrine and hematopoietic cells of various organs and tissues. CDV infection of
Keywords:
Canine distemper virus dogs is characterized by a systemic and/or nervous clinical course and viral persistence in
Cell tropism selected organs including the central nervous system (CNS) and lymphoid tissue. Main
Lymphoid tissues manifestations include respiratory and gastrointestinal signs, immunosuppression and
Central nervous system demyelinating leukoencephalomyelitis (DL). Impaired immune function, associated with
Immunopathology depletion of lymphoid organs, consists of a viremia-associated loss of lymphocytes,
Neuroimmunology especially of CD4+ T cells, due to lymphoid cell apoptosis in the early phase. After clearance
of the virus from the peripheral blood an assumed diminished antigen presentation and
altered lymphocyte maturation cause an ongoing immunosuppression despite repopula-
tion of lymphoid organs. The early phase of DL is a sequel of a direct virus-mediated
damage and infiltrating CD8+ cytotoxic T cells associated with an up-regulation of pro-
inflammatory cytokines such as interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-a and
IL-12 and a lacking response of immunomodulatory cytokines such as IL-10 and
transforming growth factor (TGF)-b. A CD4+-mediated delayed type hypersensitivity and
cytotoxic CD8+ T cells contribute to myelin loss in the chronic phase. Additionally, up-
regulation of interferon-g and IL-1 may occur in advanced lesions. Moreover, an altered
balance between matrix metalloproteinases and their inhibitors seems to play a pivotal
role for the pathogenesis of DL. Summarized, DL represents a biphasic disease process
consisting of an initial direct virus-mediated process and immune-mediated plaque
progression. Immunosuppression is due to early virus-mediated lymphocytolysis
followed by still poorly understood mechanisms affecting antigen presentation and
lymphocyte maturation.
ß 2008 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Viral properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3. Natural host range and different strains of CDV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

* Corresponding author. Tel.: +49 511 953 8620; fax: +49 511 953 8675.
E-mail address: wolfgang.baumgaertner@tiho-hannover.de (W. Baumgärtner).

0165-2427/$ – see front matter ß 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetimm.2008.09.023
2 A. Beineke et al. / Veterinary Immunology and Immunopathology 127 (2009) 1–18

4. Protective immunity in CDV infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

5. Pathogenesis of distemper. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
5.1. Route of infection and virus spread. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
5.2. Clinical manifestations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
6. Pathology of non-nervous tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
7. Immunosuppression in canine distemper. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
7.1. Lymphotropism of CDV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
7.2. Morphological findings in lymphoid organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
7.3. Phenotypical changes in lymphoid organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
7.4. Mechanisms of immunosuppression in canine distemper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
8. Pathogenesis of nervous distemper. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
8.1. Virus entry, spread and expression within the CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
8.2. Neurotropism of CDV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
8.2.1. Oligodendroglial infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
8.2.2. Astrocytic infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
8.2.3. Neuronal infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
8.3. Pathology of nervous distemper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
8.4. Immunopathology of demyelinating leukoencephalomyelitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
8.5. Role of cytokines in the pathogenesis of demyelinating leukoencephalitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
8.6. Role of extracellular matrix in the pathogenesis of demyelinating leukoencephalitis . . . . . . . . . . . . . . . . . . . . . . . . . 12
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

1. Introduction manifestation forms of distemper, the pathogenesis of

Canine distemper is a worldwide occurring infectious
disease, caused by a morbillivirus of the family Paramyx-
oviridae. The natural host spectrum of canine distemper
virus (CDV) comprises all families of the order Carnivora
(Deem et al., 2000). Clinical and morbid anatomical signs of
canine distemper were already described in detail at the
beginning of the 19th century by Jenner (Lauder et al.,
1954). He also commented on the high incidence of
nervous complications in affected dogs.
Recent canine distemper epidemics have been observed
in France, Germany, USA, Japan and Finland demonstrating
the importance of regular vaccination as a highly efficient
and protective tool (Adelus-Neveu et al., 1991; Mori et al.,
1994; Johnson et al., 1995; Gemma et al., 1996; Ek-
Kommonen et al., 1997; Moritz et al., 2000). Furthermore,
occasional disease outbreaks can be observed in vacci-
nated dog cohorts, possible due to the introduction of
genetically different CDV strains (Mori et al., 1994;
Mochizuki et al., 1999; Simon-Martı́nez et al., 2007).
Though infection of dogs may result in a variety of
clinical forms affecting the respiratory and gastrointestinal
tract, skin and other organs and tissues, immunosuppres-
sion and demyelinating leukoencephalitis (DL) represent
the main sequel in this species (Krakowka et al., 1985;
Appel, 1987). Due to the morphological similarities of
neuropathological changes between DL and human
demyelinating diseases, such as multiple sclerosis (MS),
the canine disease represents one of the few spontaneously
occurring animal models to study the pathogenesis of
myelin loss associated with immune-mediated mechan-
isms (Baumgärtner and Alldinger, 2005). Furthermore,
lymphoid depletion and long-lasting impaired immune
functions in distemper are comparable with those
described for measles virus (MV) infection, a closely
related morbillivirus in humans. It is the aim of the present
communication to provide an overview about the different
demyelination in the central nervous system (CNS) and
mechanisms of immunosuppression in CDV infected dogs.
In addition, the perpetuating role of the misdirected
immune response especially for the development of
nervous white matter lesions is detailed.
2. Viral properties
CDV belongs to the family Paramyxoviridae (Lamb and
Kolakofsky, 2001). Beside CDV, the genus morbillivirus
comprises measles virus, dolphin morbillivirus, peste-des-
petits-ruminants virus, rinderpest virus, porpoise morbil-
livirus, and phocine distemper virus (Pringle, 1999; Lamb
and Kolakofsky, 2001).
CDV is an enveloped, negative-sense, single-stranded
RNA virus (Lamb and Kolakofsky, 2001), which contains six
structural proteins, the nucleocapsid (N), the phospho- (P),
the large (L), the matrix- (M), the hemagglutinin (H), and
the fusion (F) protein (Hall et al., 1980; Örvell, 1980; Diallo,
1990; Lamb and Kolakofsky, 2001). Additional accessory
genes, the C and V protein, are found mostly as extra
transcriptional units within the P gene (Lamb and
Kolakofsky, 2001). The lipid envelope, surrounding the
virion, contains the two surface glycoproteins F and H,
which mediate viral entry and exit from the host cell.
Furthermore, the helical nucleocapsid core, containing the
N, P and L protein, initiates intracellular replication and is
located within the envelope (Lamb and Kolakofsky, 2001).
The viral M protein connects the surface glycoproteins and
nucleocapsid during viral maturation.
3. Natural host range and different strains of CDV
The host spectrum of CDV, which traditionally included
numerous families within the order of Carnivora like
Canidae (dogs, dingos, foxes), Procyonidae (raccoons,
kinkajous, lesser panda), Mustelidae (ferret, mink, badger),
 A. Beineke et al. / Veterinary Immunology and Immunopathology 127 (2009) 1–18
Mephitidae (skunk), Hyaenidae, Ailuridae, Viverridae and
Felidae, has changed in the last years and distemper-like
diseases have been observed in large felids in the
Tanzanian Serengeti National Park in 1994 and in North
American zoos in 1991 und 1992 (Montali et al., 1987;
Appel et al., 1994; Roelke-Parker et al., 1996), collared
peccaries (Tayassu tajacu) in Arizona (Appel et al., 1991)
and in a non-human primate (Macaca fuscata) in Japan
(Gorham, 1966; Budd, 1981; Yoshikawa et al., 1989;
Machida et al., 1992, 1993; van Moll et al., 1995;
Baumgärtner et al., 2003). Furthermore, besides infection
with the closely related phocine distemper virus, seals can
become infected by CDV (Mahy et al., 1988; Kennedy et al.,
1989; Likhoshway et al., 1989; Bostock et al., 1990; Curran
et al., 1990; McCullough et al., 1991; Pringle, 1999; Müller
et al., 2000, 2002; Seibel et al., 2007; Wohlsein et al., 2007).
In some CDV outbreaks including the mass mortalities
among Baikal and Caspian seals and large felids in the
Serengeti Park, terrestrial carnivores including dogs and
wolves have been suspected as vectors for the infectious
agent (Kennedy et al., 2000; Kreutzer et al., 2008).
So far, only one serotype of CDV is recognized; however,
several co-circulating CDV genotypes of different virulence
and cell tropism have been found (McCullough et al.,
1974a,b,c; Summers et al., 1984; Shapshak et al., 1987;
Mori et al., 1994; Frisk et al., 1999a; Haas et al., 1999).
Some strains are associated with a polioencephalitis (e.g.
Snyder Hill-CDV strain) while others induce a demyelinat-
ing leukoencephalomyelitis (DL) (e.g. R252- and A75-17-
CDV strain; Krakowka and Koestner, 1977; Summers and
Appel, 1994; Orlando et al., 2008). Though sequence data
analysis of field isolates of CDV revealed several clusters of
distemper strains, overall a considerable genetic stability
of CDV even in recent outbreaks was observed. Still,
cocirculation of more than one genotype of CDV must be
considered (Haas et al., 1997; Frisk et al., 1999a). Similarly,
different genotypes of CDV have been identified in recent
isolates from dogs in disease outbreaks in Japan (Mori
et al., 1994; Mochizuki et al., 1999).
4. Protective immunity in CDV infection
An intact humoral and cell-mediated immune response
is crucial for the elimination of CDV. The humoral immune
response in canine distemper has been investigated in
naturally and experimentally infected animals (Krakowka
et al., 1980; Krakowka, 1982; Rima et al., 1991). Anti-CDV
IgM antibodies occur within the first 2 weeks of infection
(Vandevelde and Zurbriggen, 2005). Here, the magnitude
of the humoral immune response correlates with the
outcome of the disease. In general, protective humoral
immunity in canine distemper is due to the production of
anti-viral nucleoprotein antibodies, followed by the
appearance of a virus envelope protein-specific humoral
immune response (Miele and Krakowka, 1983; Rima et al.,
1991). The lack of an effective humoral immune response
leads to an acute, often fatal clinical course, while a
detectable virus-specific antibody titer at 10–14 days post-
infection (pi) promotes viral elimination or persistence,
respectively. Referring to this, viral clearance significantly
depends upon the specificity of immunoglobulins to
3
recognize viral envelope proteins (Krakowka et al.,
1975b). At this, especially anti-viral H protein specific
antibodies prevent the development of CNS lesions in
infected dogs (Rima et al., 1991). However, a temporary
lack of antibodies against the viral M protein as well as a
delayed or diminished complement-fixing antibody
response to viral envelope proteins results in persistent
neurological disease (Krakowka et al., 1975b; Miele and
Krakowka, 1983). Additionally, neutralizing antibodies and
complement-mediated humoral cytotoxicity represent
critical factors for the elimination of free viral particles
and for the prediction of the clinical outcome in CDV-
infected animals (Ho and Babiuk, 1980; Appel et al., 1982,
1984). Neutralizing antibodies prevent intracellular and
extracellular viral spread; however, prolonged antibody
exposure results in an internalization of viral surface
antigens from the cell membrane and subsequently in their
disappearance from the membrane of infected cells. Thus,
despite a reduction of viral spread, antibody-mediated
modulation of viral antigen expression might represent a
contributing factor for persistent infection in dogs,
possibly due to a diminished antigen recognition and
resultant inadequate complement-mediated humoral
cytotoxicity (Ho and Babiuk, 1979b, 1980; Alldinger
et al., 1993b). Following CDV infection, a virus-specific
humoral immune response can be detected throughout
life, while a cellular immune response can only be detected
for a short time period in convalescent dogs (Appel et al.,
1982). However, the importance of the cellular immune
response in canine distemper is highlighted by the
demonstration of a protective cellular immunity in the
absence of a detectable antibody-mediated immune
response (Gerber and Marron, 1976). Additionally, vacci-
nated and infected dogs exhibit a T cell-mediated CDV-
specific immune response, independently of the antibody
titer (Krakowka et al., 1978). Cytotoxic T cells (Krakowka
and Wallace, 1979; Shek et al., 1980; Appel et al., 1982),
Killer cells (Ho and Babiuk, 1979a), Natural Killer (NK) cells
(Ringler and Krakowka, 1985) and macrophage function
(Appel et al., 1984) have also been investigated in distemper
and revealed that a sustained antiviral lymphocyte-
mediated cytotoxicity determines the clinical outcome in
CDV-infected dogs. Thereby, a strong cellular immunity
causes viral elimination, while a lacking or a delayed
lymphocyte-mediated cytotoxicity correlates with viral
persistence in the CNS (Appel et al., 1982). However, NK
cell function seems to play a minor role in the host immune
defense against CDV (Ringler and Krakowka, 1985).
5. Pathogenesis of distemper
Great variations in duration, severity and clinical
presentation of distemper have been found in experimen-
tally infected dogs as well as in animals suffering from this
world-wide spontaneously occurring disease. The incuba-
tion period may vary from 1 to 4 weeks and depends on
viral strain, age of the animal at the time of infection and
immune status of the host. Disease manifestation ranges
from virtually no clinical signs to severe disease with
approximately 50% mortality (Appel, 1970, 1987; Kra-
kowka et al., 1980; Moritz et al., 2000; Schwab et al., 2007).
4 A. Beineke et al. / Veterinary Immunology and Immunopathology 127 (2009) 1–18
5.1. Route of infection and virus spread
The virus is shed primarily by oro-nasal secretion,
however, any discharge and secretion can carry the virus.
CDV infects susceptible dogs primarily by inhalation of
airborne viruses or via infective aerosol droplets (Kra-
kowka et al., 1980) followed by virus replication in
lymphoid tissues of the respiratory tract. Tissue macro-
phages and monocytes located in or along the respiratory
epithelium and tonsils represent the first cell type to pick
up and propagate the virus (Appel, 1970). Following this
local burst of virus replication the pathogen is then
disseminated by lymphatics and blood to distant hema-
topoietic tissues during the first viremic phase.
First clinical signs are characterized by lethargy,
dehydration, anorexia, and weight loss followed by a more
pronounced clinical manifestation depending on the pre-
dominantly affected organ. Development of a biphasic fever
represents another characteristic clinical finding (Wright
et al., 1974). A transient fever and the onset of lymphopenia
can be observed 3–6 days pi (Krakowka et al., 1980) and
coincide with the first viremia that results in a generalized
infection of all lymphoid tissues including spleen, thymus,
lymph nodes, bone marrow, mucosa-associated lymphatic
tissues (MALT) and macrophages in the lamina propria of the
gastrointestinal tract (Appel, 1970; Wright et al., 1974), as
well as hepatic Kupffer cells (Appel, 1987). Viremia occurs
by spread of cell free virus as well as leukocyte and
thrombocyte associated infectious pathogens. The second
viremia follows several days later, frequently associated
with high fever, and results in infection of parenchymal and
tissue cells throughout the body (Appel, 1969; Appel and
Gillespie, 1972; Blixenkrone-Møller, 1989; Blixenkrone-
Møller et al., 1989; Okita et al., 1997). Thus, CDV can be
found in cells of the respiratory, gastrointestinal and urinary
tract, endocrine system, lymphoid tissues, central nervous
system and vasculature including keratinocytes, fibroblasts,
thrombocytes and different lymphoid cell subsets, as well as
bronchial, endothelial, epithelial and neuroectodermal cells
(Baumgärtner et al., 1989; Gröne et al., 2004a,b; Koutinas
et al., 2004).
5.2. Clinical manifestations
According to clinical features a catarrhal and nervous
form or a combination of both, also termed acute systemic
form, and a chronic nervous manifestation can be distin-
guished. In addition, various unusual manifestations,
including old dog encephalitis and hard pad disease are
recognized (Krakowka et al., 1985; Baumgärtner, 1993;
Moritz et al., 1998, 2000, 2003). At the acute stage, virus is
found in every secretion and excretion of the body. This
phase is accompanied by various dramatic clinical signs
including onset of a cutaneous rash, serous nasal and ocular
discharge, conjunctivitis and anorexia, followed by gastro-
intestinal and respiratory signs, which are often compli-
cated by secondary bacterial infections and neurological
disturbances (Krakowka et al., 1985). Nervous signs are
diverse and progressive (Parker, 1978; Greene and Appel,
1998) and include myoclonus, nystagmus, ataxia, postural
reaction deficits and tetraparesis or plegia (Koutinas et al.,
2002; Vandevelde and Zurbriggen, 2005; Amude et al.,
2007).
In some cases, an improved immune response espe-
cially an increased production of virus-specific neutraliz-
ing antibodies can promote the recovery of the animal.
However, despite elimination of the virus from several
organs and the peripheral blood, CDV can persist in certain
tissues including uvea, CNS, lymphoid organs and footpads
(Appel, 1970, 1987; Zurbriggen et al., 1995a,b; Greene and
Appel, 1998; Gröne et al., 2003a; Schobesberger et al.,
2005). Moreover, some infected animals display a delayed
progression of the disease and a moderate immune
response with subtle early clinical signs. Later, as a
consequence of viral persistence in the CNS, overt CNS
disturbances can be observed resulting in the nervous form
of canine distemper. Dogs with nervous signs usually die,
but some recover, and may display lifelong residual signs
such as a persistent myoclonus.
6. Pathology of non-nervous tissues
Following pathogen spread, virus antigen and asso-
ciated lesions including cytoplasmic and intranuclear
inclusion bodies can be found in various organs and
tissues. Respiratory manifestation results in serous to
mucopurulent rhinitis, interstitial pneumonia and necro-
tizing bronchiolitis, which is often complicated by a
suppurative bronchopneumonia due to secondary bacter-
ial infection (Caswell and Williams, 2007). Enteral infec-
tion leads to catarrhal enteritis with depletion of Peyer’s
patches (Krakowka et al., 1985; Greene and Appel, 1998;
Decaro et al., 2004). In naturally infected dogs, a pustular
dermatitis, also termed distemper exanthema, of thighs,
ventral abdomen and the inner surface of ear pinnae can be
found. Histologically, hyper- and parakeratosis with
vesicle and pustule formation as well as multinucleated
syncytial giant cells are present (Baumgärtner, 1993;
Maeda et al., 1994). Hard pad disease represents an
uncommon cutaneous manifestation of distemper and is
characterized by hyperkeratosis of the footpads and nasal
planum (MacIntyre et al., 1948; Moritz et al., 2000).
Though the pathogenesis of this unusual manifestation
remains undetermined, it seems that CDV causes a
disturbance of keratinocyte differentiation and it has been
speculated whether epidermal infection is also a conse-
quence of a restricted viral infection (Gröne et al., 2003b,
2004a). During permanent tooth development CDV also
infects tooth buds and ameloblasts causing enamel
hypoplasia (Dubielzig et al., 1981; Bittegeko et al., 1995).
A persistence of the primary spongiosa in the meta-
physes of long bones also termed metaphyseal osteo-
sclerosis or growth retardation lattice has been described
in young dogs suffering from systemic distemper (Baum-
gärtner et al., 1995a,b, 1996). Pathogenetically, osteoclast
necrosis due to direct virus infection has been assumed to
represent the primary defect resulting in loss of bone
modeling and subsequent persistence of the primary
spongiosa. Morphological similarities between this CDV-
associated lesion and the human Paget’s disease of bone
have been pointed out and a common etiology has been
assumed (Gordon et al., 1991, 1992; Mee, 1999). However,
 A. Beineke et al. / Veterinary Immunology and Immunopathology 127 (2009) 1–18
these findings could not be substantiated by others
(Ralston et al., 1991; Helfrich et al., 2000; Ooi et al., 2000).
Cytoplasmic and intranuclear inclusion bodies are
found frequently in many organs in distemper.
Additionally, a generalized depletion of lymphoid
organs and an associated immunosuppression represents
an important and common manifestation of canine
distemper and will be discussed in the following chapter
(Krakowka et al., 1975a; Krakowka, 1982).
7. Immunosuppression in canine distemper
Similar to other morbilliviruses, such as measles and
rinderpest virus, CDV is a lymphotrophic and highly
immunosuppressive infectious agent. However, exacerba-
tion of disease depends upon the virus strain as well as the
age and immune status of the animal at the time of
infection. As described above, adequate antiviral immunity
leads to an abortive clinical course or recovery, respec-
tively. However, once manifested, infection causes a long
lasting and profound inhibition and impairment of cellular
and humoral immune functions characterized by immu-
nosuppression, lymphocyte loss, and leukopenia, render-
ing animals highly susceptible to opportunistic infections
(Krakowka et al., 1975a; Cerruti-Sola et al., 1983).
Experimental infection of gnotobiotic dogs has substan-
tially increased our understanding of the CDV-induced
immune response and immunosuppression (Krakowka
et al., 1980). Additionally, natural and experimental CDV
infection causes an acute systemic, often fatal and severely
immunosuppressive disease in ferrets. Leukopenia and an
inhibited cellular immune response, such as suppressed
delayed type hypersensitivity and a decreased lymphocyte
proliferation activity as well as a reduced humoral immune
response have been reported in experimentally CDV-
infected ferrets. Thus, CDV infection of ferrets represents a
reliable animal model for the investigation of morbilli-
virus-induced immunosuppression and the development
of new vaccine strategies for human measles and canine
distemper, respectively (Kauffman et al., 1982; Stephensen
et al., 1997; Welter et al., 2000; von Messling et al., 2003).
7.1. Lymphotropism of CDV
Several studies have focused on the tropism of CDV for
lymphocytes (Appel et al., 1982; Cerruti-Sola et al., 1983).
Experimental infection of ferrets with recombinant CDV
expressing green fluorescent protein and dogs with
virulent CDV strains, demonstrated an initial infection of
lymphoid cells in spleen, lymph nodes, MALT and thymus
as well as circulating T and B lymphocytes preceding
epithelial cell invasion (Appel, 1969, 1970; von Messling
et al., 2004). Current in vitro investigations demonstrated
that lymphotropism of CDV is presumably based on the
binding of CD150 (signaling lymphocyte activation molecule,
SLAM) by the viral H protein, followed by cell entry of the
agent. SLAM is constitutively expressed in a variety of
organs in healthy dogs. However, upon CDV infection,
SLAM is markedly up-regulated in lymphoid cells of
affected dogs, indicating a possible strategy to increase
virus amplification in the host (Wenzlow et al., 2007). In
5
humans, SLAM is permanently expressed on immature
thymocytes, memory T cells and a portion of B cells.
Furthermore, its surface expression is rapidly induced on
peripheral T and B lymphocytes, monocytes and mature
dendritic cells upon activation. Similar to other morbilli-
viruses, the selective destruction or impairment of infected
SLAM-expressing cells might be an important mechanism
of immunosuppression induced by CDV. Since SLAM is
expressed on mature dendritic cells and activated mono-
cytes, antigen presentation might also be affected (Tatsuo
et al., 2001; Tatsuo and Yanagi, 2002; Lan et al., 2005).
Referring to this, SLAM recognition is essential for
lymphocyte infection, viral dissemination and induction
of immunosuppression in CDV-infected ferrets (von
Messling et al., 2006). In vitro experiments have demon-
strated an infection and subsequent induction of apoptosis
of SLAM-expressing canine lymphoid cell lines. In this
context, infection of canine neoplastic B and T cells by CDV
might represent a potential novel treatment of canine
lymphoma by attenuated CDV strains (Suter et al., 2005;
Yamaguchi et al., 2005; Puff et al., 2008).
The role of the CD46 surface antigen in CDV infection,
an alternative viral receptor in SLAM-negative canine cells,
as described for measles, remains to be determined. So far,
CD46 has only been detected in neoplastic lymphoid cells
of dogs (Nussbaum et al., 1995; Suter et al., 2005).
CD9, a tetraspan transmembrane protein is associated
with CDV-induced cell–cell fusion and syncytial cell
formation but not virus–cell fusion (Schmid et al., 2000).
However, since no direct binding of this transmembrane
protein by the virus has been demonstrated, CD9 is
regarded as a cofactor for viral infection, possibly as a part
of the receptor complex for CDV or due to intracellular
signaling, enhancing the expression or activity of a
receptor molecule (Löffler et al., 1997; Schmid et al.,
2000; Singethan et al., 2006).
7.2. Morphological findings in lymphoid organs
Systemic CDV infection causes gross changes in multi-
ple lymphoid tissues of affected dogs, such as lymph node
swelling, depletion of MALT and reduced thymus size. In
the acute phase, lesions are microscopically characterized
by a generalized depletion of T and B cell compartments in
spleen, lymph nodes, MALT and tonsils as well as
hyperplasia of reticular cells in the medullary region of
lymph nodes (Fig. 1). Thymic atrophy is associated with a
decreased corticomedullary ratio, indistinct demarcation
between cortex and medulla as well as a reduction of
Hassall’s corpuscles (Krakowka et al., 1980). Furthermore,
syncytia formation and cell death of immune cells is
predominantly observed in lymphoid follicles (Iwatsuki
et al., 1995), leading to a complete loss of secondary
follicles in dogs suffering from acute distemper infection
(Fig. 1; Jacoby and Griesemer, 1970; Krakowka and
Koestner, 1977; Iwatsuki et al., 1995). Characteristic
eosinophilic cytoplasmic viral inclusion bodies are present
in reticular cells and lymphocytes of affected dogs.
Immunosuppression during the acute phase of distemper
is further accompanied by a lack of recirculating T
lymphocytes in the marginal sinus of lymph nodes
6 A. Beineke et al. / Veterinary Immunology and Immunopathology 127 (2009) 1–18

Fig. 1. Histological and phenotypical characteristics found in the spleen of control animals (I, a–c) and dogs with acute (II, d–f) and chronic distemper (III, g–
i). (I) Normal lymphocyte distribution and compartment formation of B (follicle) and T cell (PALS) areas (a, HE). B lymphocytes (IgG) are the predominant
cell type in the follicle and marginal zone (b). Nearly all lymphocytes in the PALS and single cells in the marginal zone and follicle represent T lymphocytes
(c, CD3). (II) Severe splenic depletion consisting of a severe loss of most cell types (d, HE) including B lymphocytes (e, IgG) in lymphoid follicles and T cell (f,
CD3) in the PALS. (III) Repopulation of the spleen (g, HE) is associated with increasing numbers of B lymphocytes (h, IgG) and T cells (i, CD3) in the PALS.
F = lymphoid follicle; G = germinal center; M = marginal zone, P = PALS; C = corona; Z = central artery; HE = hematoxylin-eosin stain (a, d, g, HE) and
immunohistochemistry, ABC method (IgG, CD3, b, c, e, f, h, i; B and T cell specific antibodies). Bars = 100 mm.

(Krakowka et al., 1980). However, morphological altera-
tions of the immune system during the acute disease phase
are partially reversed by repopulation and germinal center
formation of lymphoid tissues in persistently infected and
convalescent dogs, respectively (Fig. 1; McCullough et al.,
1974b; Krakowka et al., 1980; Friedlander et al., 1985).
Overall, the degree of the lymphoid depletion is positively
correlated to the amount of CDV antigen in the affected
organ (Wünschmann et al., 1999; Kumagai et al., 2004).
Histological examination of the bone marrow of CDV
infected dogs revealed no or only non-specific changes,
such as hypercellularity and granulocytic hyperplasia
(Breuer et al., 1998) or necrosis of hematopoietic cells
(Baumgärtner et al., 1995a,b).
7.3. Phenotypical changes in lymphoid organs
The diminished immune function in the early phase of
the disease is associated with viremia and partially a
consequence of lysis of lymphocytes and macrophages.
Viral antigen is located in T-cell-dependent areas (Fig. 2)
and in the follicles during the acute phase (Iwatsuki et al.,
1995). Although different lymphocyte subsets, such as T
and B cells as well as macrophages become infected, CD4+
lymphocytes are most frequently affected by CDV during
the acute disease phase (Iwatsuki et al., 1995). Accordingly,
the loss of CD4+ cells in lymphoid organs prevails the
depletion of CD8+ cells (Wünschmann et al., 2000). After
initial lymphoid depletion in the incipient stage of CDV
infection, regeneration of lymphoid organs in the chronic
disease phase leads to the observed CD4- and CD8-
dominated inflammatory response in the CNS (McCullough
et al., 1974a,c; Krakowka et al., 1980; Krakowka and
Koestner, 1977; Wünschmann et al., 1999, 2000). Never-
theless, morphologically intact appearing compartments
after lymphoid repopulation and virus clearance from
lymphoid tissues will not result in complete functional
regeneration of the immune response (Krakowka et al.,
1975a, 1980; Cerruti-Sola et al., 1983). Lymphoid repo-
pulation in dogs with chronic distemper is characterized
by a complete reconstitution of CD3-, CD4- and CD8-
expressing lymphocyte subsets in T cell areas (Fig. 1).
However, phenotypically, the numbers of CD5- and IgG-
expressing cells are still reduced and viral antigen can be
found in dendritic cells and rarely in lymphocytes
(Wünschmann et al., 2000). In the chronic stage, dendritic
cells seem to serve as the primary host cells for the virus
(Fig. 2). This change of cell tropism is suspected to be a
consequence of the immune response and might represent a
mechanism of viral persistence as described for neurons and
oligodendrocytes (Wünschmann et al., 2000). Furthermore,
terminal differentiation of dendritic cells into mature
effector cells might be inhibited by the virus, possibly
leading to a persistent immune depressive effect by a
delayed repopulation of peripheral lymphoid tissues with
functional dendritic cells. In addition, CDV infection of
thymic epithelial dendritic cells may result in a compro-
mised maturation and selection of T cells, promoting the
 A. Beineke et al. / Veterinary Immunology and Immunopathology 127 (2009) 1–18
Fig. 2. Distribution of canine distemper virus (CDV) in the spleen of dogs with acute (a) and chronic distemper (b). During the acute disease phase, viral
antigen can be detected in many lymphoid cells (a), while CDV infection seems to be restricted to dendritic cells in follicles in chronic distemper (b). CDV-N-
specific monoclonal antibody, ABC-method. Bars = 50 mm.
release of immature CD5-negative T cells, including
potentially autoreactive cells (Wünschmann et al., 2000).
7.4. Mechanisms of immunosuppression in canine distemper
During the acute phase of distemper, lymphopenia is
characterized by a transient depletion of CD4+ T helper,
CD8+ cytotoxic T and CD21+ B cells from the peripheral
blood. Reduced numbers of circulating immune cells in CDV
might be a sequel of an impaired cellular output from
primary and secondary lymphoid organs as well as
apoptosis of peripheral blood leukocytes. As demonstrated
in lymphoid tissues, programmed cell death can be detected
in a considerable number of uninfected lymphocytes,
indicating the existence of additional virus-independent
mechanisms of apoptosis (Moro et al., 2003a; Kumagai et al.,
2004; Schobesberger et al., 2005). Therefore, mechanisms
other than directly virus-induced apoptosis, such as an over-
activation of the innate immune system or Fas-mediated
activation-induced apoptosis of lymphoid cells must be
considered as possible mechanisms of lymphocyte cell
death in canine distemper (Moro et al., 2003b; Schobes-
berger et al., 2005; Kajita et al., 2006). In addition, remaining
peripheral blood lymphocytes of naive dogs suffering from
CDV infection showed an extensively suppressed lympho-
proliferative response to mitogens (Krakowka et al., 1975a;
Krakowka and Wallace, 1979). The mechanisms of CDV-
induced immunosuppression are not completely under-
stood and are most likely caused by various factors. Firstly,
only a proportion of cells are infected and, secondly, long
lasting immunologic abnormalities are still present after
viral clearance from peripheral blood leukocytes in con-
valescent dogs. Accordingly, mechanisms of immune cell
impairment that do not involve direct viral infection of
lymphoid cells must be present (Krakowka et al., 1975a,
1980; Krakowka and Koestner, 1977). Consequently,
Krakowka (1982) discussed the existence of a mononuclear
suppressor cell population leading to the observed long
lasting immunosuppression in canine distemper. However,
the phenotypical classification of this cell type and
mechanisms leading to immune cell inhibition remain to
be determined. Furthermore, CDV modulates monocyte
7
function by inhibition of interleukin (IL)-1, while prosta-
glandin E2 release is increased in this cell population of
viremic dogs (Krakowka et al., 1987b). Therefore, besides
viral infection of B cells, an impaired antigen presentation by
monocytes might contribute to a diminished B cell
differentiation, plasma cell formation and immunoglobulin
production in CDV infected dogs (Fig. 3; Krakowka et al.,
1987b). Additionally, immunomodulatory properties of the
CDV N protein by engagement of the Fcg receptor (CD32)
expressed on B cells also leads to a suppression of uninfected
cells (Fig. 3; Kerdiles et al., 2006). Furthermore, diminished T
helper cell function in persistently infected dogs might
contribute to disturbed germinal center formation and a
reduced class switch from IgM to IgG (Winters et al., 1983).
Possibly, CDV N protein acts indirectly on T cell function by
modulating antigen presentation of dendritic cells via
interaction with CD32 and subsequent diminished IL-12
production, as described for measles virus (Schneider-
Schaulies and Dittmer, 2006). Using recombinant CDV in
ferrets, it has been demonstrated, that the viral V protein
sustains viral replication in lymphocytes and T cell
depletion. Furthermore, the viral V protein is an essential
interferon antagonist and cytokine response inhibitor in
CDV-infected ferrets (von Messling et al., 2006). Further-
more, a lack of detectable cytokine expression in peripheral
blood leukocytes is associated with a high viral load and
viremia, indicating that an overwhelming virus infection
may suppress cytokine production in lymphoid cells (Gröne
et al., 1998). Similarly, severe immunosuppression and the
lethal outcome in CDV-infected ferrets are correlated with a
lack of cytokine expression in peripheral blood leukocytes.
In comparison, surviving ferrets exhibited a robust and
sustained cytokine production with an initial Th1- and later
Th2-biased immune response (Svitek and von Messling,
2007).
Further studies have to focus on the importance of
virus-dependent and -independent mechanisms leading to
a reduced humoral and cellular immune response. This
includes questions upon mechanisms of viral persistence
in lymphoid organs with special emphasis upon the role of
dendritic cells as a potential mediator for the long lasting
immunosuppression.
8 A. Beineke et al. / Veterinary Immunology and Immunopathology 127 (2009) 1–18
Fig. 3. Mechanisms of immunosuppression in CDV-infected dogs. Viral infection and viral N protein/CD32 engagement leads to diminished antigen
presentation as well as disturbed dendritic cell and B cell maturation within germinal centers. Subsequently, plasma cell formation and immunoglobulin
production is significantly reduced.
8. Pathogenesis of nervous distemper
Occurrence of CNS lesions depend on virus strain, age
and immune status of the affected animal and can be
classified in different subtypes (Campbell, 1957; Krakowka
and Koestner, 1976; Summers et al., 1984; Pearce-Kelling
et al., 1990; Raw et al., 1992). Generally, a polio- and a
leukoencephalitis, characterized by different distribution
patterns of the lesions and pathogenesis, can be distin-
guished.
8.1. Virus entry, spread and expression within the CNS
Neuroinvasion of CDV occurs predominantly via the
hematogenous route (Fig. 4; Krakowka et al., 1987a;
Krakowka, 1989). Studies tracking the route of CDV within
the brain showed ependymal and subependymal white
matter infection indicating CNS spread along the cere-
brospinal fluid (CSF) pathway (Vandevelde et al., 1985b). In
addition, virus spread via the olfactory nerve has been
described in experimentally infected ferrets. Whether this
applies to natural infection of dogs needs to be confirmed
(Rudd et al., 2006). In addition, a direct spread from
meningeal cells of the pia mater has been assumed (Fig. 4;
Baumgärtner et al., 1989). Both a cell-free viremia during
the first days following experimental infection and a cell-
associated virus spread have been described (Axthelm and
Krakowka, 1987; Summers and Appel, 1987; Higgins et al.,
1982a,b). However, free infectious plasma virus cannot be
observed after the appearance of virus-neutralizing anti-
bodies (Rockborn, 1958). The leukocyte-associated viremia
is believed to represent the major source of hematogenous
infectivity (Rockborn, 1958; Summers et al., 1979). Viral
antigen is first detected within CNS capillaries and venular
endothelia at 5 and 6 days pi and/or in perivascular
lymphocytes, astrocytic foot processes and pericytes at 8
days pi (Axthelm and Krakowka, 1987; Summers and
Appel, 1987). Furthermore, a productive CDV infection of
the choroid plexus epithelium with release of progeny
virus into the cerebrospinal fluid followed by ependymal
infection and spread of the virus to the subependymal
white matter can be observed at 10 days pi (Appel and
Gillespie, 1972; Higgins et al., 1982a; Vandevelde et al.,
1985b).
Detailed studies on virus spread within the CNS indicate
that a rare and brief phase of gray matter disease prior to
the development of DL can be noticed (Vandevelde and
Kristensen, 1977; Summers et al., 1984). Based on this
observation, it has been proposed that any CDV infection
may pass through a sequence of CNS entry causing gray
matter and later white matter disease. The endpoint is
mainly determined by the virus strain (Summers et al.,
 A. Beineke et al. / Veterinary Immunology and Immunopathology 127 (2009) 1–18 9

Fig. 4. Canine distemper virus (CDV) spread within the CNS. Cross-section of the cerebellum: blue arrows: viral spread via infected meningeal cells; black
circles: viral spread via infected leukocytes and endothelial cells; yellow arrows: viral spread via infected choroid plexus epithelial cells; red arrows: viral
spread via infected ependymal cells. Inserts display detection of CDV antigen. CDV-N-specific monoclonal antibody, ABC-method. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of the article.)

1984). This is supported by findings of subpial lesions in
the cerebellum and the presence of CDV antigen positive
cells within the pia mater and subjacent gray matter in the
early phase of infection (Fig. 4).
The presence of viral core proteins in cell processes
suggests a possible spread of the virus along this pathway
in order to escape immune surveillance (Alldinger et al.,
1993a,b) as has been shown in cell culture studies
(Zurbriggen et al., 1995a,b). Furthermore, antigenic
modulation due to viral protein specific antibodies
may contribute to restricted expression of viral
proteins and virus persistence in DL (Alldinger et al.,
1993a,b). In the gray matter, however, more CDV-RNA
than protein is found, suggestive of impaired viral
translation and a possible mode of viral persistence
(Müller et al., 1995).
Viral persistence seems to be an important factor for the
induction of immune-mediated mechanisms in the chronic
phase of DL (Gaedke et al., 1995, 1997, 1999). In this
regard, factors favoring persistence include non-cytolytic
infection and little shedding of progeny virus, hereby
limiting the exposure of virus antigen to local immune
cells (Zurbriggen et al., 1995a,b).
8.2. Neurotropism of CDV
Within the CNS, astrocytes, microglia, oligodendro-
cytes, neurons, ependymal cells, choroid plexus cells and,
as indicated by most recent findings, aldynoglia can get
infected (Orlando et al., 2008). The term aldynolgia has
been used to define a family of growth-promoting glia and
includes several specialized macroglia with Schwann cell-
like properties. Recent in vitro studies indicated a
preferential infection of these cells by various CDV strains
(Orlando et al., 2008).
8.2.1. Oligodendroglial infection
Studies focusing on the role of CDV infection and
oligodendrocytes revealed inconsistent and controversial
results. Using light and transmission electron microscopy,
a direct oligodendrocyte infection, albeit only few cells
harbored the virus, was found (McCullough et al., 1974a;
Raine, 1976; Blakemore et al., 1989). In contrast to these
findings, various in vitro and in vivo investigations failed to
demonstrate oligodendrocyte infection (Vandevelde et al.,
1983, 1985a; Zurbriggen and Vandevelde, 1983; Zurbrig-
gen et al., 1986, 1987a,b). However, Zurbriggen et al.
(1993) demonstrated a restricted infection of oligoden-
drocytes, characterized by the presence of CDV nucleic acid
sequences and lack of viral antigen. Recently, it has been
shown, that a significant amount of oligodendrocytes is
still present in chronic lesions, and thus demyelination
seems to precede oligodendrocyte loss in DL (Schobes-
berger et al., 1999, 2002). However, in contrast to
multipolar mature oligodendrocytes that are rarely
infected a subset of bipolar oligodendrocyte precursor
10 A. Beineke et al. / Veterinary Immunology and Immunopathology 127 (2009) 1–18
cells expressing galactocerebroside exhibited a high
susceptibility for some CDV strains at least in vitro
(Pearce-Kelling et al., 1990, 1991). Similarly, another
bipolar cell population obtained from adult canine brains,
expressing the neurotrophin receptor p75NTR revealed an
increased susceptibility for CDV in in vitro studies (Orlando
et al., 2008). A phenotypically similar cell type can be
detected within CNS lesions of DL (W. Baumgärtner,
personal observation). It seems CDV infection of oligoden-
drocytes represents a still poorly understood process that
at least in part, depends upon the stage of cellular
differentiation at the time of infection.
8.2.2. Astrocytic infection
Astrocytes are the main cell population infected by CDV
in early plaques. In non-inflammatory lesions 64% of
astrocytes within a lesion are infected and this cell
population represents 95% of all infected cells (Mutinelli
et al., 1989). A recent study revealed the importance of
vimentin-positive cells during the spatio-temporal devel-
opment of distemper lesions. Vimentin-positive astrocyte-
like cells are present in the center and towards the
periphery of advanced lesions, whereas reactive astro-
gliosis of GFAP-positive cells was only present at the
periphery of the lesion. Vimentin-positive astrocyte-like
cells represent the main cell type harboring the pathogen
in advanced lesions, indicating a change in cell tropism
and/or susceptibility of glial cells during lesion progression
in DL. In addition, in vitro studies showed a higher
vulnerability and reduced susceptibility of mature,
GFAP-positive astrocytes for CDV compared to immature
astrocytes. Alternatively, these in vivo and in vitro findings
could indicate a reversion of a mature to an immature
phenotype (Seehusen et al., 2007).
8.2.3. Neuronal infection
CDV antigen and RNA expression in neurons, which
seems to occur in any type of nervous distemper during the
early phase and most prominent in distemper polioence-
phalitis is associated with a disproportional low amount of
viral protein compared to mRNA (Nesseler et al., 1999). A
reduced amount of detectable viral M protein indicated a
restricted CDV infection similar to findings in oligoden-
drocytes in DL and subacute sclerosing panencephalitis, a
rare complication of human measles virus infection
(Nesseler et al., 1997).
8.3. Pathology of nervous distemper
Polioencephalitis including old dog encephalitis, inclu-
sion body encephalitis and postvaccinal encephalitis is a
rare manifestation of CDV infection and is predominantly
located in the cortical areas and nuclei of the brain stem.
Neurons and protoplasmic astrocytes represent the pre-
dominantly affected cell populations (Nesseler et al.,
1999). Histologically, lesions consist of neuronal necrosis
and neuronophagia characterized by infiltrating microglia/
macrophages and T lymphocytes, perivascular cuffs as well
as intranuclear inclusion bodies in neurons and astrocytes
(Nesseler et al., 1997). Studies upon the pathogenesis of
distemper polioencephalitis support the hypothesis of a
restricted gray matter infection as a mechanism of viral
persistence (Nesseler et al., 1999).
In contrast to polioencephalitis, DL represents the most
common CNS manifestation of distemper. Due to simila-
rities distemper represents a useful model to study the
pathogenesis of human demyelinating immune mediated
diseases including MS (Koestner, 1975; Cook et al., 1978;
Dal Canto and Rabinowitz, 1982; Hodge and Wolfson,
1997; Baumgärtner and Alldinger, 2005).
DL lesions are found in the cerebellar and, less
frequently, in the cerebral white matter and in the spinal
cord (Baumgärtner et al., 1989; Bathen-Nöthen et al.,
2008). Changes are present most frequently in fiber tracts
in close proximity to the ventricles, within the cerebellar
velum, cerebellar peduncles and optic tracts (Summers
and Appel, 1994). Changes in DL can be categorized as
acute, subacute non-inflammatory, subacute inflamma-
tory, chronic and sclerotic lesions (McCullough et al.,
1974a,b; Raine, 1976; Summers et al., 1979; Higgins et al.,
1982b; Krakowka et al., 1985; Summers and Appel, 1987;
Gaedke et al., 1999). Several plaque types may occur
simultaneously in the brain of affected dogs (Baumgärtner
et al., 1989; Alldinger et al., 1993a,b; Gaedke et al., 1999;
Wünschmann et al., 1999).
Plaque genesis has been intensely studied in naturally
and experimentally CDV-infected dogs (McCullough et al.,
1974a,b; Raine, 1976; Summers et al., 1979; Higgins et al.,
1982b; Summers and Appel, 1987). Acute lesions consist of
focal vacuolization of the white matter, mild gliosis with few
activated astrocytes and macrophages/microglia. In experi-
mentally infected dogs, primary changes such as vacuoliza-
tion due to an intramyelin edema of the subependymal and
subpial white matter are observed as early as 24 days. These
vacuoles grow in number and size to form a demyelinated
plaque with micro- and astrogliosis and formation of
multinucleated giant cells. However, mononuclear infil-
trates are rare in the initial phase. Remyelination of denuded
axons is a rare event in older demyelinating lesions
(Wisniewski et al., 1972; McCullough et al., 1974c; Raine,
1976; Higgins et al., 1982b; Summers and Appel, 1987).
Subsequent inflammatory changes are characterized by
progressive perivascular mononuclear infiltrations. Sclero-
tic changes, so-called ‘‘burned out’’ plaques consist of
astrocytic scars characterized by isomorphic astrogliosis
(Vandevelde et al., 1981, 1982). In spite of a strong virus
specific intrathecal immune response early lesions without
overt inflammation can be observed in the vicinity of
chronic changes with prominent inflammation and reduced
or lacking virus expression (Alldinger et al., 1993b).
However, recent immunohistochemical studies revealed
the presence of immune cells already in acute and subacute
non-inflammatory lesions, indicating that this subdivision
of DL lesions represents a rather arbitrary approach to
classify distemper plaques (Tipold et al., 1999; Wünsch-
mann et al., 1999).
8.4. Immunopathology of demyelinating
leukoencephalomyelitis
DL represents be a biphasic event (Baumgärtner and
Alldinger, 2005). The initiation of this process is ascribed to
 A. Beineke et al. / Veterinary Immunology and Immunopathology 127 (2009) 1–18
a direct virus-mediated action, whereas plaque progres-
sion seems to be an immunopathological process (Baum-
gärtner et al., 1989; Summers and Appel, 1994; Alldinger
et al., 1996). Studies about the cellular immune response
revealed only a minimal but still substantial influx of
inflammatory cells in the CNS during the early phase of
infection (acute and subacute, non-inflammatory lesions),
and a dramatic increase of blood-borne inflammatory cells,
simultaneously with the recovery and repopulation of the
peripheral lymphoid tissues during the chronic phase
(McCullough et al., 1974a,b; Vandevelde et al., 1981;
Summers and Appel, 1994; Wünschmann et al., 1999). In
early lesions, infiltrating lymphocytes displayed predomi-
nantly the CD8+ phenotype indicating that antibody-
independent cytotoxicity is maybe involved in viral
clearance and possibly early lesion development (Wünsch-
mann et al., 1999). Initial T cell migration is suspected to be
mediated by microglia-derived chemokines, such as IL-8
(Tipold et al., 1999). An accumulation of immune cells in
the CNS during the early stages of the disease may facilitate
the later development of an intrathecal immune response
and associated immunopathological complications (Tipold
et al., 1999). An up-regulation of major histocompatibility
complex class II (MHC class II) molecules during the
progression of DL lesions, and a simultaneous reduction of
virus protein expression within the CNS, indicates an
involvement of non-viral antigens as a trigger of immune-
mediated processes in the chronic phase of demyelination
(Alldinger et al., 1996). MHC class II is mainly expressed on
microglia, endothelial, meningeal, choroid plexus epithe-
lial, ependymal and intravascular cells as well as most
perivascular blood-borne cells. Activated macrophages/
microglial cells exhibit beneficial and detrimental effects
during lesion development. Though myelin loss in early
lesions has been ascribed to directly or indirectly virus-
mediated processes, demyelination in chronic lesions may
occur as collateral damage and could develop due to
overactive macrophages/microglia. Chronic inflammatory
demyelination may be due to so-called bystander mechan-
isms resulting from interactions between macrophages/
microglia and antiviral antibodies (Vandevelde and
Zurbriggen, 1995). The term ‘‘bystander demyelination’’
is used to describe the myelin damaging effect of
proteolytic enzymes released by stimulated macro-
phages/microglia in the absence of obvious oligodendro-
cyte infection (Cammer et al., 1978). Activation of these
cells characterized by increased MHC class II and adhesion
molecule expression, results in release of toxic factors,
increased phagocytic activity and oxygen radical produc-
tion after induction of the respiratory burst (Bürge et al.,
1989; Griot et al., 1989a,b, 1990). Therefore, CDV-induced
phagocyte activation could contribute to oligodendrocyte
and/or myelin damage (Vandevelde and Zurbriggen, 2005;
Stein et al., 2006, 2007). Additionally, a humoral antiviral
immune response could lead to the destruction of
oligodendrocytes as ‘‘innocent bystanders’’. Also auto-
reactive T cells might be responsible for the observed
induction of myelin-specific cellular immunity via epitope
spreading secondary to myelin damage in the CNS
(Krakowka et al., 1973; Wünschmann et al., 2000).
However, the precise role of this self-reactive immune
11
response and autoantibodies in the pathogenesis of CNS
demyelination remains undetermined. In other words,
compared to autoimmune demyelinating diseases includ-
ing experimental allergic encephalitis (EAE), the anti-
myelin response might represent an epiphenomenon
rather than a contributing factor for myelin loss in DL
(Krakowka et al., 1973; Cerruti-Sola et al., 1983).
Coinciding with the recovery of the immune system at
approximately 7 weeks pi, perivascular mononuclear
cellular infiltrates are found within brain lesions. During
the recovery of the immune system, infiltration of CD4+
cells is followed by plasma cells and a strong intrathecal
antibody synthesis (Vandevelde and Zurbriggen, 2005).
Here, B cells producing predominately IgG antibodies are
mainly found in perivascular infiltrates and scattered
throughout the lesions (Vandevelde et al., 1981, 1982).
Furthermore, a prominent anti-CDV-specific humoral
immune response can be found in the serum and
cerebrospinal fluid. The decline of the intrathecal antiviral
antibody production and the simultaneously occurring
clinical improvement suggests a deleterious role of the
humoral immune response on the disease process.
Increased intrathecal antibody production due to intracer-
ebral B cells is suspected to accelerate myelin destruction
in chronic DL (Vandevelde et al., 1981, 1982, 1986;
Alldinger et al., 1996). Hence, a complement-dependent
antibody mediated humoral cytotoxicity is discussed as a
possible mechanism for demyelination (Vandevelde et al.,
1982, 1986). Furthermore, the simultaneous occurrence of
a high number of intralesional and perivascular CD8+
lymphocytes and perivascular B and CD4+ cells is
suggestive of an antibody-dependent T-cell mediated
cytotoxicity (Vandevelde et al., 1982; Alldinger et al.,
1996; Wünschmann et al., 1999). Furthermore, immuno-
phenotypical changes are indicative of a delayed-type
hypersensitivity reaction in advanced lesions (Wünsch-
mann et al., 1999).
In addition, anti-myelin specific antibodies have been
detected in the CSF (Krakowka et al., 1973; Vandevelde
et al., 1986; Rima et al., 1991). However, the significance of
anti-myelin antibodies remained uncertain as high titers of
these antibodies are also detected in dogs with resolving
lesions (Vandevelde et al., 1986).
8.5. Role of cytokines in the pathogenesis of demyelinating
leukoencephalitis
Cytokines were investigated to clarify cell-to-cell
communication and the interaction between immune
cells and resident brain cells in DL. It seems that cytokine
expression can be directly induced by the virus or due to
autocrine and paracrine regulatory loops during CDV
infection (Bencsik et al., 1996; Gröne et al., 2002; Markus
et al., 2002). Tsai et al. (1982) found interferon (IFN) in the
CSF of dogs with CNS CDV infection, indicating IFN as a
valid marker to determine CDV persistence in the CNS.
However, the type of IFN playing such an essential role
during DL remains to be determined. Referring to this, IFN-
g is not expressed in peripheral blood leukocytes of dogs
with canine distemper, whereas IL-8 can be found in blood
samples of CDV-infected animals with and without
12 A. Beineke et al. / Veterinary Immunology and Immunopathology 127 (2009) 1–18
viremia as well as in non-infected dogs (Gröne et al., 1998).
Though there is no obvious correlation between cytokine
mRNA transcripts and respiratory or gastrointestinal tract
manifestation, CNS demyelination is frequently associated
with increased expression of IL-1, IL-12, tumor necrosis
factor (TNF)-a and transforming growth factor (TGF)-b in
the blood of dogs with natural CDV infection (Gröne et al.,
1998). Furthermore, and indicative of disease initiation, IL-
6 transcripts can be detected in blood samples of animals
with early CNS lesions, while peripheral TGF-b expression
seems to play a role in chronic demyelination (Gröne et al.,
1998). The simultaneous occurrence of pro- and anti-
inflammatory cytokines in the blood of dogs with
distemper indicates a complex, most likely disease
stage-dependent orchestrated or dysregulated cytokine
expression (Fig. 5; Gröne et al., 1998). Within the CNS, the
chemokine IL-8 might be crucial for early T cell attraction
and antiviral cytotoxicity during acute distemper (Tipold
et al., 1999). In the CSF of naturally infected dogs, TNF-a
and IL-6 mRNA, associated with disease exacerbation, and
IL-10 and TGF-b mRNA, indicative of remission, are often
observed simultaneously and cannot be assigned to a
specific disease stage. However, detection of IL-10 mRNA
in the CSF might represent a stage of inactivity in dogs with
DL (Frisk et al., 1999b). Moreover, IFN-g can be found in
association with chronic lesions. A semiquantitative RT-
PCR study displayed a prominent up-regulation of the pro-
inflammatory cytokines IL-6, IL-8, IL-12 and TNF-a in early
distemper CNS lesions. In contrast, immunomodulatory
cytokines such as IL-10 and TGF-b were not significantly
up-regulated following CDV infection. In addition, IL-1, IL-
2 and IFN-g were not detected (Markus et al., 2002).
Therefore, the lack of or an inappropriate response of anti-
Fig. 5. Schematic display of the proposed pathogenesis of early lesions in demyelinating distemper leukoencephalitis. Following virus infection of astrocytes
and macrophages/microglia an increased influx of CD8+ lymphocytes associated with increased expression of IL-6, IL-8, IL-12 and TNF-a is observed. In
contrast, immunomodulatory cytokines including IL-10 and TGF-b remain unchanged.
inflammatory cytokines in the presence of an intense up-
regulation of pro-inflammatory cytokines might contri-
bute to initiation and progression of lesions, possibly due
to a derailment of the immune response in the early phase
of DL (Markus et al., 2002).
In addition, increased IL-1 and IL-6 expression can be
found in all types of distemper lesions and is predomi-
nately detected in CD3+ T lymphocytes and macrophages/
microglia in early plaques as well as in cells comprising
perivascular cuffs in chronic lesions (Fig. 6). TNF-a is
predominantly expressed by astrocytes and seems to play
a crucial role in the pathogenesis of early demyelination, as
demonstrated for MS (Gröne et al., 2000). Furthermore,
TNF-a as well as IL-1 and IL-6 represent crucial factors for
endothelial cell adhesion molecules induction and sub-
sequent leukocyte extravasation. Therefore, initial produc-
tion of TNF-a by astrocytes may drive a vicious circle of
attracting inflammatory cells to CNS lesions, leading to the
synthesis of more cytokines and development of the
chronic stage of DL. IL-12 production within demyelinating
lesions in CDV-infected brains is supposed to promote a
Th1-biased immune responses (Gröne et al., 2000;
Wünschmann et al., 2000).
8.6. Role of extracellular matrix in the pathogenesis of
demyelinating leukoencephalitis
In addition to cytokines, disruption of the blood–brain-
barrier by proteolytic enzymes is regarded as a pivotal
factor for the influx of inflammatory cells and lesion
progression in demyelinating diseases. Referring to this,
astrocytes represent the main target cells of CDV (Mutinelli
et al., 1989), form the blood–brain-barrier and are a major
 A. Beineke et al. / Veterinary Immunology and Immunopathology 127 (2009) 1–18 13

Fig. 6. Schematic display of the proposed pathogenesis of chronic lesions in demyelinating distemper leukoencephalitis. Following virus infection, reduced
or lacking virus expression and an increased influx of CD8+, CD4+ and B cells can be noticed. CD4+ and B cells are found predominantly in the perivascular
space. In addition, infiltrating peripheral or resident brain cells exhibit an increased MHC class II expression and a moderate to severe up-regulation of IL-6,
IL-8-, IL-12 and TNF-a. The role of IL-1 and IFN-g remains to be determined.

source of extracellular matrix (ECM) proteins and cell
mediators. Furthermore, astrocytes play an essential role
in the maintenance of structural integrity of the CNS
(Montgomery, 1994). The importance of the ECM is
stressed by the observation, that a plaque-associated
CD44 up-regulation, paralled by astrocyte activation is
found in acute and subacute demyelinating distemper
lesions. CD44 plays a dual role for the progression of the
demyelination process. Ligation of this cell surface
receptor for hyaluronate might induce chemokine and
cytokine activation and hence initiates and perpetuates the
inflammatory process. In chronic lesions, a reduction of
CD44 associated with a loss of astrocytes is noticed.
However, CD44 can still be found on the cell membrane of
perivascular mononuclear cells. Therefore, CD44 might
also represent a marker for the activation of immune cells
in the late phase of the infection (Alldinger et al., 2000,
2006).
Matrix metalloproteinases (MMPs) are zinc- and
calcium-dependent endopeptidases that are capable to
cleave the extracellular matrix, and thus are able to open
the blood–brain-barrier and affect myelin (Lo et al., 2002;
Ulrich et al., 2006; Puff et al., 2008). Furthermore, certain
MMPs are crucial for the activation of cytokine and
chemokine functions, as demonstrated for MS (Chandler
et al., 1997; van Lint and Libert, 2007). Their inhibitors,
TIMPs (tissue inhibitors of matrix metalloproteinases), are
most prominently up-regulated in acute and subacute
non-inflammatory lesions and are expressed mostly by
astrocytes and macrophages/microglia as demonstrated by
immunohistochemistry (Miao et al., 2003). It seems that
they play a crucial role in lesion initiation. In advanced
plaques, the expression of MMPs and TIMPs declined apart
from MMP-11, MMP-12 and MMP-13 and invading
perivascular mononuclear cells express also MMPs and
TIMPs in addition to astrocytes and macrophages/micro-
glia (Miao et al., 2002, 2003), indicating a disease phase-
dependent expression of MMPs and TIMPs in DL. Similarly,
in situ hybridization studies revealed a MMP-9, MMP-14
and TIMP-1 expression predominantly in astrocytes and
activated macrophages/microglial cells in early lesions,
whereas activated macrophages/microglia and infiltrating
lymphocytes are the main source of these enzymes and
proteins in advanced DL (Gröters et al., 2005). Overall the
number of cells expressing MMPs seems to exceed the
amount of cells with TIMP expression. These findings favor
the hypothesis of an altered MMP–TIMP balance as an
additional key factor for lesion initiation and progression
(Miao et al., 2003; Gröters et al., 2005; Alldinger et al.,
2006).
Acknowledgments
The authors wish to thank Annette Artelt, Petra Grünig,
Danuta Waschke and Julia Schirrmeier for excellent
technical support. Dr. Frauke Seehusen received a scholar-
ship from Bayer AG, Animal Health Care, Leverkusen,
14 A. Beineke et al. / Veterinary Immunology and Immunopathology 127 (2009) 1–18
Germany. Dr. Christina Puff received an award from the
Frauenförderplan of the University of Veterinary Medicine,
Hannover. Studies were supported by the Deutsche
Forschungsgemeinschaft and the Hertie-Foundation.
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