Canine and feline viral dermatoses with a particular emphasis to

Transcription

University of Zurich
Zurich Open Repository and Archive
Winterthurerstr. 190
CH-8057 Zurich
http://www.zora.uzh.ch
Year: 2007
Canine and feline viral dermatoses with a particular emphasis to
papillomavirus infections
Favrot, C
Favrot, C. Canine and feline viral dermatoses with a particular emphasis to papillomavirus infections. 2007,
University of Zurich, Vetsuisse Faculty.
Postprint available at:
Posted at the Zurich Open Repository and Archive, University of Zurich.
Originally published at:
University of Zurich, Vetsuisse Faculty, 2007.
Vetsuisse-Fakultät- Universität Zürich
Klinik für Kleintiermedizin
(Direktorin: Prof. Dr. Claudia Reusch)
Canine and feline viral dermatoses with a particular
emphasis to papillomavirus infections
(Virale Dermatosen bei Hunden und Katzen unter
spezieller Berücksichtigung der PapillomavirusInfektionen)
HABILITATIONSSCHRIFT
Zur Erlangerung der Venia Legendi
an der Vetsuisse-Fakultät
der Universität Zürich
vorgelegt von
Dr. med.vet. Claude Favrot
Diplomate, European College of Veterinary Dermatology
Masters in Sciences
Zürich, 2007
Chapter 1
General introduction - Aims and scope of the thesis
Chapter 2
Virale Dermatosen bei Hunden und Katzen
11
Chapter 3
Parvovirus infection of keratinocytes as a cause of
canine erythema multiforme
24
Chapter 4
Two cases of FeLV-associated dermatoses
28
Chapter 5
Evaluation of papillomaviruses associated with
cyclosporine-induced hyperplastic verrucous lesions
in dogs
35
Detection of novel papillomaviruses in canine mucosal,
cutaneous and in situ squamous cell carcinomas
42
Detection of novel papillomavirus-like DNA sequences
in paraffine embedded samples of feline invasive
and in situ squamous cell carcinomas
52
Clinical, histological and immunohistochemical study
of feline viral plaques and bowenoid in situ carcinomas
59
Chapter 9
Summarizing discussion and further studies
68
Chapter 10
Zusammenfassung und weitere Studien
76
Chapter 6
Chapter 7
Chapter 8
Acknowledgments
The studies in this thesis were conducted at the University of Zurich, Switzerland
and at the Clinique Vétérinaire de Ferrette, France. They were financially
supported by these institutions as well as the Waltham Foundation and the
European College of Veterinary Dermatology.
2
3
Chapter 1
General introduction
Aims and scope of the thesis
3
Skin lesions are a predominant feature of many viral diseases in humans and large
domestic mammals [1, 2]. In comparison, viral dermatoses are rarely described in dogs
and cats [3]. However, they may be underdiagnosed due to the difficulty in detecting
viruses. The development and increased availability of diagnostic techniques such as
electron microscopy, immunohistochemistry, viral amplification (PCR) is making
detection more routine [4]. To complement advances in diagnosis, several effective
antiviral agents for treating at least some viral dermatoses have been developed in the last
few years [1]. Consequently, making the correct diagnosis may actually have important
consequences for therapy as well as for prognosis.
Definition and classification:
Viruses form a diverse group of non-cellular infectious agents that share a distinct
composition and a unique mode of replication. These agents lack much of the enzymatic
machinery necessary for their multiplication. They are consequently obligate intracellular
parasites that multiply inside cells and use the synthetic apparatus of the host cell to
produce their own components [1].
The animal viruses are divided in several families according to their shape, structure of
virion and the type of nucleic acid within it [5-7]. In fact, in the virion, the genome of the
virus consists of only one type of nucleic acid (DNA or RNA). The viral particle (virion)
is made of the nucleic acid surrounded by a protective protein core called the capsid.
Some viruses do additionally possess an envelope which play a major in the infection of
host cells [5-7].
Viral Replication and host response:
Viruses make use of the host-cell machinery to synthesize and assemble viral particles
(replication) because they do not possess the required enzymes. Viruses encode for structural
(capsid) proteins and non structural proteins that usually regulate the viral replication. Some
4
of these non-structural proteins (transforming proteins) are responsible of the oncogenic
potential of tumour viruses, like high-risk papillomavirus [8, 9]. Some viruses, like
papillomaviruses depend on epithelial cell differentiation for completion of their replication
[10]. After an initial inoculation in the basal layer of the epithelium, non-structural (early)
proteins are expressed in the suprabasal layers and in the stratum spinosum. Capsid (late)
proteins are subsequently produced in the stratum spinosum and the viral particles assembled
and released in the upper stratum granulousm and stratum corneum, respectively [10].
Epithelial host-cells are infected by papillomaviruses through inoculation but for most other
epithelium-infecting viruses, the replication cycle begins with an attachment phase called
adsorption [1, 11]. This attachment requires specific interaction between host-cell receptors
and virus. Cells lacking virus-specific receptors are not susceptible to infection.
Following adsorption and penetration, viral envelopes and capsids are destroyed (uncoating)
and viral genome can therefore instruct the host-cell machinery to produce its own proteins.
Viral particles of most viruses infecting epithelial cells (herpes, papillomavirus, poxvirus) are
produced inside the cells and released after cell death or cytolysis [1].
Each virus has its own site of replication: Herpesviruses and papillomaviruses replicate in the
nuclei whereas poxviruses multiply in the cytoplasm.
Viral replication usually causes gross cytopathic changes and host-cells sometimes die. These
cytopathic effects may be pathognomonic of one specific viral infection (viral inclusions and
pseudo-inclusions, syncytium formation (herpesvirus, retrovirus, paramyxovirus), modified
keratinisation process (papillomaviruses).
Some viruses, like papillomaviruses or distemper virus, can however, at least in some
instances, replicate without causing irreversible damage to the host keratinocytes (true
commensality, chronic infections)[12-15].
Another form of non cytocydal infection is the latency (herpesviruses, papillomaviruses). In
this instance, very few or no virion are produced in the infected cells but reactivation of the
infection can occur at any moment.
Last but not least, some viruses (papillomaviruses, retroviruses) are able to induce host-cell
immortalization and neoplastic transformation. Most of the time, transformed host-cells loose
their ability to sustain productive infection [8, 9].
5
Viral infection and skin lesions:
Virus-induced skin lesions are usually the direct consequence of the virus replication.
Examples of these direct effects are wart formations associated with papillomaviruses
infections, pock formation in poxviruses infections or vesiculation associated with
herpesvirus infections. Some skin lesions are however due to the host response or to the
interaction of replication and host response. Erythema multiforme, for example, are
exanthematous skin lesions associated with herpesvirus infection in humans and cats and may
be, with parvovirus infections in dogs [16-18]. This reaction is considered to be due to the
destruction of infected keratinocytes by cytotoxic T-cells. Finally, viruses may also modify
skin biology and cause indirect changes, like in the so-called “hard pad disease” [15, 19-21].
Diagnosis of viral infections:
Several approaches are now available to diagnose viral infection: virus isolation and culture,
microscopy, serology or detection of viral antigens or nucleic acids. As these techniques
demonstrate increasing sensitivity, results should always be interpreted in the context of the
clinical and histological setting. In fact, one must always keep in mind that virus infection
can be fortuitous and unrelated to the disease.
Cultivation of the virus and/or direct identification (pathognomonic cytopathic effects) from
the clinical material represent the “gold standard” for viral diagnosis because they establish at
the same time that the virus is present and actively replicates in the lesional sample. These
techniques are however limited by the low sensitivity and the difficulty to cultivate some
viruses like papillomaviruses.
The recent development of techniques that allow the amplification and multiplication of viral
nucleic acids (PCR) has dramatically increased the sensitivity of virus detection. The main
pitfall of PCR is however its great sensitivity itself, as false-positive assays may result from
the amplication of minute amount of nucleic acid of viruses unrelated to the disease.
Electron microscopy and immunohistological identification of viral antigens in lesional
samples are also available. As they detect productive infections, these techniques may be
regarded as more specific. They are however less sensitive than PCR techniques.
6
Serologic studies to detect antiviral antibodies are important for epidemiological studies, to
determine the prevalence of one specific virus in a population, and to detect individuals that
have been previously affected by the condition or in contact with the virus.
Aims and scope of the thesis:
As mentioned above, viral dermatoses are rarely reported in domestic carnivores [3, 22]. The
aim of this thesis was to report some unusual aspects of viral infections of the skin of
domestic carnivores and to show that at least some of these viral cutaneous infections
remained under-diagnosed. Furthermore we aimed to broaden our knowledge of the genetic
diversity of carnivores papillomaviruses and to demonstrate that these latter viruses may
contribute to the development of skin cancer in these species.
We have first shown that canine parvovirus 2 is able to induce, in some instances, clinical
and histological changes that mimic human erythema multiforme (EM) [17]. Similar changes
have already been described in dogs but were, most of time, attributed to drug reactions [23].
On the contrary, true EM is virtually always associated in Man with herpesvirus infections
and almost never with drug reaction [16]. Feline EM has also been shown to be due to
herpesvirus infections [24]. The disclosure of canine EM associated with virus infection
should encourage veterinary dermatologists to look for virus antigens or nucleic acids in skin
samples of dogs affected by this condition.
We have described and studied two cases of FeLV-induced skin conditions [25]. We have
first shown that FeLV, like other retroviruses, is able to induce syncytium formation in the
skin of infected cats. Similar cases have already been described but with a very different
clinical phenotype [26]. More interestingly, FeLV antigens and nucleic acids were uncovered
in cutaneous lymphoma samples in a serologically negative cat. These findings suggest that
focal skin FeLV replication may occur in some instances.
Dogs treated with cyclosporine A sometimes develop lichenoid plaques and warts of
unknown origin. We have evaluated such lesions in nine affected dogs and demonstrated that
the majority of these plaques are not papillomavirus-induced. Some however harbour
papillomavirus DNA and antigens and are probably due to the reactivation of a latent PVinfection of the skin [27].
Anecdotal reports have suggested that PV could play a role in the development of skin
squanous cell carcinomas in dogs and cats [28-33]. We have consequently tried to amplify
7
PV DNA from skin samples of canine and feline squamous cell carcinomas and demonstrated
that nucleic acids of these viruses are present in a significant amount of such samples [34,
35]. Furthermore, amplified PV sequences revealed that these samples are infected by PV of
great genetic diversity. These findings suggest that PV could play an active role in the
development of such cancers in dogs and cats and that domestic carnivore, like humans, may
be infected my numerous different PVs. A last study carried out on a subset of feline
squamous cell carcinomas (Bowenoid in situ carcinomas: BISC) in situ has shown that BISC
are often infected by PVs and that these viruses actively replicate in such lesion [36]. These
results further suggest an active role of these viruses in the development of such lesions.
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Editor. 2001, W.B. Saunders: Philadelphia. p. 517-542.
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Murphy, F.A., et al., The Nature of Viruses as Etiologic Agents of Veterinary and
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Harwood, C.A. and C.M. Proby, Human papillomaviruses and non-melanoma skin
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P.M., Editor. 2001, Lippincott, Williams & Wilkins: Philadelphia. p. 2231-2264.
Antonsson, A., et al., Prevalence and type spectrum of human papillomaviruses in
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1881-1886.
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haired skin, nasal mucosa, and footpad epithelium: a method for antemortem
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Assier, H., et al., Erythema multiforme with mucous membrane involvement and
Stevens-Johnson syndrome are clinically different disorders with distinct causes.
Arch Dermatol, 1995. 131(5): p. 539-543.
Favrot, C., et al., Parvovirus infection of keratinocytes as a cause of canine erythema
multiforme. Vet Pathol, 2000. 37(6): p. 647-649.
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Grone, A., M.G. Doherr, and A. Zurbriggen, Up-regulation of cytokeratin expression
in canine distemper virus-infected canine footpad epidermis. Vet Dermatol, 2004.
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Grone, A., P. Engelhardt, and A. Zurbriggen, Canine distemper virus infection:
Proliferation of canine footpad keratinocytes. Vet Pathol, 2003. 40(5): p. 574-578.
Koutinas, A.F., et al., Histopathology and Immunohistochemistry of Canine
Distemper Virus-induced Footpad Hyperkeratosis (Hard Pad Disease) in Dogs with
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Praxis, 2006. 24 (K): p. 307-318
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Prost, C., A case of exfoliative erythema multiforme associated with herpesvirus 1
infection in a cat (abstract). Vet Dermatol, 2004. 15 (Suppl. 1): p. 51.
Favrot, C., et al., Two cases of FeLV-associated dermatoses. Vet Dermatol, 2005. 16
(6): p. 407-412.
Gross, T.L., et al., Giant cell dermatosis in FeLV-positive cats. Vet. Dermatol., 1993.
4(3): p. 117-122.
Favrot, C., et al., Evaluation of papillomaviruses associated with cyclosporineinduced hyperplastic verrucous lesions in dogs. Am J Vet Res, 2005. 66(10): p. 17641769.
LeClerc, S.M., E.G. Clark., and D.M. Haines,. papillomavirus infection in association
with feline cutaneous squamous cell carcinoma in situ (Abstract). in AAVD/ACVD
Meeting. 1997: p. 125-126.
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cutaneous mucosa and the transitional epithelium in dogs: an immunolocalization
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Zaugg, N., et al. Detection of novel papillomaviruses in canine mucosal, cutaneous
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10
Chapter 2
Virale Dermatosen bei Hunden und Katzen
C. Favrot1, S. Wilhelm1
Tierärtzlich Praxis, 2006, 34 (5): 307-318
1
Klinik für Kleintiermedizin, Dermatologie, Vetsuisse-Fakultät der Universität Zürich,
Schweiz
11
Virale Dermatosen bei Hund und Katze
C. Favrot, S. Wilhelm
Aus der Klinik für Kleintiermedizin, Dermatologie (Leiter: Dr. C. Favrot), Vetsuisse Fakultät, Universität Zürich, Schweiz
Schlüsselwörter:
Virus – Dermatose –
Hund – Katze
Zusammenfassung:
Virale Dermatosen kommen bei Kleintieren nur selten vor und werden vermutlich auch häufig übersehen.
Ziel dieser Übersichtsarbeit ist, die klinischen und histologischen Veränderungen viraler Dermatosen bei
Hunden und Katzen darzustellen. Berücksichtigung finden insbesondere Veränderungen, die direkt oder
indirekt von Viren der Gattungen und Familien Papillomavirus, Ortho- und Parapoxvirus, Herpesvirus, Retrovirus, Lentivirus, Morbillivirus und Parvovirus verursacht werden. Neueste Techniken wie DNA-Amplifikation machen den Nachweis kleinster viraler DNA- und RNA-Mengen möglich und vereinfachen so die
Diagnosestellung. Allerdings darf nicht vergessen werden, dass der Nachweis viraler DNA in Hautläsionen
noch keinen Beweis für das Vorliegen einer viralen Dermatose darstellt. Demzufolge ist es unerlässlich,
zwischen Veränderungen, die direkt aufgrund einer Virusinfektion entstanden sind, und solchen, die mit
viralen Erkrankungen nur assoziiert sind, zu unterscheiden. Auf Letztere wird im vorliegenden Artikel nur
dann eingegangen, wenn besagte Assoziationen in der Literatur häufig erwähnt werden. Einige Dermatosen, die indirekt durch Viren verursacht werden, zeichnen sich durch veränderte Proliferisationseigenschaften oder Antigenität der Hautzellen aus. Solche Modifikationen können mit Kanzerogenese oder immunologischen Reaktionen, wie beispielsweise einem Erythema multiforme, einhergehen.
Key words:
Virus – dermatosis –
dog – cat
Summary:
Viral dermatoses are considered rare in domestic animals but are probably often underdiagnosed. The purpose of this review is to present the clinical and histological features of viral dermatoses in domestic animals. It will focus on conditions directly or indirectly caused by viruses of the genera Papillomavirus, Orthoand Para-Poxvirus, Herpesvirus, Retrovirus, Lentivirus, Morbillivirus and Parvovirus. New techniques such
as nucleic acid amplification enable the detection of minute amounts of viral DNA and RNA. Diagnoses
have consequently been facilitated. However, one must keep in mind that detection of viral nucleic acid in
skin lesions does not prove that the virus is the cause of the disease. It is mandatory to distinguish diseases
that are directly caused by viruses and conditions that are sometimes associated with viruses. The latter
will only be mentioned when they are frequently reported in the literature. Some dermatoses are caused indirectly by viruses that modify the proliferation properties or the antigenicity of skin cells. These modifications may be associated with cancerization or immunologic reactions such as erythema multiforme.
Viral dermatoses of the dog and cat
Einleitung
Virale Dermatosen kommen bei Kleintieren selten vor (86). Da
die Erreger schwer zu identifizieren sind, wurde diese Krankheitsbilder vermutlich lange übersehen. Durch bessere Nachweismethoden gelingt es häufiger, Viren nachzuweisen und mit dem
bestehenden klinischen Bild in Verbindung zu bringen (89).Allerdings kann dadurch noch keine Kausalität bewiesen werden.Auch
der Nachweis viraler Antigene oder Nukleinsäuren in der betroffenen Läsion ist kein ausreichender Beweis (18, 45). Üblicherweise wird die Diagnose mittels Histologie – dem Vorhandensein
direkter zytopathischer Effekte – gestellt. Viren können das Gewebe aber auch indirekt schädigen. Dies geschieht vor allem in
solchen Fällen, in denen die Viren die Antigenizität von Epidermalzellen modifizieren oder die Wachstumsrate der Hautzellen
erhöhen. In ersterem Fall werden die Keratinozyten durch das
Eingegangen: 07.04.2005; akzeptiert: 05.01.2006
lokale Immunsystem als fremd erkannt und es kommt zu einer
Apoptoseinduktion (Erythema multiforme). Im zweiten Fall besteht bei der schnell wachsenden Epidermalzellpopulation die
Gefahr einer karzinomatösen Entartung. Nicht zuletzt können
Viren auch das lokale Immunsystem schädigen.
In der folgenden Übersichtsarbeit liegt das Schwergewicht auf
der klinischen Präsentation, der Histologie und den Behandlungsmöglichkeiten der häufigsten nachgewiesenen viralen Dermatosen bei Hunden und Katzen.
Papillomaviren
Ätiologie und Pathogenese
Papillomaviren (PV) gehören zu den DNA-Viren und zeigen einen ausgeprägten Plattenepithel-Tropismus (52). Das Virus ist
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HUND/KATZE
© 2006 Schattauer GmbH
Tierärztl Prax 2006; 34 (K): 307-18
HUND/KATZE
308
normalerweise speziesspezifisch, ansteckend und wird oft durch
Mikroläsionen übertragen (74). Bei Hunden wird die Mehrheit
der Infektionen durch das kanine orale PV (COPV) verursacht.
Allerdings ließen sich auch andere Stämme aus kaninen Hautläsionen isolieren (60, 103). Bei Katzen ist diese Krankheit selten
und wird durch feline oder bovine Papillomaviren hervorgerufen
(85, 98). Bisher konnte erst bei einem kaninen (COPV) und einem
felinen (FdPV-1) Papillomavirus das Genom aufgeschlüsselt werden (99, 102).
Die Replikation des Virus läuft parallel zur Differenzierung
des Plattenepithels ab und kann in eine „Early Phase“ (E) und eine „Late Phase“ (L) unterteilt werden. Während der E-Phase im
Stratum spinosum und der suprabasalen Schicht werden die viralen Proteine synthetisiert. Im Stratum granulosum findet während
der L-Phase die Synthese der Kapsidproteine statt. Erst im oberen
Bereich des Stratum granulosum wird das Kapsid zusammen-
gebaut. Die Freisetzung des Virions erfolgt gleichzeitig mit der
Desquamation (52).
Papillomaviren induzieren auch die Synthese der so genannten transformierenden Proteine E6 und E7, die für die proliferativen und karzinogenen Eigenschaften des Virus verantwortlich
sind (45).
Eine Infektion durch ein unproduktives Papillomavirus ist
ebenfalls möglich und wird mit der Entwicklung eines felinen
Sarkoids oder eines Fibropapilloms assoziiert (85). Es wird angenommen, dass es sich hierbei um das Pendant des equinen
Sarkoids handelt. In der Mehrzahl der Fälle gelang es, in solchen
Läsionen PV-DNA nachzuweisen, wobei die Blast-Analyse der
L1-Sequenz starke Homologien mit einem bovinen PV aufwies
(42, 85, 105).
Klinik
Hund
Abb. 1 Typische virale Warzen (kanine orale Papillomatose)
Abb. 2 Persistierende Warzen am Schwanz einer erwachsenen
Deutschen Dogge (Papillomatose des adulten Hundes)
13
Es existieren verschiedene klinische Präsentationen, die unterschiedlichen Viren zugeschrieben werden, deren komplettes Genom jedoch noch nicht entschlüsselt ist. Dennoch ist es möglich,
dass jede einzelne klinische Präsentation (z. B. orale Infektion
oder invertiertes Papillom) mit einem spezifischen PV assoziiert
ist (9, 30).
Kanine orale Papillomatose (COPV): Hierbei handelt es
sich um die klassischste und auch häufigste transiente virale
Hauterkrankung beim Hund. Betroffen sind meist junge Hunde.
Eine Rassen- oder Geschlechtsprädisposition gibt es nicht. Die
Läsionen befinden sich auf der Schleimhaut der Maulhöhle, der
Lippen, des Planum nasale und/oder der Konjunktiven (87). Ausnahmsweise können auch die peribukkale Haut, die Augenlider
oder der obere Gastrointestinaltrakt betroffen sein (Abb. 1). In der
Regel sind es mehrere Läsionen, die durch ihre wuchernde, hyperplastische Struktur das Tier stören können. Der Durchmesser variiert von 2–3 mm bis zu über 3 cm. Ihr exophytisches Aussehen,
ihre Größe, die blumenkohlartige Oberfläche und die Lokalisation der Läsionen lassen oft bereits eine klinische Diagnose zu (6).
Kanine orale Papillome bilden sich in den meisten Fällen innerhalb von einem bis drei Monaten spontan zurück (14). Bei Vorliegen einer Immundefizienz wurden persistierende PapillomavirusInfektionen beschrieben (63, 73).
Papillomatosen des adulten Hundes: Bei diesen Läsionen
kommt es zu keiner spontanen Rückbildung. Hunde jeden Alters
können betroffen sein. Die Veränderungen treten oftmals an mukokutanen Übergängen, vor allem in der Maulhöhle (hier am häufigsten und gravierendsten), dem Gesicht und zwischen den Zehen auf (Abb. 2). Es handelt sich um multiple typische Warzen,
die mit einer dicken Keratinschicht bedeckt sein können (Abb. 3).
In vielen Fällen sind die Läsionen pigmentiert, manchmal stehen
sie dicht nebeneinander stehend und können bis in den Pharynx
reichen (117), wo sie zu Schluckstörungen führen. Die Ätiologie
ist noch ungeklärt, doch werden vielfach Immundefizienzen als
Ursache angenommen (7, 63, 73, 75, 100, 117).
Invertierte Papillome: Diese ungewöhnlichen Hautveränderungen treten vor allem am Abdomen und am Kinn junger adulter
Hunde auf (Abb. 4). Eine Streuung über den gesamten Körper ist
ebenfalls möglich. Die aufgewölbten Veränderungen weisen einen Durchmesser von 1–2 cm auf und besitzen im Zentrum eine
Pore (9). Das ursächliche PV scheint sich vom COPV-1 zu unterscheiden (9).
Multiple pigmentierte papillomatöse Plaques (Abb. 5): Sie
sind selten, kommen jedoch bei einigen Rassen (z. B. Mops) häufiger vor und wurden mit immunsuppressiven Therapien in Zusammenhang gebracht (60, 72, 96, 111). In manchen Fällen ähneln diese Plaques klinisch und histopathologisch der humanen
Epidermodysplasia verruciformis (72, 111).Andere dagegen weisen einzigartige histologische Strukturen mit eosinophilen zytoplasmatischen Granula auf (60). Bei nicht immunsupprimierten
Hunden konnte eine spontane Abheilung beobachtet werden. Bei
manchen Hunden führte das Absetzen der immunsuppressiven
Therapie zur kompletten Abheilung, wiederum bei anderen kam
es zu einer malignen Transformation (54, 72, 96, 111).
Plattenepithelkarzinom (Abb. 6): In der Veterinärmedizin
finden sich nur wenige Berichte über eine maligne Transformation
von COPV-induzierten Veränderungen in Plattenepithelkarzinome
(101, 104, 112). Bei einem Hund wird über ein multizentrisches
Karzinom in situ (Bowen’s Disease) berichtet (35). Eine virale
Ätiologie ist zwar anzunehmen, wurde bis dato aber nicht bestätigt.
Bei den meisten Katzen äußert sich diese Krankheit in Form von
schuppigen, pigmentierten Plaques (Abb. 7) (12, 20, 62, 98). Perserkatzen und immunsupprimierte Tiere sind prädisponiert. Bis
heute wurde erst ein einziges katzenspezifisches PV entdeckt und
sein Genom komplett entschlüsselt (FdPV-1) (102).
Bei Katzen kommt es nur selten zu oralen Läsionen. Diese üblicherweise multifokalen, festsitzenden und durch leichte Erhebungen charakterisierten Veränderungen finden sich in der unteren Gesichtshälfte und auf der Zunge (98). Ansonsten existiert in
der Literatur nur der Fallbericht einer Katze mit exophytischen
Papillamovirus-induzierten Warzen an den Augenlidern (13).
Abb. 3 Warze am Pfotenballen mit Hornbildung
Abb. 4 Invertiertes Papillom (Abb.: D. Carlotti)
Abb. 5 Papillomavirus-induzierte pigmentierte Plaques
Abb. 6 Papillomavirus-induziertes Plattenepithelkarzinom in situ
Katze
14
309
HUND/KATZE
HUND/KATZE
310
Regelmäßig wird über Katzen mit multizentrischen Karzinomen in situ berichtet (Abb. 8) (2, 67). In einer diesbezüglichen
Arbeit wurde PV bei bis zu 40% der untersuchten Proben nachgewiesen (59). Solche Veränderungen werden auch mit DemodexInfektionen in Zusammenhang gebracht (38).
Die letzte Form der felinen PV-Hautinfektionen ist das Fibropapillom oder feline Sarkoid (42, 85, 105). Diese Läsionen im
Gesicht oder an den Extremitäten bestehen aus festen Knoten in der
Dermis,wahrscheinlichaufgrundeinernichtproduktivenPV-Infektion.
Diagnosestellung
Oft genügt bei jungen Hunden mit oraler Papillomatose bereits
die klinische Untersuchung. In fraglichen Fällen und bei adulten
Hunden unterscheidet die histologische Untersuchung zwischen
einer echten Papillomatose und einer nichtviralen warzenähnlichen Läsion. Histopathologisch erkennt man eine papillomatöse
epidermale Hyperplasie mit ballonierender Degenereration von
Zellen im Stratum spinosum (Koilozytose). Zusätzlich kommt es
zu einer Hypergranulose mit prominent verklumpten Keratohyalingranula (118). Eosinophile intranukleäre virale Einschlusskörperchen und basophiles intrazytoplasmatisches fibrilläres
Material bestätigen die ätiologische Diagnose (118). Fehlen virale Einschlusskörperchen, kann eine virusinduzierte Papillomatose nicht ausgeschlossen werden. Zum Nachweis der Ätiologie
sind auch immunhistochemische Techniken und DNA-Amplifikation nützlich.
Epidermale Hypermelanose, unregelmäßige Akanthose und
Hypergranulose mit verklumpten Keratohyalingranula werden
normalerweise bei Vorliegen von kaninen multiplen pigmentierten papillomatösen Plaques gesehen (72, 96, 111). In einigen Fällen konnten virale Einschlusskörperchen nachgewiesen werden
(60, 96). Bei Katzen liegen ähnliche histopathologische Veränderungen vor, wobei virale Einschlusskörperchen häufiger beobachtet werden (12, 20, 62, 98).
Histologische Untersuchungen von felinen Sarkoiden ergaben eine pseudokarzinomatöse Akanthose und dicht gepackte
mesenchymale Zellen, die Kollagenbündel umgaben (85, 105).
Therapie
Abb. 7 Feline Papillomavirus-induzierte pigmentierte Plaques
(Abb.: C. Mege)
Abb. 8 Felines
Papillomavirus-induziertes Plattenepithelkarzinom in
situ (Ohrgrund)
15
Es existieren verschiedene Therapievorschläge, deren Erfolg jedoch eher auf spontaner Regression als auf echter Heilung beruht
(74). Bei starkem Verdacht auf eine spontan abheilende COPVEntzündung ist es durchaus vernünftig, als Erstes abzuwarten und
die Läsionen zu beobachten (87). Am effektivsten erweist sich ein
chirurgisches Vorgehen, besonders in Form von Kryochirurgie
und Lasertherapie, doch wurde die chirurgische Vorgehensweise
auch mit Resistenz und Rezidiven assoziiert (61). Zusätzlich gibt
es Protokolle für Behandlungen mit Retinoiden und Interferon,
über deren Wirkung bisher aber noch keine ausführlichen Studien
publiziert wurden (114, 115).
Beim Menschen, inklusive immunsupprimierten Patienten, werden vireninduzierte Neoplasien derzeitig mit Imiquimod und anderen Imidazoquinolonen therapiert (92). Diese Medikamente regen
die Toll-like-Rezeptoren an und führen so zur Freisetzung von Zytokinen vom Typ Th1 und von Interferon-α. Über die erfolgreiche Anwendung von Imiquimod beim Hund liegen nur wenige anekdotische Berichte vor.Aufgrund seiner spezifischen antiviralenWirkung
wird beim Menschen auch Cidofovir eingesetzt (79). Über einen
möglichen Einsatz bei Hunden ist noch nichts bekannt.
Aktuelle Fortschritte in der Prävention und der Immuntherapie
der humanen Papillomatose, speziell beim Papillomavireninduzierten zervikalen Karzinom der Frau, könnten als Basis für immuntherapeutische Behandlungen von PV-Infektionen dienen (61).
Bis dato wurde nicht über eine erfolgreiche Behandlung kaniner pigmentierter Plaques berichtet, doch kann eine spontane Regression auftreten. Zusätzlich empfiehlt es sich, die Ursache der
HUND/KATZE
Immunsuppression bei nicht prädisponierten Rassen zu identifizieren, da es bei deren Therapie zur Abheilung der Hautläsionen
kommen kann (96).
Die übrigen Formen kaniner und feliner PapillomavirenInfektionen sollten chirurgisch behandelt werden.
Pockenviren
Ätiologie
Pockenviren sind große DNA-Viren, die einen starken kutanen
Tropismus aufweisen. Bis dato wurden Pockenviren-Infektionen
bei zahlreichen Spezies beschrieben, einige davon scheinen zoonotisches Potenzial zu besitzen. Das Genus Orthopoxvirus umfasst verschiedene eng verwandte pathogene Erreger von Mensch
und Tier. Einige von diesen (z. B. das Kuhpockenvirus) sind in der
Lage, eine ganze Bandbreite an Spezies zu infizieren (Menschen,
Wiederkäuer, Fleischfresser und Nagetiere) (66). Hunde sind nur
selten betroffen. Bisher wurde auch noch kein hundespezifisches
Poxvirus identifiziert. Mehrere Studien konnten bei Füchsen
Anti-Orthopoxviren-Antikörper nachweisen, weshalb man auf
die Existenz eines an Karnivoren adaptierten Poxvirus schloss
(65, 69). Es sind nur zwei Fallberichte über Orthopoxviren-Infektionen beim Hund bekannt (88, 109).
Im Gegensatz zu Hunden gibt es bei Katzen in den meisten europäischen Ländern diverse Fallberichte über Infektionen mit
Kuhpockenviren (4, 10, 27, 37, 50, 76, 106, 108, 110). Auch in
Deutschland scheint die Inzidenz relativ hoch zu sein, und in Norwegen waren bis zu 10% der getesteten Katzen positiv (78, 108).
Über Zoonosen, die auf Kontakt mit einer infizierten Katze zurückzuführen waren, wurde sporadisch berichtet (17, 21, 47, 94).
Über eine Parapoxviren-Infektion (Orf, Ecthyma contagiosum) beim Hund findet sich nur ein einziger Fallbericht (116).
Pathogenität der Kuhpocken-Infektion
Wildlebende Nagetiere scheinen diesem Erreger als Reservoir zu
dienen und auch die meisten der infizierten Tiere halten vorher
Kontakt zu Nagern oder Wiederkäuern (4, 10, 27, 37, 50, 76, 106,
109, 110). Die Ansteckung erfolgt üblicherweise über den perkutanen oder oralen Weg (27). Nach der Infektion beginnt das
Virus mit der lokalen Replikation und produziert so eine primäre
„Pockenläsion“ (Abb. 9). Anschließend verteilt es sich via Lymphe und führt zu multiplen Veränderungen (27).
Klinik der Pockenviren-Infektion
Hund
Die mit Parapoxvirus infizierten Hunde wiesen sekundäre und
unspezifische Läsionen (Ulzerationen, Krusten) auf. Die Diagnose wurde mithilfe von histopathologischen Untersuchungen ge-
311
Abb. 9 Feline Kuhpocken-Dermatitis (Abb.: O. Fischer)
stellt (116). Zwei an einer Orthopoxviren-Infektion erkrankte
Hunde zeigten knotige und ulzerierende Veränderungen im Gesicht. Bei beiden wurde eine Infektion aufgrund eines Kontakts
mit infizierten Katzen oder Nagern vermutet. Bei einem Tier bildeten sich die Läsionen spontan zurück, beim anderen mussten sie
chirurgisch entfernt werden (91, 109).
Kuhpocken-Infektion bei Katzen
Die Primärläsion besteht aus einem einzelnen Knoten auf dem
Kopf, im Nacken oder an den Gliedmaßen. Bereits nach kurzer
Zeit treten Ulzerationen auf. Innerhalb von sieben bis 12 Tagen
entwickeln sich auf dem ganzen Körper sekundäre Papeln und
Knötchen (4, 10, 27, 37, 50, 76, 106, 109, 110). Auch diese ulzerieren in der Regel rasch. Gelegentlich können systemische Anzeichen wie Anorexie und Fieber beobachtet werden. Teilweise
treten Husten und Konjunktivitis als Begleiterscheinung auf. In
wenigen Fällen wurde eine Involvierung der Schleimhäute beschrieben (27). Gewöhnlich heilen die Läsionen innerhalb weniger Wochen ab. Bei immunsupprimierten, hauptsächlich FeLVoder FIV-infizierten Katzen sind Fälle tödlich verlaufender Pneumonien bekannt (4, 5). Die Wahrscheinlichkeit einer humanen Infektion ist gering. Trotzdem sollten die Besitzer einer infizierten
Katze über das Risiko, das für immunsupprimierte Personen besteht, informiert werden (47, 94).
Diagnose von Pockenviren-Infektionen
Üblicherweise reicht eine histologische Untersuchung von Hautbioptaten aus (76). Die typischen Kennzeichen umfassen eine hydropische Degeneration der Keratinozyten (inklusive derer der
Haarfollikel), Präsenz großer intrazytoplasmatischer Einschlusskörperchen, Mikrovesikelformation und eventuell Nekrose. Die
zytologische Untersuchung eines Feinnadelaspirats oder eines
Abklatschpräparats kann eventuell pathognomonisch sein (Präsenz intrazytoplasmatischer Einschlusskörperchen) (76). Zur
Bestätigung eines klinischen Verdachts kommen weiterhin Viruskulturen, serologische Tests und elektronenmikroskopische Untersuchungen in Betracht (76).
16
HUND/KATZE
312
Therapie
Eine spezifische Behandlung gibt es zwar nicht, doch heilen die
Läsionen heilen meist innerhalb weniger Wochen ab. Immunsupprimierte Tiere können allerdings systemische Anzeichen entwickeln. Hin und wieder ist eine Behandlung sekundärer bakterieller Infektionen notwendig.
Herpesviren-Infektionen
Ätiologie
Diese Erreger gehören zu den Doppelstrang-DNA-Viren, die bei
zahlreichen Spezies die verschiedensten Erkrankungen hervorrufen können. Herpesviren zeigen einen Tropismus für Nervengewebe, Haut, lymphatische Zellen und respiratorisches Epithel sowie für Epithelien der Genitalregionen (81). Das Charakteristikum aller bekannten Herpesviren ist ihre Eigenschaft, latent in ihren Wirten zu verharren. In Zellen, die Viren im Latenzstadium
beherbergen, werden nur wenige virale Gene exprimiert. Solche
Viren behalten ihre Replikationsfähigkeit bei, wodurch es bei einer Reaktivierung zum Rezidiv kommen kann (81). Bei kaninen
und felinen Herpesviren handelt es sich um α-Herpesviren. Beide
weisen große genetische Homologien auf (11). Infizierte Hündinnen sind normalerweise asymptomatisch, doch kann es während
der Trächtigkeit oder durch Immunsuppression zu einer Reaktivierung der latenten Infektion kommen. In der Folge wird das Virus über nasale, genitale, okuläre und orale Sekretion freigesetzt
(11). Die primäre Replikation bei infizierten Welpen geschieht in
den Epithelzellen der oronasalen Mukosa. Anschließend erfolgt
die virämische Phase (in Makrophagen). Die Mehrheit der betroffenen Welpen, die durch maternale Antikörper geschützt werden,
überlebt, bleibt aber chronisch infiziert. Bei ungeschützten Welpen werden durch das Virus nach und nach verschiedene Organe
(z. B. Lymphknoten, Milz, Niere, Leber und Lungen) befallen.
Die meisten Hunde überleben eine solche Infektion nicht (11).
Infektionen mit dem felinen Herpesvirus (felines Rhinotracheitisvirus) geschehen durch den direkten Kontakt mit akut infizierten Tieren, mit latent infizierten Katzen während einer Reaktivierungsphase oder über die kontaminierte Umgebung. Die Infektionswege sind üblicherweise oral, nasal oder okulär und die
Replikation erfolgt in der Mukosa der Nase und derTonsillen. Das
Auftreten einer Virämie ist selten. Bei latent infizierten Tieren
zieht sich das Virus in die Ganglien des Nervus trigeminus und
auch in epitheliale Gewebe zurück (28).
Kanine Herpesvirus-Infektion auf Haut und
Schleimhaut
Kanine Herpesviren zeigen einen ausgeprägten genitalen Tropismus. Nebst Abort, Unfruchtbarkeit und letalen neonatalen Septikämien können Herpesviren auch vesikulopapilläre Läsionen an
den Genitalien, am Abdomen und auf der Maul- und Genitalschleimhaut hervorrufen (11, 46, 49).
Aujeszky'sche Krankheit/Pseudotollwut
Die Aujeszky’sche Krankheit wird durch ein um ein α-Herpesvirus verursacht, das einen Tropismus für Nervengewebe aufweist.
Das Hauptreservoir dieses Virus sind Schweine. Hunde infizieren
sich durch denVerzehr von kontaminiertem Schweinefleisch (86).
Der Großteil der erkrankten Hunde weist einen intensiven Juckreiz im Gesicht auf, wobei es zu schweren selbst induzierten Läsionen kommt (51, 68). Zusätzliche Symptome sind Speicheln
und neurologische Ausfälle. Die Krankheit führt unweigerlich
zum Tod.
Feline Herpesviren-Infektionen
Das feline Herpesvirus 1 (FHV-1) verursacht bei Katzen Rhinotracheitis und Konjunktivitis (28). Nach einer Inkubationsperiode
von üblicherweise weniger als einer Woche entwickeln die betroffenen Katzen eine schwere Apathie, Fieber, Augen- und Nasenausfluss. In der Folge treten Konjunktivitis und Rhinotracheitis
auf (28). Im Gegensatz zu Calicivirus-Infektionen kommt es selten zu oralen Ulzerationen (24). Es wurden auch faziale erosive
Dermatitiden beschrieben (Abb. 10) (25, 43). Bei durch FHV-1
bedingten Hauterkrankungen treten Vesikel, Erosionen, Ulzerationen, Krusten und Stomatitis auf. Histopathologisch ist neben
den ulzerösen und verkrusteten Läsionen eine ausgeprägte eosinophile dermale Infiltration zu erkennen. Einige Keratinozyten
weisen intranukleäre Einschlüsse auf (43, 44). Die Diagnose wird
üblicherweise mittels Immunhistochemie und/oder PCR-Amplifikation der viralen DNA gestellt (97). Ebenso ist eine kulturelle
Anzüchtung des Virus möglich (28).
Abb. 10 Feline Herpesvirus-Dermatitis (Abb.: L. Beco)
17
Felines Erythema multiforme bedingt durch
Herpesviren
Erythema multiforme ist eine immunologische Antwort auf die
Präsenz von Fremdantigen in der Epidermis. Es tritt beim Menschen in der Regel wenige Tage nach einer Infektion mit Herpessimplex-Viren auf (54, 113). Bei Katzen wurden insgesamt zwei
Fälle publiziert (Abb. 11) (77, 80). Ein paar Tage nach einer Rhinotracheitis entwickelten sich kutane Veränderungen in Form einer exfoliativen Dermatose. Histologische Untersuchungen zeigten eine typische lymphozytäre Interface-Dermatitis, lymphozytäre Exozytose, Apoptose und Satellitose. Im einen Fall führte
eine Azyklovir-Therapie zu einer klinischen Verbesserung, im
anderen kam es zu einer Spontanheilung (77, 80).
Retroviren-Infektionen
Ätiologie
Diese häufigen Erkrankungen bei Katzen werden vor allem durch
zwei Viren verursacht: das feline Leukämievirus (FeLV, Genus
Gammaretrovirus) und das feline Immundefizienzvirus (FIV, Genus Lentivirus). FIV induziert eine Immundefizienz. Die meisten
der FIV-assoziierten Hautveränderungen sind jedoch nicht spezifisch, weshalb diese Krankheit im vorliegenden Artikel nur kurz
erwähnt wird. Im Gegensatz dazu wurde in der veterinärmedizinischen Literatur häufig über FeLV-assoziierte Dermatosen und
Hauttumoren berichtet. Die meisten der folgenden Aussagen beziehen sich auf das FeLV-Virus.
Infektion
Die Übertragung des felinen Leukämievirus geschieht primär
über den Speichel. Andere Körperflüssigkeiten können ebenfalls
kontaminiert sein. Bei Katzenwelpen besteht zudem die Möglichkeit einer transplazentaren Infektion (16). Die ersten Symptome
sind Fieber und Lymphadenopathie. Schafft es das Immunsystem
HUND/KATZE
Impfungen haben zu einer drastischen Abnahme der Prävalenz und der Schwere der felinen Herpesvirus-Infektion beigetragen.Trotzdem kann es zu Infektionen bei Katzenwelpen kommen,
wenn sie entweder von einer Katze ohne Herpesvirus-spezifische
Immunität geboren werden oder vor ihrer ersten Impfung keine
maternalen Antikörper mehr haben (28). In Fällen akuter Infektion oder der Reaktivierung einer latenten Infektion sind
Antibiotika zur Kontrolle der sekundären bakteriellen Infektion
hilfreich. Antivirale Medikamente wie Azyklovir, Ganzyklovir,
Penzyklovir oder Cidofivir sowie dieTherapie mit α- und ω-Interferon, L-Lysin und bovines Laktoferrin zeigten ihre Wirkung in
In-vitro- oder in limitierten experimentellen Studien (3, 38, 64,
82, 83, 95).
313
Abb. 11 Felines Herpesvirus-induziertes Erythema multiforme
(Abb.: C. Prost)
nicht, diese Infektion zu beseitigen, kann das Virus das Knochenmark befallen und dort persistieren. Das betroffene Tier wird
in der Folge über längere Zeit transient, permanent oder gar
nicht virämisch, abhängig von der Reaktion des Immunsystems
(16). Über die fokale Replikation bei virämischen oder aber auch
bei nichtvirämischen Katzen in Milz, Knochenmark, Lymphknoten, Haut oder Dünndarm gibt es diverse Publikationen (24,
43, 58).
Klinik (FeLV)
Über FeLV-induziertes Lymphom, Leukämie und Knochenmarksuppression liegen ausführliche Berichte vor (16). Im vorliegenden Artikel werden diese Erkrankungen nicht näher besprochen.
Retroviren-assoziierte Hautveränderungen bei
Katzen
Kutanes Lymphom. Bei bis zu 75% der Lymphome von Katzen
konnte FeLV-Antigen oder -DNA nachgewiesen werden (56).
Auch kutane T-Zell-Lymphome sind häufig FeLV-positiv (56, 87,
107). Interessanterweise gibt es auch Berichte über FeLV-positive
Neoplasien bei serologisch FeLV-negativen Katzen (107). Da zumindest bei einem Teil dieser Tumoren virales Kapsidantigen entdeckt wurde, darf eine lokale kutane Replikation des Virus vermutet werden (24, 48). Latenten oder nicht produktive Infektionen können jedoch nicht ausgeschlossen werden (107). An kutanen Lymphomen erkranken normalerweise ältere Katzen. Die Läsionen können solitär oder multizentrisch auftreten (87, 107). Die
klinische Symptomatik ist sehr variabel und umfasst erythematöse Plaques, Knoten und Ulzerationen (Abb. 12). Häufig treten
auch systemische Symptome auf. Histologisch werden diese Neoplasien weiter in epitheliotrope und nichtepitheliotrope T- und
B-Zell-Lymphome unterteilt.
18
Hautveränderungen assoziiert mit hoher Rate an
FeLV/FIV-positiven serologischen Befunden
HUND/KATZE
314
Plasmazell-Pododermatitis. Bei dieser sehr schmerzhaften Erkrankung sind ein oder mehrere metakarpale oder metatarsale
Pfotenballen betroffen. Neben einer Schwellung treten mit der
Zeit auch Ulzera auf. Die Diagnose wird mittels zytologischer
oder histologischer Untersuchung gestellt. Häufig sind FIV-positive Katzen betroffen (39, 88). Die Ursache ist bis heute unbekannt. Glukokortikoide, Doxycyclin (10 mg/kg KM/Tag) und
auch chirurgische Eingriffe werden als Therapiemöglichkeiten
vorgeschlagen (19, 39, 40, 84).
Rezidivierende Plasmazell-Polychondritis. Hierbei handelt
es sich um eine sehr seltene Krankheit, die durch eine asymmetrische Schwellung der Pinna, gefolgt von einer permanenten Eindrehung oder Deformation des Ohrknorpels charakterisiert wird
(8, 29, 88). Auch diese Plasmazell-Polychondritis tritt häufig bei
FeLV- oder FIV-positiven Katzen auf.
Abb. 12 FeLV-induziertes kutanes Lymphom
Hautveränderungen assoziiert mit Immundefizienz
FIV-positive Katzen leiden häufig unter verschiedenen infektiösen Krankheiten wie zum Beispiel Abszesse, Pyodermien, Dermatophytosen, Kryptokokkose und Demodikose (86).
Diagnose retroviraler Infektionen
Die Diagnose einer FeLV-Infektion beruht auf dem Nachweis des
FeLV-Kapsidproteins p27, normalerweise mittels ELISA. Virämische Katzen weisen ein positives Testergebnis auf. Bei transient
virämischen oder latent infizierten Tieren kann das Resultat
falsch negativ sein (16). Zusätzlich besteht die Möglichkeit, mit
Gewebe von Katzen, einschließlich des vorher formalinfixierten
und paraffinisierten, eine PCR durchzuführen (16).
Abb. 13 FeLV-induzierte Riesenzell-Dermatitis
Prävention und Therapie einer FeLV-Infektion
Riesenzell-Dermatitis. Eine Riesenzell-Dermatitis wurde
bei verschiedenen an FeLV erkrankten Katzen beschrieben (22,
36). Klinisch lagen Erosionen, Ulzerationen, Krusten, Schuppen
und Alopezie vor (Abb. 13). Die Tiere zeigten Juckreiz, systemische Symptome (Fieber, Anorexie, Apathie) und auch hämatologische Veränderungen (Anämie).Alle betroffenen Katzen wurden
euthanasiert oder starben innerhalb weniger Wochen. Das Auffälligste der histologischen Untersuchung waren ein KeratinozytenSynzytium und eine Dyskeratose. Bei allen beschriebenen Fällen
konnte FeLV-Antigen oder -DNA nachgewiesen werden. Zurzeit
ist noch ungeklärt, ob es sich bei dieser Synzytium-Formation um
eine neoplastische Anordnung handelt.
Kutane Hörner. Im Zusammenhang mit FeLV-Infektionen
wurden auch kutane Hörner beschrieben. In der darunter liegenden Epidermis konnte virales Antigen nachgewiesen werden
(86).
19
Zur Prävention einer FeLV-Infektion wurden verschiedene wirksame Impfstoffe entwickelt. Bei allen handelt es sich um Totoder Vektorvakzinen (basiert auf genetisch entwickeltem Protein
gp70).
Zur Behandlung von FeLV-induzierten Lymphomen und
FeLV-induzierter Knochenmarksuppression existieren verschiedene Arbeiten, auf die im vorliegenden Artikel nicht weiter eingegangen wird.
Staupe und “Hard Pad Disease”
Ätiologie und Pathogenese
Staupe wird durch ein RNA-Virus der Familie Paramyxoviridae
verursacht (70). Der breite Einsatz von wirksamen Impfprogrammen hat zu einer drastischen Reduktion des Auftretens dieser
Krankheit geführt. Nach wie vor ist es jedoch wichtig, auch die
kutanen Manifestationen dieser Infektion zu erkennen.
Die Ansteckung erfolgt in der Regel über den Respirationstrakt mit einer ersten viralen Replikation in den Lymphknoten der
oberen Atemwege und den Tonsillen (70). Die Inkubation dauert
zirka eine Woche, wobei es zu einer Verteilung des Virus in den
gesamten primären und sekundären lymphatischen Organen sowie im Verdauungstrakt kommt (70). Der weitere Verlauf ist von
der Synthese neutralisierender Antikörper abhängig. Bei deren
Vorhandensein kann der Hund die Infektion kontrollieren, ohne
dass Symptome auftreten (71). Fehlen diese Antikörper, kann das
Virus sämtliche epitheliale Gewebe befallen: Respirationstrakt,
Verdauungstrakt, Harnwege, Augen und Haut. Erst kürzlich ließ
sich nachweisen, dass eine transiente Infektion der basalen Keratinozyten des Epithels für eine vorübergehende Proliferierung
von Keratinozyten verantwortlich ist (33, 35). Der Befall von Nerven erfolgt später, jedoch nicht immer. Neurologische Symptome
sind somit eine Spätfolge der Staupe (70).
Abb. 14 Staupe beim Hund: nasale Hyperkeratose
Klinik
Die akute und subakute Form der Staupe ist durch Fieber, Apathie,
Anorexie, Dehydratation, respiratorische Symptome (Husten, Bronchitis, Bronchopneumonie), gastrointestinale Symptome (Erbrechen, Durchfall), okuläre Symptome (Konjunktivitis, Iridozyklitis)
und eventuell auch durch neurologische Symptome (Verhaltensänderungen, Krämpfe) gekennzeichnet (71). Bei Welpen kann sich
die Infektion in Form eines Impetigo manifestieren. In vielen Fällen
werden Papeln und Pusteln entdeckt; ein direkter Zusammenhang
oder ein viraler zytopathischer Effekt konnte bisher nicht nachgewiesen werden. Mittels immunologischer Techniken wurde vor
kurzem bestätigt, dass das Virus nahezu bei allen an Staupe erkrankten Hunden auch in der Haut vorkommt (41). Nach einiger Zeit, mit
oder ohne Vorgeschichte von Staupe, können bei manchen Tieren
neurologische („Old Dog Encephalitis“) und/oder dermatologische
Symptome auftreten (71, 86). Bei den Hautveränderungen handelt
es sich um Hyperkeratose des Planum nasale (Abb. 14) und der Pfotenballen (Hard Pad Disease, Abb. 15) (86). „Hard Pad Disease“ tritt
auch bei Hunden auf, die bei einer Infektion mit dem Staupevirus
über einen unvollständigen Impfschutz verfügen.
Diagnose
Ohne passende Anamnese kann die „Hard Pad Disease“ als Autoimmunkrankheit (Pemphigus, Lupus), Malnutrition (zinkresponsive Dermatitis), kongenitale Erkrankung (familiäre idiopathische nasodigitale Hyperkeratose), Papillomatose oder hepatokutanes Syndrom fehldiagnostiziert werden. Histopathologisch erkennt man eine deutliche ortho- oder parakeratotische Hyperkeratose des Pfotenballens und des Planum nasale mit azidophilen zytoplasmatischen und nur selten intranukleären Einschlüssen von
variabler Größe und Form (Lentz Bodies) in den Keratinozyten
auf follikulärer Ebene. Teilweise können auch synzytiale Riesenzellen beobachtet werden (119). Da virale Einschlüsse nicht im-
315
HUND/KATZE
Abb. 15 Staupe beim Hund: Hard Pad Disease
mer vorhanden sind, werden auch andere Techniken wie RT-PCR
zur Diagnosestellung herangezogen (26, 57).
Staupeimpfungen
Da im Fall einer akuten Infektion keine effektive Behandlung
existiert und nur die Möglichkeit einer symptomatischen Therapie besteht, ist eine prophylaktische Impfung unumgänglich. Experimentelle Studien zeigten, dass eine Vakzinationen mit inaktivierten Viren den Hund nicht ausreichend schützen kann. Deshalb
werden in den zurzeit verwendeten Produkten nur die Hüllproteine verwendet. Maternale Antikörper, die transplazentar oder mit
dem Kolostrum vom Welpen aufgenommen werden, verhindern
eine Immunisierung. Im Allgemeinen wird ein Welpe zur Grundimmunisierung zwischen der sechsten und 16. Lebenswoche
zweimal im Abstand von vier Wochen geimpft.
Parvovirose
Bei einer zweimonatigen Dogge wurde im Zusammenhang mit
Parvovirose vom Auftreten eines Erythema multiforme (EM) be-
20
HUND/KATZE
316
Abb. 16
Kutane Parvovirose:
Ulkus am Pfotenballen
richtet (23). Bei EM werden die Antigene der Keratinozyten modifiziert, worauf durch eine anschließende Lymphozyteninfiltration eine selektive Apoptose induziert wird (1, 55). Neben den
klassischen Symptomen einer Parvovirose zeigte der Welpe vesikulopapuläre kutane und mukokutane Veränderungen im Gesicht,
Maul, an derVulva, an der ventralen Körperhälfte, den Pfoten (mit
fokalen Ulzerationen an den Pfotenballen) und über den Knochenvorsprüngen (Abb. 16). Erythematöse Plaques waren am
Kinn und Abdomen sichtbar (23). Da der Hund nicht überlebte,
konnte die Entwicklung der Hautveränderungen nicht weiter beobachtet werden. Histopathologisch wurden eine regenerative nekrotisierende Enteritis, vereinbar mit Parvovirose, und kutane Läsionen ähnlich einem Erythema multiforme major beschrieben.
Die Hautveränderungen setzten sich aus einer lymphozytären Interface-Dermatitis, Apoptose und Satellitose, verteilt über die gesamte Epidermis, sowie aus apoptotischen Keratinozyten mit intranukleären basophilen viralen Einschlüssen zusammen. Die immunhistochemische Untersuchung bestätigte das Vorhandensein
von kaninem Parvovirus CPV-2 in der Haut und in den intestinalen Drüsen.
Danksagung
Wir danken Ingrid Wilhelm-Dotter herzlich für die Durchsicht des Manuskripts.
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112. Watrach AM, Small E, Case MT. Canine papilloma: progression of oral papilloma to carcinoma. J Nat Cancerol Instit 1970; 45: 915–20.
113. Weston WL, Morelli JG. Herpes simplex virus-associated erythema multiforme in prepubertal children. Arch Pediatr Adolesc Med 1997; 151 (10):
1014–6.
114. White SD. Newly introduced drugs in veterinary dermatology. In: Proc
World Congress Vet Dermatol, Edinborough 1996.
115. White SD, Rosychuk RA, Scott KV, Trettien AL, Jonas L, Denerolle P. Use
of isotretinoin and etretinate for the treatment of benign cutaneous neoplasia and cutaneous lymphoma in dogs. J Am Vet Med Assoc 1993; 202 (3):
387–91.
116. Wilkinson GT, Prydie J, Scarnell J. Possible „orf“ (contagious pustular dermatitis, contagious ecthyma of sheep) infection in the dog.Vet Rec 1970; 87
(25): 766–7.
117. Withrow SJ, Mac Ewen EG. Viral papillomatosis. In: Clinical Veterinary
Oncology. Withrow SJ, Mac Ewen EG, eds. Philadelphia: Lippincott 1989.
118. Yager JA, Wilcock BP. Squamous papilloma. In: Color Atlas and Text of
Surgical Pathology of the Dog and Cat. Dermatopathology and Skin Tumors.
Yager JA, Wilcock BP, eds. London: Wolfe 1994; 251–2.
119. Yager JA, Wilcock BP. Perivascular dermatitis with diffuse orthokeratotic
hyperkeratosis. In: Color Atlas and Text of Surgical Pathology of the Dog
and Cat. Dermatopathology and Skin Tumors. Yager JA, Wilcock BP, eds.
London: Wolfe 1994; 66–70.
Claude Favrot, DVM, MsSc, Dip. ECVD
Klinik für Kleintiermedizin, Dermatologie
Vetsuisse Fakultät, Universität Zürich
Winterthurerstrasse 260
CH-8057 Zürich
E-Mail: [email protected]
Chapter 3
Parvovirus Infection of Keratinocytes as a Cause of Canine
Erythema Multiforme
C. Favrot1, T. Olivry2, S. M. Dunston2, F. DegorceRubiales3 and J. S. Guy2
Veterinary Pathology, 2000: 37 (6): 647-649
1
2
Clinique Vétérinaire de Ferrette, Ferrette, Haut-Rhin, France
Department of Clinical Sciences, College of Veterinary Medicine, North Carolina
State University, Raleigh, NC, USA
3
LAPVSO, Toulouse, France
24
Vet Pathol 37:647–649 (2000)
BRIEF COMMUNICATIONS AND CASE REPORTS
Parvovirus Infection of Keratinocytes as a Cause of
Canine Erythema Multiforme
C. Favrot, T. Olivry, S. M. Dunston,
F. Degorce-Rubiales AND J. S. Guy
Abstract. Erythema multiforme major was diagnosed in a dog with necrotizing parvoviral enteritis. Skin
lesions consisted of ulceration of the footpads, pressure points, mouth, and vaginal mucosa; vesicles in the oral
cavity; and erythematous patches on the abdomen and perivulvar skin. Microscopic examination of mucosal
and haired skin specimens revealed lymphocyte-associated keratinocyte apoptosis at various levels of the epidermis. Basophilic cytoplasmic inclusions were seen in basal and suprabasal keratinocytes. Immunohistochemical staining, performed with canine parvovirus-2–specific monoclonal antibodies, confirmed the parvovirus
nature of the inclusions in the nucleus and cytoplasm of oral and skin epithelial cells. This is the first case of
canine erythema multiforme reported to be caused by a viral infection of keratinocytes. This case study indicates
that the search for epitheliotropic viruses should be attempted in cases of erythema multiforme in which a drug
cause cannot be identified.
Key words:
Canine parvovirus-2 (CPV-2); dog; immunology; infection; skin; virus.
In humans, the classification of erythema multiforme
(EM) variants recently has been revised with an emphasis
on clinical manifestations.2 The relevance of this modified
clinical nosology has been supported by subsequent epidemiologic and pathologic case studies. Dermatoses described
clinically as EM minor and major most commonly seem to
be caused by viral infections leading to lymphocyte-mediated keratinocyte apoptosis.1,3,6,7 Human EM generally is
caused by herpes simplex virus,3,6,7 but it also can be triggered by other infectious agents such as parvovirus B19.8
In 1998, the consensus clinical classification used for human EM was adapted to the canine species.4 That case study
established that, in contrast to previous reports,10 canine cases of EM (minor or major) rarely were associated with previous drug exposure.4 In non–drug-related cases, offending
causes could not be determined but a viral etiology was considered plausible. The purpose of the present paper is to
describe a canine case of EM major in which parvovirus
infection of epidermal and mucosal keratinocytes led to lymphocyte-associated apoptosis and clinical signs of EM major.
A 2-month-old female Great Dane puppy was presented,
3 days after adoption, with acute-onset diarrhea, vomiting,
dehydration, and skin lesions. Because parvovirus enteritis
had been diagnosed recently at the facility of the dog’s
breeder, parvovirus was suspected as the cause of diarrhea.
However, 6 days before the initial presentation, the dog had
received a tetravalent vaccine (distemper, parvovirus, parainfluenza, and hepatitis). Dermatologic examination revealed well-demarcated ulceration of the footpads (Fig. 1)
and pressure points, as well as mouth and vaginal mucosae.
Vesicles were seen in the oral cavity. Erythematous patches
were present on the abdomen and chin. In spite of fluid therapy and intravenous cephalexin and metoclopramide, the
dog died 2 days after presentation.
A necropsy was performed and necrotic lesions were seen
throughout all intestinal sections. Histopathologic analysis of
small intestine specimens consisted of severe segmental necrotizing enteritis suggestive of an acute infection due to canine parvovirus-2 (CPV-2). Viral inclusions were not identified in the intestinal specimens, presumably because of the
severe necrosis of the digestive epithelium. Skin biopsy
Fig. 1
ent.
647
25
Skin, footpad; dog. A sharp-edged ulcer is pres-
648
Brief Communications and Case Reports
Vet Pathol 37:6, 2000
Fig. 2 Haired skin; dog. Clusters of lymphocytes (white arrowheads) are located in the immediate vicinity of apoptotic
keratinocytes (black arrows). Viral inclusions are visible in an intracellular vacuole (black arrowhead). H.E. Scale bar 5
18 mm.
Fig. 3 Haired skin; dog. A lymphocyte (white arrowhead) is situated near an apoptotic keratinocyte (black arrow) that
contained viral inclusions (black arrowheads). H.E. Scale bar 5 4 mm.
Fig. 4 Haired skin, abdomen; dog. Dark-staining viral inclusions fill the cytoplasm of basal and juxtabasal keratinocytes.
Viral aggregates are occasionally present in the superficial dermis. Small inclusions are visible within keratinocyte nuclei
(black arrow). Avidin–biotin–peroxidase immunohistochemistry, aminoethylcarbazole chromogen, hematoxylin counterstain, CPV2c2A parvovirus-specific monoclonal antibodies. Scale bar 5 11mm.
specimens were obtained from lesional skin and oral mucosa. Focal mononuclear interface gingivitis was identified
in biopsy samples collected from the gum. Additionally, confluent basal keratinocyte vacuolation progressing to vesiculation with epithelial ulceration and neutrophil accumulation
was observed. Prominent lymphocyte exocytosis was present
in preblistered mucosal epithelium. Keratinocyte apoptosis,
often in close contact with lymphocytes (e.g., satellitosis),
was observed at all levels of the epithelium. In some specimens, basophilic inclusions were observed in the cytoplasm
of basal and suprabasal keratinocytes. Examination of haired
skin specimens revealed varying degrees of the same pathologic process. The epidermis exhibited focal hyperplasia,
crusting, and erosion. Lymphocyte exocytosis and keratino-
cyte apoptosis with satellitosis were restricted to sites of epidermal hyperplasia (Figs. 2, 3). Numerous basophilic cytoplasmic inclusions were seen in the lower third of the hyperplastic epidermis (Figs. 2, 3).
To verify the viral origin of cytoplasmic inclusions, a
three-step avidin–biotin–peroxidase immunohistochemical
technique was performed as previously described.9 Immunostaining of paraffin-embedded sections was done with two
monoclonal antibodies specific for CPV-2 (CPV2c2A and
CPV103B10A, 1 2,000 dilution, Mérial, Lyon, France). Examination of negative controls, consisting of sections immunostained with irrelevant monoclonal antibodies, was unremarkable. In mucosal specimens, multiple intracellular
parvovirus inclusions were seen throughout the epithelium.
26
Vet Pathol 37:6, 2000
Brief Communications and Case Reports
In haired skin samples, parvovirus inclusions were seen most
commonly coalescing in basal and juxtabasal keratinocytes
of hyperplastic epidermis (Fig. 4). The smallest viral inclusions were identified in the keratinocyte’s nucleus (Fig. 4).
Inclusions further aggregated and filled-up the cytoplasm of
epithelial cells leading to displacement of the nucleus to the
cell’s periphery and subsequent cell degeneration. Immunostaining of digestive specimens similarly demonstrated CPV2 particles in the epithelial crypts of the small intestine. Furthermore, viral inclusions of skin and mucosal sections were
negative when immunohistochemistry was performed using
monoclonal antibodies specific for either distemper virus (1 :
50, Mérial, Lyon, France) or papillomavirus-group–specific
antigens (AR087–5R, undiluted, Biogenex, San Ramon,
CA).
According to the recently proposed classification of canine
EM, the skin lesions exhibited by this patient fit the criteria
for a clinical diagnosis of EM major (erythematous patchy
lesions with ulcerations on less than 10% of the body surface
and with more than one mucosa affected).4 Our histologic
and immunohistochemical investigations suggested CPV-2
infection of mucosal and epidermal keratinocytes as the primary cause of EM in this dog. Remarkably, most parvoviral
inclusions were identified in the cytoplasm of keratinocytes,
whereas only rare viral particles were seen in cell nuclei.
However, these observations are identical to those described
in glossal specimens of dogs naturally infected with CPV2.5 Indeed, viral replication initially occurs in the nucleus
but large virion clusters appear as cytoplasmic aggregates.
However, these inclusions still are surrounded by the nuclear
membrane and should be referred to as pseudocytoplasmic.5
A viral infection of keratinocytes suggests a logical pathogenesis of EM lesions in this dog. We hypothesize that an
infection of stem cells and transient amplifying keratinocytes
most likely occurred following hematogenic dissemination
of the parvovirus. Viral peptides could be presented by class
I major histocompatibility complex molecules at the surface
of epithelial cells. Recognition of viral antigens by T-lymphocytes, possibly sensitized by the previous parvovirus
vaccination, would trigger these cytotoxic cells to induce the
apoptosis of virus-infected keratinocytes.
The present case study supports the concept that a viral
etiology is possible in some forms of canine EM. We propose that a search for epitheliotropic viruses (e.g., distemper,
papilloma-viruses, parvoviruses, and herpesviruses) should
be attempted in cases of canine EM in which a causative
drug cannot be clearly established.
649
Lyon, France) for providing distemper- and parvovirus-specific monoclonal antibodies.
References
ACKNOWLEDGEMENTS
1 Assier H, Bastuji-Garin S, Revuz J, Roujeau JC: Erythema multiforme with mucous membrane involvement and
Stevens–Johnson syndrome are clinically different disorders with distinct causes. Arch Dermatol 131:539–543,
1995
2 Bastuji-Garin S, Rzany B, Stern RS, Shear NH, Naldi L,
Roujeau J-C: Clinical classification of cases of toxic epidermal necrolysis, Stevens–Johnson syndrome and erythema multiforme. Arch Dermatol 129:92–96, 1993
3 Brice SL, Leahy MA, Ong L, Krecji S, Stockert SS, Huff
JC, Weston WL: Examination of non-involved skin, previously involved skin, and peripheral blood for herpes
simplex virus DNA in patients with recurrent herpesassociated erythema multiforme. J Cutan Pathol 21:408–
412, 1994
4 Hinn AC, Olivry T, Luther PB, Cannon AG, Yager JA:
Erythema multiforme, Stevens–Johnson syndrome and
toxic epidermal necrolysis in the dog: clinical classification, drug exposure and histopathological correlations.
J Vet Allergy Clin Immunol 6:13–20, 1998
5 Hullinger GA, Hines ME, Styer EL, Frazier KS, Baldwin
CA: Pseudocytoplasmic inclusions in tongue epithelium
of dogs with canine parvovirus-2 infections. J Vet Diagn
Invest 10:108–111, 1998
6 Imafuku S, Kokuba H, Aurelian L, Burnett J: Expression
of herpes simplex virus DNA fragments located in epidermal keratinocytes and germinative cells is associated
with the development of erythema multiforme lesions. J
Invest Dermatol 109:550–556, 1997
7 Kokuba H, Imafuku S, Aurelian L, Burnett JW: Erythema multiforme lesions are associated with expression of
a herpes simplex virus (HSV) gene and qualitative alterations in the HSV-specific T-cell response. Br J Dermatol
138:952–964, 1998
8 Lobkowicz F, Ring J, Schwarz TF, Roggendorf M: Erythema multiforme in a patient with acute human parvovirus B19 infection. J Am Acad Dermatol 20:849–850,
1989
9 Olivry T, Moore PF, Naydan DK, Puget BJ, Affolter VK,
Kline AE: Antifollicular cell-mediated and humoral immunity in canine alopecia areata. Vet Dermatol 7:67–79,
1996
10 Scott DW, Miller WH: Erythema multiforme in dogs and
cats: literature review and case material from the Cornell
University College of Veterinary Medicine (1988–1996).
Vet Dermatol 10:297–309, 1999
We thank Dr. Guaguère for his comments in the initial
phase of the study and Drs. Latour and Soulier (Mérial,
Request for Reprints from Dr. C. Favrot, Clinique Vétérinaire, 32 rue de Mulhouse, F-68300 St. Louis (France).
27
Chapter 4
C. Favrot1, S. Wilhelm1, P. Grest2, M. L. Meli3, R.
Hofmann-Lehmann3 and A. Kipar4
Veterinary Dermatology, 2005, 16:407-412
1
Clinic for Small Animal Internal Medicine, Dermatology Unit, Vetsuisse Faculty,
University of Zurich, Zurich, Switzerland
2
Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Zurich,
Switzerland
3
Clinical Laboratory, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland
4
Department of Veterinary Pathology, Faculty of Veterinary Science, University of
Liverpool, Liverpool, UK
28
Veterinary Dermatology 2005, 16, 407– 412
Case report
Blackwell Publishing Ltd
C. FAVROT*, S. WILHELM*, P. GREST†, M. L. MELI§, R. HOFMANN-LEHMANN§
and A. KIPAR‡
*Clinic for Small Animal Internal Medicine, Dermatology Unit, †Institute of Veterinary Pathology,
and §Clinical Laboratory, Vetsuisse Faculty, University of Zürich, Zürich, Switzerland, ‡Department
of Veterinary Pathology, Faculty of Veterinary Science, University of Liverpool, Liverpool, UK
(Received 25 July 2005; accepted 10 July 2005)
Abstract Two cases of feline leukaemia virus (FeLV)-associated dermatosis are described. The first cat was
affected by an ulcerative dermatitis identified as a giant-cell dermatosis. The second case was a cutaneous
lymphoma. In both cases, FeLV antigens and FeLV genome were demonstrated in the affected skin immunologically
and with polymerase chain reaction, respectively. The first case suggests that, like other retroviruses, at least some
strains of FeLV can induce syncytium formation. As FeLV antigens and genome were demonstrated in a
serologically negative cat, the second case suggests that focal skin FeLV replication may occur. FeLV-associated
dermatoses are rare skin conditions that may be under-diagnosed.
I N T RO D U C T I O N
M AT E R I A L A N D M E T H O D S
Feline leukaemia virus (FeLV), a member of the oncornavirus subfamily of retroviruses, occurs worldwide
and replicates in many tissues including respiratory
epithelium, salivary gland and bone marrow.1 It causes
approximately one-third of feline lethal cancers and
numerous cats die of anaemia or infectious diseases
as a consequence of the immunosuppressive effects of
the virus.1 FeLV infection has also been associated with
numerous infectious dermatoses of fungal, parasitic
and/or bacterial aetiology.2 A direct cytopathic effect
of the virus in the skin, however, has been rarely
demonstrated, but is associated with two different
syndromes: giant-cell dermatosis and epidermal horns.3,4
Lymphoma accounts for about 90% of the haematopoietic tumours in cats and is often a consequence of
FeLV infection.5 Cutaneous lymphomas, however, are
rare and usually occur in older FeLV-negative cats.5
The purpose of this article is to present two new
cases of FeLV-induced dermatoses with evidence of
viral antigens and proviral sequences in the skin: one
of T-cell lymphoma in a serologically negative cat and
one case of giant-cell dermatosis in a serologically
positive cat.
Animals
Two castrated domestic indoor–outdoor male cats
(the first aged 3 and the second aged 15 years), in
reduced general condition and with skin plaques and
ulcerations, were presented at the Clinic for Small
Animal Internal Medicine, Dermatology Unit, Vetsuisse
Faculty, Zürich. Both cats were given clinical and
dermatological examinations.
Serological examination
The presence of plasma FeLV p27 antigen as a measure
for viraemia was determined using double-antibody
sandwich enzyme-linked immunosorbent assay (ELISA)
as previously described.6
Plasma samples were tested for feline immunodeficiency virus (FIV) by ELISA, measuring antibodies
against the FIV transmembrane protein.7
Histological and immunohistological examination
Skin samples for histopathological examination
were taken (during the first examination of both cats) by
biopsy from the lesions with a 6-mm skin punch, fixed
in 4% neutral buffered formalin and embedded in
paraffin wax. Sections were cut and either stained with
haematoxylin and eosin or used for immunohistological
examination.
Skin lesions were examined immunohistologically
for FeLV antigens using a cocktail of mouse monoclonal
antibodies against the FeLV envelope protein gp70
and the group-specific protein p27 (clones C11D82i
and PF12J-10A; Custom Monoclonals, Sacramento,
CA, USA). The peroxidase–antiperoxidase method
Correspondence: Claude Favrot, Clinic for Small Animal Internal
Medicine, Vetsuisse Faculty, Winterthurerstrasse 260, 8057 Zürich,
Switzerland. E-mail: [email protected]
The clinical cases have been reported at the 5th World Congress of
Veterinary Dermatology, Vienna, 25 –28 August 2004 (case 1) and
20th North America Veterinary Dermatology Forum, Sarasota, 6–10
April 2005 (Case 2).
© 2005 European Society of Veterinary Dermatology
407
29
408
C Favrot et al.
was applied as previously described.8 In case 2, the
skin lesions were also stained for the pan-T-cell marker
CD3 and the pan-B-cell marker CD45R as previously
described.9
Real-time polymerase chain reaction (real-time
PCR) for exogenous FeLV and feline herpesvirus-1
(FHV-1)
Polymerase chain reaction (PCR) analysis was performed on skin with lesions after deparaffinization of
two 20-µm thick sections of the same lesional tissues
as mentioned previously and DNA extraction using
the DNeasy tissue kita. FeLV provirus was detected by
real-time PCR with primers that recognize the unique
region (U3) of the long-terminal repeat (LTR) of
exogenous FeLV-A,-B,-C as described previously.10,11
Examination for feline herpesvirus (FHV-1) sequences
was undertaken as described elsewhere.12
R E S U LT S
Case 1
The cat was presented with a pruritic dermatosis of
3 months’ duration and a previous history including
vaccination for FeLV during the first year of life and
booster injection in the second year. Physical examination revealed well-demarcated ulcerative lesions
of the head, limbs and paws (Fig. 1). The cat was also
depressed and febrile (39.7 °C). A staphylococcal infection was identified by cytological and bacteriological
examination (Staphylococcus intermedius), but multiple
skin scrapings were negative for parasites and fungal
culture was negative. A nonregenerative normochrome
normocytic anaemia (hematocrit: 18% (reference range:
33 – 45%) reticulocytes: 0.6%) was diagnosed. The ELISA
for FeLV antigen was positive, whereas that for FIV
antibodies was negative. The cat was started on cefalexin
(25 mg kg−1 twice daily) therapy.
Figure 2. Case 1. A skin lesion. Folliculitis (red arrow) and
syncytium formation (black arrow) of epithelial cells of a hair
follicle. Haematoxylin and eosin stain.
Histological examination revealed an ulcerative
dermatitis with folliculitis, dyskeratotic keratinocytes
and syncytia formation within the epidermis and the
sebaceous glands (Figs 2 and 3). The epidermis was
acanthotic and hyperkeratotic. Multiple giant keratinocytes and scattered apoptotic cells were present in
the superficial epidermis, sebaceous glands and hair
follicles. In the dermis, a severe perifollicular to diffuse
inflammatory infiltration with numerous lymphocytes,
plasma cells and neutrophils was present.
Immunohistological analyses revealed numerous
epithelial cells that expressed viral proteins with variable intensity (Fig. 4) in the epidermis of the skin surface, hair follicles and sebaceous glands. Lymphocytes
in the dermal infiltrates were often positive as well. A
131-bp long proviral FeLV DNA was amplified from
skin samples of this cat. Skin samples evaluated for
FHV-1 DNA were deemed negative. A diagnosis of
FeLV-induced giant-cell dermatosis was made.
Despite the treatment and a marked but temporary
improvement of the skin lesions, the general condition
deteriorated and the cat was euthanized. Necropsy was
not permitted.
Case 2
The cat was presented with a dermatosis of 2 months’
duration and weight loss. It had previously been treated
with megestrol acetate and prednisolone (variable dosages) on the basis of a tentative diagnosis of eosinophilic
plaques. The cat was depressed and febrile (39.6 °C). Physical examination of the skin revealed multiple nodules
Figure 1. Case 1. Well-demarcated ulcerated lesion on the face.
a
QIAGEN, Hombrechtikon, Switzerland
© 2005 European Society of Veterinary Dermatology, Veterinary Dermatology, 16, 407– 412
30
FeLV-induced dermatoses
409
Figure 5. Case 2. Ulcerated nodule on the carpus.
Figure 3. Case 1. Sebaceous glands. Syncytium formation (black
arrow) in epithelial cells. Haematoxylin and eosin stain.
Figure 6. Case 2. Histology of the cutaneous nodule in Fig. 5.
Superficial ulceration (black arrow) and diffuse infiltration of
the dermis by neoplastic round cells (red arrow). Haematoxylin and
eosin.
Figure 4. Case 1. Epithelial cells in a hair follicle express FeLV
antigen with variable intensity (arrows). Immunohistological
demonstration of FeLV gp70, peroxidase antiperoxidase method,
Papanicolaou’s haematoxylin counterstain.
results a diagnosis of cutaneous non-epitheliotropic T-cell
lymphoma was made. Immunohistology for FeLV antigen
revealed variably intense viral protein expression by numerous neoplastic cells and weak expression by epidermal
cells in all layers (Fig. 9). A 131-bp long proviral FeLV
DNA fragment was amplified from skin samples of this cat.
Despite Lomustine therapy (10 mg once daily)
started after histological diagnosis, the cat’s general
condition deteriorated and it was euthanized. Necropsy
was not permitted so the nodular liver and pulmonary
lesions could not be further evaluated.
and ulcerated lesions that affected the face, feet and
abdomen (Fig. 5). Multiple skin scrapings were negative
for parasites. Additionally, the cat was anaemic (Ht. 23%)
and lymphopenic (170 lymphocytes per microlitre). Serum
ELISA tests for FeLV antigens and FIV antibodies were
both negative. Radiographic examination of the thorax
revealed a nodular opacity in the left lung. Sonographic
examination of the abdomen showed the presence of a
small nodule in the liver. Fine needle aspirates of pulmonary and liver nodules were, however, unremarkable.
Histological examination of the skin lesions revealed
extensive superficial ulceration and focally extensive
dense dermal infiltration by pleomorphic round cells,
resembling lymphoblasts with round to indented nuclei
containing fine chromatin and one single medium-sized
nucleolus (Fig. 6). Cellular atypia such as anisocytosis
and anisocaryosis were observed, multiple large nucleoli
and abnormal mitoses were also seen (Fig. 7). A large
proportion of neoplastic cells exhibited peripheral and /or
cytoplasmic CD3 expression (Fig. 8). Based on these
DISCUSSION
This report describes two FeLV-associated skin conditions in cats, presenting clinically as dermatoses with
poor response to treatment: giant-cell dermatosis and
cutaneous lymphoma. The presence of proviral FeLV
sequences as well as FeLV antigens in the skin with
© 2005 European Society of Veterinary Dermatology, Veterinary Dermatology, 16, 407–412
31
410
C Favrot et al.
Figure 7. Case 2. The neoplastic infiltrate is composed of
pleomorphic round cells, resembling lymphoblasts. They exhibit
variably chromatin-dense nuclei (red arrows) and distinct nucleoli
(black arrow). Haematoxylin and eosin.
Figure 9. Case 2. Neoplastic cells (arrowheads) and epidermal
keratinocytes (arrow) express FeLV antigens. Immunohistological
demonstration of FeLV gp70 and p27, peroxidase antiperoxidase
method, Papanicolaou’s haematoxylin counterstain.
carcinoma), infectious (alpha-herpesvirus infections)
and immunologic disorders such as lupus erythematosus, Hailey–Hailey disease and psoriasis.13 Retroviruses
including human lentiviruses such as human immunodeficiency virus and feline gammaretroviruses (e.g. FeLV),
possess fusion proteins and are sometimes seen to induce
syncytium formation in lymphoid tissues.14–16 However syncytial keratinocytes are mainly observed in
AIDS patients that are concomitantly infected with
herpesvirus or papillomavirus.17,18 Gross and coworkers
suggested that syncytium formation observed in feline
cases is not a consequence of a direct cytopathic effect
of the virus but of carcinomatous transformation of
the epidermis.4 HIV-induced squamous cell carcinoma,
however, has rarely been observed in humans.18 The
oncogenic potential of FeLV is much greater than that
of HIV but Rohn and coworkers have also demonstrated
that FeLV variants do exhibit various pathogenic and
cytopathic effects, including syncytium formation in
one strain.16 It thus appears possible that FeLV-induced
giant-cell dermatosis is the result of a specific and
probably rare viral variant. Confirmation of this
hypothesis needs further investigation.
Lymphomas are frequent neoplasms in cats and
often a consequence of FeLV infection.5 Cutaneous
lymphomas, however, are rare and usually occur in
older serologically FeLV-negative cats.5 Attempts to
identify FeLV genomic sequences and antigens in
epitheliotropic and nonepitheliotropic cutaneous
Figure 8. Case 2. Neoplastic cells exhibit a variably intense
peripheral and/or nuclear reaction for the pan T-cell marker CD3
(arrowheads). Blood vessels and hair follicles are negative.
Peroxidase–antiperoxidase method, Papanicolaou’s haematoxylin
counterstain.
lesions of both cats was demonstrated by PCR and
immunohistological analysis, respectively.
So far, six cases of FeLV-induced giant-cell dermatosis
have been described in the literature.4 Most presented
as scaling and crusting dermatoses affecting mainly
the face and the neck. Vesicular and ulcerative lesions
were also reported with involvement of the footpads
and mucous membranes.4 The clinical and histological
presentation of this case was similar to those previously
described, although more ulcerative and less hyperkeratotic. Mucous membranes were unremarkable.
Affected cats usually decline quickly with death or
euthanasia days to weeks after initial presentation.
The histological hallmark of giant-cell dermatosis
is the presence of syncytial keratinocytes and dyskeratotic cells. The former have also been observed in
FeLV-infected cats in association with cutaneous
horns.3 Multinucleated keratinocytes are observed in
humans in association with neoplastic (squamous cell
32
FeLV-induced dermatoses
lymphomas are occasionally successful and confirm
FeLV involvement in the development of at least some
cutaneous lymphomas in cats.19,20 Tobey and coworkers suggested defective or latent infection as they
did not detect FeLV antigens in the neoplastic cells of a
cutaneous lymphoma.21 In the case reported here, both
proviral genome and antigens were demonstrated. The
presence of viral antigens in neoplastic cells and keratinocytes but not in the peripheral blood suggests focal
productive infection of both cell types. As the greater
sensitivity of RT-PCR detects proviral DNA in the
serum of cats with undetectable antigenaemia, negative
ELISA does not rule out generalized infection. Localized FeLV replication has been reported after experimental infection with viral antigens in the spleen, bone
marrow, lymph nodes or small intestine.22 Restricted,
localized FeLV replication was also shown in another
study in naturally infected cats. The same organs
were examined but no viral antigen was found.19 This
case report supports the hypothesis that infections
restricted to the skin may sometimes occur in cats and
subsequently induce cutaneous lymphomas.
As PCR assays and immunohistology for FeLV are
not routinely carried out on cutaneous lymphomas,
the frequency of this association is unknown and may
be underestimated. It is possible, however, that FeLV
tumorigenesis of dermal T cells is rare and caused of
particular FeLV variants. A previous study failed to
detect FeLV nucleic acid in a portion (20%) of feline
lymphoma samples and suggested that FeLV lymphomagenesis can be associated with clearance of viral nucleic
acids from cancer cells.23 This ‘hit and run’ mechanism
has already been associated with other conditions induced
by retroviruses.24 This study was, however, carried out
with a single-round PCR system23 that has a lower diagnostic sensitivity than the more recently developed FeLVspecific nested and TaqMan PCR systems.10,11 As real-time
TaqMan PCR can detect fewer nucleic acid copies
compared to conventional PCR we may be able to detect
FeLV in a larger portion of lymphosarcoma cases.
In conclusion, this report suggests that FeLV-induced
dermatoses are probably due to particular viral variants
and their frequency might be underestimated.
411
2. Scott DW, Miller WH, Griffin CE. Viral, rickettsial
and protozoal diseases. In: Muller and Kirk’s Small
Animal Dermatology. Philadelphia: W.B. Saunders, 2001:
517–42.
3. Center SA, Scott DW, Scott FW. Multiple cutaneous
horns on the foot pads of a cat. Feline Practice 1982; 12:
26–30.
4. Gross TL, Clark EG, Hargis AM et al. Giant cell dermatosis in FeLV-positive cats. Veterinary Dermatology
1993; 4: 117–22.
5. Vonderhaar MA, Morisson WB. Lymphosarcoma. In:
Morisson WB ed. Cancer in Dogs and Cats. Jackson,
Wyoming: Teton New Media, 2002: 641–70.
6. Lutz H, Pedersen NC, Durbin R et al. Monoclonal antibodies to three epitopic regions of feline leukemia virus
p27 and their use in enzyme-linked immunosorbent assay
of p27. Journal of Immunological Methods 1983; 56:
209–20.
7. Calzolari M, Young E, Cox D et al. Serological diagnosis
of feline immunodeficiency virus infection using
recombinant transmembrane glycoprotein. Veterinary Immunology and Immunopathology 1995; 46: 83–
92.
8. Kipar A, Kremendahl J, Grant CK et al. Expression of
viral proteins in feline leukemia virus-associated enteritis.
Veterinary Pathology 2000; 27: 129–36.
9. Kipar A, Bellmann S, Kremendahl J et al. Cellular composition, coronavirus antigen expression and production
of specific antibodies in lesions in feline infectious peritonitis. Veterinary Immunology and Immunopathology
1998; 65: 243–57.
10. Tandon R, Cattori V, Gomes-Keller MA et al. Quantitation of feline leukaemia virus viral and proviral loads by
TaqMan((R)) real-time polymerase chain reaction.
Journal of Virological Methods 2005; 130(1–2): 124–32.
11. Hofmann-Lehmann R, Huder JB, Gruber S et al. Feline
leukaemia provirus load during the course of experimental infection and in naturally infected cats. Journal of
General Virology 2001; 82: 1589–96.
12. Vogtlin A, Fraefel C, Albini S et al. Quantification of
feline herpesvirus 1 DNA in ocular fluid samples of clinically diseased cats by real-time TaqMan PCR. Journal
of Clinical Microbiology 2002; 40: 519–23.
13. Kimura SK, Hatano H. Multinucleated epidermal cells
in non-neoplastic dermatoses. British Journal of Dermatology 1978; 99: 485–9.
14. White JM. Membrane fusion. Science 1992; 258: 917–24.
15. Fenyo EM, Morfeldt-Manson L, Chiodi F et al. Distinct
replicative and cytopathic characteristics of human immunodeficiency virus isolates. Journal of Virology 1988; 62:
4414–9.
16. Rohn JL, Moser MS, Gwynn SR et al. In vivo evolution
of a novel, syncytium-inducing feline leukemia virus
variant. Journal of Virology 1998; 72: 2686–96.
17. Fagan WA, Collins PC, Pulitzer DR. Verrucous Herpes
virus infection in human immunodeficiency virus patients.
Archives of Pathology and Laboratory Medicine 1996;
120: 956–8.
18. Orem J, Otieno MW, Remick SC. AIDS-associated cancer
in developing nations. Current Opinion in Oncology
2004; 16: 468–76.
19. Kovacevic S, Kipar A, Kremendahl J et al. Immunohistochemical diagnosis of feline leukemia virus infection in
formalin-fixed tissue. European Journal of Veterinary
Pathology 1997; 3: 13–9.
AC K N OW L E D G E M E N T S
Thanks to the Centre for Clinical Studies, the Vetsuisse
Faculty of the University of Zürich for use of logistics
and financial support from the Swiss National Science
Foundation (31-65231). R.H.-L. is the recipient of a
professorship from the Swiss National Science Foundation (PP00B-102866).
REFERENCES
1. Cotter SM. Feline viral neoplasia. In: Green CG ed.
Infectious Diseases of the Dog and Cat. Philadelphia:
W.B. Saunders Co, 1998: 71– 84.
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feline leukemia virus infection in cats. American Journal
of Veterinary Research 1992; 53: 604–7.
23. Jackson ML, Haines DM, Meric SM et al. Feline leukemia virus detection by immunohistochemistry and
polymerase chain reaction in formalin-fixed, paraffineembedded tissue of cats with lymphosarcoma. Canadian
Journal of Veterinary Research 1993; 57: 269–76.
24. Nath A. Pathobiology of human immunodeficiency virus
dementia. Journal of Biology and Chemotherapy 1999;
19: 113–27.
20. Jackson ML, Wood SL, Misra V et al. Immunohistochemical identification of B and T lymphocytes in
formalin-fixed, paraffin-embedded feline lymphosarcomas: relation to feline leukemia virus status, tumor site,
and patient age. Canadian Journal of Veterinary
Research 1996; 60: 199 –204.
21. Tobey JC, Houston DM, Breur GJ et al. Cutaneous
T-cell lymphoma in a cat. Journal of the American
Veterinary Medical Association 1994; 204: 606 –9.
22. Hayes KA, Rojko JL, Mathes LE. Incidence of localized
Résumé Deux cas de dermatoses associées au FeLV sont rapportées. Le premier chat présentait une dermatite
ulcérative identifiée comme une dermatose à cellules géantes. Le second cas était un lymphome cutané. Dans les
deux cas, des antigènes du FeLV et du génome ont été mis en évidence immunologiquement et par PCR respectivement. Le premier cas suggère que comme d’autres rétrovirus, au moins certaines souches de FeLV peuvent
provoquer la formation de syncytium. Comme les antigènes et l’ADN du FeLV ont été retrouvés chez un chat
séronégatif, le second cas suggère qu’une réplication focale du FelV peut se dérouler dans la peau. Les dermatoses
associées au FeLV sont des dermatoses rares peut être sous diagnostiquées.
Resumen Describimos dos casos de dermatosis en gatos asociados con la presencia del virus de la Leucemia
Felina (FeLV). El primer gato presentó una dermatitis ulcerativa con presencia de células gigantes. El segundo
caso fue un linfoma cutáneo. En ambos casos se demostró la presencia de antígeno de FeLV y del genoma
de FeLV en la piel afectada, mediante una prueba inmunológica y con una reacción de polimerasa en cadena,
respectivamente. Las características del primer caso sugieren que, al igual que otros retrovirus, al menos algunas
variantes de FeLV pueden inducir la formación de células sincitiales. En el segundo caso, ya que tanto el antígeno
como el genoma de FeLV fueron detectados en un gato serológicamente negativo, se sugiere que puede ocurrir
una replicación local del virus de la Leucemia Felina. Las dermatosis asociadas con la presencia de FeLV son
procesos raros, y tal vez no suficientemente reconocidos.
Zusammenfassung Zwei Fälle einer FeLV-assoziierten Dermatose sind beschrieben. Die erste Katze hatte eine
ulzerierende Dermatitis, die identifiziert wurde als eine Riesenzell-Dermatose. Der zweite Fall zeigte kutanes
Lymphom. In beiden Fällen wurden FeLV Antigene immunologisch, sowie FeLV Genom mittels Polymerase
Chain Reaction nachgewiesen. Der erste Fall lässt darauf schließen, dass zumindest einige FeLV Stämme, sowie
andere Retroviren, eine Synzytium Formation induzieren können. Da FeLV Antigene und Genom auch in einer
serologisch negativen Katze demonstriert wurden, weist der zweite Fall darauf hin, dass eine fokale FeLV
Replikation in der Haut vorkommen kann. FeLV-assoziierte Dermatosen sind seltene Erkrankungen der Haut,
die möglicherweise zu selten diagnostiziert werden.
34
Chapter 5
Evaluation of papillomaviruses associated with
cyclosporine-induced hyperplastic verrucous lesions in
dogs
C. Favrot 1, T. Olivry2, A.H. Werner3, G. Nespecca1, A.
Utiger1, P. Grest4, M. Ackermann5
American Journal of Veterinary Research, 2005, 66; 10:
1764-1769
1
2
Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State
University, Raleigh, NC, USA
3
Valley Veterinary Speciality Services, Studio City, CA, USA
4
Pathology Institute, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland
5
Virology Institute, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland
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Evaluation of papillomaviruses associated
with cyclosporine-induced hyperplastic
verrucous lesions in dogs
Claude Favrot, DrVet, MSc; Thierry Olivry, DrVet, PhD; Alexander H. Werner, DVM;
Gilles Nespecca; Anna Utiger, DVM; Paula Grest, DVM; Mathias Ackermann, DVM, PhD
helper T lymphocytes. In humans, cyclosporine A has
been used for more than a decade to prevent transplant
rejection and for treatment for dermatologic conditions
that include severe psoriasis and atopic dermatitis.1
The usefulness of cyclosporine A in treatment for
atopic dermatitis in dogs has been reported,2-6 and the
drug has also been approved for treatment for immunemediated conditions such as perianal fistulae and sebaceous adenitis.7,8
Hyperplastic skin lesions are known adverse
effects of long-term treatment with cyclosporine A in
humans. Most lesions appear to be papillomavirusinduced verruca vulgaris, but malignant carcinomatous transformations are also possible.9 Similar lesions
have also been described in dogs,10,11 but evidence for
causative involvement by papillomavirus is lacking.
Most lesions in dogs resemble those reported as psoriasiform lichenoid dermatosis, and skin nodules usually regress spontaneously or in response to antimicrobial treatment.10,11 Because papillomavirus has been
detected in most cyclosporine A-induced hyperplastic
skin lesions in humans, a similar role for papillomavirus in the development of similar lesions in dogs
warrants investigation. We observed that these druginduced nodules appear to be heterogeneous in nature.
Most lesions are numerous and resemble those of psoriasiform lichenoid dermatosis, with staphylococci in
the stratum corneum and absence of detectable papillomavirus DNA and antigens. In 2 dogs, however,
cyclosporine A administration was associated with the
eruption of few skin nodules diagnosed as viral papillomas. The objective of this study was to determine
whether cyclosporine A-induced hyperplastic skin
lesions in dogs contained papillomavirus DNA and
genus-specific structural antigens.
Objective—To determine whether cyclosporine Ainduced hyperplastic skin lesions of dogs were associated with papillomavirus infections.
Animals—9 dogs that were treated with cyclosporine
A and developed hyperplastic skin lesions.
Procedure—History and clinical and histopathologic
data were collected. Paraffin-embedded skin biopsy
specimens from hyperplastic skin lesions were
immunostained for common papillomavirus genusspecific structural antigens by use of a polyclonal rabbit anti-bovine papillomavirus type 1 antiserum.
Sections from each tissue block underwent DNA
extraction, and polymerase chain reaction (PCR)
assays were performed with several sets of primers
to amplify a wide range of papillomavirus DNA from
humans and other animals.
Results—In 7 of 9 dogs, there were more than 10
hyperplastic skin lesions that microscopically resembled those of psoriasiform lichenoid dermatosis. In
those dogs, results of testing for papillomavirus via
immunohistochemical analyses and PCR assays were
negative. In the other 2 dogs, there were only 1 and
3 verrucous lesions, and in those dogs, histologic
evaluation revealed koilocytes and nuclear viral inclusions that were immunoreactive for papillomavirus
antigens. Papillomavirus DNA was amplified from
both dogs. One of the sequences was characteristic
for the canine oral papillomavirus, whereas the other
had similarities with the recently described canine
papillomavirus 2.
Conclusions and Clinical Relevance—In dogs,
hyperplastic skin lesions occasionally develop during
treatment with cyclosporine A. Most of the lesions
resemble those of psoriasiform lichenoid dermatosis,
although papillomavirus can be detected in some
instances. (Am J Vet Res 2005;66:1764–1769)
Materials and Methods
C
yclosporine A is a potent immunosuppressive
agent that acts primarily by selectively inhibiting
History and clinical information regarding 9 dogs that
were treated with cyclosporine A and developed verrucous
skin lesions were recorded retrospectively. Limited clinical
and pathologic information regarding 1 of the dogs has
already been published.2
Received December 16, 2004.
Accepted February 2, 2005.
From the Clinic for Small Animal Internal Medicine, Dermatology
Unit (Favrot, Nespecca, Utiger), the Pathology Institute (Grest),
and the Virology Institute (Ackermann), Vetsuisse Faculty,
University of Zürich, Winterthurerstrasse 260, CH 8057 Zurich,
Switzerland; the Department of Clinical Sciences, College of
Veterinary Medicine, North Carolina State University, Raleigh, NC
27606 (Olivry); and Valley Veterinary Specialty Services, 13125
Ventura Blvd, Studio City, CA 91604 (Werner).
Presented in part at the Annual Meeting of the American Academy
of Veterinary Dermatology and American College of Veterinary
Dermatology, Monterey, Calif, April 2003.
Address correspondence to Dr. Favrot.
Histologic and immunohistochemical evaluations—
Punch biopsy specimens of the skin were obtained from dermatologic lesions of the 9 dogs. Biopsy specimens were fixed in
formalin, embedded in paraffin, and processed routinely for
histologic assessment. Five-micrometer-thick skin sections
were stained with H&E and Gram stains by use of standard
methods.
For the detection of papillomavirus antigens, a 3-step
immunohistochemical method was used. The primary
immunoreagent was a polyclonal rabbit antiserum directed
against chemically disrupted bovine papillomavirus type 1.a
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This reagent detects papillomavirus genus-specific common
structural antigens regardless of the host species. The primary antiserum was used at a 1:200 dilution, and other reagents
(ie, biotinylated goat anti-canine rabbit IgG and streptavidin
peroxidaseb) were diluted at 1:40. Diaminobenzidine was used
as a chromogen. The positive control consisted of paraffinembedded sections from a dog with canine oral papillomavirus (COPV)-induced oral papillomas, whereas the negative control antiserum consisted of normal rabbit serum.
Skin sections stained with H&E, Gram stain, and
immunostain for papillomavirus were coded and evaluated by
an author (TO) who was unaware of the origins of the specimens. Sections stained with H&E were examined for changes
typically associated with papillomavirus infection in dogs,
and those changes were recorded as present or absent, including epidermal dysplasia, hypergranulosis, coalescing keratohyalin granules, koilocytes, intranuclear viral inclusions in
the stratum spinosum and stratum granulosum, intrafollicular or intraepidermal pustules, and bacteria in the stratum
corneum. Sections stained with Gram stain were evaluated for
epidermal bacteria. Sections immunostained for papillomavirus group-specific antigens were evaluated for staining in
keratinocytes of the stratum spinosum, stratum granulosum,
and stratum corneum. Transmission electron microscopy was
performed on the stratum corneum of 1 specimen.
both genomes. The forward primer was 5’-ATGGCGGMTARAAAAGGTA-3' and the reverse primer was 5’-AACAGCTGYTTTTTARCYTTTTT-3'. Internal control was made by use
of the same forward primer with the reverse primer PapE1 5’ACAGTTGCAGGGAAAGTC-3' to amplify an internal 184-bp
fragment. The PCR reactions were performed in 30-µL volumes containing 1 µL of genomic DNA, 50mM KCl, 3mM
KCl2, 200µM of each dNTP, 0.3µM each of consensus sense
and antisense primers, and 2.5 units of DNA polymerase.e The
amplification involved an initial denaturation at 95oC for 4
minutes and 30 cycles at 95oC for 1 minute, 50oC for 1 minute,
and 74oC for 1 minute, with a final elongation step at 74oC for
5 minutes. Reaction mixture with no DNA served as a negative
control, and COPV-positive papilloma DNA samples and feline
papilloma-positive DNA samples were used as positive controls. The PCR products were resolved via electrophoresis in
2% agarose gel stained with ethidium bromide. Amplified
DNA was sequenced by use of fluorescent sequencing and fluorescent dye terminator.f
Detection of papillomavirus with CP4, C5P, and PPF1
primers—To amplify the DNA of as many different papillomaviruses as possible, the CP4, CP5, PPF1 primer set was
selected because of its ability to detect the nucleic acids of up
to 64 human papillomaviruses.12 The PCR reactions were performed in 30-µL volumes containing 1 µL of genomic DNA,
50mM KCl, 3mM KCl2, 200µM of each dNTP, 0.45µM of the
CP4 and CP5 primers, 0.3µM of the PPF1 primer, and
2.5 units of DNA polymerase.d Amplification involved an initial denaturation at 95oC for 10 minutes and 40 cycles at 95oC
Polymerase chain reaction assays—Amplification of
papillomavirus DNA via polymerase chain reaction (PCR)
assays was performed on formalin-fixed paraffin-embedded
specimens. Thirty-micrometer-thick sections were cut from
tissue blocks by use of a new disposable
microtome blade for each block to avoid
cross-contamination between samples. Each
section was deparaffinized twice with 1.2 mL
of xylene at 20oC for 10 minutes, washed
with 100% ethanol, and air-dried. Desiccated
samples were suspended in a lysis buffer
(50mM Tris-HCl [pH, 8.5], 1mM EDTA,
2.8% sodium dodecylsulfate, and 20 mg of
proteinase K/mL) and incubated for 10 hours
at 56oC on a rocking platform. After lysis,
samples were transferred to a spin columnc
and centrifuged to reduce viscosity. Viral
DNA was precipitated with absolute ethanol
and extracted by use of a commercially available kit.d
Phylogenetic studies have revealed that
COPV and feline papillomavirus are closely
related and that this group of viruses is closer Figure 1—Photomicrographs a section of an exophytic papilloma from a dog. Notice
to some genera of human papillomavirus than hypergranulosis, koilocytes, and intranuclear viral inclusions (arrowheads) in kerto papillomaviruses in other animals, includ- atinocytes of the stratum spinosum, stratum granulosum, and lower stratum
ing bovine papillomavirus. Therefore, 2 sets corneum. H&E stain; bar = 1 mm (A) and 25 µm (B).
of primers were designed. The first set of
primers (PapE1-forward and PapE1-reverse)
amplified DNA from COPV and feline papillomavirus, whereas the second set of primers
(CP4, CP5, and PPF1) amplified human
papillomaviruses, including the oncogenic
strains. To amplify genomic sequences of
canine, feline, or closely related papillomaviruses in clinical samples, nucleotide
sequences conserved among known canine
and feline papillomaviruses were reviewed
and sequences encoding the E1 early gene
were found to be the most highly conserved.
Therefore, E1 sequences of feline (LOCUS
AF480454) and canine (LOCUS NC001619)
Figure 2—Photomicrographs of a section of a papilloma in a dog. Notice papillomapapillomaviruses were aligned with the aim of tous epidermal hyperplasia with prominent koilocytosis and intranuclear viral includesigning consensus primer pairs able to sions (arrowheads) predominantly in the stratum spinosum. H&E stain; bar =
amplify an approximate 341-bp fragment of 0.1 mm (A) and 25 µm (B).
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Eight breeds were represented, and there were 2 West
Highland White Terriers; 8 dogs were male, and 1 dog
was female. Age at the time hyperplastic skin lesions
developed ranged from 6 months to 9 years (median, 3
years). Two dogs (dogs 1 and 2) had 1 to 3 lesions with
the typical appearance of a papilloma. The other dogs
(dogs 3 to 9) had numerous variably pigmented, slightly raised verrucous papules on the trunk and limbs. In
dog 1, the skin nodules were removed surgically. In 5
dogs, the lesions regressed after the dose of cyclosporine
A was reduced and administration of antibimicrobials
was instituted. In the remaining 3 dogs, lesions
regressed spontaneously after cyclosporine A administration was discontinued or the dose was tapered.
for 1 minute, 47oC for 1 minute, and 74oC for 1 minute, with
a final elongation step at 75oC for 5 minutes. Reaction mixture with no DNA served as a negative control, and COPVpositive papilloma DNA samples and feline papilloma-positive DNA samples were used as positive controls. The PCR
products were resolved via electrophoresis in 2% agarose gel
stained with ethidium bromide. Amplified DNA was
sequenced on an automated sequencer with fluorescent dye
terminator,e and sequences were compared with those included in the GenBank database by use of alignment software.g
Samples were considered to have positive results for
detection of papillomavirus DNA if they had a band of the
expected size after gel electrophoresis and if amplified DNA
was sequenced and the protein encoded by the sequence had
homology with the E1 protein of a previously established
papillomavirus sequence. Comparisons were made with
alignment software.f
Histopathology—In dogs 1 and 2, examination of
H&E-stained sections of biopsy specimens revealed
severe focal epidermal hyperplasia and dysplasia,
koilocytosis, and intranuclear viral inclusions with
margination of chromatin (Figures 1 and 2). Focal
hypergranulosis and coalescing keratohyalin granules
also were observed in 1 of those dogs. Such changes
were absent in sections from the other dog, suggesting
that the differing cytopathic effects seen in the 2 dogs
resulted from infection with different viruses.13
Among the remaining 7 dogs, microscopic findings in H&E-stained sections were similar. Findings
included epidermal acanthosis without dysplasia,
hypergranulosis, variable lymphocyte exocytosis,
intraepidermal or intrafollicular pustules, and bacteria
in the stratum corneum (Figure 3). The upper portion
of the dermis contained bands of lymphocytes and
plasma cells, although a true interface dermatitis was
not detected. These features were considered similar to
changes referred to as psoriasiform lichenoid dermatitis.14-16 Examination of stained sections revealed gramnegative rods in epidermal crypts in 1 dog and clusters
of gram-positive cocci in the stratum corneum of hair
follicle infundibula in dogs 3 to 9. Transmission electron microscopy revealed bacteria with features similar
to those of staphylococci.
Results
Clinical information—In 8 of the 9 dogs, administration of cyclosporine A at the median dosage of
5 mg/kg every 24 hours was associated with the eruption
of multiple hyperplastic and verrucous skin lesions. A
single lesion developed in the other dog. The duration of
treatment with cyclosporine A before development of
lesions varied from 1 to 24 months (median, 4 months).
Figure 3—Photomicrographs of a section of a hyperplastic verrucous skin
lesion in a dog. Notice irregular epidermal hyperplasia with luminal (open
arrowheads) and mural (solid arrowhead) folliculitis and a band of lymphocytes and plasma cells in the superficial portion of the dermis.
Lymphocytes are also in the lower epidermal layers. H&E stain; bar = 0.1
mm (A) and 25 µm (B).
Figure 4—Photomicrograph of a portion of an exophytic papilloma from a dog. Notice that intranuclear inclusions in keratinocytes in the stratum granulosum are immunohistochemically stained for papillomavirus group-specific antigens.
Diaminobenzidine chromogen with hematoxylin counterstain;
bar = 10 µm.
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Figure 5—Photomicrographs of a section of a hyperplastic verrucous skin lesion in a dog. Notice intracorneal clusters of bacteria
immunohistochemically stained (inset [arrowheads]) with an antiserum specific for papillomavirus antigens (A) but not with irrelevant
control rabbit serum (B). Diaminobenzidine chromogen with hematoxylin counterstain; bar = 0.1 mm.
canine COPV E1 gene. In dog 1, papillomavirus
DNA was amplified with the CP4, CP5, PPF1, and
PapE1 primers (Figure 6). The amplified sequence
was 98% homologous with that of COPV. In dog 2,
papillomavirus DNA was amplified with the CP4,
CP5, and PPF1 primers. The amplified sequence was
97% homologous with that of a recently described
canine papillomavirus (GenBank No. AY725239).
Moreover, this sequence was homologous at the predicted amino-acid level with E1 protein of human
papillomavirus 63 (76% homology), bovine papillomavirus 3 (74% homology), and feline papillomavirus (72% homology). Papillomavirus DNA was
not amplified from any other specimens from dogs
3 to 9.
Discussion
In humans, administration of cyclosporine A is
often associated with numerous cutaneous adverse
effects.9,17,18 Most of those changes are associated with
the development of viral, bacterial, or fungal
infections. Papillomaviruses are the most frequently
reported virus detected in the associated infections,
but herpes simplex and molluscum contagiosum virus
infections also have been recorded.9,17,19 Noninfectious
changes such as hyperpigmentation, skin tags, lichen
simplex, acne, cysts, and sebaceous hyperplasia are
reported less frequently.17,18,20 Follicular dystrophy,
increased hair growth (hypertrichosis),21,22 and gingival
hyperplasia
are
frequently
recorded.23
Furthermore, compared with the general population,
the incidence of squamous cell carcinoma is higher in
human patients treated with cyclosporine A for longer
than 2 years.24
In dogs, lesions resembling psoriasiform lichenoid
dermatitis have been reported in association with
administration of cyclosporine A.2,10,25 Gingival hyperplasia and hypertrichosis have been reported to be rare
adverse drug events.7,11,25,26 Results of our study indicated that psoriasiform lichenoid dermatitis was the most
common diagnosis for hyperplastic verrucous skin
lesions in 9 dogs treated with cyclosporine A. It has
Figure 6—Gel electrophoretogram of a polymerase chain reaction assay performed with CP4, CP5, and PPF1 primers for
detection of papillomaviruses in verrucous skin lesions in 7
dogs. Notice a 450-bp amplicon that indicates papillomavirus
DNA in extracts of skin biopsy specimens from 2 dogs (lane 3
[dog 1] and lane 4 [dog 2]) and a positive control specimen (pos
[canine oral papillomavirus]). A similar amplicon is not evident
in a negative control specimen (neg) or lanes 5 to 9 (dogs 3 to
7). Lanes 1 and 11 represent the ladder of molecular weight
markers.
Immunohistochemical analyses—In dogs 1 and
2, the results of immunohistochemical staining confirmed the intranuclear keratinocyte viral inclusions
to be derived from papillomavirus. In dog 1, papillomavirus inclusions were in nuclei in the stratum granulosum and lower stratum corneum, whereas in dog 2,
the inclusions were in cells from the stratum spinosum to the lower stratum corneum (Figure 4). In
dogs 3 to 9, intrakeratinocyte staining with the papillomavirus-specific antiserum was not detected.
However, there was bacterial uptake of stain in the
stratum corneum (Figure 5).
PCR assay—The PCR assay detected papillomavirus DNA from sections of the 3 positive controls with both sets of primers, and the sequence of
amplicons was 99% homologous with that of the
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cyclosporine A-induced skin lesions develop most frequently in humans and dogs treated with high doses,
for extended periods of time, or with a combination of
immunosuppressive drugs.1,25 Most of those changes
regress after treatment is discontinued.
Our results suggested that hyperplastic and verrucous skin lesions observed in dogs after cyclosporine A
administration may have multiple causes. In most
dogs, the lesions are typical of those characterized as
lichenoid psoriasiform dermatosis, but infection by
papillomaviruses may develop in some dogs. In all
instances, decreasing or discontinuing administration
of cyclosporine A, with or without concurrent administration of antimicrobials, resulted in regression of the
lesions.
been hypothesized10 that this reaction is induced by
staphylococcal infection, a premise that was supported
by the observation of cocci in 6 of 9 specimens in our
study. However, in 3 of 8 dogs, all lesions regressed
without the administration of antimicrobials after discontinuation or decreasing the administration of
cyclosporine A. Immunohistochemical staining for
papillomavirus with the polyclonal antiserum stained
bacteria in the stratum corneum. In 1 dog with such
bacteria, transmission electron microscopy revealed
bacteria with features consistent with staphylococci in
the stratum corneum but no papillomavirus. Thus,
care must be taken in the interpretation of immunostaining for papillomavirus with this reagent.
Several pharmacologic properties of cyclosporine
A can explain the development of hyperplastic lesions
in the skin of dogs and humans. Cyclosporine A modifies cytokine secretions in several cell types.27 In
humans, development of gingival hyperplasia is caused
by increased production of extracellular matrix in association with secretion of transforming growth
factor-β.28 Moreover, inhibition by cyclosporine A of
the calcineurin-nuclear factor of activated T-cell 1
pathway in follicular keratinocytes stimulates hair
growth and induces hypertrichosis.21
Results of our study also suggested that
cyclosporine A-induced verrucous skin lesions can
sometimes be associated with infections with various
papillomaviruses, for which emergence might be promoted by suppression of cell-mediated immunity.
However, it is not known whether treatment with
cyclosporine A favors recurrence of a latent infection
or promotes de novo infection. In dog 1, clinical findings, histologic findings, and results of immunohistochemical and PCR testing were consistent with those
typically associated with infection with COPV29
(LOCUS NC001619). In dog 2, results of histologic
and immunohistochemical staining were different from
those in dog 1, mainly with regard to koilocytes in the
stratum spinosum and the absence of hypergranulosis
or coalescing keratohyalin granules.13 These differences
may have indicated infection with a virus other than
COPV. Moreover, DNA from papillomavirus was only
amplified by use of the CP4, CP5, PPF1 set of primers;
the subsequent amplification indicated infection with a
recently described papillomavirus (GenBank No.
AY725239). In humans, the use of degenerated primers
has broadened the spectrum of capability of PCR
assays and enabled detection of unknown papillomaviruses.30 It is possible that the epidermis of affected
dogs may be infected by papillomaviruses that are difficult to detect by use of traditional techniques that
amplify COPV DNA. Likewise, it is possible that the
cyclosporine A-induced hyperplastic skin lesions in
dogs 3 to 9 might have been caused by papillomaviruses that were undetectable with available techniques. In humans, DNA of papillomavirus is often
amplified from lesions of psoriasis, a dermatologic condition with similarities to canine psoriasiform
lichenoid dermatosis. It has been hypothesized31 that
papillomavirus induces autoimmune reactions in the
epidermis and could play a role in the development of
such lesions. Finally, it must be kept in mind that
a.
b.
c.
d.
e.
f.
g.
B0580, Dako Corp, Carpinteria, Calif.
Biogenex, San Ramon, Calif.
QIA shredder, Qiagen, Basel, Switzerland.
QIAamp DNA mini kit, Qiagen, Basel, Switzerland.
Pfu Turbo DNA polymerase, Stratagene, La Jolla, Calif.
Applied Biosystems, Foster City, Calif.
Blast, version 2.2.11. Available at: www.ncbi.nlm.gov/BLAST.
Accessed Jan 15,0205.
References
1. Gupta AK, Brown MD, Ellis CN, et al. Cyclosporine in dermatology. J Am Acad Dermatol 1989;21:1245–1256.
2. Olivry T, Rivierre C, Jackson HA, et al. Cyclosporine
decreases skin lesions and pruritus in dogs with atopic dermatitis: a
blinded randomized prednisolone-controlled trial. Vet Dermatol
2002;13:77–87.
3. Olivry T, Steffan J, Fisch RD, et al. Randomized controlled
trial of the efficacy of cyclosporine in the treatment of atopic dermatitis in dogs. J Am Vet Med Assoc 2002;221:370–377.
4. Fontaine J, Olivry T. Treatment of canine atopic dermatitis
with cyclosporine: a pilot clinical study. Vet Rec 2001;148:662–663.
5. Olivry T, Mueller RS. Evidence-based veterinary dermatology: a systematic review of the pharmacotherapy of canine atopic
dermatitis. Vet Dermatol 2003;14:121–146.
6. Steffan J, Alexander D, Brovedani F, et al. Comparison of
cyclosporine A with methylprednisolone for treatment of canine
atopic dermatitis: a parallel, blinded, randomized controlled trial. Vet
Dermatol 2003;14:11–22.
7. Mathews KA, Sukhiani HR. Randomized controlled trial of
cyclosporine for treatment of perianal fistulas in dogs. J Am Vet Med
Assoc 1997;211:1249–1253.
8. Linek M, Boss C, Haemmerling R. Effects of cyclosporine A
on clinical and histological abnormalities in dogs with sebaceous
adenitis. J Am Vet Med Assoc 2005;226:59–64.
9. Iraji F, Kiani A, Shahidi S, et al. Histopathology of skin
lesions with warty appearance in renal allograft recipients. Am J
Dermatopathol 2002;24:324–325.
10. Werner AH. Psoriasiform-lichenoid-like dermatosis in three
dogs treated with microemulsified cyclosporine A. J Am Vet Med
Assoc 2003;223:1013–1016.
11. Seibel W, Sundberg JP, Lesko LJ, et al. Cutaneous papillomatous hyperplasia in cyclosporine-A treated beagles. J Invest
Dermatol 1989;93:224–230.
12. Iftner A, Klug SJ, Garbe C, et al. The prevalence of human
papillomavirus genotypes in nonmelanoma skin cancers of nonimmunosuppressed individuals identifies high-risk genital types as possible risk factors. Cancer Res 2003;63:7515–7519.
13. Croissant O, Breitburd F, Orth G. Specificity of cytopathic effect
of cutaneous human papillomaviruses. Clin Dermatol 1985;3:43–55.
14. Gross TL, Halliwell RE, McDougal BJ, et al. Psoriasiform
lichenoid dermatitis in the springer spaniel. Vet Pathol 1986;23:76–78.
15. Mason KV, Halliwell RE, McDougal BJ. Characterization of
lichenoid-psoriasiform dermatosis of springer spaniels. J Am Vet Med
Assoc 1986;189:897–901.
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16. Yager JA, Wilcock BP. Interface dermatitis. In: Yager JA,
Wilcock BP, eds. Color atlas and text of surgical pathology of the dog
and cat dermatopathology and skin tumors. London: Mosby Year Book
Inc, 1994;85–106.
17. Euvrard S, Kanitakis J, Cochat P, et al. Skin diseases in children with organ transplants. J Am Acad Dermatol 2001;44:932–939.
18. Lugo-Janer G, Sanchez JL, Santiago-Delpin E. Prevalence
and clinical spectrum of skin diseases in kidney transplant recipients. J Am Acad Dermatol 1991;24:410–414.
19. Van der Leest RJ, Zachow KR, Ostrow RS, et al. Human
papillomavirus heterogeneity in 36 renal transplant recipients. Arch
Dermatol 1987;123:354–357.
20. Bencini PL, Montagnino G, Sala F, et al. Cutaneous lesions
in 67 cyclosporin-treated renal transplant recipients. Dermatologica
1986;172:24–30.
21. Gafter-Gvili A, Sredni B, Gal R, et al. Cyclosporin Ainduced hair growth in mice is associated with inhibition of calcineurin-dependent activation of NFAT in follicular keratinocytes.
Am J Physiol Cell Physiol 2003;284:C1593–C1603.
22. Heaphy MR Jr, Shamma HN, Hickmann M, et al.
Cyclosporine-induced folliculodystrophy. J Am Acad Dermatol
2004;50:310–315.
23. Wysocki GP, Gretzinger HA, Laupacis A, et al. Fibrous
hyperplasia of the gingiva: a side effect of cyclosporin A therapy. Oral
Surg Oral Med Oral Pathol 1983;55:274–278.
24. Paul CF, Ho VC, McGeown C, et al. Risk of malignancies in
psoriasis patients treated with cyclosporine: a 5 y cohort study.
J Invest Dermatol 2003;120:211–216.
25. Rosenkrantz WS, Griffin CE, Barr RJ. Clinical evaluation of
cyclosporine in animal models with cutaneous immune-mediated disease
and epitheliotropic lymphoma. J Am Anim Hosp Assoc 1989;25:377–384.
26. Seibel W, Yahia NA, McCleary LB, et al. Cyclosporineinduced gingival overgrowth in beagle dogs. J Oral Pathol Med 1989;
18:240–245.
27. Esposito C, Fornoni A, Cornacchia F, et al. Cyclosporine
induces different responses in human epithelial, endothelial and
fibroblast cell cultures. Kidney Int 2000;58:123–130.
28. Stabellini G, Carinci F, Luigi Bedani P, et al. Cyclosporin A
and transforming growth factor beta modify the pattern of extracellular glycosaminoglycans without causing cytoskeletal changes in
human gingival fibroblasts. Transplantation 2002;73:1676–1679.
29. Yager JA, Wilcock BP. Squamous papilloma. In: Yager
J,Wilcock BP, eds. Color atlas and text of surgical pathology of the dog
and cat: dermatopathology and skin tumors. London: Mosby Year Book
Inc, 1994;251–252.
30. Meyer T, Arndt R, Christophers E, et al. Importance of
human papillomaviruses for the development of skin cancer. Cancer
Detect Prev 2001;25:533–547.
31. Majewski S, Jablonska S, Favre M, et al. Papillomavirus and
autoimmunity in psoriasis. Immunol Today 1999;20:475–476.
1769
41
Chapter 6
Detection of Novel Papillomaviruses in Canine Mucosal,
Cutaneous and in situ Squamous Cell Carcinomas
N. Zaugg1, G. Nespeca1, B. Hauser2, M. Ackermann3, C.
Favrot1
Veterinary Dermatology, 2005; 16: 290-298
1
University of Zurich, Zurich, Switzerland.
2
Institute for Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Zurich,
Switzerland
3
The study has been funded by grants of the Waltham Foundation and The
European Society of Veterinary Dermatology (ESVD)
42
Veterinary Dermatology 2005, 16, 290– 298
Detection of novel papillomaviruses in canine mucosal, cutaneous
and in situ squamous cell carcinomas
Blackwell Publishing, Ltd.
N. ZAUGG*, G. NESPECA*, B. HAUSER†, M. ACKERMANN‡ and C. FAVROT*
*Clinic for Small Animal Internal Medicine, Dermatology Unit, †Institute for Veterinary Pathology and
‡Institute for Virology, Vetsuisse Faculty, University of Zürich, Switzerland
(Received 31 January 2005; accepted 20 May 2005)
Abstract Papillomavirus (PV) DNA is frequently uncovered in samples of human skin squamous cell carcinomas
(SCC). However, the role of these viruses in the development of such cancers in canine species remains controversial. While approximately 100 human PVs are known, only one single canine oral PV (COPV) has been identified and studied extensively. Therefore, we applied a narrow-range polymerase chain reaction (PCR) suitable
for the detection of classical canine and feline PVs, as well as a broad-range PCR, which has been used for the
detection of various novel PVs in humans, in order to analyse 42 paraffin-embedded samples, representing three
different forms of canine SCCs. Ten samples of skin tissues with various non-neoplastic conditions served as controls. While none of the negative controls reacted positively, PV DNA was discovered in 21% of the tested SCC
samples. Interestingly, the classical COPV was amplified from only one sample, while the other positive cases were
associated with a variety of thus far unknown PVs. This study suggests that a fraction of canine SCC is infected
with PVs and that a genetic variety of canine PVs exists. Therefore, these results will facilitate the future study
of the role of PVs in the development of canine skin cancers.
I N T RO D U C T I O N
The aetiology of canine skin and mucous membrane
SCC remains largely unknown, although environmental
factors such as sunlight exposure or burns have been
implicated.1,2 The role of papillomaviruses (PV) also
remains unclear and very few reports have confirmed
the association between PV and SCC in dogs.10–13
Additionally, the role of these viruses in the development of cancer has not been established and the
genomes of potentially causative viruses have not yet
been cloned and analysed.
Similarly, the role of human PVs (HPV) in the development of human skin SCC remains controversial.
HPV DNA is, however, frequently uncovered in at least
three types of human skin SCCs: Epidermodysplasia
verruciformis, Bowen’s disease and Bowenoid papulosis.7,9,14,15 Additionally, links between HPVs and cervical and anal human SCCs have been well demonstrated
and the causality established.16–18
Papillomaviruses are host-specific epitheliotropic
DNA viruses that infect skin and mucous membranes. The complete genome and the biological properties of only one canine papillomavirus are well
known.19–21 However, the existence of up to six different types has been suggested.4,21–23
In contrast, classification of HPVs has recently
been reviewed, and nearly 100 HPV types have been
described based on isolation and sequencing of complete
genomes.15 de Villiers has additionally proposed criteria to define genera, species, types, subtypes and variants within the papillomaviridae family.15 It has also
recently been shown that phylogenetic classification
based on the L1 gene of PVs correlates, at least partially, with the biological and pathological properties.15
Squamous cell carcinomas (SCC) are malignant
tumours that arise from the squamous epithelium of
the skin and the mucous membranes. Squamous cell
carcinomas account for up to 5% of the skin tumours
in dogs and are the most frequent malignant canine
tumours of the digits, tongue and gingiva.1,2 Cutaneous SCCs may be either exophytic or ulcerative,
whereas mucous membrane SCCs are usually exophytic.1,2 Aside from these two classical forms of invasive SCC, a case of multifocal in situ SCC arising from
pigmented papules and plaques has been described in a
dog.3 In this case, tumoral cells were confined to the
epidermis, while the basal membrane remained
unaffected. Furthermore, cases of canine multiple
pigmented plaques that evolved into SCC have been
reported.4,5
In humans, solar keratosis, Bowen’s disease and
Bowenoid papulosis are regarded as forms of in situ
SCC.6–9 Some of them subsequently develop into invasive SCCs. Mucosal forms of SCCs also affect the
cervical, anal and oral mucous membranes. Furthermore, Epidermodysplasia verruciformis is a rare genetic
predisposition to develop viral warts with high risk of
carcinomatous transformation.
The study has been funded by grants of the Waltham Foundation and
The European Society of Veterinary Dermatology (ESVD).
Correspondence: C. Favrot, Clinic for Small Animal Internal
Medicine, Dermatology Unit, Vetsuisse Faculty Zürich,
Winterthurerstrasse 260, CH-8057 Zürich, Switzerland. E-mail:
[email protected]
290
© 2005 European Society of Veterinary Dermatology
43
Detection of novel papillomaviruses
Papillomavirus infection may either be subclinical,
or induce microlesions or benign neoplasias.14,15,24,25
Additionally, a subset of HPVs and animal PVs is
clearly implicated in the development of cancer in
humans and animals.14,18,26–28 In humans, these PVs
cause mucosal carcinomas and are referred to as highrisk PVs.14
The nature and the biological properties of canine
PVs, as well as the aetiology of canine SCC, remain
largely unknown.2 As HPVs that induce benign warts
in humans are usually different from those that induce
cancer, it can be speculated that the well known canine
oral PV (COPV) does not usually induce SCC in dogs
and that some other unknown canine PVs, the canine
counterparts of the high-risk HPVs, may be responsible or contribute to such development.21
The purpose of our study was therefore to detect a
broad spectrum of PV DNAs in samples from three
forms of canine SCC (cutaneous invasive, cutaneous
in situ and mucosal invasive) and to sequence the
amplified DNA.
291
to a QIA shredder column (Quiagen, Basel, Switzerland) and centrifuged in order to reduce viscosity.
Desoxyribonucleic acid was precipitated with absolute
ethanol and extracted with the QIAamp® DNA Mini
Kit (Quiagen).
Papillomavirus detection and sequencing
Phylogenetic studies have shown that COPV and feline
PV are closely related and that this group is closer to
some human PVs, including oncogenic PVs, than to
PV in other animals, such as bovine PV.15 The investigators consequently selected two sets of primers: the
first one (PapE1-Forward, PapE1-Reverse) is designed
to amplify specifically COPV and FdPV DNA and the
second one (CP4, CP5, PPF1) is designed to amplify
various HPV DNA, especially the oncogenic ones.
Narrow-range PCR with PapE1 primers
All samples were coded before being assayed. The
sequences encoding E1 are most highly conserved
amongst canine, feline or closely related PVs.29 Therefore,
the E1 sequences of feline [LOCUS AF480454] and
canine [LOCUS NC001619] papillomaviruses were
aligned with the aim of designing degenerated consensus primer pairs able to amplify an estimated 341-bp
fragment from both phylogenetically related genomes.
The forward primer used was 5 ′ -ATGGCGGMTARAAAAGGTA-3′ and the reverse primer used
was 5′-AACAGCTGYTTTTTARCYTTTTT-3′. To
amplify an internal 184-bp fragment using the same
forward primer, a second reverse primer 5′-GAAACAGTTGCAGGGAAAGTC-3′ was designed.
The PCR reactions were performed in 30-µL volumes,
containing 1 µL of genomic DNA, 50 m KCl,, 3 m
KCl2, 200 µ of each dNTP, 0.3 µ each of consensus
sense and antisense primers and 2.5 U of PfuTurboDNA polymerase (Stratagene, CA, USA). Polymerase chain reaction amplification involved an initial
denaturation step at 95 °C for 4 min, followed by 30
cycles at 95 °C for 1 min, 50 °C for 1 min and 74 °C for
1 min, with a final elongation step at 74 °C for 5 min.
Reaction mixture with no DNA served as negative control, and COPV-positive papilloma DNA samples and
feline papilloma positive DNA samples were used as
positive controls. The PCR products were resolved by
electrophoresis in 2% agarose gel stained with ethidium bromide. Amplified DNA was sequenced using
AB-3100-based fluorescent sequencing and BigDye
terminator chemistry.
M AT E R I A L S A N D M E T H O D S
Materials
Fifty-seven samples of paraffin-embedded skin were
included in the study:
• seventeen samples of canine invasive cutaneous
SCC;
• twenty-three samples of canine mucosal SCC;
• two samples of canine in situ SCC;
• ten samples of canine skin with various nontumoral
conditions;
• three samples of virus-induced canine wart;
• one sample of virus-induced feline in situ SCC; and
• one sample of virus-induced bovine fibropapilloma.
Except for one case (#2 provided by Dr T. L. Gross),
all samples were selected by one board-certified pathologist (BH) at the Institute of Veterinary Pathology of
the University of Zürich, Switzerland.
Methods
The study was carried out using a PCR technique on
formalin-fixed, paraffin-embedded samples. Thirty-µmthick sections of each sample were cut from each tissue
block, using a new disposable microtome blade for
each block. Rigorous precautions were taken in order
to avoid cross-contamination between samples.
Broad-range PCR with CP4, CP5 & PPF1 primers
DNA extraction. Each section was deparaffinized twice
with 1.2 mL xylene at room temperature for 10 min,
washed with ethanol 100% and then dried. The desiccated
samples were suspended in an ATL lysis buffer (50 m
Tris-HCl, pH 8.5 1 m  ethilenediaminetetraacetic
acid, 2.8% sodium dodecylsulphate and 20 mg mL−1
Proteinase K) and incubated at 56 °C on the rocking
platform overnight. After lysis, samples were transferred
In order to amplify as many different PVs as possible,
the CP4, CP5, PPF1 was selected, because of its ability
to uncover up to 64 different HPVs.29
The PCR reactions were performed in 30-µL volumes, containing 1 µL of genomic DNA, 50 m KCl,
3 m KCl2, 200 µ of each dNTP, 0.45 µ of the CP4
and CP5 primers and 0.3 µ of the PPF1 primer,
and 2.5 U of PfuTurboDNA polymerase (Stratagene).
Polymerase chain reaction amplification involved an
44
292
N. Zaugg et al.
initial denaturation step at 95 °C for 10 min, followed
by 40 cycles at 95 °C for 1 min, 47 °C for 1 min and
74 °C for 1 min, with a final elongation step at 75 °C
for 5 min. Reaction mixture with no DNA served as
negative control, and COPV-positive papilloma DNA
samples and feline papilloma positive DNA samples
were used as positive controls. The PCR products were
resolved by electrophoresis in 1% agarose gels stained
with ethidium bromide. Amplified DNA was sequenced
using AB-3100-based fluorescent sequencing and
BigDye terminator chemistry, and obtained sequences
were compared with entries in the GenBank database.
Interpretation of the results and sequence analyses
Samples were deemed positive if the two following
criteria were fulfilled:
• samples exhibited a band of the expected size after
gel electrophoresis;
• the amplified DNA exhibited a significant homology with DNA coding for the E1 protein of a previously established PV. Comparisons were made
with the BLAST software (GenBank – National
Center of Biotechnology Information: NCBI).
Figure 2. Histological section from lesion in Fig. 1. H&E. Canine
SCC in situ. Irregular acanthosis (white arrow), hyperpigmentation
and pigmentary incontinence (blue arrow), follicular involvement
(green arrow). The basement membrane is intact (yellow arrow).
Bar: 200 µm.
Sequences were subsequently compared to each
other on the amino acid sequence level in order to
establish their homology on the protein level.
R E S U LT S
Clinical and histological criteria
Available SCC cases were assigned to clinical and histological groups according to the following criteria.
Two dogs (cases 1 and 2) exhibited numerous hyperpigmented, scaly maculae, plaques and nodules. One of
the plaques of case 1 ulcerated and was subsequently
biopsied (Fig. 1). Case 2 exhibited several ulcerated
nodules that were biopsied. Histopathological examination of the two cases revealed marked acanthosis,
Figure 3. Histological section from lesion in Fig. 1. H&E. Canine in
situ SCC. Proliferation of basaloid cells (red arrow). Clumped
keratohyalin granules (white arrow), hyperpigmentation (blue arrow),
anisocaryosis (double arrow). Bar: 50 µm.
orthokeratotic hyperkeratosis and hypergranulosis
(Fig. 2) with keratohyalin granule clumping (Fig. 3).
The epidermis was disorganized, with numerous atypical cells and premature keratinization. Proliferation of
basaloid cells was observed in some areas but most of
the atypical cells were of the squamous type (Fig. 3).
Atypia consisted of macrokaryosis, anisokaryosis
(Fig. 3), hyperchromasia, prominent nucleoli, multinucleated cells and abnormal mitoses (Fig. 4). However,
the basement membrane was intact and the dermis
was not affected (Figs 2, 3 and 4). Therefore, these dogs
Figure 1. In situ squamous cell carcinoma. Hyperpigmented plaque.
Case 1.
45
Figure 4. Histological section from lesion in Fig. 1. H&E. Canine in
situ SCC. Intact basement membrane (green arrow). Numerous
atypias: giant tumour cells/abnormal mitosis (red arrow),
prominent nucleoli (yellow arrow), multinucelated cells (blue arrow).
Bar: 50 µm.
293
Figure 6. Histology from lesion in Fig. 5. H&E. Canine invasive
SCC: cords (red arrow) of keratinocytes invade the dermis. The
epidermis is ulcerated (left, white arrow) and hyperkeratotic (right,
blue arrow). Horn pearls (green arrow). Bar: 200 µm.
Figure 7. Histology from lesion in Fig. 5. H&E. Canine invasive
SCC. Cords of atypical keratinocytes. Several malignancy criteria
are present: numerous mitoses (white arrows), macrokaryosis (green
arrow), prominent nucleoli (blue arrow). Bar: 50 µm.
Figure 5. Invasive squamous cell carcinoma. Clawbed.
were considered to represent cases of in situ SCC
(Table 1).
Seventeen additional cases represented the group of
skin-derived invasive SCC. Some cases exhibited a
proliferative, exophytic and invasive pattern (Fig. 5),
whereas others were also invasive but more ulcerative
(Table 1). Histologically, they all consisted of cords
or islands of atypical cells that invaded the dermis
(Fig. 6). Large nuclei with prominent nucleoli were
present in all cases, as well as numerous mitoses
(Fig. 7). Premature keratinization and intercellular
bridges were also present in most of the samples.
Finally, 23 cases of mucous membrane-derived invasive
SCC were available, which histologically all exhibited
similar changes as those described above for skinderived SCCs. Seventeen of the latter lesions arose
from the gingiva, four from the tongue, one from the
nasal mucosa and one from the lips.
Three canine papillomas, one feline squamous cell
carcinoma in situ and one bovine fibropapilloma were
included as positive controls. The bovine fibropapilloma
and the canine papillomas exhibited changes typical of
papillomavirus infections, such as koilocytosis, clumping of the keratohyalin granules and viral inclusion
bodies (data not shown). The feline in situ SCC (data
not shown) had previously been shown by immunochemistry to react positively with papillomavirusspecific antibodies.
PCR studies
Papillomavirus DNA was detected in the positive control tissues but not in the negative control tissues. The
narrow-range PCR, optimized to detect known feline
and canine papillomaviruses, reacted positively with
extracts from the canine papillomas as well as with the
immunohistologically positive case of feline in situ
SCC. In contrast, this set of primers was unable to
discover bovine papillomavirus in extracts from the
bovine fibropapilloma tissue. However, the broad-range
PCR detected papillomavirus DNA in both carnivorous
and bovine positive control tissues.
46
294
N. Zaugg et al.
Table 1. Identification and properties of clinical SCC cases
Case #
Group
Breed
Sex
Age (years)
Localization
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
In situ
In situ
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Mucous membrane
Mucous membrane
Mucous membrane
Mucous membrane
Mucous membrane
Mucous membrane
Mucous membrane
Mucous membrane
Mucous membrane
Mucous membrane
Mucous membrane
Mucous membrane
Mucous membrane
Mucous membrane
Mucous membrane
Mucous membrane
Mucous membrane
Mucous membrane
Mucous membrane
Mucous membrane
Mucous membrane
Mucous membrane
Mucous membrane
Rhodesian ridgeback
Retriever mixed
German shepherd dog
Flatcoat retriever
Golden retriever
Giant schnauzer
Schnauzer
Giant schnauzer
Bernese mountain dog
Weimaraner
Golden retriever
Flatcoat retriever
Giant schnauzer
Siberian husky
Mongrel
Giant schnauzer
Mongrel
Weimaraner
Labrador retriever
English cocker
Yorkshire terrier
Golden retriever
Airedale terrier
Standard poodle
Standard poodle
Yorkshire terrier
Golden retriever
Cocker spaniel
Old English sheepdog
Maltese
Irish setter
Irish wolfhound
West Highland white terrier
German Wachtelhund
Pekingese
Mongrel
Golden retriever
Pekingese
Schnauzer
Briard
Appenzeller
Golden retriever
M
F
F
F
M
F
M
F
M
M
M
M
M
F
M
F
M
M
M
M
F
M
M
F
M
M
M
M
F
F
M
F
F
F
F
M
M
F
M
M
F
M
4
5
13
12
13
10
Unknown
8
7
8
12
8
7
11
10
9
10
8
4
7
8
10
9
10
7
12
5
14
11
10
8
7
12
11
11
13
11
9
13
6
12
4
Diffuse
Abdomen, limbs
Limb
Clawbed
Nasal planum
Clawbed
Clawbed
Clawbed
Dorsum
Flank
Limb
Limb
Carpus
Carpus
Limb
Clawbed
Clawbed
Face
Clawbed
Nose
Gingiva
Gingiva
Gingiva
Tongue
Gingiva
Tongue
Gingiva
Gingiva
Gingiva
Gingiva
Gingiva
Lips
Gingiva
Tongue
Gingiva
Gingiva
Gingiva
Gingiva
Gingiva
Gingiva
Tongue
Gingiva
Table 2. Papillomavirus-positive cases
Case #
Group*
Breed
Sex
Age
Localization
Related to†
1
2
11
18
20
33
36
40
42
In situ
In situ
Invasive skin
Invasive skin
Mucous membrane
Mucous membrane
Mucous membrane
Mucous membrane
Mucous membrane
Rhodesian ridgeback
Retriever mix
Golden retriever
Weimaraner
Cocker
West Highland white terrier
Mongrel
Briard
Golden retriever
M
F
M
M
M
F
M
M
M
4
5
12
8
7
12
13
6
4
Diffuse
Abdomen, limbs
Limb
Face
Nose
Gingiva
Gingiva
Gingiva
Gingiva
HPV65‡
BPV1
HPV85
HPV59
COPV§
HPV65
HPV59
HPV65
HPV4
*Clinical SCC type; †closest relative detected by NCBI-BLAST analysis; ‡unless otherwise stated, detected exclusively by broad-range PCR;
§detected by both narrow-range and broad-range PCR.
The results of the test samples included in this
study are presented in Table 2. Interestingly, the narrowrange PCR detected only one case of SCC associated
with a papillomavirus infection, namely a mucous
membrane-derived SCC (case 20). However, this case
and eight additional cases were detected with the
broad-range technique, including 2/2 samples from in
situ SCC, 2/17 invasive skin SCCs, and 5/23 cases of
mucous membrane-derived invasive SCC.
Sequencing studies
The above PCR results strongly suggested that thus far
unknown papillomaviruses had been detected with the
broad-range PCR. To address this issue, the nucleotide
47
295
the use of formalin-fixed, paraffin-embedded tissue
decreases the amplification rate of viral DNA.31 Our
study demonstrates that COPV DNA is rarely present
in SCC samples.
The absence of PV DNA in the nontumoral skin in
the present study suggests that dogs, unlike humans,
are rarely affected by occult infections. This conclusion
is corroborated by Antonnsson et al. who have uncovered PV DNA in the skin of various healthy animals
but not in dogs and cats.24
Invasive skin SCCs appear to be infrequently
infected by PVs (2/17: 12%). These results are in
contrast with those of mucous membrane SCCs that
appeared to be more frequently affected (5/23: 22%).
Last but not least, the two cases of skin in situ SCC
were deemed positive. The viral aetiology of this latter
condition has already been suggested by two different
case reports.4,5 However, the presence of PV DNA in
the two tested samples of canine in situ SCC are not
proof that these viruses are directly responsible for the
development of these tumours. In fact, establishing
causality between papillomaviruses and skin cancers
remains problematic. Criteria for causality were first
proposed by zur Hausen and have been modified by
Harwood.16,17 According to these authors, epidemiological evidence that the viral infection represents a
risk factor, regular presence of the viral nucleic acid,
stimulation of proliferation upon cross-infection of the
viral genome in tissue culture cells and demonstration
that induction of proliferation depends on functions of
the viral DNA are essential criteria to demonstrate
causality. At the present time, not all these criteria are
fulfilled by the canine in situ SCCs.
The clinical relevance of the presence of PVs in invasive mucosal and skin SCC lesions is another important question. Assuming that PVs play a role in the
development of canine SCC, the absence of PV DNA
in numerous canine SCC can be explained by the ‘hit
and run’ model, which postulates an initial transformation of the infected cell and a subsequent loss of
PV-DNA.32 Interestingly, in a previous study in canines,
a PV antigen-negative SCC arose from a PV antigenpositive Epidermodysplasia verruciformis-like lesion.4
This model, however, implies a deletion of viral genes
during integration of the viral DNA in the host chromosomes. Such a deletion has only been demonstrated
in humans with the E2 gene.33 As human high-risk
papillomaviruses are genetically different from lowrisk HPVs one can postulate that canine high-risk PV
(provided they do exist) should be genetically different
from COPV. As we have only used sets of primers that
were designed to uncover COPV DNA and high-risk
HPV DNA, we cannot rule out that the DNA of some
unknown canine PV remained undetectable.
The amplified DNA sequences suggest the presence
of several different PVs in the canine SCCs. Furthermore, eight out of nine positive samples were infected
by these unknown PVs. Moreover, our findings confirm that dogs, as well as humans, can be infected with
PVs of great genetic diversity.
sequences of the amplification products of the individual PCRs were determined and compared by BLAST
analysis to known papillomavirus sequences. If available (7/42 dogs), several independent samples from each
dog were used for PCR and sequencing, and sequencing results were identical for different locations of
the lesions from each individual dog. However, the
sequences differed largely between different dogs. As
expected, the sequence obtained from case 20 turned
out to be closely related to the published sequence of
canine oral papillomavirus.21 In contrast, the remaining eight positive cases were more closely related to
other papillomaviruses, such as human and bovine
papillomaviruses (Table 2). Although the classification
of papillomavirus is based on the L1 sequences, these
results strongly support that novel canine papillomaviruses were detected in canine SCCs throughout this
study. However, 78% of the tested samples still remained
negative, which indicated that either a large proportion
of canine SCC is not associated to papillomavirus
infection or that the corresponding virus strains are
still more different, which would mean that the range
of PCR detection would still need to be further
enlarged.
Breed, sex and age distribution
These data are included in Tables 1 and 2. Although
schnauzers appeared to be over-represented in the total
sample, retrievers (flatcoated and golden) (19% of the
sample but 33% of papillomavirus-positive individuals)
emerged as a possible breed group with a tendency to
SCC caused by papillomavirus. The total sex distribution resulted in 60% male and 40% female dogs. However, 70% of the papillomavirus-positive dogs were
male. The average age of dogs with SCC was 9.25 years,
with in situ SCC, 4.5 years, with invasive skin SCC,
9.38 years, and of dogs with mucous SCC, 9.57 years.
The average age of papillomavirus-positive dogs with
SCC was 7.89 years.
DISCUSSION
In this study, it was possible to uncover PV DNA in
9/42 samples of canine SCC (21.4%) with a broad-range
PCR-assay and in 1/42 (2.3%) with a narrow-range
PCR-assay designed to amplify DNA from COPV,
FdPV and closely related PVs. Interestingly, the last
figure is in line with that of the only other study which
used a PCR technique:12 Teifke et al. used a narrowrange PCR and amplified COPV DNA in 3/53 SCC
samples (6%). These numbers are, however, lower than
those obtained in previously published immunohistochemistry studies. In one of these studies, PV antigens
were demonstrated in 10/100 canine SCC samples, with
17 additional questionable positive results (10–27%).11
In the second one, PV antigens were uncovered with an
immunoperoxidase technique in 6/20 (30%) samples.30
It is, however, important to consider that the PCR
technique cannot amplify the DNA of all PVs and that
48
296
N. Zaugg et al.
The detection of PV DNA in SCC tissues and not in
normal skin or skin affected by other conditions might
result from the increased replication of latent virus in
response to tumoral cell cytokine secretions.34
Mitsuishi et al. have uncovered HPV DNA in 74 and
67% of the samples of human actinic keratosis and
Bowen’s diseases, respectively.9 In the same study, no
differences in p53, p21, Ki 67 and PCNA were found
between HPV-positive and HPV-negative samples. This
set of data suggests that HPVs probably play a role in
the pathogenesis of both conditions but that the viruses
alone are not able to induce cancer transformation.
Human SCCs also occur many years after the initial
infection, which usually has a benign course, even with
oncogenic HPV types.14 Malignant transformation
thus implies the presence of continuing infection and
prolonged E6/E7 oncogene expression.35 Such chronic
infections occur in human anal or cervical PV infections and Epidermodysplasia verruciformis but have
not been demonstrated with canine PV infections.20
Although the present results cannot be regarded as
sufficient proof for the carcinogenic potential of canine
PVs, the detection of novel members of this large
family of viruses is important for further research on
this issue.
Importantly, the novel papillomaviruses were uncovered in the two cases of SCC in situ. These two dogs
were younger than the average age of the available dogs
with SCC, which also favours a viral aetiology for this
type of lesions. Indeed, the presence of PVs in such
lesions has been established previously.4 Additionally,
one of the lesions described by this author subsequently developed into invasive SCC and the similarities
between these cases and the human Epidermodysplasia
verruciformis has already been emphasized.4 The presence of specific oncogenic PVs in a significant number
of such canine lesions consequently warrants further
investigation.
The study has also demonstrated the great diversity
of the canine PVs. Cloning these new viruses will allow
phylogenetic comparison with human commensal and
high-risk PVs. These comparisons can eventually
provide clues for the interpretation of past and future
studies.
3. Gross TL, Fau-Brimacomb BH. Multifocal intraepidermal carcinoma in a dog histologically resembling Bowen’s
disease. American Journal of Dermatopathology 1986;
8: 509–15.
4. Nagata M, Nanko H, Moriyama A et al. Pigmented
plaques associated with papillomavirus infection in dogs:
is this epidermodysplasia verruciformis? Veterinary
Dermatology 1995; 6: 179–85.
5. Stokking LB, Ehrhart EJ, Lichtensteiger CA et al.
Pigmented epidermal plaques in three dogs. Journal of
the American Animal Hospital Association 2004; 40:
411–17.
6. Anwar J, Wrone DA, Kimyai-Asadi A et al. The development of actinic keratosis into invasive squamous cell
carcinoma: evidence and evolving classification schemes.
Clinical Dermatology 2004; 422: 189–96.
7. Arlette JP, Trotter MJ. Squamous cell carcinoma in situ
of the skin: history, presentation, biology and treatment.
Australasian Journal of Dermatology 2004; 45: 1–
11.
8. Cockerell CJ. Pathology and pathobiology of the actinic
(solar) keratosis. British Journal of Dermatology 2003;
149: 34–6.
9. Mitsuishi T, Kawana S, Kato T et al. Human papillomavirus infection in actinic keratosis and Bowen’s disease:
comparative study with expression of cell-cycle regulatory proteins p21waf1/cip1, 53, pcna, ki-67, and bcl-2 in
positive and negative lesions*1. Human Pathology 2003;
34: 886–92.
10. Sundberg JP, Junge RE, Lancester WD. Immunoperoxidase localization of papillomaviruses in hyperplastic and
neoplastic epithelial lesions in animals. American Journal
of Veterinary Research 1984; 45: 1441–6.
11. Schwegler K, Walter JH, Rudolph R. Epithelial neoplasms of the skin, the cutaneous mucosa and the transitional epithelium in dogs: an immunolocalization study
for papillomavirus antigen. Journal of Veterinary
Medicine A 1997; 44: 115–23.
12. Teifke JP, Lohr CV, Shirasawa H. Detection of canine
oral papillomavirus-DNA in canine oral squamous cell
carcinomas and p53 overexpressing skin papillomas of
the dog using the polymerase chain reaction and nonradioactive in situ hybridization. Veterinary Microbiology
1998; 60: 119–30.
13. Watrach AMSe, Case MT. Canine papilloma: progression of oral papilloma to carcinoma. Journal of National
Cancerology Institute 1970; 45: 915–20.
14. Lowy DRH, Howley PM. Papillomaviruses. In: Knipe DM,
Howley PM eds. Fields Virology, 4th edn. Philadelphia:
Lippincott Williams & Wilkins, 2001: 2231–64.
15. de Villiers E-M, Fauquet C, Broker TR et al. Classification of papillomaviruses. Virology 2004; 324: 17–27.
16. Harwood CA, Proby CM. Human papillomaviruses and
non-melanoma skin cancer. Current Opinion in Infectious
Diseases 2002; 15: 101–14.
17. zur Hausen H. Papillomavirus infections – a major cause
of human cancers. Biochimica et Biophysica Acta 1996;
1288: F55–78.
18. zur Hausen H. Papillomaviruses and cancer: from basic
studies to clinical application. National Review of
Cancer 2002; 2: 342–50.
19. Chambers VC, Evans CA. Canine oral papillomatosis. I.
Virus assay and observations on the various stages of the
experimental infection. Cancer Research 1959; 19: 1188–
95.
AC K N OW L E D G E M E N T S
The authors would like to acknowledge Dr Thelma Lee
Gross who has provided one of the samples (case #2).
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Morrison WB ed. Cancer in Dogs and Cats. Jackson:
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2. Thomas RCF, Fox LE. Tumors of the skin and the
subcutis. In: Morisson WB ed. Cancer in Dogs and Cats,
2nd edn. Jackson: Teton New Media, 2002: 473 – 88.
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20. Nicholls PK, Stanley MA. Canine papillomavirus – A
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21. Sundberg JP, O’Banion MK, Schmidt-Didier E et al.
Cloning and characterization of a canine oral papillomavirus. American Journal of Veterinary Research 1986;
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22. Le Net JL, Orth G, Sundberg JP et al. Multiple pigmented cutaneous papules associated with a novel canine
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23. Campbell KL, Sundberg JP, Goldschmidt MH et al.
Cutaneous inverted papillomas in dogs. Veterinary
Pathology 1988; 25: 67 –71.
24. Antonsson A, Hansson BG. Healthy skin of many animal
species harbours papillomaviruses which are closely
related to their human counterparts. Journal of Virology
2002; 76: 12537 – 42.
25. Antonsson A, Erfurt C, Hazard K et al. Prevalence and
type spectrum of human papillomaviruses in healthy skin
samples collected in three continents. Journal of General
Virology 2003; 84: 1881– 6.
26. Pfister H. Human papillomavirus and skin cancer. Journal
of National Cancer Institute Monographs 2003: 52– 6.
27. Saveria Campo M. Animals models of papillomavirus
pathogenesis. Virus Research 2002; 89: 249 – 61.
28. Smith KT, Campo MS. Papillomaviruses and their
involvement in oncogenesis. Biomedical Pharmacotherapy 1985; 39: 405 –14.
29. Iftner A, Klug SJ, Garbe C et al. The prevalence of
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Sundberg JP, Smith EK, Herron AJ et al. Involvement of
canine oral papillomavirus in generalized oral and cutaneous verrucosis in a Chinese Shar Pei dog. Veterinary
Pathology 1994; 31: 183–7.
Albini S, Zimmermann W, Neff F et al. Identification
and quantification of ovine gammaherpesvirus 2 DNA in
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Smith KT, Campo MS. ‘Hit and run’ transformation of
mouse C127 cells by bovine papillomavirus type 4: the
viral DNA is required for the initiation but not for maintenance of the transformed phenotype. Virology 1988;
164: 39–47.
Ordonez RM, Espinosa AM, Sanchez-Gonzalez DJ
et al. Enhanced oncogenicity of Asian-American human
papillomavirus 16 is associated with impaired E2 repression of E6/E7 oncogene transcription. Journal of
General Virology 2004; 85: 1433–44.
de Villiers EM, Ruhland A. Do specific human papillomavirus types cause psoriasis? Archives of Dermatology
2001; 137: 384.
Duensing S, Münger K. Mechanisms of genomic instability in human cancer: insights from studies with human
papillomavirus oncoproteins. International Journal of
Cancerology 2004; 109: 157–62.
Résumé L’ADN de papillomavirus (PV) est fréquemment retrouvé dans des achantillons de carcinome épidermoïde (SCC) chez l’homme. Cependant le rôle de ces virus dans le développement de ces cancers est controversé.
Alors qu’environ une centaine de PV sont recensés chez l’homme, un seul PV canin oral (COPV) a été identifié
et étudié. Nous avons utilisé une technique de PCR spécifique des PV canin et félin, ainsi qu’une technique de
PCR utilisée pour la détection des nouveaux PV humains, afin d’analyser 42 biopsies paraffinées, représentant
trois formes différentes de SCC canins. 10 échantillons de tissus affectés par des maladies non néoplasiques ont
servi de contrôle. Aucun des témoins négatifs n’a réagi positivement, et de l’ADN de PV a été retrouvé cajs 21%
des prélèvements testés de SCC. Le COPV classique n’a été amplifié qu’une seule fois, alors que les autres cas
positifs étaient associés à la présence d’une variété inconnue de PV humain. Cette étude suggère qu’une fraction
des SCC canins est infectée par le PV, et qu’il existe une variété génétique des PV canins. Ces résultats vont faciliter
les études futures qui s’intéresseront au rôle des PV dans le développement des cancers cutanés du chien.
Resumen El ADN del virus papiloma (PV) se descubre de forma frecuente en muestras de carcinoma de células
escamosas (SCC) de la piel humana. Sin embargo, el papel de estos virus en el desarrollo de estas neoplasias en
el perro es aún controvertido. Mientras que se conocen aproximadamente 100 virus papiloma en humanos, tan
sólo un virus papiloma canino oral (COPV) ha sido identificado y estudiado de forma exhaustiva. Por ello, para
analizar 42 muestras incluidas en parafina que representaban tres formas diferentes de carcinomas de células escamosas en perros, aplicamos una reacción de polimerasa en cadena (PCR) de estrecho rango, válida para detectar
virus papiloma clásicos caninos y felinos; y también una PCR de amplio rango, que ha sido utilizada para la detección de nuevos virus papiloma en humanos. Diez muestras de piel con lesiones no neoplásicas se utilizaron como
controles. Mientras que ninguno de los controles negativos dio resultado positivo, ADN de virus papiloma se
encontró en un 21% de las muestras de carcinoma de células escamosas. Curiosamente, el clásico virus papiloma
oral canino solo se amplificó de una muestra, mientras que los otros casos positivos se asociaron con variedades
hasta ahora desconocidas de virus papiloma. Este estudio sugiere que una fracción de carcinomas de células escamosas caninos está infectada con el virus papiloma, y que existe una diversidad genética de virus papiloma caninos. Por lo tanto, estos resultados facilitarán futuros estudios sobre el papel del virus papiloma en el desarrollo
de cáncer de piel en perros.
Zusammenfassung Papillomavirus (PV) DNA wird häufig in Hautproben von Plattenepithelkarzinomen des
Menschen gefunden. Beim Hund bleibt die Rolle dieser Viren bei der Entstehung derartiger Tumoren allerdings
50
298
N. Zaugg et al.
umstritten. Während etwa 100 humane Papillomaviren bekannt sind, wurde erst ein einziges canines orales Papillomavirus (COPV) identifiziert und umfangreich untersucht. Daher haben wir eine ‘narrow-range’ Polymerase
Chain Reaction (PCR) angewendet, die passend ist für den Nachweis von klassischen caninen und felinen Papillomaviren, sowie eine ‘broad-range’ PCR, die verwendet worden war für den Nachweis von verschiedenen neuen
Papillomaviren beim Menschen, um 42 in Paraffin eingebettete Proben zu analysieren, die drei unterschiedliche
Formen von caninem Plattenepithelkarzinom repräsentierten. Zehn Hautproben von verschiedenen nichtneoplastischen Zuständen dienten als Kontrollen. Während keine der Negativkontrollen positiv war, wurde PV
DNA in 21% der untersuchten Proben der Plattenepithelkarzinome gefunden. Interessanterweise wurde das
klassische COPV nur aus einer Probe isoliert, während die anderen positiven Fälle im Zusammenhang mit einer
Variation von bisher unbekannten PVs gefunden wurden. Diese Studie weist darauf hin, dass ein Teil der caninen
Plattenepithelkarzinome mit PV infiziert ist und dass eine genetische Variation der caninen Papillomaviren
besteht. Daher werden diese Ergebnisse zukünftige Studien über die Rolle von Papillomaviren bei der Entstehung
von caninen Hauttumoren erleichtern.
51
Chapter 7
Detection of novel papillomavirus-like DNA sequences in
paraffine-embedded samples of feline invasive and in situ
squamous cell carcinomas
G. Nespeca1, P. Grest2, W. S. Rosenkrantz3, M.
Ackermann4, C. Favrot1
American Journal of Veterinary Research, 2006; 67 (12):
2036-2041
1
2
Institute for Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Zurich,
Switzerland
3
Animal Dermatology Clinic, San Diego, CA, USA
4
Funded by grants from the Waltham Foundation and the European Society of
Veterinary Dermatology
52
Detection of novel papillomaviruslike sequences
in paraffin-embedded specimens of invasive
and in situ squamous cell carcinomas from cats
Gilles Nespeca, Med Vet; Paula Grest, DVM; Wayne S. Rosenkrantz, DVM;
Mathias Ackermann, DVM, PhD; Claude Favrot, DrVet, MS
Objective—To detect and partially characterize papillomavirus (PV) DNA in squamous cell carcinoma
(SCC) tumor specimens from cats.
Sample Population—54 formalin-fixed paraffinembedded skin biopsy specimens were examined.
Specimens originated from Bowenoid in situ SCC
(BISC; n = 21), invasive SCC (22), and skin affected by
miscellaneous nonneoplastic conditions (11).
Procedures—Samples from each tissue block underwent DNA extraction after deparaffinization, and PCR
assays were performed. Two sets of primers derived
from PV E1 were used. The first set of primers was
designed for the narrow-range PCR assay and was
able to generate amplification products of feline PV
(FePV), canine oral PV, or closely related PVs. The second set of primers was selected for the broad-range
PCR assay because of its ability to amplify DNA from
64 human PVs. Sequence analysis of each amplified
DNA was performed.
Results—1 of the 21 specimens of BISC was positive
for PV DNA on the basis of narrow-range PCR assay
results, whereas all the other specimens (BISC, invasive SCC, and controls) had negative results for PV
DNA. In contrast, 5 of 21 BISC specimens and 4 of 22
invasive SCC specimens were positive for PV DNA on
the basis of broad-range PCR assay results.
Sequence analysis revealed that only 1 specimen was
infected by a virus closely related to classic FePV. In
the 8 other specimens positive for PV DNA, DNA of
unknown PVs was uncovered.
Conclusions and Clinical Relevance—Bowenoid in
situ SCC and invasive SCC of cats may be associated
with PVs of genetic diversity. (Am J Vet Res
2006;67:2036–2041)
AK
SCC
PV
HuPV
FePV
BISC
CaPV
BoPV
ABBREVIATIONS
Actinic keratosis
Squamous cell carcinoma
Papillomavirus
Human PV
Feline PV
Bowenoid in situ SCC
Canine PV
Bovine PV
AK, a precancerous skin growth associated with sun
exposure.3 However, AK is often regarded as a form of
SCC, which is confined to the epidermis; thus, AK is
also referred to as in situ SCC.3-6 A second form of in
situ SCC, precancerous dermatitis (termed Bowen’s disease), presents as 1 or more flat red scaly patches up to
several centimeters wide, often found in large numbers.1,4,6 In situ SCC can persist as such; regress; or
develop into a third, even more malignant form, invasive SCC. Similar skin cancers are also observed in veterinary medicine, specifically in cats.7 Squamous cell
carcinoma has been linked to a variety of causative
associations, which include exposure to UV or ionizing
radiation; arsenic ingestion; toxic exposure to tars and
oils; immunosuppression from drugs such as corticosteroids, azathioprine, and cyclosporine; and last but
not least, to PV infection.1,8-10
Papillomaviruses are host-specific epitheliotropic
DNA viruses that infect skin and mucous membranes.
In general, PV infections are benign, result in a latent
infection, or induce microlesions or benign neoplasias.11-14 However, a subset of HuPVs and other animal PVs is clearly implicated in the development of
cancer.10, 14-17 Human PVs that cause mucosal and skin
carcinomas in humans are referred to as high-risk PVs
or epidermodysplasia verruciformis–associated HuPV
types, respectively.14
Close to 100 HuPV types have been described on
the basis of isolation of complete genomes.13
Knowledge on the combination of biological properties
and sequence similarities led to the definition of new
criteria to define genera, species, types, subtypes, and
variants within the Papillomaviridae family.13
In contrast to the numerous HuPVs, only a single
FePV has been identified.18 However, the existence of a
few other FePVs has been suggested on the basis of
findings from several clinical and immunohistochemical studies.19-22
Until now, little has been known about the presence of PVs in SCCs of cats. Investigators in 1 study23
S
quamous cell carcinoma is, after basal cell carcinoma, the second most common cancer of the skin in
humans.1,2 Squamous cell carcinoma involves cancerous
changes to the cells of the middle portion of the epidermal skin layer. This cancer may begin in normal skin;
in skin at the site of a burn, injury, or scar; or at a site
of chronic inflammation.1 Most often, it originates from
Received June 29, 2006.
Accepted July 31, 2006.
From the Clinic for Small Animal Internal Medicine, Dermatology
Unit (Nespecca, Favrot), the Institute for Veterinary Pathology
(Grest), and the Virology Institute (Ackermann), Vetsuisse
Faculty, University of Zürich, Winterthurerstrasse 260, CH 8057
Zurich, Switzerland; and the Animal Dermatology Clinic, 5610
Kearny Mesa Rd, Ste B, San Diego, CA 92111 (Rosenkrantz).
Supported by grants from the Waltham Foundation and the
European Society of Veterinary Dermatology.
Address correspondence to Dr. Favrot.
2036
AJVR, Vol 67, No. 12, December 2006
53
failed to uncover PV antigen in SCCs of cats, whereas
findings in another study24 revealed the presence of PV
antigens in 44% of tumor specimens of BISC from cats.
Furthermore, PV DNA has been uncovered in tumor
specimens from fibropapillomas, another type of cutaneous proliferative disease, of cats.25
Similar to the human disease types, 3 varieties of
SCC have been described for cats, which are AK, BISC,
and invasive SCC.26 Actinic keratosis usually occurs as
a solitary lesion on sun-exposed, lightly haired areas,
such as ear tips, external nares, or eyelids. White cats
are predisposed for the development of such lesions.
On the other hand, BISC is characterized usually by
multiple well-circumscribed, hyperpigmented lesions
that occur frequently on the face, neck, and limbs.27,28
To our knowledge, comparative studies of these 2 early
forms of cancer have not been performed in cats.
The purpose of the study reported here was to
detect PV DNA in specimens representing the various
types of SCC in cats and in specimens from feline skin
with various nontumor conditions. We wanted to test
whether tumor specimens from cats with SCC were
more often infected by PVs than nontumor skin specimens. Two types of PCR assays, narrow and broad
range, were applied to extend the range of targeted PVs
as far as possible.
AACAGCTGYTTTTTARCYTTTTT-3′) for narrow-range PCR
assay, which is able to generate amplification products of
approximately 341 bp of FePV, CaPV, or closely related PVs.
The second set of primers (ie, CP4, CP5, and PPF1
primers), also derived from E1, was selected for broad-range
PCR assay with the objective of amplifying as many PVs as
possible. With this set of primers, up to 64 HuPVs are identifiable.30 The expected size of the PCR product was approximately 450 bp.
PCR assay and agarose gel electrophoresis—
Polymerase chain reaction conditions for PapF and PapR
were performed. Volumes of 30 mL were used. Each reaction
contained 1 µL of genomic DNA, 200µM of each deoxynucleoside triphosphate, 0.3µM of each of the sense and antisense primers, and 2.5 units of a DNA polymerase.c After an
initial denaturation step at 95oC for 4 minutes, PCR assay
was performed for 30 cycles at 95oC for 1 minute, 50oC for 1
minute, and 74oC for 1 minute, with a final elongation step
at 74oC for 5 minutes. Deoxyribonucleic acid extracted from
canine warts served as positive control, whereas DNA- and
RNA-free water was used as negative control.
The PCR mix with CP4, CP5, and PPF1 primers was
identical to the mix for narrow-range PCR assay, except that
0.45µM of the CP4 and CP5 primers and 0.3µM of the PPF1
primer were used. The PCR assay consisted of a denaturation
step at 95oC for 10 minutes, followed by 40 cycles at 95oC for
1 minute, 47oC for 1 minute, and 74oC for 1 minute, with a
final elongation step at 75oC for 5 minutes. An extract from 1
bovine fibropapilloma served as an additional positive control.
Polymerase chain reaction products were segregated by
agarose gel electrophoresis, and bands were viewed under UV
light after ethidium bromide staining. Bands on the gel were
excised, and DNA was extracted with a gel extraction kit.d
Amplified DNA was sequenced by use of fluorescent
sequencing and terminator chemistry.e
Materials and Methods
Tissue specimens—Tissues were obtained from the collections of the Prairie Diagnostics Services, Western College
of Veterinary Medicine, University of Saskatchewan,
Saskatoon, SK, Canada; the Institut de Pathologie et de génétique, Loverval, Belgium; Rest associates, London; and, the
Pathology Institute, Vetsuisse Faculty, University of Berne,
Berne, Switzerland. Fifty-four formalin-fixed paraffinembedded skin biopsy specimens were examined. Specimens
originated from BISC (n = 21), invasive SCC (22), and miscellaneous skin conditions other than skin cancers (eg, allergic dermatitis; 11) that were used as negative controls.
Specimens from white cats or from locations typical for AK
such as ear tips and eyelids were excluded from this study.
Thirty-micrometer-thick sections were cut from each tissue
block, with a new disposable microtome blade for each
block, before DNA extraction. Two canine warts, which had
histopathologic characteristics of typical PV-induced inclusion bodies, and 1 bovine fibropapilloma served as positive
controls.
Sequence analysis—Samples were considered positive
for PV DNA if they met the following requirements: they
had a band of the expected size after gel electrophoresis and
the sequenced DNA had homology with E1 of previously
sequenced PVs. Homologous DNA sequences were searched
for by use of the National Center for Biotechnology
Information GenBank via a BLAST search.f Sequence
alignments and phylogenetic trees were made from
the clustal algorithm obtained by use of a software
program.g
Results
Macro- and microscopic analysis—A careful
macroscopic selection and microscopic confirmation
of affected specimens was a major prerequisite prior
to the virologic analysis. Specimens representing
invasive SCC had been resected from sun-exposed or
white areas of 21 domestic shorthaired cats and 1
Persian cat (12 males and 10 females). Twenty-two
specimens from ear tips (n = 11), nose (3), eyelids
(3), digits (3), and lips (2) met criteria required for
invasive SCC. These criteria included the macroscopic presence of scaly-to-crusty and erosive-to-plaquelike or ulcerative lesions (Figure 1). The growth
process was always endophytic. Histologically, cords
or islets of infiltrative cells were detected in all specimens. Furthermore, anisocytosis; anisokaryosis;
large, hyperchromatic nuclei; prominent nucleoli;
increased mitotic index; and abnormal mitoses were
encountered in all specimens with variable intensity
and in variable proportion. In addition, keratin pearls
DNA extraction—The protocol of Albini et al29 was used
for DNA extraction. Briefly, each section was deparaffinized
twice with 1.2 mL of xylene at room temperature (approx
20oC) for 10 minutes, washed with 100% ethanol, and then
dried at 37oC for 30 minutes. Desiccated samples were suspended in a tissue lysis buffer (50mM Tris-HCl [pH 8.5],
1mM ethilenediaminetetraacetic acid, and 2.8% sodium
dodecylsulfate) and proteinase K (20 mg/mL) and incubated
at 56oC on a rocking platform overnight. After lysis, samples
were transferred to a columna and centrifuged to reduce viscosity. The DNA was precipitated with absolute ethanol and
extracted with a commercial DNA kit.b
Primers—Two sets of primers were used for the PCR
assay. Because the sequences encoding E1 are highly conserved, the E1 regions of FePV (GenBank accession No.
AF480454) and CaPV (GenBank accession No. NC001619)
were aligned to design a set of consensus primers (ie, PapF,
5′-ATGGCGGGMTARAAAAGGTA-3′ and PapR, 5′AJVR, Vol 67, No. 12, December 2006
2037
54
BISC were available for virologic analysis by PCR assay
and sequencing.
and intercellular bridges were present in 16 and 12
specimens, respectively.
A second group of 21 tumor specimens met the
criteria for BISC. These specimens were obtained
from the face (n = 16), neck (12), and limbs (3) or
were scattered (2). Thirteen domestic shorthair cats,
3 domestic longhair cats, 2 Siamese, 1 Persian, 1
Himalayan, and 1 Cornish Rex were affected.
Macroscopically, the lesions were squamous crustosus
and grossly circular. Two lesions had a single center,
but 19 were multicentric (Figure 1). Microscopically,
the following criteria were met for BISC: moderate-tosevere parakeratotic hyperkeratosis, acanthosis with
papillomatous hyperplasia (n = 1) or irregular hyperplasias (20), loss of polarity, and scattered dyskeratotic keratinocytes atypia in all layers of the epidermis
and usually also in the infundibulum and reaching
the isthmus. Furthermore, the following types of
atypia were recorded: enlarged nuclei, anisokaryosis,
monster cells (bizarre multinucleated giant cells),
and abnormal mitotic figures. Hyperpigmentation
was found in all but 3 specimens. Fifteen of the 21
specimens had clumped keratohyalin granules, which
were considered as suggestive for PV infection.
However, other signs such as koilocytosis and nuclear
inclusion bodies were not detected.
With the exception of the ulcerated lesions, the
dermis of all samples was considered normal and not
heavily inflamed (Figure 1). Thus, a total of 22 samples
representing invasive SCC and 21 samples representing
PCR assays—The narrow-range PCR assay amplified PV DNA extracted from canine warts but not DNA
extracted from bovine fibropapilloma (Figure 2). In
contrast, the broad-range PCR assay amplified PV DNA
from canine warts and bovine fibropapilloma. It was
concluded that both PCR assays were able to specifically amplify selected PV DNAs.
The narrow-range PCR assay was applied to samples from invasive SCC and BISC specimens; 1 BISC
sample (BISC sample No. 15; Appendix; Figure 3) had
positive results for PV DNA, whereas the others had
negative results. Next, the broad-range PCR assay was
applied to the same samples. Interestingly, 5 of 21 BISC
samples (BISC sample Nos. 2, 5, 6, 10, and 15) as well
as 4 of 22 SCC samples (SCC sample Nos. 15, 24, 28,
and 29) had positive results for PV DNA (Figure 2).
One of the samples that had positive results for PV
DNA on the broad-range PCR assay (BISC sample No.
15) also had positive results for PV DNA on the narrow-range PCR assay. These results suggested that the
broad-range PCR assay was indeed able to uncover PVs
that were different from the known FePV and CaPVs.
4c
Figure 1—Macroscopic and microscopic lesions of SCCs in cats.
A—Photograph of invasive SCC in a cat with ulceration of the
eyelid. B—Photomicrograph of a section of the invasive SCC
from panel A at low magnification. Notice invasive proliferation
of atypical keratinocytes with pearl formation (white arrow) and
the cornified layer (red arrow). The basal membrane is not discernible. The dermis (long arrow) is invaded by cords of atypical
keratinocytes (short black arrow, pointing towards such an invasive site). H&E stain; bar = 200 µm. C—Photomicrograph of a
section of the invasive SCC from panel A at high magnification.
Notice islets of keratinocytes with features of malignancy, such
as anisokaryosis, anisocytosis, multinucleated cells (short
arrow), and abnormal mitosis (long arrow). H&E stain; bar = 50
µm. D—Photograph of BISC in a cat with circular, crusted, erosive, and hyperpigmented plaques (arrow). E—Photomicrograph
of a section of the BISC from panel D at low magnification. The
basal membrane (white arrow) is intact, and the dermis is not
invaded. Irregular acanthosis (long black arrow) is obvious.
Notice that hair follicles are affected (short arrow). H&E stain;
bar = 200 µm. F—Photomicrograph of a section of the BISC
from panel D at high magnification. Notice acanthosis, hyperpigmentation (brown cells), clumped keratohyalin granules
(white arrow), loss of polarity, and anisokaryosis (branched
arrow). H&E stain; bar = 50 µm.
Figure 2—Establishment of broad-range and narrow-range PCR
assays for detection of carnivore PVs. Polymerase chain reaction
products were loaded on agarose gels and stained with ethidium bromide. A—Amplification of cloned DNA by either broadrange (450 bp) or narrow-range PCR assay (341 bp). Lane 1 =
Water in place of DNA added to the reaction. Lane 2 = DNA
from a commercially available phagemid.h Lane 3 = DNA from
oral CaPV cloned into the phagemid.h Lane 4 = DNA from CPV3
(GenBank accession No. DQ295066) cloned into the phagemid.h
M1 = Molecular weight marker (100-bp ladder). B—The DNA
was extracted from tissues before being amplified by either the
narrow range or the broad-range PCR assays. M2 = 1-kilobase
ladder. M1 = 100-bp ladder. Lane 1 = Negative control with no
DNA added to the reaction. Lane 2 = Extract from canine wart
tissue, which had typical PV-induced inclusion bodies on histologic examination. Lane 3 = Extract from a tumor specimen of a
cat with SCC (GenBank accession No. DQ085784). Lane 4 =
Extract from SSC sample No. 39.
2038
55
Discussion
The purpose of our study was to detect and partially characterize PV DNA in samples representing in
situ and invasive types of SCC in cats to learn more
about PV variants in cats and about possible associations of these viruses with individual forms of SCC in
cats. Two types of PCR-assays, a narrow range and a
broad-range PCR, were applied to extend the range of
targeted PVs as far as possible.
Careful macroscopic selection and microscopic
confirmation resulted in the identification of 22 samples representing invasive SCC and 21 samples representing BISC, which were available for virologic analysis by PCR assay. Papillomavirus DNA was detected in
4 of 22 samples representing invasive SCC and in 5 of
21 samples representing BISC, whereas all nontumor
control samples had negative results for PV DNA. Only
1 (BISC sample No. 15) of the 9 viral DNAs had been
revealed by the narrow-range PCR assay. However, the
same narrow-range PCR assay amplified PV DNA
extracted from canine warts, which was expected
because the primers had been chosen for their homology with conserved sequences within E1 of FePV and
CaPV. Yet, the restricted range of these primers was
confirmed, as they proved unable to amplify DNA from
the more distantly related bovine fibropapilloma virus.
These results indicate that the remaining samples with
positive results for PV DNA did not harbor conventional FePV or CaPV.
Eight samples, which had negative results for PV
DNA on narrow-range PCR assay, had positive results
for PV DNA on broad-range PCR assay. The broadrange PCR assay made use of a second set of primers
that were also derived from E1 but known to uncover
a large variety of HuPVs.30 In our study, this second set
of primers also amplified DNA from bovine fibropapilloma virus as well as viral DNA from canine warts.
Sequencing of the amplification products obtained
from the 8 samples revealed novel PV-related DNAs,
although relations to HuPV, FePV, CaPV, rat PV, and
BoPV were evident. A phylogenic tree drawn from the
aligned sequences divided the new sequences into 4
clusters. Three of those clusters had close relationship
to CaPV, FePV, and HuPV. Interestingly, the fourth cluster, represented by 6 amplification products, was clearly distinct from BoPV type 5 and from HuPV type 71,
FePV, and CaPV. Judging from the limited sequence
information available, it appeared as if this fourth cluster represented a novel group of FePVs that had not
been detected previously and that may be associated
with SCC in cats. Notably, all PVs detected in association with invasive SCC were found to belong to this
novel cluster.
This represents, to our knowledge, the first evidence of thus far unknown PV-like sequences associated with SCC in cats. Interestingly, some of the novel
sequences were found in association with invasive
SCC. Notably, previous attempts to detect conventional PV antigens in such lesions had failed,23 which led to
the hypothesis that invasive carcinomas of cats are
probably not virally induced, whereas instances of
Bowen’s disease in cats are probably PV-induced.7 Our
findings clearly challenge the former opinion, although
Figure 3—Phylogenetic relationships of the newly detected PV
sequences with known PV E1 sequences (ie, CaPV, FePV, HuPV,
and BoPV are represented by GenBank accession Nos. D55633,
AF377865, AY330623, and AJ620206, respectively). IS = BISC.
SCC= Invasive SCC. Units indicate the number of substitution
events (percentage of nucleotides),
Sequence analysis—The nucleotide sequence of
the amplified DNA was determined and the resulting
sequences were compared to assess whether novel PVlike sequences had been detected. Indeed, use of the
basic local alignment search toole revealed relatedness to
PV E1 sequences for all 9 samples that were positive for
PV DNA. Relation to HuPV, FePV, CaPV, rat PV, and
BoPV was evident. Clustal alignmentsg revealed the various degrees of relationship of the newly determined
sequences among each other as well as in comparison
with known PVs. Overall, CaPVs and FePVs were most
closely related to the newly detected viral sequences,
with a relative amino acid identity of 56% to 71%.
Among the HuPVs, types 4, 55, 63, 65, 71, and 74 were
aligned most frequently but type 71 most often had the
closest relationship to the new sequences with 58% to
61% amino acid identity. Among the BoPVs, type 5 was
the closest relative, having 55% to 62% amino acid identity. A phylogenic tree drawn from the aligned sequences
divided the new sequences into 4 clusters (Figure 3). In
cluster 1, BISC sample No. 15 was situated most closely
with CaPV and FePV. Cluster 2 was occupied by BISC
sample No. 10 and was between FePV and HuPV type
71. Cluster 3 was represented by BISC sample No. 2 and
found close to HuPV type 71. The remaining sequences
(SCC sample Nos. 15, 24, 28, and 29 and BISC sample
Nos. 5 and 6) represented a fourth cluster, which was
clearly distinct from BoPV type 5 on the most distant
side and HuPV type 71, FePV, and CaPV on the less distant side. These results suggested the presence of thus
far unidentified PVs in tissues representing invasive SCC
and BISC. Interestingly, sequences obtained from specimens of 3 cats with invasive SCC (SCC sample Nos. 15,
24, and 28) had identical sequences. Furthermore, it
was observed that not a single sample from invasive SCC
specimens had been associated with the more classic
FePV and CaPV. However, because the classification of
PVs is based on the sequence of L1, the exact taxonomic position of these novel PV-like sequences could not be
assigned.
2039
56
a causative correlation between the disease and the
novel PV strains has not yet been shown. Results of
another study24 did reveal PV antigens in 44% of BISCs.
Although the proportion of BISC samples with positive
results for PV DNA in our study is lower (5 of 21 BISC
samples), it should be kept in mind that the broadrange PCR assay may not be able to reveal all variants
of FePVs. Furthermore, it is well-known that PCR
detection of viral nucleic acids in formalin-fixed and
paraffin-embedded tissues may be decreased in comparison to fresh tissue.29 Finally, the absence of PV
DNA in SCC samples can also be explained by the socalled hit-and-run model, which postulates an initial
transformation of the infected cell and a subsequent
loss of PV DNA.31
Full proof of the existence of the novel PVs that
are predicted through the results of our study still
needs to be provided. However, we suggest that a
great diversity of FePVs may exist that is in need of
detection and characterization. The future use of
the technique applied here will help in identifying
more affected cats with papilloma-associated diseases. Virologic studies can be initiated with the
aim to better characterize these novel viruses.
Cloning and sequencing of the entire genomes of
these viruses will allow phylogenetic comparisons
with HuPVs as well as discrimination between
benign and high-risk variants. Such studies can
eventually provide insights into the molecular pathways underlying the pathogenesis of these viruses
in cats.
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cutaneous squamous cell carcinoma and its variants. Semin Cutan
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3. Anwar J, Wrone DA, Kimyai-Asadi A, et al. The development of actinic keratosis into invasive squamous cell carcinoma: evidence and evolving classification schemes. Clin Dermatol
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(solar) keratosis. Br J Dermatol 2003;149:34–36.
6. Mitsuishi T, Kawana S, Kato T, et al. Human papillomavirus
infection in actinic keratosis and bowen’s disease: comparative
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2040
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Appendix
New papillomaviruslike sequences.
GenBank
accession No.
Sample type
and No.
DQ085782
BISC 15
1 ATGGTACAAT GGGCATTTGA CAATAAGTAC ACAGATGAAG CAGAGATAGC TTTTCATTAT
61 GCACGTTTGG CAGAGGAGGA TGCAAATGCA GAGGCTTGGT TAAAAAGCAA CTCCCAAGCT
121 AAATATGTCC GAGATTGTGC GCAAATGGTG AAGCTGTATC TTAGACAAGA AATGAGGCAG
181 ACTACTATTT CTGAATGGAT TGACAAGTGC TGCCAGTCAG TGACAGAGGA CGGTGACTGG
241 GGGGATATTA TGCGCTTCTT AAAATATCAG CAAGTTAATT TCACTCAGTT TTTAACTGCC
301 ATGAGAAATG CTTTAGAGGG TAAACCTAAA AAAAACTGCT TAGTATTTTA TGGGCCTCCA
361 GATACTGGCA AGTCATATTT CTGCTTTAGT TTGGTTAGTT TTATGCAGGG GAAAGTGGTG
421 AATTTTATGA ATAGCAA
DQ085783
BISC 2
1 ANGAGGAACG ATATAGCCTA CCACTATGCA TTGCTAGCCG ACGAGGACAC AAATGCAGCG
61 GCATGGCTAG GTACAAACTC ACAGGCCAAG CATGTCAGGG ACTGCGCAGT GATGGTCAAG
121 CATTACAGGC GTGCCATAAT GTCTGCCATG AGTATGTCCG AATGGATAAA CAGACGAATG
181 GGCCTGATAG AGGAGGAAGG AGACTGGAAA AACATAGGCA ATTTCCTCAG ATACCAGGGT
241 ATAGAGGTTA TTACATTTAT AGGGGCGCTG AGGGACATGT TAAAGGGCAT TCCAAAAAGG
301 ACATGTATGT GTATAGTGGG ACCACCAGAC ACAGGGAAAT CAGCGTTTTG CCTTAGCCTG
361 CTAGACTTCT TCGGGGGTAG GGTACTGTCA TTCACCAATT ACAAAAGCCA TTTTTGNTGN
421 CCNACCCTCA A
DQ085784
SCC 15, 24, and 28
1 TTATGGTACA NGTGGGCATT TGACAATAAG TACACAGATG AAGCAGAGAT AGCTTTTCAT
61 TATGCACGTT TGGCAGAGGA GGATGCAAAT GCAGAGGCTT GGTTAAAAAG CAACTCCCAA
121 GCTAAATATG TCCGAGATTG TGCGCAAATG GTGAAGCTGT ATCTTAGACA AGAAATGAGG
181 CAGACTACTA TTTCTGAATG GATTGACAAG TGCTGCCAGT CAGTGACAGA GGACGGTGAC
241 TGGGGGGATA TCATGCGCTT CTTAAAATAT CAGCAAGTTA ATTTCACTCA GTTTTTAACT
301 GCCATGAGAA ATGCTTTAGA GGGTAAACCT AAAAAAAACT GCTTAGTATT TTATGGGCCT
361 CCAGATACTG GCAAGTCATA TTTCTGCTTT AGTTTGGTTA GTTTATGCAT GGAAAGTGGA
421 TTTNA
DQ085785
SCC 29
1 TTATGCACGT TTGGCAGAGG AGGATGCAAA TGCAGAGGCT TGGTTAAAAA GCAACTCCCA
61 AGCTAAATAT GTCCGAGATT GTGCGCAAAT GGTGAAGCTG TATCTTAGAC AAGAAATGAG
121 GCAGACTACT ATTTCTGAAT GGATTGACAA GTGCTGCCAG TCAGTGACAG AGGACGGTGA
181 CTGGGGGGAT ATTATGCGCT TCTTAAAATA TCAGCAAGTT AATTTCACTC AGTTTTTAAC
241 TGCCATGAGA AATGCTTTAG AGGGTAAACC TAAAAAAAAC TGCTTAGTAT TTTATGGGCC
301 TCCAGATACT GGCAAGTCAT ATTTCTGCTT TAGTTTGGTT AGTTTATGCT TGAAAGTGGA
DQ085786
BISC 6
1 TTTTATGGTA CAGTGGGCAT TTGACAATGA ATACTTTGAG GAAAGTGAGA TAGCATATCA
61 GTATGCATGC CTTGCAGAAA CAGAAGAAAA TGCTGCAGCC TTCCTAAATT CTAACAGCCA
121 AGCTAAGCAT GTCAGGGACT GTGCAACTAT GTGCAGATAT TATAAGAGAG CAGAAATGCA
181 GAGAATGTCA ATGTCCGCCT GGATTCACAA GAGATGTAAG GAGACCAGCC TGCAGGGAGA
241 TTGGAAAGAA ATAGTCAAGT TTCTTAGACA TCAAAGTGTA GAGTTTATTA CCTTTCTCTG
301 CAGCTTCAAG AAATTTCTCA GGGGTGTGCC TAAAAAAAAT TGCATGCTTT TCTGGGGTCC
361 TCCTAACACA GGCAAATCTA TGTTTTGCAT GAGCTTACTT TCTTTCCTAA AGGCANAGAT
421 TCTTTANC
DQ085788
BISC 5
1 TTCTTATGGT ACAGTGGGCA TTTGACAATA AGTACACAGA TGAAGCAGAG ATAGCTTTTC
61 ATTATGCACG TTTGGCAGAG GAGGATGCAA ATGCAGAGGC TTGGTTAAAA AGCAACTCCC
121 AAGCTAAATA TGTCCGAGAT TGTGCGCAAA TGGTGAAGCT GTATCTTAGA CAAGAAATGA
181 GGCAGACTAC TATTTCTGAA TGGATTGACA AGTGCTGCCA GTCAGTGACA GAGGACGGTG
241 ACTGGGGGGA TATTATGCGC TTCTTAAAAT ATCAGCAAGT TAATTTCACT CAGTTTTTAA
301 CTGCCATGAG AAATGCTTTA GAGGGTAAAC CTAAAAAAAA CTGCTTAGTA TTTTATGGGC
361 CTCCAGATAC TGGCAAGTCA TATTTCTGCT TTAGTTTGGT TAGTTTATGC AGGGAAAGTG
421 TATTTAAAA
DQ085789
BISC 10
1 TTTTTNTGGT NNCCAGTGGC NTACGATAAC GACTTCCGTG ACGAGTGCCA AATTGCCTAC
61 GAATATGCAC GGCTTGCCAC GGAGGACAGC AATGCATTGG CATGGTTGGA ATGCAATAAT
121 CAGGCCAAAT TTGTCAAAGA CTGTGCACGT ATGGTCGGGT ACTATAAGCG CGCTGAAATG
181 CAAAATATGT CTATCTCTGC TTGGATACNT AAGCAAATTA AAGATAGGCA GTGCACTACC
241 GATTGGAAAG TAATTNTGAA TTTTCNTAAG TTTCANCATG TGGAGGTTAT AATTTTTTTA
301 AATGCAATGA TGCATTTGCT CCGTGGCACG CCAAAGAAAA ATTGTCTGGT TCTGTACGGT
361 CCCCCAAATA CAGGGAAATC CATGTTCGCA ATGAGCTTAA TTCAGTGTCT GAAAGGACGT
421 GTATTGTNGT ATGTGAATTC ACGTAGTCAG TTNTGGTTGC ANCCCTTGGC AGATGCAAAA
481 ATAGCACTGC TGGACGATGC AACCAGACCA TGCTGGGAAC TATATAGATA TTTATTGAGA
541 AATGCATTGG ATGGTAATCC TATATGCCTG ACTAANCNAG C
Sequences
2041
58
Chapter 8
Clinical, histological and immunohistochemical study of
feline viral plaques and bowenoid in situ carcinomas
S. Wilhelm1, F. Degorce-Rubiales2, D. Godson3, C. Favrot1
Veterinary Dermatology, 2006; 17: 424-431
1
2
LAPVSO, Toulouse, France
1
Prairie Diagnostic Service, Saskatoon, Saskatchewan, Canada
59
Clinical, histological and immunohistochemical study of
feline viral plaques and bowenoid in situ carcinomas
Blackwell Publishing Ltd
Sylvia Wilhelm*, Frederique Degorce-Rubiales†,
Dale Godson‡ and Claude Favrot*
cats in the FVP + BISC group. On the other hand, only
one of the nine BISC cats was positive. The presence
of both FVP and BISC lesions in some cats and the high
detection rate of PV antigens in the FVP and FVP +
BISC groups suggest that both conditions might have
the same viral cause and that some BISC may evolve
from FVP. The low rate of viral antigen detection in the
BISC group indicates another cause or a loss of viral
replication during the cancerogenesis.
*Clinic for Small Animal Internal Medicine, Dermatology Unit,
Vetsuisse-Faculty University of Zurich, Zurich, Switzerland
†LAPVSO, Toulouse, France
‡Department of Veterinary Microbiology, Western College of
Veterinary Medicine, University of Saskatchewan, Saskatoon,
Saskatchewan, Canada
Correspondence: C. Favrot, Clinic for Small Animal Internal Medicine,
Dermatology Unit, Vetsuisse-Facility, University of Zurich,
Winterthurerstrasse 260, CH-8057 Zurich, Switzerland. Tel. +41 44
635 81 12; Fax: +41 44 635 89 20; Email: [email protected];
Email: swilhelm@vetclinics,unizh.ch
Accepted 31 August 2006
Introduction
What is known about the topic of this paper
• Reports of papillomavirus-induced dermatitis in cats are
rare.
• Lesions of feline viral plaques have been described as
feline hyperpigmented plaques and are clinically indistinguishable from lesions of bowenoid in situ carcinomas.
• Feline bowenoid in situ carcinoma could be, like feline
viral plaques, papillomavirus-induced.
Papillomaviruses (PV) are highly diverse viruses that usually
induce benign skin or mucous membrane proliferation
in mammals and birds but can also cause squamous cell
carcinomas.1 In humans, the PVs that induce benign hyperplasia and those that induce cancers are phylogenetically
different.1 Benign hyperplasias (warts) usually regress
after a few months, a regression associated with the
development of cell-mediated immunity.2
In contrast with dogs, where PV infections are frequently
observed, reports of PV-induced dermatoses are rare in
cats.3–7 Lesions are usually flat and hyperpigmented, rather
than exophytic and flesh colour warts, and spontaneous
regression is rare.3–7 These lesions are usually, but not
always, multiple and have been described as feline viral
plaques (FVP).8
Feline multicentric in situ squamous cell carcinomas
also usually occur as multiple hyperpigmented plaques
that resemble those of human Bowen’s disease.9,10 Gross
and coworkers, however, recently remarked that there are
major differences between the human and the feline
diseases, and have coined the term ‘bowenoid in situ
carcinoma’ (BISC) to describe the feline condition.8 As
FVP clinically resembles BISC, it was suggested that both
conditions may have the same cause, and one report
mentions the association of both FVP and BISC on the
same cat.11,12 Furthermore, it has been shown immunohistologically that up to 47% of feline BISC samples are
positive for PV antigen, suggesting that BISC is virally
induced and that FVP could be, at least in some instances,
precursory lesions of feline BISC.11
Using records of the clinical, histological and immunohistological features of 26 cases of feline dermatoses
clinically described as pigmented plaques and with an initial
histological diagnosis of FVP and/or BISC, the hypotheses
that both lesions are often associated in the same samples,
and that PV antigens are present in the majority of these
lesions, were tested.
What this paper adds to the field of veterinary
dermatology
• Clinically, feline viral plaques and feline bowenoid in situ
carcinomas are indistinguishable.
• Feline viral plaques and feline bowenoid in situ carcinomas
might have the same viral cause.
• Feline viral plaques could be a precursory lesion of feline
bowenoid in situ carcinoma.
Abstract
Feline viral plaques (FVP) induced by papillomavirus
(PV) are often hyperpigmented and flat warts. The fact
that up to 47% of bowenoid in situ carcinomas (BISC),
which also usually occur in the form of hyperpigmented
plaques, are positive for PV antigen in immunochemistry
suggests that BISC could evolve from FVP.
The relationship between the presence of PV antigens and the clinical and histological features of 26
cases of feline dermatoses (clinically described as
pigmented plaques and with histological diagnosis of
FVP and/or BISC) was therefore determined. The
cases were classified into one of the three following
groups: FVP, FVP + BISC or BISC. Immunohistological
detection of papillomavirus group-specific antigen
was performed using a polyclonal rabbit antibovine
papillomavirus antiserum.
Of the seven cases in the FVP group, six were
deemed positive by immunohistology as were all 10
424
© 2006 The Authors. Journal compilation © 2006 European Society of Veterinary Dermatology. 17; 424–431
60
FVP and BISC in cats with hyperpigmented plaques
for 20 min at 42 °C and treated with a 1 : 2000 dilution of rabbit
antibovine papillomavirus type-1 antibody (Dako Diagnostics Canada
Inc., Missisauga, ON, Canada). A goat-biotinylated antirabbit IgG
(Vector Laboratories Inc., Burlington, ON, Canada) was used at a
1 : 400 dilution as the secondary antibody. Replicate sections were
stained as above without protease digestion, and additional sections
were stained with a normal rabbit antiserum as the primary antibody to
provide negative control. A positive control tissue, canine cutaneous
papilloma, was included in each assay run.
Both diaminobenzidine (DAB) (Electron Microscopy Sciences,
Fort Washington, PA, USA) and Nova Red (Vector Laboratories Inc.,
Burlington, ON, Canada) were used as chromogens on two different
sections for each sample.
Materials and methods
Animals
History and clinical information was obtained from 26 cats with hyperpigmented plaques. Cats were included, provided that a histological
diagnosis of FVP and/or BISC had been made previously, and clinical
data (including concurrent diseases, immunosuppressive therapy and
evolution of the lesions, when available) were subsequently analysed
for each of the three histological groups: FVP, FVP + BISC and BISC.
Statistical analysis
Data were analysed using nonparametric statistical methods
(GraphPad PRISM® for Windows, version 4.0; GraphPad Software,
Inc., San Diego, CA, USA). Kruskal–Wallis one-way ANOVA by ranks and
the Dunn’s post-test for multiple comparisons were used to compare
ages among the three histological groups.
Results
Clinical information
The clinical data are summarized in Table 2. Differences
between ages of cats in FVP, FVP + BISC and BISC groups
(median 11.5, 12 and 13, respectively) were not statistically
significant. The sizes of the groups did not allow a proper
evaluation of potential breed or sex predispositions.
On clinical examination, FVP and BISC lesions were
often indistinguishable and usually presented as solitary or
multiple grey, tan to black papules or small flat plaques
(Figs 1 and 2). Some, more frequently the BISC, appeared
ulcerated (Fig. 2). Solitary lesions were observed in only
three of the 26 cats. The face, neck and limbs were mostly
affected by BISC. FVP occurred mostly on the trunk, even
if other areas, including face and neck, were also affected.
Cats with both conditions usually presented lesions on
more than one body area and all body regions could be
affected. Very little follow-up information was available but
cases of transformation of FVP into BISC after the initial
histological diagnosis were not recorded. None of the
affected cats had a known history of immunosuppressive
drug administration or concurrent disease.
Histological evaluation
Archival specimens of all 26 cats were compiled. These samples have
been previously collected by biopsy from all 26 cats, fixed in formalin,
and processed routinely to paraffin wax for histological assessment.
Sections (5 µm) were cut, routinely processed and stained with haematoxylin and eosin. The following criteria were systematically
assessed: severity and nature of the acanthosis, hypergranulosis and
size of the keratohyalin granules, premature keratinization, involvement of the hair follicle in the pathological process, disorderly or
abnormal maturation of the epidermis, atypia (pleomorphic or abnormally large nuclei, multinucleate cells), mitoses more than three cell
layers above the basal cell layer, koilocytosis, clear cells and presence
of intracytoplasmic pseudo-inclusions and intranuclear inclusions.
Koilocytes were defined as keratinocytes with swollen cytoplasms
and shrunken nuclei.8 Clear cells were defined as keratinocytes
with swollen cytoplasm but rather enlarged, vesicular nuclei. These
modified keratinocytes (clear cells and koilocytes) have been reported
to be also regularly associated with human PV infection.13 When
observed, the margins of the lesions were checked for changes
suggestive of viral infection such as koilocytes and clear cells, pseudoinclusions, and clumped keratohyalin granules.
Samples were subsequently classified into one of three groups:
FVP, FVP + BISC (when both lesions were present on the same cat or on
the same section) or BISC in accordance with standard criteria (Table 1)
for the diagnosis of FVP and BISC.8 When changes overlapping typical
FVP and BISC lesions were observed, lesions were designated as
FVP, provided that the acanthosis remained moderate and atypia was
absent. Lesions were classified as BISC if the acanthosis was marked
and loss of polarity as well as atypia was evident.
Histological examination
The results are summarized in Table 3.
FVP
The diagnosis of FVP was made in seven cases (Table 3).
Lesions consisted of well-demarcated epidermal hyperplasia with acanthosis, hyperpigmentation, hypergranulosis
with clumped keratohyalin granules and numerous koilocytes
(Fig. 3). Some of these keratinocytes contained bluegrey fibrillar pseudo-inclusions (one of seven). Larger and
compact amphophilic intracytoplasmic pseudo-inclusions
were present in four cases (Fig. 3). In one case, both
pseudo-inclusion types were present in the same sample
and compact ones (present in the stratum granulosum)
Immunohistochemical analysis
Papillomavirus antigen was detected (at the Immunology Laboratory
of Prairie Diagnostic Services, Saskatoon, Saskatchewan, Canada)
using an avidin–biotin complex technique adapted for an automated
slide stainer (Codon Histomatic Stainer, Fisher Scientific, Edmonton,
AB, Canada) as previously described.14 This method has already been
validated for the detection of feline PV antigens.7 Briefly, sections
from each tissue block were mounted on slides (Codon Slides, Fisher
Scientific, Edmonton, AB, Canada) coated with 0.1% poly-D-lysine,
digested with protease XIV (Sigma Chemical Co., St. Louis, MO, USA)
Table 1. Histological features of feline viral
plaque and bowenoid in situ carcinoma
Acanthosis
Follicular involvement
Differentiation
Clumped keratohyalin granules
Koilocytes
Intracytoplasmic pseudo-inclusions
Atypia
Mitotic activity
Feline viral plaque
Bowenoid in situ carcinoma
Mild to moderate
Sometimes
Normal
Yes
Yes
Yes
No
No
Moderate to severe
Yes
Dysplastic epidermis, loss of polarity
Yes
Yes
Yes
Yes
Moderate
© 2006 The Authors. Journal compilation © 2006 European Society of Veterinary Dermatology.
61
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Wilhelm et al.
Table 2. Clinical findings
Case
Breed
Sex
Age (years)
Lesions
Multiple/Solitary
Localization
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
DSH
Cornish Rex
DSH
DSH
DSH
DSH
DSH
American curl
DSH
DSH
Sphynx
DSH
DSH
DSH
DSH
DSH
DLH
DSH
DLH
Himalayan
DSH
DSH
DLH
DSH
DSH
DSH
M
M
F
F
14
12
11
15
18
12
13
14
13
12
6
3
16
8
13
13
9
12
11
8
15
11
17
11
16
12
Crusty plaques
Papillary plaques
Plaque
Plaques, papules
Papules
Crusts, plaques
Crusts, plaques
Crusts, plaques
Papules
Papules
Macules
Ventral papules
Plaque
Papules
Crusts, erythema, erosion
Papules, crusts
Scaly papules
Erythematous papules
Erythematous plaques
Crusty plaque
Crusty plaque
Crusty plaques
Plaques
Crusty plaques
Crusty plaques
Crust papules
Multiple
Multiple
Solitary
Multiple
Multiple
Multiple
Multiple
Multiple
Multiple
Multiple
Multiple
Multiple
Solitary
Solitary
Multiple
Multiple
Multiple
Multiple
Multiple
Multiple
Multiple
Multiple
Multiple
Multiple
Multiple
Multiple
Thorax, face, shoulder
Shoulder, paw, trunk, abdomen
Neck
Face, feet, abdomen
Flank
Face and digits
Eyelids, face
Face, neck
Unknown
Flank
Neck
Abdomen
Abdomen
Dorsum
Axilla, feet
Face, neck
Face
Neck, face
Neck, face
Dorsum, neck
Leg, toe
Face, lip
Dorsum
Face, lip
Face, neck, shoulders, foot pads
Face, neck
M
F
M
F
F
F
M
M
F
F
M
F
F
M
F
F
F
M
F
M
M
DSH, domestic shorthair cat; DLH, domestic longhair cat.
epidermis with irregular acanthosis and broad rete ridges.
Irregular acanthosis frequently descended around hair
follicles. The epidermis was disorganized with a marked
loss of cellular polarity and loss of normal stratification of
the stratum basale and spinosum in all cases (wind-blown
appearance). Keratinocytes with a hyperchromatic
nucleus were present throughout the whole epidermis.
Atypia was variable in nature and intensity (anisocytosis,
anisocryosis and rare binucleated keratinocytes). Rare
mitotic figures were present in all samples. Scattered
apoptotic keratinocytes were present in four BISC samples. Koilocytes were present in all of them (Fig. 6). Other
clear cells with rather enlarged vesicular nuclei were also
observed. The cells (koilocytes and clear cells) contained
sometimes intracytoplasmic additional blue-grey fibrillar
pseudo-inclusions (three of nine cases). Clumped keratohyalin granules were seen in one of nine BISC cases. Erosions or ulcerations were present in five of nine cases.
Figure 1. Cat no. 6. Pigmented plaque on the head diagnosed as
feline viral plaque: Note the slightly raised and hyperpigmented lesion
with a small central ulceration. Courtesy of Catherine Mège.
Immunohistochemical examination
Results are summarized in Table 3. Of the seven cases of
the FVP group, six were positive for PV antigen. Interestingly, all of the 10 samples with BISC and FVP lesion types
were positive (Fig. 7). Only one of the nine BISC cases
was deemed positive (11%). PV antigens were always visualized in the nucleus of the koilocytes; intracytoplasmic
pseudo-inclusions remained unstained (Fig. 4).
seemed to result from the condensation of fibrillar ones
(more prevalent in the stratum spinosum) (Fig. 4). Intranuclear inclusions were not observed.
FVP + BISC
Interestingly, both BISC and FVP changes were present in
10 cats, sometimes in the same, sometimes in different,
skin samples (Fig. 5a,b). Transition lesions exhibiting both
FVP and BISC features were also sometimes observed.
Discussion
The clinical resemblance between BISC and FVP and the
presence of both lesions in some cats suggest that some
BISC evolve from FVP. Furthermore, despite the absence
BISC
The diagnosis of BISC was made on nine cases. These
lesions consisted of sharply demarcated expansion of the
426
62
Figure 3. Cat no. 10. Histology of a feline viral plaque. Note the
presence of clear cells (ballooned cytoplasm, rather swollen nucleus
(black arrow) with intracytoplasmic pseudo-inclusions (red arrow).
Haematoxylin and eosin. Magnification ×10. Bar = 200 µm.
Figure 2. Cat no. 26. Pigmented plaques at the base of the ear and
the pinna diagnosed as feline bowenoid in situ carcinoma. Note the
slightly raised and ulcerated lesions partially covered by crusts.
Table 3. Histopathological findings
Case
Margins?
Hyperpig.
Koilocytes/clear cells
Dyskerat.
Comp. ps. incl.
Fibr. ps. incl.
KH Gran.
Diagnosis
PV-Ag
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Yes
Yes
No
Yes
No
Yes
No
Yes
No
No
No
No
No
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Yes
No
No
No
Yes
No
Yes
No
No
Yes
Yes
Yes
Yes
No
No
No
No
No
No
No
Yes
No
No
No
Yes
No
No
Yes
No
No
No
No
No
No
Yes
No
No
No
No
No
No
No
Yes
No
No
No
No
No
Yes
No
No
No
No
No
Yes
Yes
Yes
No
Yes
No
No
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
No
Yes
Yes
Yes
No
No
Yes
Yes
Yes
Yes
BISC
BISC + FVP
FVP
BISC + FVP
BISC
BISC + FVP
BISC
BISC
FVP
FVP
FVP
FVP
FVP
FVP
BISC
BISC + FVP
BISC
BISC
BISC + FVP
BISC + FVP
BISC
BISC
BISC + FVP
BISC + FVP
BISC + FVP
BISC + FVP
Neg
Pos
Pos
Pos
Neg
Pos
Neg
Neg
Pos
Neg
Pos
Pos
Pos
Pos
Neg
Pos
Neg
Pos
Pos
Pos
Neg
Neg
Pos
Pos
Pos
Pos
Margins?, presence of lesional margins; Hyperpig., hyperpigmentation; Dyskerat., dyskeratosis; Comp. ps. incl., compact pseudo-inclusions;
Fibr. ps. inclus., fibrillar pseudo-inclusions; KH Gran., clumped keratohyalin granules; PV-Ag, papillomavirus antigen.
Pos, positive; Neg, negative.
63
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Wilhelm et al.
Figure 4. Cat no. 9. Immunohistochemical analysis of a feline viral
plaque. Note the presence of positive nuclei (black arrow). The fibrillar
(red arrow) and the solid (green arrow) intracytoplasmic inclusions
remained unstained. Diaminobenzidine. Magnification ×40.
Bar = 50 µm.
Figure 6. Cat no. 21. Histology of a feline bowenoid in situ
carcinoma. Note the marked acanthosis (black stars: acanthotic
epidermis), the follicular involvement (black points), the loss of
polarity and the presence of numerous koilocytes (arrow).
Haematoxylin and eosin. Magnification ×10. Bar = 200 µm.
of statistically significant difference, cats affected by FVP
tended to be younger than those affected by BISC: this
could imply that FVP are precursor lesions of BISC. However, while BISC affected the face, neck or the limbs in
most cases, FVP lesions were more often present on the
trunk even if other areas, including neck and face, were
affected. This finding does not seem to support the
hypothesis that BISC evolve from FVP but the discrepancy
could be explained by a higher cancerization rate of lesions
located on the face and neck, for example as a result of
increased ultraviolet radiations exposure, compared to
those in other regions of the body.
Figure 5. Cat no. 16. Histology of two lesions present on the same biopsy sample. Haematoxylin and eosin. Magnification ×40. Bar = 50 µm. (a)
Feline viral plaque. Note the moderate acanthosis. The stratification and the differentiation of the epidermis are conserved. Koilocytes and clumped
keratohyalin granules are the most obvious papillomaviruses’ cytopathic characteristics on this lesion. (b). Early bowenoid in situ carcinoma. Note
the acanthosis, the obvious disorganization of the epidermis and the abnormal differentiation of most keratinocytes. Clumped keratohyalin granules
and one single koilocyte are the only papillomavirus cytopathic effects noticed on this lesion.
428
64
acids are often uncovered in normal mammalian skin.
However, genome copy number is usually very low and
productive infection rarely occurs in such cases.15,16
Establishing causality between the presence of viruses in
skin lesions and oncogenesis remains problematic, and
the presence of replicating viruses cannot be regarded as a
sufficient proof. In vitro studies are mandatory to establish
such causality.17
Almost all cats affected by BISC were deemed negative
by IHC. These findings might suggest that BISC has two
distinct causes and that only a subgroup of BISC is virally
induced. A loss of viral replication during the cancerization
process could also explain these findings. In fact latent PV
infection or infection with minimal replication may remain
undetected by IHC, because of the relatively low sensitivity
of such techniques. The ‘hit and run’ model, which postulates an initial cellular transformation by the virus and a
subsequent loss of viral genome, could account for the
negative IHC in some BISC lesions.18 Furthermore, it was
recently demonstrated that PVs maintained productive
infections in precursory lesions of cervical cancer but that
capsid antigens were no longer produced in late cervical
cancers.19 In conclusion, a loss of viral protein expression
in advanced cases of BISC seems likely.
Feline BISC has long been considered the counterpart
of human Bowen’s disease (BD) – an in situ squamous cell
carcinoma that presents as solitary, well-circumscribed,
erythematous plaques and occurs on the face, extremities
and genitalia.20,21 Koilocytes are usually not present in such
lesions.21 Human bowenoid papulosis is characterized
by genital pigmented verrucous papules or plaques.21 This
condition is also histologically characterized by in situ SCC
lesions but, in contrast to BD, bowenoid papulosis lacks
full-thickness epidermal atypia. PV DNA is uncovered
in virtually all samples of bowenoid papulosis but data
concerning the presence of PV in human BD remain
contradictory.22–25 Furthermore, PVs that infect human
bowenoid papulosis and BD are usually to mucosal and
not to cutaneous strains.23,24 These data show that feline
BISC lesions display substantial differences from both
human conditions and justify the use of a specific denomination, as emphasized by Gross and coworkers.8
The results of the present study support the hypothesis
that some BISC evolve from FVP lesions and the causative
role of PV. However, evidence that these PVs are able to
induce cancerization in mammalian skin is lacking and
further studies are warranted. Nucleic acids amplification
techniques could establish which PVs are present in FVP
and BISC lesions and whether BISC samples without FVP
are really sterile or infected by dormant PV. As well, in vitro
studies addressing the transforming potential of feline PV
are required to better understand the role that these
viruses play in this condition.
Figure 7. Cat no 19. Immunohistochemical analysis of a feline bowenoid in situ carcinoma. Note the presence of numerous koilocytes and
clear cells (red arrow) with positive nuclei. Novared.
Magnification ×10. Bar = 200 µm.
FVP usually conserved the general organization of the
epidermis and atypia was absent, whereas BISC lesions
were disorganized and abnormal keratinocytes were present
throughout the epidermis. However, both conditions share
numerous histological features: irregular acanthosis with
rete ridges formations, presence of clumped keratohyalin
granules, koilocytes and clear cells. The presence of
koilocytes or clear cells in all BISC lesions (including IHCnegative ones) might be regarded as a proof of presence
of the virus. These cells with vacuolated cytoplasm and
shrunken, pycnotic nuclei are usually considered highly
suggestive of PV infections.8,13 All the authors who have
studied feline BISC have recognized these cells, but two
of three have not used the term ‘koilocyte’ to describe
them.8–10 In situ hybridization studies could be helpful to
determine if these cells actually harbour PV nucleic acids
and if the term ‘koilocyte’ is appropriate.
In both FVP and BISC samples, fibrillar and compact
pseudo-inclusions were seen. In one case both were
present in the same sample, and compact ones (more
present in the stratum granulosum) seemed to result from
the condensation of fibrillar ones (more prevalent in the
stratum spinosum) (Fig. 4). This condensation has already
been described by Carney and coworkers.3
Our study demonstrates that the association between
FVP and BISC is frequent and occurs sometimes on the
same skin lesion. Additionally, cases of overlapping BISC
and FVP lesions have been detected. This association was
already described before.11,12 These similarities support
the hypothesis that FVP could be precursory lesions of
BISC.
All except one FVP and FVP + BISC cases were positive
for PV antigen by immunohistochemistry (IHC). As
pseudo-inclusions were present in the negative case, it
can be considered that all these samples were infected by
PV. Furthermore, as IHC detects capsid antigens, it can be
concluded that productive infection occurred in all positive
samples (all FVP lesions and positive BISC). These findings
support the hypothesis that PVs play an active role in the
development of such lesions. It must, however, be borne
in mind that PVs are sometimes commensal, and nucleic
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Résumé Les plaques virales du chat (FVP) induites par les papillomavirus (PV) se présentent souvent
comme des plaques hyperpigmentées. Le fait que jusqu’à 47% des carincomes in situ bowenoides (BISC),
qui se présentent aussi sous la forme de plaques hyperpigmentées, sont positifs pour l’antigène de PV par
immunohistochimie suggère que les BISC pourraient provenir de FVP. La relation entre la présence
d’antigènes de PV et les données cliniques et histologiques de 26 cas de dermatoses félines cliniquement
répertoriées comme des plaques hyperpigmentées avec un diagnostic histologique de FVP et/ou de
BISC a été recherchée. Les cas ont été classés en trois groupes : FVP, FVP + BISC ou BISC. La recherche
immunohistochimique de papillomavirus a été réalisée en utilisant un antisérum polyclonal de lapin
anti-bovin. Sur les sept cas du groupe FVP, six étaient positifs à l’immunohistochimie, un seul des neuf
BISC était positif. La présence de lésions de FVP et de BISC chez certains chats, et la fréquence importante
de découverte d’antigènes de PV dans les groupes FVP et FVP + BISC suggère que ces deux maladies
ont une même cause virale, et que certains BISC peuvent provenir de FVP. Le faible taux de détection
d’antigène viral dans le groupe BISC indique une autre cause, ou la perte de la réplication virale pendant
la cancérogénèse.
Resumen Las placas virales felinas (FVP) inducidas por el virus papiloma son a menudo verrugas
hiperpigmentadas y planas. El hecho de que hasta un 47% de los carcinomas Bowenoides in situ (BISC),
que también ocurren como placas hiperpigmentadas, son positivos al antígeno del virus papiloma mediante
inmunohistoquímica sugiere que los BISC pueden evolucionar a partir de placas virales felinas. Se determinó
la relación entre la presencia de antígenos del virus del papiloma y las características clínicas e histológicas
de 26 casos de dermatosis (clínicamente descritas como placas pigmentadas y con diagnostico histológico
de FVP y/o BISC). Los casos se clasificaron en uno de los tres grupos siguientes: FVP, FVP + BISC o BISC.
La detección inmunohistológica de antígeno especifico del grupo del virus papiloma se realizó utilizando
un antisuero policlonal de conejo frente al papiloma bovino. De los siete caso en el grupo FVP, seis fueron
considerados positivos mediante inmunohistoquímica así como los diez gatos del grupo FVP + BISC. Por
otro lado, solo uno de los nueve gatos con BISC fue positivo. La presencia de ambas lesiones FVP y BISC
en algunos gatos y el elevado nivel de detección de antígenos del virus papiloma en los grupos FVP y
FVP + BISC sugiere que ambas condiciones podrían tener la misma causa vírica y que algunos BISC podrían
430
66
progresar desde FVP. El bajo porcentaje de detección de antígeno vírico en el grupo BISC sugiere otra causa
o una pérdida de replicación viral durante el proceso de carcinogénesis.
Zusammenfassung Feline virale Plaques (FVP), die von Papillomavirus (PV) verursacht werden, sind oft
hyperpigmentierte und flache Warzen. Die Tatsache, dass bis zu 47% der ‘Bowen’-ähnlichen in situ
Karzinome (BISC), die normalerweise auch in Form von hyperpigmentierten Plaques erscheinen, mittels
Immunchemie positiv sind für PV-Antigen, weist darauf hin, dass BISC sich aus FVP entwickeln könnte.
Der Zusammenhang zwischen dem Auftreten von PV Antigenen und den klinischen und histologischen
Erscheinungsbildern von 26 Fällen von felinen Dermatosen (die klinisch als pigmentierte Plaques
beschrieben und histologisch als FVP und/oder BISC diagnostiziert wurden) wurde daher bestimmt. Die Fälle
wurden in eine der drei folgenden Gruppen eingeteilt: FVP, FVP + BISC oder BISC. Die immunhistologische
Bestimmung des gruppenspezifischen Papillomavirus Antigens wurde mit einem polyklonalen Kaninchen
Antiserum gegen bovines Papillomavirus durchgeführt. Von den sieben Fällen in der FVP Gruppe wurden
sechs mittels Immunhistologie als positiv angesehen, genauso wie alle 10 Katzen in der FVP + BISC Gruppe.
Andererseits war nur eine der neun BISC Katzen positiv. Das Vorhandensein von beiden, FVP und BISC
Läsionen bei manchen Katzen und das häufige Auftreten von PV-Antigenen in den FVP und FVP + BISC
Gruppen ist ein Hinweis darauf, dass beide Formen dieselbe virale Ursache haben und einige BISC sich aus
den FVP entwickeln könnten. Das seltene Auftreten von viralem Antigen in der BISC Gruppe bedeutet, dass
eine andere Ursache vorliegt oder der Verlust von viraler Replikation während der Kanzerogenese besteht.
67
431
Chapter 9
Summarizing discussion and further studies
68
Viruses replicate inside cells by synthesizing their own proteins and assembling them into
virions. This replication is associated with various cytopathic effects, which are, usually,
typical or pathognomonic of one specific virus. Poxviruses infections are associated with
large intracytoplasmic inclusions and herpesvirus infections with intranuclear inclusions, for
example. Aside from these cytopathic effects, viruses induce macroscopic changes which are
sometimes, easily recognizable. Papillomaviruses induce cauliflower-like lesions, the socalled warts, which are virtually pathognomonic. Poxviruses and herpesviruses induce pock
lesions and vesicles, respectively, which are very typical of these infections. These virusassociated changes have long been described in dogs and cats [1]. Viruses may also induce
some less obvious changes, which are described in humans but remained often undescribed in
canine and feline. These changes may be due to various pathogenic states like minimal viral
replication, latency or non-productive infections.
This thesis aims to describe some of these undescribed virus-induced skin changes in dogs
and cats and, especially, papillomavirus-induced ones.
We first described a case of canine erythema multiforme presumably associated with
parvovirus infection [2]. We hypothesized that an infection of stem cells and primary
amplifying keratinocytes occurred following hematogenic dissemination of the parvovirus.
Viral antigens could have been presented by class I major histocompatibility complex
molecules at the surface of the keratinocytes. Recognition of the viral antigens by Tlymphocytes, possibly sensitised by a previous parvovirus vaccination would have triggered
these cytotoxic T-cells to induce apoptosis of infected keratinocytes. In this case, clinical and
pathological lesions are not due to the cytopathic effect of the virus itself but to the T
lymphocyte-induced cytolysis. Interestingly, virus infections (especially herpes simplex
infection but also B19 parvovirus infections) are the most frequent causes of erythema
multiforme in humans [3, 4]. This case was the first report of virus-associated erythema
multiforme in dogs. This report leaves however some moot questions that warrant some
further studies. The most important question is to know whether canine parvovirus usually
replicates in the skin of affected dogs without causing any cytopathic effects or if the skin
contamination reported in this study was incidental or due to a specific parvovirus strains.
Second, as parvovirus antigens have been uncovered in the affected skin, one cannot exclude
a direct effect of the virus infection associated to a secondary lymphocytic reaction.
69
The second article of this thesis aimed to describe some previously unknown cutaneous
consequences of FeLV infection.
FeLV is a member of the oncornavirus subfamily of retroviruses, which replicates in many
tissues like bone marrow, salivary glands and respiratory epithelium. Its replication in the
feline skin was already described by Gross and coworkers and associated with the so-called
giant cell (multinucleated keratinocytes) dermatosis and horn formation [1, 5].
Multinucleated keratinocytes are sometimes observed in humans in association with
neoplastic conditions, infectious diseases like herpesvirus infection and immunologic
disorders [6]. Retroviruses also possess fusion proteins, which are able to induce syncytium
formation in infected tissues [7, 8]. However, although FeLV infection is a frequent disease,
syncytium are rarely observed in the affected skin of infected cats [5]. We described another
case of FeLV-associated giant cell dermatosis with an ulcerative phenotype and demonstrated
the presence of both FeLV antigens and proviral sequences in the lesional skin. Gross and
coworkers suggested that these cytopathic effects were not the direct consequence of FeLV
infection but the early stage of carcinomatous transformation [5]. The presence of FeLV
antigens in the affected skin of the cat we observed, suggested an active replication of the
virus and supported the hypothesis of a direct cytopathic effect. Furthermore, Rohn and
coworkers demonstrated that FeLV variants do possess various pathogenic and cytopathic
effects[9]. All in all, we considered more likely that these changes are the direct consequence
of infection with a specific and rare variant of FeLV. This hypothesis however warrants
further investigation.
Feline internal lymphomas are often the consequence of FeLV infection. Cutaneous
lymphomas, however, usually occur on FeLV-negative cats [10]. FeLV genomic sequences
have however already been sometimes amplified from cutaneous lymphomas [11]. The
originality of the case we reported lies on the fact that FeLV antigens have been
demonstrated in the affected skin of a serologically negative cat. These findings suggest that
productive FeLV infection may in some instances occur and may be restricted to the skin.
Further studies are needed to demonstrate the existence of multiple FeLV strains with various
physiologic and pathologic properties.
Papillomaviruses (PV) are host-specific epitheliotropic viruses that infect the skin and
mucous membranes. As these viruses do not possess the enzymatic machinery required for
replication, they depend upon host-cell machinery to achieve this process and upon host-cell
70
differentiation for completion of their life cycle [12]. As more than 150 different PV have
been isolated from the human lesional or healthy skin, only a few PV have been identified in
carnivores [13, 14]. In this thesis, we have demonstrated the existence of new
papillomavirus-like sequences in various canine and feline lesions, including cyclosporine Aassociated exophytic lesions, in situ and invasive carcinomas [15, 16]. We have used two sets
of primers designed for the amplification of a sequence of the E1 gene of PV. The narrowrange set of primers was supposed to amplify canine and feline PV and their close relatives
[16]. The broad range PCR system was designed to amplify up to 64 human PVs and several
animal PV such as canine and feline PV[16, 17]. These studies have shown the existence of
at least six feline and five canine unknown papillomavirus-like sequences. As the
classification of papillomaviruses is based upon L1 gene, the amplification of sequences of
the E1 gene does not allow proper evaluation of these sequences and classification of the
newly uncovered PVs but these results suggested however that canine and feline lesional skin
can be infected by PV of great genetic diversity. It would be of great interest to amplify and
clone these novel canine and feline PVs. Fortunately, a new technique, the rolling-circle
amplification (RCA) technique, was recently introduced to amplify and isolate circular DNA
and, especially human and animal PVs [18, 19]. RCA is a multiple random primed, sequenceindependent amplification of circular DNA. Furthermore, the amplification is as effective as
PCR. Therefore, only minute amount of crudely isolated DNA from tissue can be used for
amplification of papillomavirus genomic DNA.
RCA analysis of canine and feline skin samples will permit to determine whether healthy
skin harbors PVs and to sequence PVs that are present in lesional skin. This descriptive study
is the mandatory initial step for a better understanding of the role that play PV in the
development of skin lesion in dogs and cats, and, especially, in the development of skin
cancers. We have already applied this new technique to the isolation and cloning of a new
canine PV (CPV3)[20].
In mammals and birds, PV induce a wide range of cutaneous and mucous changes such as
exophytic and flat warts, precancerous and cancerous lesions. They are considered important
carcinogens in humans and some high-risk PVs are directly responsible for the development
of cervical cancers in women[21-23]. Even though the link between cervical carcinoma and
human PV is clear, the role of PVs in the development of cutaneous squamous cell carcinoma
(SCC) is not as definite [24]. There is however emerging epidemiological evidence to suggest
71
that PV might play an important role in skin cancerogenesis, especially in epidermodysplasia
(EV) associated-one. Establishing causality between the presence of PV in a skin lesion and
the development of the lesion is nevertheless problematic [25]. Criteria have been proposed
to establish this relationship but difficulties in culturing PVs have made their fulfillment
often impossible [22, 25]. Additionally the use of extremely sensitive nested polymerase
chain reaction (PCR) makes possible the detection of minute amounts of viral DNA (even
0.05 viral genome per cell). The presence of such an amount of PV nucleic acid does not
indicate a productive infection and can also be found in healthy skin [26, 27]. Evidence also
suggests that ultraviolet (UV) radiation contributes to the cancerization of some PVassociated skin cancers [25, 28]. All in all, the role of PV in the development of skin cancer
in humans remains questionable.
Some animals models support the causative role of PV in the induction of skin SCC: A few
decades ago, it was demonstrated that cottontail-rabbit PV (CRPV) are able to induce skin
cancers in rabbit [29-30]. Other studies have also established that attenuated life canine oral
PV (COPV) vaccine induce SCC in Beagles [32]. Additionally, canine and feline can be
affected by skin conditions that share some similarities with human EV and cancerization has
been reported in some patients [33]. Epidemiologic studies have demonstrated association
between carnivores SCC and PV but causality has never been established [16, 33-40].
This thesis has confirmed the epidemiologic association between some groups of feline and
canine skin cancers and PV infections and the genetic diversity of carnivore PVs [16, 20, 34,
40]. Aside from the identification and cloning of these new PVs, the most important studies
to carry out would be to determine the relative prevalence of each new carnivore PV and to
demonstrate in vitro that, at least some of them, are able to induce keratinocyte
transformation and immortalization.
We have, for example, detected, cloned, and sequenced a novel PV (CPV3), which was
associated with a case of canine epidermodysplasia verruciformis [20]. The affected dog
developed multiple plaques and one single interdigital lesion of in situ SCC [20]. DNA of
CPV3 was uncovered in each lesion tested (including SCC) but was not present in intact skin
of the same dog. Sequence-independent, multiply primed rolling-circle amplification was
used to amplify, clone, and sequence the entire genome of CPV3. Indeed, analysis of the
cloned and sequenced canine papilloma genome allowed its classification as a member of a
new papillomavirus genus (GenBank accession DQ295066). Additionally, mRNA for the
putative transforming protein E6 was discovered in each tested lesion: These findings
72
demonstrated that CPV3 was transcriptionally active in the mentioned skin lesions and
supported the hypothesis of a causative role of CPV3 in the pathogenesis of canine EV [41].
Complementary DNA of the p53 transcript of the affected dog was cloned from blood, intact
skin as well as skin lesions and its nucleotide sequence was found not to differ from the wild
type canine p53 sequence. This finding suggested that the development of malignancy could
not be attributed to UV-dependent mutagenesis. Therefore, the hypothesis was supported that
transforming proteins of CPV3 induced cancer development by different mechanism than
human EV-associated PVs [41].
As well, we are currently studying the relative prevalence of CPV3 in canine sera. We have
cloned CP3-L1 gene (codes for major CPV3 capsid protein) and generated antisera against
this protein. Our goal would be to establish an ELISA test and to evaluate 500 already
collected canine sera.
All in all, the findings in this thesis, the subsequent and future studies open new avenues to
study PV-induced skin cancerogenesis. As at least some carnivore conditions bear major
resemblances with human ones, these breakthroughs will reveal helpful for both veterinary
and human oncology.
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Chapter 10
Zusammenfassung und weitere Studien
76
Viren replizieren sich innerhalb von Zellen indem sie ihre eigenen Proteine synthetisieren
und diese zu Virionen assemblieren. Diese Replikation wird von verschiedenen
zytopathischen Effekten begleitet, die in der Regel typisch oder pathognomonisch für ein
bestimmtes Virus sind. Zum Beispiel werden Pockenvirusinfektionen von grossen
intrazytoplasmischen Inklusionen begleitet und Herpesvirusinfektionen von
intranuklearen Inklusionen. Abgesehen von diesen zytopathischen Effekten rufen Viren
auch makroskopische Veränderungen hervor, die manchmal einfach zu erkennen sind.
Papillomaviren rufen blumenkohlähnliche Läsionen hervor, sogenannte Warzen, die
nahezu pathognomonisch sind. Pocken- und Herpesviren rufen Pockenpusteln bzw.
Blasen hervor, die sehr typisch sind für diese Infektionen. Diese Viren begleitenden
Veränderungen wurden schon lange für Hunde und Katzen beschrieben [1]. Viren können
auch weniger offensichtliche Veränderungen hervorrufen, die für Menschen beschrieben
sind, für Hunde und Katzen aber grösstenteils nicht. Diese Veränderungen können auf
unterschiedlichen pathogenischen Zuständen beruhen, wie minimale virale Replikation,
Latenz oder nicht-produktive Infektion. Ziel dieser Arbeit ist es, einige der von Viren
hervorgerufenen Hautveränderungen zu beschreiben, die bei Katzen und Hunden bisher
nicht bekannt waren, besonders die von Papillomaviren verursachten.
Wir waren die ersten, die einen Fall von Erythema multiforme (EM) bei Hunden
beschrieben haben, das mit einer Parvovirusinfektion assoziiert war [2]. Wir haben
angenommen, dass die Infektion von Stammzellen und primären amplifizierenden
Keratinozyten nach einer hämatogenen Ausbreitung des Parvovirus auftrat. Virale
Antigene wurden demnach von Haupthistokompatibilitätskomplex-Molekülen der Klasse
I auf der Oberfläche von Keratinozyten präsentiert. Wir gingen davon aus, dass die
Erkennung des viralen Antigens von T-Lymphozyten, die möglicherweise durch eine
vorgängige Parvovirus-Impfung sensibilisiert wurden, diese zytotoxischen T-Zellen dazu
gebracht haben, die Apoptose von infizierten Keratinozyten herbeizuführen. In diesem
Fall sind die klinischen und pathologischen Läsionen nicht auf den zytopathischen Effekt
des Virus selber zurückzuführen, sondern auf die von T-Lymphozyten hervorgerufene
Zytolyse. Interessanterweise sind virale Infektionen (besonders die Herpes simplex
Infektion, aber auch die B19 Parvovirus Infektion) die häufigste Ursache von EM bei
77
Menschen [3,4]. Unser Bericht war der erste veröffentlichte Fall von Virus assoziiertem
EM bei Hunden. Er liess aber einige Fragen offen, die weitere Untersuchungen
rechtfertigen. Die wichtigste Frage war, ob Parvoviren von Hunden sich in der Haut von
betroffenen Hunden ohne zytopathische Effekte replizieren oder ob die Kontaminierung
der Haut, von der wir in unserer Studie berichteten, zufällig oder auf einen spezifischen
Stamm von Parvoviren zurückzuführen war. Zweitens konnten wir eine direkte Wirkung
des mit einer sekundären lymphotischen Reaktion assoziierten Virus nicht ausschliessen,
weil Parvovirus-Antigen in der befallenen Haut gefunden wurde.
Das Ziel des zweiten Teils dieser Arbeit war es, einige vorgängig unbekannte kutane
Folgen der FeLV-Infektion zu beschreiben.
FeLV ist ein Mitglied der Oncornavirus-Unterfamilie von Retroviren und repliziert sich
in vielen Geweben wie zum Beispiel im Knochenmark, in Speicheldrüsen und im
Atemwegsepithel. Seine Replikation in feliner Haut wurde schon von Gross und
Mitarbeitern beschrieben und ist von Riesenzellen (multinukleare Keratinozyten)
Dermatose assoziiert. Mulitnukleare Keratinozyten werden manchmal bei Menschen im
Zusammenhang mit neoplastischen Konditionen, infektiösen Krankheiten wie
Herpesvirus Infektion und immunologischen Störungen beobachtet [6]. Retroviren
besitzen auch Fusionsproteine, die in infizierten Geweben die Bildung von Synzytium
herbeiführen können [7,8]. Dennoch werden Synzytia selten in der Haut von infizierten
Katzen beobachtet, obwohl die FeLV Infektion eine häufige Krankheit ist. Wir haben
einen Fall von FeLV assoziierter Riesenzellen-Dermatosis mit einem ulzerativen
Phänotyp beschrieben und haben sowohl die Präsenz von FeLV-Antigenen als auch von
proviralen Sequenzen nachgewiesen. Gross und Mitarbeiter haben vorgeschlagen, dass
diese zytopathischen Effekte nicht die direkte Folge einer FeLV Infektion sind, sondern
ein frühes Stadium von karzinomatöser Transformation [5]. Das Vorhandensein von
FeLV-Antigenen in den Hautläsionen der Katze in unserer Studie legte die aktive
Replikation des Virus nahe und stützte die Hypothese eines zytopathischen Effektes.
Ferner haben Rohn und Mitarbeiter gezeigt, dass FeLV-Varianten verschiedene
pathogenische und zytopathische Effekte haben [9]. Angesichts dieser Ergebnisse haben
wir vermutet, dass es wahrscheinlicher ist, dass die Veränderungen die direkte Folge
78
einer Infektion mit spezifischen und seltenen Varianten von FeLV sind. Diese
Auffassung bedarf jedoch weiterer Untersuchungen.
Feline Lymphome sind oft das Resultat einer FeLV-Infektion. Ein kutanes Lymphom tritt
jedoch normalerweise bei FeLV-negativen Katzen auf [10]. Feline Genomsequenzen von
Leukämieviren wurden manchmal durch kutane Lymphome amplifizert [11]. Die
Einzigartigkeit unseres Falles liegt in der Tatsache, dass FeLV-Antigene in den
Hautläsionen einer serologisch negativen Katze nachgewiesen wurden. Diese
Erkenntnisse legen nahe, dass eine produktive FeLV-Infektion in gewissen Fällen
auftreten und auf die Haut beschränkt sein kann. Weitere Studien sind nötig um zu
untersuchen, ob multiple FeLV-Stämme mit verschiedenen physiologischen und
pathologischen Eigenschaften existieren.
Papillomaviren sind wirtespezifische epitheliotrope Viren, die Haut und Schleimhäute
infizieren. Weil diese Viren keine enzymatische Ausrüstung besitzen, die für eine
Replikation benötigt wird, sind sie von Ausrüstungen der Wirtszellen abhängig, um
diesen Prozess zu vollbringen, von einer Differenzierung der Wirtszelle, um ihren
Lebenszyklus zu vollenden [12]. Obwohl mehr als 150 verschiedene Papillomaviren von
gesunder und kranker menschlicher Haut isoliert wurden, wurden nur wenige in
Karnivoren identifiziert [13,14]. In der vorliegenden Studie wurden neue Papillomaähnliche Sequenzen in verschiedenen caninen und felinen Läsionen nachgewiesen,
einschliesslich Cyclosporin A-assoziierte exophytische Läsionen und in-situ und invasive
Karzinome [15,16]. Es wurden zwei Sets von Primern verwendet, die für die
Amplifikation einer Sequenz des E1-Gens des Papillomavirus entwickelt wurden. Das
Set von Primern mit begrenzter Wirkung wurde entwickelt, um canine und feline
Papillomaviren und deren nahe Verwandten zu amplifizieren [16]. Das umfassende PCR
System wurde entwickelt, um bis zu 64 humane Papillomaviren und mehrere
Papillomaviren von Tieren einschliesslich die caninen und felinen zu amplifizieren [16,
17]. Diese Untersuchungen haben die Existenz von mindestens sechs felinen und fünf
caninen zuvor unbekannten Papillomavirus-ähnlichen Sequenzen gezeigt. Weil die
Klassifikation der Papillomaviren auf dem L1-Gen basiert, erlaubt die Amplifikation von
Sequenzen des E1-Gens keine einwandfreie Evaluation dieser Sequenzen oder
79
Klassifikation der neu entdeckten Papillomaviren. Diese Resultate haben jedoch
nahegelegt, dass canine und feline Hautläsionen von Papillomaviren von beträchtlicher
genetischer Diversität infiziert werden können. Es wäre von enormem Interesse, diese
canine und feline Papillomaviren zu amplifizieren und zu klonen. Glücklicherweise
wurde kürzlich eine neue Technik, das Rolling-circle Amplifikationsverfahren (rollingcircle amplification, RCA) eingeführt, um zirkuläre DNA und insbesondere
Papillomaviren von Menschen und Tieren zu amplifizieren und isolieren [18,19]. Das
Rolling-circle Amplifikationsverfahren ist eine multiple „random primed“, Sequenz
unabhängige Amplifikation von zirkulärer DNA. Zudem ist diese Amplifikation so
wirkungsvoll wie PCR. Daher werden für die Amplifikation von Papilloma genomischer
DNA nur kleinste Mengen von grob isolierter DNA benötigt. Das Rolling-circle
Amplifikationsverfahren kann angewendet werden um zu bestimmen, ob gesunde Haut
Papillomaviren in sich trägt und um Papillomaviren zu sequenzieren, die in Hautläsionen
von Hunden und Katzen vorhanden sind. Diese beschreibende Studie ist der
unumgängliche erste Schritt für ein besseres Verständnis der Rolle, die Papillomaviren in
der Entwicklung von Hautläsionen bei Hunden und Katzen spielen, besonders in der
Entwicklung von Hautkrebsen. Wir haben diese neue Technik bereits bei der Isolation
und beim Klonen eines neuen caninen Papillomavirus angewandt (CPV3) [20].
Bei Säugetieren und Vögeln führen Papillomaviren ein grosses Spektrum an kutanen
Veränderungen und Veränderungen der Schleimhaut herbei, wie zum Beispiel
exophytische und Flachwarzen und präkanzeröse und kanzeröse Läsionen. Sie werden für
wichtige Karzinogene bei Menschen gehalten und einige risikoreiche Papillomaviren sind
direkt für die Entwicklung von Gebärmutterhalskrebs bei Frauen verantwortlich [21-23].
Obwohl die Verbindung zwischen zervischen Karzinomen und humanen Papillomaviren
klar ist, ist die Rolle von Papillomaviren in der Entwicklung von kutanem PlattenepithelKarzinom (SCC) nicht so klar [24]. Es gibt jedoch vermehrte epidemiologische
Evidenzen, die nahelegen, das Papillomaviren eine wichtige Rolle bei der
Hautkanzerogenese spielen, besonders bei der Epidermodysplasia verruciformis (EV). Es
ist dennoch problematisch, eine Kausalität zwischen dem Vorhandensein von
Papillomaviren in Hautläsionen und der Entwicklung der Läsionen herzustellen [25]. Es
80
wurden Kriterien vorgeschlagen, um diese Beziehung zu bestätigen, Schwierigkeiten bei
der Kultivierung von Papillomaviren haben deren Erfüllung aber grösstenteils unmöglich
gemacht [22, 25]. Zusätzlich erlaubt der Gebrauch von extrem sensitiver Nested-PCR den
Nachweis einer winzigen Menge von viraler DNA (0.05 virale Genome per Zelle). Die
Anwesenheit von kleinen Mengen von Nukleinsäure des Papillomavirus induziert keine
produktive Infektion und kann auch in gesunder Haut vorkommen [26, 27]. Vieles deutet
auch darauf hin, dass ultraviolette Strahlung (UV) zur Kanzerisation von einigen
Papillomavirus assoziierten Hautkrebsen beiträgt [25, 28]. Alles in allen bleibt angesichts
all dieser Ergebnisse die Rolle von Papillomaviren bei der Entwicklung von Hautkrebs
beim Menschen unklar.
Einige Tiermodelle stützen die kausative Rolle von Papillomaviren bei der Induktion des
Squamosazell-Karzinoms (SCC): Vor einigen Jahrzehnten wurde gezeigt, dass Cottontail
rabbit Papillomaviren (CRPV) in der Lage sind, Hautkrebs bei Hasen zu induzieren [2931].
Andere Studien haben auch festgestellt, dass attenuierter Lebend-Impfstoff für Canines
orales Papillomavirus (COPV) bei Beagle SCC induziert [32]. Hinzu kommt, dass Hunde
und Katzen von Hautkonditionen betroffen sein können, die einiges gemein haben mit
EV von Menschen und bei einigen Patienten wurde von einer Kanzerisation berichtet
[33]. Einige Studien haben eine Verbindung zwischen SCC und Papillomaviren bei
Karnivoren gezeigt, es wurde aber keine Kausalität hergestellt [16, 34-40]. Diese Arbeit
hat die epidemiologische Verbindung zwischen einigen Gruppen von felinem und
caninem Hautkrebs und Papillomavirus-Infektion sowie die genetische Diversität von
karnivoren Papillomaviren bestätigt [16, 20, 34, 40]. Nebst der Identifikation und dem
Klonen dieser neuen Papillomaviren sind die wichtigsten Untersuchungen diejeinigen,
die die relative Prävalenz von jedem neuen karnivoren Papillomavirus feststellen und in
vitro zeigen, dass zumindest einige davon in der Lage sind, die Transformation und
Immortalisation von Keratinozyten zu induzieren.
Wir haben zum Beispiel ein neuartiges Papillomavirus (CPV3) entdeckt, geklont und
sequenziert, das mit einem Fall von Caniner Epidermodysplasia verruciformis assoziiert
war [20]. Der befallene Hund entwickelte multiple Plaques und eine einzige interdigitale
Läsion von in-situ SCC [20]. In jeder untersuchten Läsion (einschliesslich SCC) wurde
81
DNA von CPV3 isoliert, diese war aber nicht vorhanden in der intakten Haut desselben
Hundes. Es wurde Sequenz-unabhängige, „multiple primed“ RCA verwendet, um das
ganze CPV3 Genom zu amplifizieren, klonen und sequenzieren. Tatsächlich liess die
Analyse des geklonten und sequenzierten caninen Papilloma-Genoms seine
Klassifikation als Mitglied einer neuen Gattung von Papillomaviren (GenBank-Eintrag
DQ295066) zu. Zusätzlich wurde in jeder untersuchten Läsion mRNA für das
mutmassliche transformierende Protein E6 gefunden. Diese Resultate zeigten, dass CPV3
in den erwähnten Hautläsionen transkriptional aktiv war und stützten die Hypothese einer
kausativen Rolle von CPV3 in der Pathogenese von Caniner EV [41]. Komplementäre
DNA des p53 Transkriptes des betroffen Hundes wurde sowohl von intakter Haut als
auch von Hautläsionen geklont, und seine Nukleotid-Sequenz wich nicht von der
Wildtyp-caninen p53-Sequenz ab. Dieses Ergebnis deutete darauf hin, dass die
Entwicklung eines bösartigen Tumors nicht auf UV-abhängige Mutagenese
zurückzuführen war. Folglich wurde die Hypothese gestützt, dass transformierende
Proteine von CPV3 die Entwicklung von Krebs mit Mechanismen induzieren, die anders
sind als die Mechanismen, die bei humanen EV-assoziierten Papillomaviren angestrengt
werden [41].
Wir untersuchen gegenwärtig auch die relative Prävalenz von CPV3 in caninen Seren.
Wir haben die CP3-L1 Gene geklont (Kodes für CPV3 Haupt-Kapsidprotein) und
Antiseren gegen dieses Protein generiert. Unser Ziel ist es, einen ELISA Test zu
etablieren und 500 vorgängig gesammelte canine Seren zu evaluieren.
Schliesslich öffnen die Resultate dieser Arbeit and hoffentlich zukünftiger Studien neue
Zugänge zur Erforschung der Papillomavirus-induzierten Hautkanzerogenese. Das sollte
sich sowohl für die veterinär- als auch die human-medizinische Onkologie als hilfreich
erweisen, wenn man bedenkt, dass es mehrere ähnliche Hautkonditionen bei Karnivoren
und Menschen gibt.
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