EcPV2 DNA in equine genital squamous cell carcinomas and normal genital mucosa

Squamous cell carcinoma (SCC) represents the most common genital malignant tumor in horses. Similar to humans, papillomaviruses (PVs) have been proposed as etiological agents and recently Equine papillomavirus type 2 (EcPV2) has been identified in a subset of genital SCCs. The goals of this study were (1) to determine the prevalence of EcPV2 DNA in tissue samples from equine genital SCCs, penile intraepithelial neoplasia (PIN) and penile papillomas, using EcPV2-specific PCR, (2) to examine the prevalence of latent EcPV2 infection in healthy genital mucosa and (3) to determine genetic variability within EcPV2 and to disentangle phylogenetic relationships of EcPV2 among PVs. EcPV2 DNA was detected in all but one penile SCC (15/16), in all PIN lesions (8/8) and penile papillomas (4/4). Additionally, EcPV2 DNA was demonstrated in one of two metastasized lymph nodes, one contact metastasis in the mouth, two vaginal and one anal lesion. In healthy horses, EcPV2 DNA was detected in 10% (4/39) of penile swabs but in none of vulvovaginal swabs (0/20). This study confirms the presence of EcPV2 DNA in equine genital SCCs and shows its involvement in anal lesions, a lymph node and contact metastases. Latent EcPV2 presence was also shown in normal male genital mucosa. We found that different EcPV2 variants cocirculate among horses and that EcPV2 is related to the Delta+Zeta PVs and is only a very distant relative of high-risk human PVs causing genital cancer. Thus, similar viral tropism and similar malignant outcome of the infection do not imply close evolutionary relationship.     

An evaluation of the Irish Single Reactor Breakdown Protocol for 2005-2008 inclusive and its potential application as a monitor of tuberculin test performance

The number of recognized papillomavirus (PV) species and potential PV genera has dramatically been increasing throughout the past decade. It seems that every host species might potentially harbour a large set of PVs, while the PVs of each species appear to belong to only a few genera. In horses at least three conditions beside the equine sarcoid have been described, being supposedly PV induced namely classical equine papillomas, genital papillomas and aural plaques. We were able to identify the DNA of novel equine PVs (EcPVs) in the two latter disorders where PV involvement had been predicted. Both PV genomes were entirely cloned and sequenced. Both EcPV genomes, one derived from a penile papilloma, the other derived from an ear papilloma contain the characteristic open reading frames (ORFs) E6, E7, E1, E2, L2 and L1, a large non-coding region between the late and early region as well as a small non-coding region between the early and the late region. The viruses were consequently designated as EcPV2 and EcPV3. The genomes of the three equine PVs were analysed and compared with each other and further PVs. Upon phylogenetic analyses the equine PVs group well together. Pairwise alignment of the L1 nucleotide sequences reveals that EcPV1 shares 54.9% identities with EcPV2 and 53.2% with EcPV3. EcPV2 and EcPV3 share 51.3% identities. As the three EcPVs share less than 60% of nucleotide identities in L1, they may be regarded as belonging to different genera.     

Phylogenetic and structural studies of a novel equine papillomavirus identified from aural plaques

Papillomaviruses (PVs) infect a wide range of animal species and show great genetic diversity. To date, excluding equine sarcoids, only three species of PVs were identified associated with lesions in horses: Equus caballus papillomavirus 1 (EcPV1-cutaneous), EcPV2 (genital) and EcPV3 (aural plaques). In this study, we identified a novel equine PV from aural plaques, which we designated EcPV4. Cutaneous samples from horses with lesions that were microscopically diagnosed as aural plaques were subjected to DNA extraction, amplification and sequencing. Rolling circle amplification and inverse PCR with specific primers confirmed the presence of an approximately 8 kb circular genome. The full-length EcPV4 L1 major capsid protein sequence has 1488 nucleotides (495 amino acids). EcPV4 had a sequence identity of only 53.3%, 60.2% and 51.7% when compared with the published sequences for EcPV1, EcPV2 and EcPV3, respectively. A Bayesian phylogenetic analysis indicated that EcPV4 clusters with EcPV2, but not with EcPV1 and EcPV3. Using the current PV classification system that is based on the nucleotide sequence of L1, we could not define the genus of the newly identified virus. Therefore, a structural analysis of the L1 protein was carried out to aid in this classification because EcPV4 cause lesion similar to the lesion caused by EcPV3. A comparison of the superficial loops demonstrated a distinct amino acid conservation pattern between EcPV4/EcPV2 and EcPV4/EcPV3. These results demonstrate the presence of a new equine PV species and that structural studies could be useful in the classification of PVs.     

Cutaneous habronemiasis in a 9‐year‐old Arab gelding in the United Kingdom

Cutaneous habronemiasis causes ulcerative granulating lesions in the skin of equids. Dramatic exuberant tissue may be seen, which becomes traumatised by the horse. To the authors' knowledge, cutaneous habronemiasis has not previously been documented on the distal limb of horses in the UK. Cutaneous habronemiasis should be considered as a potential differential diagnosis for ulcerative granulating skin lesions on the distal limb of the horse.     

Cutaneous and ocular habronemiasis in horses: 63 cases (1988-2002)

OBJECTIVE: To describe clinical manifestations of cutaneous and ocular habronemiasis in horses and evaluate outcome of treatment. DESIGN: Retrospective study. ANIMALS: 63 horses. PROCEDURE: The diagnosis was made on the basis of history, clinical signs, and identification of calcified concretions (sulfur granules) in lesions. Histologic examination of biopsy specimens was used to confirm the diagnosis. Case horses were compared with a control population of 12,720 horses examined during the same period. RESULTS: Arabians, gray horses, and horses with diluted coat colors were overrepresented; Thoroughbreds were underrepresented. Lesions were identified most often during the summer and early fall. The medial canthus of the eye, male genitalia, third eyelid, and distal portions of the extremities were the most commonly affected locations. Twenty-five lesions were biopsied, and results of histologic examination were consistent with a diagnosis of habronemiasis. However, nematode larvae were seen in only 11 (44%) biopsy specimens. Treatment consisted of surgical removal (7 horses) or medical treatment (56) consisting of debulking granulation tissue and topical, intralesional, or systemic treatment with corticosteroids. All horses were treated with ivermectin. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that cutaneous and ocular habronemiasis should be considered when examining a horse during the summer months with a proliferative, moist, granulomatous lesion. Treatment should be aimed at decreasing the size of the lesion, reducing inflammation, and preventing recurrence. In general, the prognosis was good, and healing occurred within a few weeks. Fly control and regular deworming with ivermectin are recommended to reduce the incidence of habronemiasis.     

Cutaneous inverted papillomas in dogs

Inverted papillomas of the skin occurred in five dogs. Lesions were 1-2 cm, circumscribed, flask-like structures below the level of the surrounding normal skin. Walls of the structures consisted of hyperplastic epidermis, forming thin papillary projections on thin fibrovascular stalks. Cells in the stratum granulosum had clear cytoplasm, numerous keratohyalin-like granules of various sizes, and poorly defined intranuclear inclusions. These cells stained positively for papillomavirus group-specific antigens by both the peroxidase-antiperoxidase and avidin-biotin methods. Virions with a mean diameter of 35.7 nm were present within nuclei in cells of the stratum granulosum when examined by electron microscopy. In situ DNA hybridization, using a canine oral papillomavirus probe, localized papillomavirus DNA in canine oral papillomas, but not in canine cutaneous squamous or inverted papillomas, suggesting that a different papillomavirus type was present in the latter lesions. Although these lesions resembled intracutaneous cornifying epitheliomas (keratoacanthomas), they appear to be a distinct lesion, probably with a different etiology.     

Papillomas and carcinomas associated with a papillomavirus in European harvest mice (Micromys minutus)

Papillomaviruses, group-specific papillomavirus antigens, or extrachromosomal papillomavirus DNA were detected in cutaneous, mucocutaneous, and pulmonary tumors affecting a colony of European harvest mice (Micromys minutus). Skin lesions were classified as acanthomatous hyperplasia, epidermal inclusion cysts, squamous papillomas, inverted papillomas, trichoepitheliomas, and sebaceous carcinomas. Cutaneous horns (hyperkeratotic papillomas) were on mucocutaneous junctions of one animal. One mouse, with a cutaneous sebaceous carcinoma, had multiple pulmonary keratinaceous cysts. Papillomavirus antigens, detected by the avidin-biotin technique, were in 20 of 31 lesions tested. In contrast, by Southern blot hybridization all 28 lesions tested contained papillomavirus DNA. Papillomavirus DNA was demonstrated in two of ten benign cutaneous lesions by in situ hybridization.     

GRANULOMATOUS AND NEOPLASTIC DISEASES OF THE SKIN OF HORSES

Lesions encountered in a clinicopathological study of cutaneous lesions resembling 'swamp cancer' from horses in North Queensland included 37 cases of subcutaneous phycomycosis, 5 of which were also infected with Habronema sp larvae. In addition 9 cases of primary cutaneous habronemiasis, 58 sarcoids, 12 fibromas, 8 fibrosarcomas, 10 squamous cell carcinomas, 4 haemangiomas, 3 melanomas, 1 papilloma, 1 palpebral adenocarcinoma and 7 cases of simple granulation were diagnosed. Subcutaneous phycomycosis resulted in the most extensive lesions. These were rapidly growing and provided the poorest prognosis of all conditions studied. The incidence of phycomycosis in north Queensland was high in comparison to more temperate areas and appeared to be related to the wetter seasons. Young horses were more commonly affected. A fungus isolated from 3 phycomycotic lesions was tentatively identified as Hyphomyces destruens as no sporulation was observed.     

Evidence for papillomaviruses in ocular lesions in cattle

Negatively stained preparations of various ocular lesions, generally considered to be precursors to bovine ocular squamous cell carcinoma, were subjected to electron microscopic examination for viruses. Lesions examined included five conjunctival plaques, an acanthotic lesion from an eyelid, 15 conjunctival papillomas, two papillomas of the eyelid and two keratinised elongated proliferative lesions of the eyelid (cutaneous horns). Eight lesions contained particles that resembled papillomaviruses. These consisted of a conjunctival plaque, five conjunctival papillomas, a papilloma of the eyelid and one of the samples of cutaneous horn. The particles were present in small numbers and were found only after prolonged search. The finding of these particles suggests that papillomavirus may be involved in the aetiology of bovine ocular squamous cell carcinoma.     

Cutaneous and gastrointestinal pythiosis in a dog in Brazil

The oomycete Pythium insidiosum is the pathogenic cause of pythiosis, a life-threatening disease that affects several animal species. Canines are the second most affected species, and the disease is characterized by the development of cutaneous and gastrointestinal lesions. While concomitant cutaneous and gastrointestinal lesions are rarely found in the same animal, this report documents a case of concurrent cutaneous and gastrointestinal pythiosis in an 18-month-old female Labrador. This dog had an ulcerative cutaneous lesion on the right thoracic region for 12 months that was unresponsive to itraconazole and terbinafine therapy. Two months prior to death and concurrent with the cutaneous lesion, the dog became anorexic with frequent vomiting and bloody stools. At necropsy, a cutaneous lesion that extended subcutaneously into the intercostal muscles was observed. Additionally, the large intestine contained two lesions that caused luminal narrowing. Organs were collected, routinely processed and stained using hematoxylin and eosin and Gomori methenamine silver. Histological examination of the lesions in the large intestine and on the skin revealed areas of necrosis surrounded by a pyogranulomatous infiltrate. Occasionally, black, septate, branching hyphae were detected following staining with Gomori methenamine silver. The diagnosis of pythiosis was confirmed using immunohistochemical methods. This report describes the occurrence of concomitant gastrointestinal and cutaneous lesions in a dog and highlights the therapeutic difficulties encountered with this disease.     

Canine inverted papillomas associated with DNA of four different papillomaviruses

Inverted papillomas are uncommon papillomavirus (PV)‐induced canine skin lesions. They consist of cup‐ to dome‐shaped dermal nodules with a central pore filled with keratin. Histologically they are characterized by endophytic projections of the epidermis extending into dermis. Cytopathic effects of PVs infection include the presence of clumped keratohyalin granules, koilocytes and intranuclear inclusion bodies. Different DNA hybridization studies carried out with a canine oral papillomavirus (COPV) probe suggested that a different PV than COPV might cause these lesions. Canine papillomavirus 2 (CPV2) was discovered a few years ago in inverted papillomas of immunosuppressed beagles. Two other cases, presenting with distinct clinical and histological features have also been described. This study was carried out on four dogs with clinical and histological signs of inverted papillomas. Molecular biological analyses confirmed that PV DNA was present in all four lesions but demonstrated that the sequences in each case were different. One corresponded to COPV, the second to CPV2, and the third and fourth to unknown PVs. These findings suggest that inverted papillomas are not caused by one single PV type. Similar observations have also been made in human medicine.     

Papillomavirus infection of aged Persian cats

Papillomavirus infection was confirmed in 2 Persian cats with sessile hyperkeratotic skin lesions. Skin lesions were not typical papillomas as found in other species. Papillomavirus were demonstrated in negative stain preparations of homogenized tissue and within nuclei of cells in the stratum granulosum. Papillomavirus group-specific antigens were detected within nuclei corresponding to those containing virions. Attempts to transmit this disease to other cats or propagate the virus in tissue cultures were unsuccessful. A 7.8-kilobase DNA molecule was present in low-stringency Southern blots using a bovine papillomavirus type 1 cloned DNA probe. In reverse Southern blots, the cat papillomavirus hybridized under conditions of low stringency with all papillomavirus genomes tested. Combined with limited restriction endonuclease restriction mapping, the above information indicates that the feline cutaneous papillomavirus is a unique virus type and thus expands the list of hosts known to be infected by papillomaviruses.     

The development of multiple cutaneous inverted papilloma following ovariohysterectomy in a dog

Abstract

CASE HISTORY: Ovariohysterectomy was performed on an adult Cavalier King Charles Spaniel. The skin that had been clipped for surgery was noticed to be erythematous 8 days later.

CLINICAL AND PATHOLOGICAL FINDINGS: Poorly defined patches containing multiple papules were visible bilaterally within the clipped skin. These became larger over the following 2 weeks, and samples were collected for histology. Seven days later, the lesions were multiple raised masses, up to 5 cm in diameter. Histology revealed numerous cup-shaped epidermal proliferations extending into the dermis. The presence of keratinocytes with increased quantities of blue-grey cytoplasm, and koilocytosis suggested papillomavirul infection; Canis familiaris papillomavirus (CfPV-2) DNA was amplified from two separate samples. Complete regression was observed 8 weeks after the lesions had been initially observed.

DIAGNOSIS: Multiple inverted papilloma confined to skin that had been clipped for surgery.

CLINICAL RELEVANCE: This is the first time that the development of canine cutaneous papillomas has been associated with surgery. The nature of the association between surgery and development of the papillomas is uncertain. However, it is possible that damage to superficial skin could promote the formation of papillomas. This is the first identification of CfPV-2 in New Zealand.     

Therapeutic value of partial excision of lesions combined with administration of an autogenous vaccine during an episode of cutaneous papillomatosis in cattle of Uganda

The therapeutic value of partial excision of lesions combined with administration of an autogenous vaccine in calves during an episode of cutaneous papillomatosis was evaluated. Of 10 Holstein calves naturally infected with cutaneous papillomatosis, 5 were given 20 ml of autogenous vaccine in addition to undergoing partial excision of the lesions; the other 5 calves were not given vaccine. Results indicate that partial excision combined with administration of autogenous vaccine has some therapeutic value in calves with small pedunculated papillomas, but not in calves with large confluent lesions.     

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 non-radioactive in situ hybridization

Nineteen cutaneous and mucocutaneous papillomas, as well as 29 oral and 25 non-oral squamous cell carcinomas of dogs were analyzed immunohistologically for the presence of papillomavirus (PV)-antigens. Canine oral papillomavirus (COPV)-DNA was detected in formalin-fixed, paraffin-embedded tissues by polymerase chain reaction (PCR) and non-radioactive in situ hybridization (ISH). Furthermore, the expression of the tumor suppressor protein p53 was investigated. PV-antigens were detectable in more than 50% of the oral and cutaneous papillomas, while no PV-antigens could be demonstrated in venereal papillomas. One squamous cell carcinoma was PV-antigen positive. Only two cutaneous papillomas of the head showed a strong p53-specific immunostaining, while overexpressed p53 was detectable in approximately 35% of all squamous cell carcinomas. It was possible to amplify fragments of the E6, E7 and L1 gene by polymerase chain reaction (PCR) from five of eight oral and from five of eight cutaneous papillomas as well as from three oral squamous cell carcinomas. Nine of 10 papillomas showed a strong nucleus-associated hybridization signal typical for COPV-DNA. In three squamous cell carcinomas COPV-DNA was located in nests of the epithelial tumor cells surrounding ‘horn pearls' or disseminated in the carcinoma tissue. These observations support the view that COPV may also induce non-oral papillomas in the dog and confirm the opinion that a progression of viral papillomas into carcinomas in dogs may occur.     

Papillomavirus associated skin lesions in a cat seropositive for feline immunodeficiency virus.

A cat was presented with skin lesions consisting of slightly raised pigmented plaques, 2-7 mm in diameter with a rough slightly verrucous surface. Histologically these lesions were identified as papillomas. A papillomavirus infection was demonstrated: virus-like particles were present in the nuclei of cells within the lesions, and staining with an anti-bovine papillomavirus (BPV-1) antibody was obtained. An infection with feline immunodeficiency virus was diagnosed in this cat; this condition had probably enhanced the development of papillomas. This is the first report of a papillomavirus infection in a cat in Europe.     

Cloacal papillomas in psittacines

Papilloma-like masses affecting the cloaca of 19 Amazons, 16 macaws, 3 parrots, 1 conure, and 1 parakeet were examined. Papillomatous lesions were characterized by proliferation of the lining epithelium on thin fibrovascular stalks. Carcinoma in situ was diagnosed in the cloaca of a macaw in addition to the other 16 macaws with papillomas. Papillomavirus group-specific antigens were not detected in any of the 41 lesions, using the peroxidase-antiperoxidase technique. The DNA extracts from 6 different frozen papillomas did not contain papillomavirus genomes detectable by Southern blot hybridization, using an African gray parrot cutaneous papillomavirus as a probe. Evidence of an infective agent was not found by electron microscopic examination of 8 of the papillomas. Inoculations of partially purified homogenates of a cloacal papilloma from a yellow-crowned Amazon did not induce lesion formation on cloacal mucosa of an adult yellow-crowned Amazon, green and yellow macaw, sulphur-crested cockatoo, or mollucan cockatoo.     

Digital papillomatosis in a confined Beagle.

Papillomavirus-induced papillomas were diagnosed on multiple digits of all 4 feet of a young Beagle. No other cutaneous or oral involvement was identified. Papillomavirus antigen was confirmed by immunoperoxidase localization within keratinocyte nuclei. In addition to the typical basophilic intranuclear inclusions associated with papillomavirus infections, keratinocytes within the papillomas contained large, eosinophilic cytoplasmic inclusions that previously have been described in a Boxer with cutaneous lesions associated with a papillomavirus infection. The papillomas in this Beagle regressed completely within 2 months of the initial diagnosis.     

Persistent, widespread papilloma formation on the penis of a horse: a novel presentation of equine papillomavirus type 2 infection

A 9-year-old gelding presented with approximately 100 papillomas that covered about 75% of the distal penis. Biopsy was performed, and histology showed evidence of viral cytopathic change and koilocytosis. Polymerase chain reaction using DNA extracted from biopsied tissue amplified equine papillomavirus type 2 (EcPV-2) DNA sequences. Sixteen months later, the horse was re-examined and the appearance of the papillomas was unchanged. Equine papillomavirus type 2 DNA sequences were again amplified from both biopsied tissue and swabs of the penis. Papillomavirus was localized to the lesions by immunohistochemistry and in situ hybridization. An examination 2 years after the initial presentation revealed no detectable change in the appearance of the penis. The large number of papillomas and their failure to regress over an extended period support a clinical classification of papillomatosis. To the authors' knowledge, this is the first report of papillomatosis of the equine penis. This novel clinical manifestation suggests that persistent EcPV-2 infection is possible in horses. As there is evidence that EcPV-2 may promote development of equine penile squamous cell carcinoma, understanding the natural history of EcPV-2 infections may be important in preventing equine penile neoplasia.