Bovine papillomavirus DNA in neoplastic and nonneoplastic tissues obtained from horses with and without sarcoids in the western United States

OBJECTIVE: To determine the incidence of bovine papillomavirus (BPV) type 1 or 2 in sarcoids and other samples of cutaneous tissues collected from horses in the western United States. ANIMALS: 55 horses with sarcoids and 12 horses without sarcoids. PROCEDURE: Tissue samples (tumor and normal skin from horses with sarcoids and normal skin, papillomas, and nonsarcoid cutaneous neoplasms from horses without sarcoids) were collected. Tissue samples were analyzed for BPV-1 or -2 DNA, using a polymerase chain reaction (PCR) and restriction fragment length polymorphism. The PCR products from 7 sarcoid-affected horses were sequenced to evaluate percentage homology with expected sequences for BPV-1 or-2. RESULTS: Most (94/96, 98%) sarcoids contained BPV DNA. Sixty-two percent of the tumors examined had restriction enzyme patterns consistent with BPV-2. Thirty-one of 49 (63%) samples of normal skin obtained from horses with sarcoids contained BPV DNA. All samples subsequently sequenced had 100% homology with the expected sequences for the specific viral type. All tissues from healthy horses, nonsarcoid neoplasms, and papillomas were negative for BPV DNA. CONCLUSIONS AND CLINICAL RELEVANCE: Bovine papillomaviral DNA was detected in essentially all sarcoids examined. There appears to be regional variation in the prevalence of viral types in these tumors. The fact that we detected viral DNA in normal skin samples from horses with sarcoids suggests the possibility of a latent viral phase. Viral latency may be 1 explanation for the high rate of recurrence following surgical excision of sarcoids.     

Detection of bovine papillomavirus DNA on the normal skin and in the habitual surroundings of horses with and without equine sarcoids

The purpose of the present study was to examine whether bovine papillomavirus (BPV) DNA can be detected on the normal skin and in the habitual surroundings of horses with and without equine sarcoids by means of superficially taken swabs. In affected horses, no significant difference in presence of BPV-DNA could be observed between samples obtained from the equine sarcoid surface, from normal skin close to the tumour and from a normal skin site in direct contact with the tumour. From the group of healthy horses living in contact with affected horses, 44% were BPV-DNA positive. The surroundings of affected and non-affected horses are probably not a major source of BPV-DNA contamination. It can be concluded that BPV-DNA is present on the normal skin of horses affected by equine sarcoid and to a lesser degree, on the normal skin of horses living in contact with affected horses.     

High prevalence of bovine papillomaviral DNA in the normal skin of equine sarcoid-affected and healthy horses

Bovine papillomavirus (BPV), the causative agent of papillomas in cattle, has been shown to play a major role in the pathogenesis of equine sarcoids in horses. BPV has also been detected occasionally in normal equine skin. In this study, presence and activity of BPV in normal skin and peripheral blood of 4 groups of horses were evaluated: sarcoid-affected horses, horses living in contact with sarcoid-affected horses, horses living in contact with papilloma-affected cattle and control horses. From each horse, 3 samples on 4 locations were collected: a swab of the intact skin surface and both a swab and a biopsy after decontamination. BPV DNA was found in the normal skin of 24 of 42 horses (57%). Mainly sarcoid-affected horses and horses living in contact with cattle were carriers (73%), but BPV DNA was also detected in 50% of the horses living in contact with sarcoid-affected horses and in 30% of the control population. BPV mRNA was detected in 58% of the samples positive for BPV DNA, although in a much lower quantity compared to sarcoids. In most of the BPV DNA positive samples mild acanthosis, slight basophilic cytoplasmic swelling of the epidermal layers and/or thickening of the basal membrane were noticed, but these observations were also present in several BPV DNA negative normal skin samples. BPV DNA could not be detected in peripheral blood. These findings suggest latent infection and a wide-spread occurrence of BPV in the horse population.     

Polymerase chain reaction analysis of the surgical margins of equine sarcoids for bovine papilloma virus DNA

OBJECTIVE: To examine apparently normal skin around equine sarcoids for evidence of bovine papilloma virus (BPV) DNA, and to relate this finding to the observed recurrence after surgery. STUDY DESIGN: Prospective study. ANIMALS OR SAMPLE POPULATION: Forty-one equine sarcoids from 19 horses. MATERIALS AND METHODS: The tumors were surgically excised at a measured distance of 8, 12, or 16 mm. Samples from the tumor and of the entire surrounding skin were taken at 4, 8, 12, and 16 mm from the tumor border and analyzed for the presence of BPV DNA using polymerase chain reaction (PCR) amplification. The samples were grouped per examined sarcoid, and a tumor was considered positive at a certain distance as soon as at least one of the samples at that distance was positive. The clinical outcome was recorded for each sarcoid after a minimal follow-up of 6 months. RESULTS: All sarcoids were positive for BPV(1) or BPV(2). The tumor margin was positive at 4, 8, 12, and 16 mm in, respectively, 95%, 73%, 39%, and 33% of the examined sarcoids. Local recurrence was observed in 3 sarcoids on 3 different horses. From survival analysis, there was a greater likelihood for local recurrence when sarcoids had a surgical margin that was positive for BPV DNA. CONCLUSIONS AND CLINICAL RELEVANCE: BPV DNA is often detected in visibly normal skin around sarcoids, and there is a significantly greater probability for local recurrence when the surgical margins are positive for the presence of BPV DNA.     

Phylogenetic analysis of bovine papillomavirus E5 detected in equine sarcoids in Poland

The aim of the study was to analyse a part of the sequence of the E5 gene of bovine papillomaviruses (BPV) associated with equine sarcoids in Polish horses. Samples of 40 skin lesions obtained from 29 horses were collected for molecular examination. The PCR amplicons of BPV DNA were detected in 38 specimens. After phylogenetic analysis 37 specimens were recognized as BPV-1 and one as BPV-2. Phylogenetic analysis has allowed the classification of the amplicons into two phylogenetic groups (A1,) and four separate isolates (2, 10, 16, 17).     

Bovine papillomavirus DNA can be detected in keratinocytes of equine sarcoid tumors

Bovine papillomavirus (BPV)-1 and -2 is linked to equine sarcoids, a commonly observed skin tumor in horses that is of considerable veterinary importance. Previous studies using in situ hybridization have detected BPV DNA only in fibroblasts and not in keratinocytes of sarcoids. In contrast, normal equine skin latently infected with BPV shows a dysplastic epithelium without dermal changes, similar to lesions induced by other papillomavirus types infecting the epithelium. The first goal of our study was to describe the epidermal and dermal characteristics of several stages in sarcoid development. Next, we explored whether BPV can infect epidermal cells in the horse using real-time PCR on laser-micro-dissected keratinocytes and fibroblasts. We found that latently infected normal skin samples and a subset of early stage sarcoids show dysplastic, koilocyte-like epithelial changes. BPV DNA was detected in keratinocytes in 40% of the samples with these particular epithelial properties, whereas advanced sarcoids only had BPV DNA in the fibroblasts. These data may indicate a novel and intriguing pathway of BPV infection in the horse composed of a first step of keratinocyte infection, followed by migration of viral material towards the dermis resulting in infection of sub-epidermal fibroblasts and their fully transformed phenotype. Additionally, an example of co-existence of a dermal BPV-1 and an epidermal BPV-2 infection in the same lesion is shown, indicating that horses can harbor infection with more than one BPV type at the same time.     

Cutaneous sarcoids in captive African lions associated with feline sarcoid-associated papillomavirus infection

Solitary and multiple cutaneous and mucocutaneous masses were identified in 5 of 24 captive African lions (Panthera leo) over a 6-month-period. All masses were surgically excised, and all were histologically similar to equine and feline sarcoids. DNA was extracted from formalin-fixed, paraffin-embedded tissue. Polymerase chain reaction amplified DNA sequences that had been previously detected in feline sarcoids and clinically normal bovine skin. All lions had been fed a diet that included bovine carcasses that had not been skinned. Since the cessation of feeding bovine carcasses with cutaneous lesions, no additional skin lesions have been observed within any of the lions. Herein is described the clinical, gross, and histopathological findings of sarcoids in 5 captive lions. As the causative papillomavirus most likely has a bovine definitive host, it is hypothesized that the lions were exposed to the virus by feeding on bovine carcasses with skin still attached.     

Detection of bovine papillomavirus DNA in equine sarcoids using the polymerase chain reaction (PCR)]

Unfixed and formalin-fixed frozen sections and paraffin-sections of histopathologically confirmed sarcoids of 20 horses were studied in the PCR. The used set of primers was located in the E5 open reading frame fitting both to bovine papillomavirus 1 (BPV-1) and BPV-2. Independent of the quality of the used tissues BPV-DNA was detected in all 20 sarcoids. By cleaving with restriction endonuclease Bst XI it was shown that the DNA-sequences amplified by PCR were identical with that of BPV-1. The results support the general view that BPV play an important role in equine sarcoids.     

Successful treatment of equine sarcoids by topical aciclovir application

Based on the anecdotally reported eradication of a sarcoid using aciclovir cream, the curative potential of this ointment was investigated in 22 sarcoid-affected horses referred to the Equine Clinic Tillysburg, Austria, between 2006 and 2009. Sarcoid disease was diagnosed by clinical examination and bovine papillomavirus types 1 and 2 from intact skin and tumour tissue. As nine horses had more than one lesion, a total of 47 sarcoids were treated by daily topical application of aciclovir 5 per cent cream for a period of two to six months; in four horses, surgical tumour ablation was performed before treatment. Disease parameters, including the tumour type, number, location and size, were recorded before and after aciclovir therapy. All 47 (100 per cent) of the sarcoids responded to treatment, with complete tumour regression observed for 32 (68 per cent) lesions and no recurrences reported thus far. Incomplete resolution was observed for 15 (32 per cent) lesions, probably due to their thickness. Aciclovir is proposed to be routinely used for the treatment of mild-type sarcoids and as an adjuvant therapeutic agent in combination with surgery.     

Bovine papillomavirus infection in equine sarcoids and in bovine bladder cancers

Bovine papillomavirus (BPV) type 2 is involved in carcinogenesis of the urinary bladder in cattle, while BPV-1 is commonly associated with equine sarcoid tumours. In both cases the early viral proteins are expressed, but virion is not produced. Given the similarities in BPV biology between the tumours in cattle and horses, bovine bladder cancers and equine sarcoids were compared with respect to physical status, load of viral DNA and variability of the E5 open reading frame (ORF). Rolling circle amplification demonstrated that BPV-1 and BPV-2 genomes exist as double stranded, episomal, circular forms in the two tumours. Realtime quantitative PCR revealed that equine sarcoids contained higher viral DNA loads compared to bovine bladder cancers. The BPV-1 E5 ORF showed sequence variation but BPV-2 ORF did not. The presence of BPV-1 E5 variations or their absence in the BPV-2 E5 ORF does not appear to have an effect on viral DNA load in either tumour type.     

BPV-1 infection is not confined to the dermis but also involves the epidermis of equine sarcoids

In equids, bovine papillomaviruses of type 1 (BPV-1) and less frequently type 2 induce common, locally aggressive skin tumours termed sarcoids. Whereas BPV infection in cattle usually involves the epidermis and is productive in this skin layer, infection in equids is currently thought to be abortive, with virus solely residing as multiple episomes in dermal fibroblasts. Based on recent observations that do not agree with this assumption, we hypothesised that BPV also infects equid epidermis and is active in this skin layer. To test this hypothesis, we conducted a proof-of-principle study on eight distinct sarcoids. Presence of viral DNA was addressed by qualitative and quantitative BPV-1 PCR from microdissected sarcoid epidermis, and by subsequent amplicon sequencing. Viral activity was assessed by screening sarcoid epidermis for BPV-1 protein expression using immunohistochemistry (IHC) or immunofluorescence (IF). Virus-free equine skin served as negative control throughout the assays. BPV-1 DNA was demonstrated in all sarcoid epidermis samples, with viral DNA loads ranging between 2 and 195 copies/cell. Identical BPV-1 E5 genes were identified in epidermis and dermis of each of two sarcoids, yet different E5 variants were found in individual lesions. IHC/IF revealed the presence of E5 and E7 protein in sarcoid epidermis, and L1 capsomers in the squamous layer of one lesion. These findings indicate that BPV infection also involves the epidermis, where it may occasionally be productive.     

Equine Sarcoid Associated with Cutaneous Habronemosis

Equine sarcoids are benign fibroblastic skin tumors affecting equids worldwide. Infection with bovine papillomavirus types 1 and 2 has been implicated as a major fact in the disease development; however, the cellular mechanisms underlying fibroblast transformation are still largely unknown. In the present study, a diagnosis of sarcoid was histologically assessed along with eosinophilic dermatitis. The sarcoid lesion expressed the viral oncoproteins E5 and E2, suggesting a causative role of the virus and its replication. Ribosomal DNA of the nematode Habronema muscae was also revealed in the lesion. This is the first report to describe and discuss an association of cutaneous habronemosis with equine sarcoid.     

Use of cisplatin-containing biodegradable beads for treatment of cutaneous neoplasia in equidae: 59 cases (2000-2004)

OBJECTIVE: To determine outcome for equids with cutaneous neoplasms treated with cisplatin-containing biodegradable beads, alone or in conjunction with debulking. DESIGN: Retrospective case series. ANIMALS: 56 horses, 1 zebra, 1 donkey, and 1 mule. PROCEDURES: Medical records were reviewed. Follow-up information was obtained through telephone conversations with owners and trainers of the animals. RESULTS: 22 tumors were sarcoids, 6 were fibrosarcomas, 1 was a fibroma, 2 were peripheral nerve sheath tumors, 11 were squamous cell carcinomas, 14 were melanomas (13 gray horses and 1 bay horse), 1 was a lymphosarcoma, 1 was an adenocarcinoma, and 1 was a basal cell tumor. Forty-five (76%) animals underwent conventional or laser debulking of the tumor prior to bead implantation. Forty of 48 (83%) animals for which long-term follow-up information was available were relapse free 2 years after treatment. This included 20 of 22 animals with spindle cell tumors (including 11/13 horses with sarcoids), 6 of 10 animals with squamous cell carcinomas, 13 of 14 animals with melanomas, and 2 of 3 animals with other tumor types. Adverse effects were minimal. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that implantation of cisplatin-containing biodegradable beads, with or without tumor debulking, may be an effective treatment for equidae with various cutaneous neoplasms.     

Safety and immunogenicity of BPV-1 L1 virus-like particles in a dose-escalation vaccination trial in horses

Reasons for performing study: Infection with bovine papillomaviruses types 1 and 2 (BPV‐1, BPV‐2) can lead to the development of therapy‐resistant skin tumours termed sarcoids and possibly other skin diseases in equids. Although sarcoids seriously compromise the welfare of affected animals and cause considerable economic losses, no prophylactic vaccine is available to prevent this common disease. In several animal species and man, immunisation with papillomavirus‐like particles (VLP) has been shown to protect efficiently from papillomaviral infection. Hypothesis: BPV‐1 L1 VLPs may constitute a safe and highly immunogenic vaccine candidate for protection of horses against BPV‐1/‐2‐induced disease. Methods: Three groups of 4 horses each received 50, 100 or 150 µg of BPV‐1 L1 VLPs, respectively, on Days 0, 28 and 168. Three control horses received adjuvant only. Horses were monitored on a daily basis for one week after each immunisation and then in 2 week intervals. Sera were collected immediately before, 2 weeks after each vaccination and one and 2 years after the final boost and analysed by pseudovirion neutralisation assay. Results: None of the horses showed adverse reactions upon vaccination apart from mild and transient swelling in 2 individuals. Irrespective of the VLP dose, all VLP‐immunised horses had developed a BPV‐1‐neutralising antibody titre of ≥1600 plaque forming units (pfu)/ml 2 weeks after the third vaccination. Eight of 10 trial horses still available for follow‐up had neutralising antibody titres ≥1600 pfu/ml one year and ≥800 pfu/ml 2 years after the last immunisation. Conclusion: Intramuscular BPV‐1 L1 VLP vaccination in horses is safe and results in a long‐lasting antibody response against BPV‐1. Neutralisation titres were induced at levels that correlate with protection in experimental animals and man. Potential relevance: BPV‐1 L1 VLPs constitute a promising vaccine candidate for prevention of BPV‐1/‐2‐induced disease in equids.     

Equus caballus papillomavirus‐2 (EcPV‐2): An infectious cause for equine genital cancer?

Reasons for performing study: The aetiology of genital squamous cell carcinoma (SCC) in horses remains unknown, but the similarity to the disease in man, for which papillomavirus infection has been shown to be a causal factor, requires to be investigated in horses. Hypothesis: One or more novel papillomaviruses cause equine genital SCC and its associated premalignant lesions. Methods: DNA was extracted from samples of equine genital SCC and performed rolling circle amplification, in order to identify closed circular DNA viral genomes within the samples. The amplified DNA was subcloned and sequenced and the DNA sequence compared to that of other papillomavirus genomes. Using PCR primers developed from these genomic DNA sequences, studies were then carried out in order to identify the frequency at which the viral DNA could be identified in equine genital cancer samples from horses in both the UK, Australia and Austria. Finally, in situ hybridisation using specific probes developed from this DNA sequence were used to confirm the presence of the viral RNA sequences in the neoplastic cells in these lesions. Results: The full length genome of a novel papillomavirus species was characterised from the equine genital SCC tissue and termed Equus caballus papillomavirus‐2 (EcPV‐2). Viral DNA and RNA was identified in the genital tumour samples, but not in the adjacent histologically normal tissue. EcPV‐2 DNA could not be identified in equine ocular or nasal carcinomas or within the scrotal skin or in most smegma samples obtained from tumour‐free horses. Sequencing of amplicons, generated from the archived equine genital tumours, identified variations within E1 and E6 on DNA and predicted protein level. Conclusions: A novel papillomavirus, EcPV‐2, is likely to play a causal role in the pathogenesis of equine genital epithelial tumours. Potential relevance: Identification of a papillomavirus causal for genital carcinomas in horses may lead to development of a vaccine that could be used to prevent this serious disease in horses. This would be analogous to man, where vaccination against oncogenic papillomavirus species is currently being used to help prevent cervical cancer.     

Evaluation of excision, cryosurgery and local BCG vaccination for the treatment of equine sarcoids.

Ninety-five horses with sarcoids were subjected to three types of treatment: surgical excision (conventional or carbon dioxide laser), cryotherapy or local BCG vaccination. The type of treatment was selected on the basis of the size, location and clinical appearance of the tumours. The choice between conventional and laser excision was empirical. A successful outcome was obtained in 11 of 14 (79 per cent) of the horses treated by cryosurgery, 18 of 27 (67 per cent) treated by BCG vaccination, 18 of 22 (82 per cent) treated by conventional excision, and 20 of 28 (71 per cent) treated with a carbon dioxide laser. For both excision methods, rigorous measures were taken to avoid autoinoculation and to ensure a wide margin of normal skin. The probability of local recurrence after excision was significantly higher for large sarcoids and sarcoids which had previously failed to respond to treatment. In 10 of the 31 horses with remaining sarcoids, some or all of the untreated sarcoids were observed to regress spontaneously.     

Feline cutaneous fibropapillomas: clinicopathologic findings and association with papillomavirus infection

Twenty-three feline cutaneous fibropapillomas with histologic features similar to equine sarcoids were diagnosed. They were characterized by dermal fibroblastic proliferation with overlying, often ulcerated hyperplastic epidermis. Electron microscopic findings supported the fibroblastic nature of the neoplastic cells. The 23 tumors came from 20 cats and were submitted from veterinary clinics in Wisconsin and Minnesota. These tumors occurred most commonly in young cats and were found primarily on the head, neck, and digits. Fifteen of the 17 cats for which breed was reported were domestic shorthair cats. In 11/20 cases, there was confirmed exposure to cattle. Local recurrence of the tumor following surgical excision was reported in 7 of the 18 cats for which follow-up information was available. Metastasis was not documented in any of the cases. Two of the 19 tumors tested by polymerase chain reaction (PCR) had no amplifiable DNA. The remaining 17 were positive for papillomavirus by PCR. No papillomavirus DNA was detected in three other feline skin tumors (cutaneous mast cell tumor, malignant lymphoma, and fibrosarcoma) that served as controls. This is the first report of detection of papillomavirus in feline tumors that have clinicopathologic features similar to equine sarcoids.     

BAG3 protects Bovine Papillomavirus type 1-transformed equine fibroblasts against pro-death signals

In human cancer cells, BAG3 protein is known to sustain cell survival. Here, for the first time, we demonstrate the expression of BAG3 protein both in equine sarcoids in vivo and in EqS04b cells, a sarcoid-derived fully transformed cell line harbouring bovine papilloma virus (BPV)-1 genome. Evidence of a possible involvement of BAG3 in equine sarcoid carcinogenesis was obtained by immunohistochemistry analysis of tumour samples. We found that most tumour samples stained positive for BAG3, even though to a different grade, while normal dermal fibroblasts from healthy horses displayed very weak staining pattern for BAG3 expression. By siRNA technology, we demonstrate in EqS04b the role of BAG3 in counteracting basal as well as chemical-triggered pro-death signals. BAG3 down-modulation was indeed shown to promote cell death and cell cycle arrest in G₀/G₁. In addition, we found that BAG3 silencing sensitized EqS04b cells to phenethylisothiocyanate (PEITC), a promising cancer chemopreventive/chemotherapeutic agent present in edible cruciferous vegetables. Notably, such a pro-survival role of BAG3 was less marked in E. Derm cells, an equine BPV-negative fibroblast cell line taken as a normal counterpart. Altogether our findings might suggest a mutual cooperation between BAG3 and viral oncoproteins to sustain cell survival.     

Sequences of papillomavirus DNA in equine sarcoids

DNA was extracted from 14 equine sarcoids, electrophoresed and hybridised with a radioactively labelled probe of bovine papillomavirus type I (BPV 1) DNA under conditions of low stringency. Twelve sarcoids contained sequences of DNA that hybridised with the probe and that comigrated with BPV 2 DNA. The viral DNAs in four of these sarcoids differed from BPV 1 and BPV 2 DNA on restriction endonuclease analysis. One of four cell lines derived from sarcoids also contained BPV 1 related DNA. The results confirm the frequent presence in equine sarcoids of unintegrated papillomaviral DNA and suggest a role for papillomavirus infection in this disease.