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.     

A review of equine sarcoid

Equine sarcoid is the most common tumour of horses and accounts for over half of all equine skin tumours. Six types of sarcoid based on gross appearance and clinical behaviour have been described including occult, verrucous, nodular, fibroblastic, mixed and malevolent. Common locations for sarcoid development include the periocular region, ear pinnae, lips, neck, extremities and ventrum (including groin region). Bovine papillomavirus (BPV) is causally associated with equine sarcoid with genetic haplotype, fly vectors and skin trauma identified as potential risk factors for development of the disease. Histopathology is required for definitive diagnosis of equine sarcoid but incomplete excision is thought to activate latent BPV and stimulate growth. Although there are no uniformly effective treatment options, several modalities have been successful in eliminating or managing equine sarcoid. Surgical excision, intratumoural chemotherapy, cryotherapy, hyperthermia, radiotherapy, immunotherapy and immune modulators are used with degrees of success relative to the accessibility and invasiveness of the tumour. Prevention of equine sarcoid may be facilitated by future development of vaccines against bovine papillomavirus.     

Survey of equine cutaneous neoplasia in the Pacific Northwest.

A retrospective study examined data regarding equine cutaneous and mucocutaneous neoplasms submitted to the Veterinary Diagnostic Laboratory at Oregon State University in a 3.5-year period. A total of 536 neoplasms were identified, accounting for 30% of the total equine pathology submissions. Sarcoid, squamous cell carcinoma, melanocytic tumors, papillomas, and mast cell tumors were the most common neoplasms, constituting 87.5% of all cutaneous neoplasms. Sarcoids represented 51.4% of all neoplasms and 15.18% of total equine accessions. Sarcoid was most common in paints, quarter horses, and Arabians, and was the only common tumor in donkeys and mules. Mean age at diagnosis of equine sarcoid was 9 years. Squamous cell carcinoma constituted 18.3% of all neoplasms and 5.41% of total equine accessions. Ocular squamous cell carcinoma was most common in paints and quarter horses, and penile/preputial squamous cell carcinoma was most common in appaloosas and quarter horses. The mean age of horses with ocular squamous cell carcinoma (13 years) and squamous cell carcinoma of the skin (15 years) was significantly less (P < 0.5) than that of horses with squamous cell carcinoma of the penis and prepuce (21 years) or vulva, anal, and perianal skin (19 years). Findings suggest that equine sarcoid and squamous cell carcinoma occur more frequently in the Pacific Northwest than in the northeastern United States.     

Equine leucocyte antigens in sarcoid-affected horses

The distribution of equine leucocyte antigens (ELA) in horses affected by equine sarcoid tumours was determined and compared with unaffected controls. ELA-haplotype W3,B1 occurred more frequently in affected riding horses of Irish, Swiss and French background. The combined data for the three breeds resulted in a chi 2 value of 20.35 (P less than 0.0005 after correction). Simultaneously, ELA-specificity W11 was more frequently found in horses of Irish background, while W5 was found in Swiss and French horses with sarcoids. The combined data for haplotype W3,B1 and/or W5 specificity demonstrated, in the genetically closely-related Swiss and French horses, a highly significant difference in the occurrence of these haplotypes between affected horses and healthy controls (chi 2 = 28.69, P less than 0.00001 after correction). Thus, the results strongly suggest that the predisposition of horses to sarcoid is associated with or linked to the major histocompatibility complex.     

Immediate Split-Thickness Autogenous Skin Grafts in the Horse Case Reports on the Treatment of Equine Sarcoids in 3 Horses

Three horses with equine sarcoids were treated with radical surgical excision and immediate split-thickness skin grafts. Graft take ranged from 50 to 100%, and the epithelial coverage attained resulted in an early functional repair. In no instance was there a recurrence of the sarcoid.     

Electronic brachytherapy used for the successful treatment of three different types of equine tumours

An electronic form of interstitial brachytherapy was used to treat tumours on 3 different horses. One horse was treated for an ocular lymphoma, the second for a sarcoid, and the third for melanoma. This form of brachytherapy contains inherent advantages over previously used forms of brachytherapy and provides another treatment option for equine tumours that cannot be treated with, or do not respond to, conventional tumour therapies.     

Tumour-associated antigens in bovine ocular squamous cell carcinoma: studies with sera from tumour-bearing animals

Sera from 21 cattle (10 bovine ocular squamous cell carcinoma (BOSCC) bearing and 11 controls) were tested for antibody reactivity against various cultured cells (BOSCC, normal skin and tumours other than BOSCC), by an indirect immunofluorescence technique. Most of the BOSCC sera reacted with autologous and allogeneic BOSCC cells and did not show any significant reactivity with normal skin cells (autologous or allogeneic). These sera when further tested on 7 different allogeneic or xenogeneic tumour cell types (other than BOSCC), showed significant reactivity only with cultured equine sarcoid and cutaneous papilloma cells. Three of the BOSCC sera did not react with autologous BOSCC cells but reacted indiscriminately with all other allogeneic BOSCC, normal cells and other tumour cells. Most of the control sera did not show any significant reactivity against BOSCC cells except sera from one cow bearing ocular papilloma and 2 healthy normals that were in contact with BOSCC bearing animals. This study suggests that BOSCC cells possess tumour associated antigens that are common to most (if not all) BOSCC cells.     

Molecular approaches to equine sarcoids

Sarcoids are the most commonly diagnosed skin tumours in equines. Bovine papillomaviruses (BPVs) are the primary causative agent of sarcoids. There has been intensive research to discover the molecular mechanisms that may contribute to the aetiopathogenesis of this disease and tumour suppressors and proto-oncogenes known to play a role in human neoplastic conditions have been investigated in equine sarcoids. Current approaches include the identification of gene expression profiles, characterising sarcoid and normal skin tissues, and an assessment of epigenetic alterations such as microRNA differential expression and DNA methylation status. This review focuses on selected groups of genes that contribute to the molecular mechanisms of sarcoid formation. These genes have the potential to complement current clinical examinations of equine sarcoid disease in diagnosis, prognosis, therapeutic response and screening.     

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.     

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.     

Consistent detection of bovine papillomavirus in lesions, intact skin and peripheral blood mononuclear cells of horses affected by hoof canker

Reasons for performing the study: Equine hoof canker is a chronic proliferative pododermatitis of as yet unknown aetiology. Like equine sarcoid disease, canker is a therapy‐resistant disorder characterised by hyperkeratosis, acanthosis and a marked tendency to recur. Hypothesis: There is an association of sarcoid‐inducing bovine papillomaviruses of types 1 and 2 (BPV‐1, BPV‐2) with hoof canker disease. Methods: Using PCR‐based techniques, we assessed canker tissue, intact skin and/or peripheral blood mononuclear cells (PBMCs) of 25 canker‐affected horses for the presence of sarcoid‐associated BPV‐1 and ‐2. Results: Conventional PCR revealed BPV‐1/‐2 DNA in 24/24 canker, 12/13 skin and 10/11 PBMC DNA isolates. Using inverse PCR, full‐length BPV episomes were detected in 1/5 canker specimens. Sequencing of viral early and late genes amplified from canker, intact skin and PBMC DNA of 2 cases revealed an overall identity of 98% to BPV‐1. Viral DNA loads amounted to ≤16 copies per cell in canker tissue and intact skin, and to ≤0.35 copies per PBMC, as determined by quantitative PCR. Using RT‐PCR, the viral major oncogene E5 was shown to be transcribed in 2/4 canker tissue specimens and 5/7 PBMC isolates. Immunocapture PCR from 7 canker and 6 skin extract supernatants revealed capsomere‐associated viral DNA in one canker and one skin sample. Hoof tissue, skin and PBMCs collected from 13 individuals with no signs of canker or BPV‐related malignancies scored negative throughout the experiments. Conclusion: These findings suggest that the observed presence of BPV‐1/‐2 in canker‐affected horses is not coincidental but indicative of an active contribution to hoof canker disease. Potential relevance: The use of antivirals and/or immune modulators may help improving canker therapy.     

Pathomorphological and immunohistochemical study of selected markers of tumour cell proliferation in equine sarcoids

The purpose of the study was a pathomorphological and immunohistochemical analysis of tumour cells and connective tissue in equine sarcoids. Investigations were performed using histopathological, ultrastructural, immunohistochemical (PCNA, p53, cytokeratin, vimentin) and histochemical (Ag-NORs) methods. The study was conducted on 50 sarcoids originating from 36 horses and classified as occult, verrucous, fibroblastic and a mixed type of sarcoid based on their clinical appearance. Most of the tumours were located on the girth (30%), neck (24%), head (12%), and legs (12%). The average age of the horses at the first clinical examination was 5.7 years. The sarcoids occurred on the skin of mares (61%), geldings (31%) and stallions (8%), the predominant was Wielkopolska breed (41%) and mixed breeds with Wielkopolska breed (41%). The predominant colour was bay (80%). The data showed that the presence of characteristic, microscopic features was variable but it was not consistent enough to allow differentiation of the clinical types based on histopathology. PCNA expression was not characteristic for the clinical type of sarcoid but it appeared to be a useful tool for the determination of the biological activity of the tumour and the probability of its recurrence. No relationship was found between AgNORs and cell proliferation. The study demonstrated the presence of p53 positive cells in the epidermal and fibroblastic portions. Numerous p53-positive cells were observed in the sarcoids and tended to recurrence. The staining for cytokeratin and vimentin makes the diagnosis of tumour easier. The immunohistochemical studies of PCNA, and p53 are of great significance to the prognosis.     

IRIDIUM-192 INTERSTITIAL BRACHYTHERAPY OF EQUINE SARCOID

Twenty-three equine sarcoids in 22 horses were treated, using an afterloading technique, with iridium-192 (¹⁹²Ir) interstitial radiation therapy. Previously unsuccessful treatment in 14 horses included surgical excision, cryosurgery, hyperthermia, and nonspecific immunotherapy. Twenty tumors were located in the skin of the head, and three were in the skin of the extremities. Before implantation, surgical debulking was done in six tumors, and hyperthermia (43°C, 30 min) was done in three tumors. Total implantation time varied from 4 to 14 days to give radiation doses of 52–93 Gy. The criteria for assessment of therapy were tumor response, tumor-free incidence at one year, and complications. Follow-up times ranged from six to 41 months (mean, 20 months). All tumors of the head and two tumors of the extremities had complete response to therapy. One sarcoid of the extremity had partial response to treatment. Tumor recurence was seen in the head of one horse. Of 16 horses followed for one year, the tumor-free incidence was 94% (15/16). Complications were severe local necrosis in two horses and deep infection in one horse. It was concluded that ¹⁹²Ir interstitial brachytherapy was an effective treatment of recurrent and difficult-to-manage equine sarcoid.     

Prevalence and body distribution of sarcoids in South African Cape mountain zebra (Equus zebra zebra)

There are no reports in the literature describing any tumours, and specifically sarcoids, in zebras. The equine sarcoid, a locally aggressive, fibroblastic skin tumour, is the most common dermatological neoplasm reported in horses. The Cape mountain zebra (CMZ) has been described as one of the most vulnerable mammals in South Africa with current populations existing in isolated units. All South African CMZ are descendants from no more than 30 individual animals originating from 3 populations, namely the Mountain Zebra National Park, and Kammanassie and Gamka Mountain Nature Reserves near Cradock. The possibility therefore exists that the existing populations arose from a very small gene pool and that they are considerably inbred. A reduction in major histocompatibility complex diversity due to genetic bottlenecks and subsequent inbreeding probably contributed to uniform population sensitivity and the subsequent development of sarcoid in two CMZ populations, namely in the Bontebok National Park and Gariep Nature Reserve. The entire population of CMZ in the Bontebok National Park was observed and sampled during 2002 to document the prevalence and body distribution of sarcoids. During the same year, a comparative study was carried out on an outbred population of Burchell's zebra in the Kruger National Park. The prevalence in CMZ in the Bontebok National Park was 53 %, while the Burchell's zebra in Kruger National Park had a prevalence of 1.9 %. The most common sites for sarcoid in CMZ were the ventral abdomen and limbs. Prevalence of sarcoids in horses recorded in the literature varies between 0.5 % and 2 %. The Gariep Nature Reserve recently reported a prevalence of almost 25 % in CMZ in the reserve.     

A survey of granulomatous and neoplastic diseases of equine skin in north Queensland

SUMMARY A survey of granulomatous and neoplastic diseases of the skin of horses of tropical north Queensland was carried out during the period 1970–1980. Of 338 horses affected, 46.4% suffered from equine fibrosarcoma (sarcoid), 30.2% from phycomycosis, 7.4% from squamous cell carcinoma, 6.8% from other tumours, 4.7% from cutaneous habronemiasis and 4.4% from exuberant granulation tissue. Most specimens were submitted during the first half of the year after the wet monsoonal season and significantly more diseases originated during the first quarter. There were minor variations with breed, age and sex susceptibility between each disease group. Most lesions were found on the legs and head although other parts of the body were also affected. The size and anatomical distribution of lesions varied with each disease. When the results of this survey were compared with reports from southern Queensland as well as from Florida and the United Kingdom, major differences between localities were observed. Some reasons for these differences are discussed.     

Equine skin tumours in 20 horses resembling three variants of human melanocytic naevi

Melanocytic tumours are important in horses, especially grey horses. Intradermal common melanocytic naevi, cellular blue naevi and combined cellular blue naevi are subgroups of human melanocytic tumours, which have not been reported in horses. In this study, we describe 20 horses with skin tumours similar to these naevi of humans. These tumours represented individual skin masses in male and female horses of different breeds. Tumours resembling human intradermal common melanocytic naevi were noted in 12 horses aged between 2 and 17 years. Seven horses aged between 4 and 15 years developed cutaneous lesions similar to human cellular blue naevi. A combined cellular blue naevus-like tumour was diagnosed in a 20-year-old horse. All tumour types formed expansile, well-demarcated, non-encapsulated, symmetrical masses. Tumours similar to intradermal common melanocytic naevi were composed of nests of round and spindeloid neoplastic cells, often embedded in myxomatous stroma. Lesions resembling cellular blue naevi were formed by intradermal bundles of ovoid to elongated cells separated by collagen fibres. The combined cellular blue naevus-like tumour resembled human cellular blue naevus with in addition, an overlying junctional common melanocytic naevus. Neoplastic cells in all groups contained varying amounts of melanin pigment and were immunopositive for S100. These equine skin tumours differ from the commonly recognized equine melanocytic tumours by their cytomorphological features, random location and the absence of an increased tumour frequency in grey horses. The resemblance of these tumours to three distinct subgroups of human naevi expands the complexity of equine proliferative cutaneous melanocytic lesions.     

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.     

IgE and IgG antibodies in skin allergy of the horse

In horses, allergies have been characterized by clinical signs and/or intradermal (i.d.) allergen testing. Our aim was to find the first direct evidence that immunoglobulin E (IgE) mediates equine allergy. In addition, we tested the hypothesis that immediate skin reactions in horses can also be mediated by IgG. Anti-IgE affinity columns were used to purify IgE from serum of one healthy horse and three horses affected with summer eczema, an allergic dermatitis which is believed to be induced by Culicoides midges. A modified Prausnitz-Küstner experiment was performed in four clinical healthy horses by i.d. injection of the purified serum IgE antibodies. The following day, Culicoides allergen was injected at the same sites. Skin reactions were not observed in response to allergen alone, and in two horses after stimulation at any previous IgE injection site. However, the other two horses showed an immediate skin reaction at the previous injection sites of IgE obtained from allergic horses. In addition, purified monoclonal antibodies to various equine immunoglobulin isotypes were injected i.d. into six healthy horses. Immediate skin reactions were observed in response to anti-IgE (6/6 horses) and anti-IgG(T) injections (5/6 horses). The specificities of both antibodies for IgE and IgG(T), respectively, were confirmed by enzyme linked immunosorbent assays. The results provide the first direct evidence that IgE mediates classical Type-I allergy in horses and plays a major role in the pathogenesis of summer eczema. The data also suggest that IgG(T) can bind to skin mast cells and might contribute to clinical allergy.     

Evaluation of plasma alpha-2-macroglobulin and interactions with tumour necrosis factor-alpha in horses with endotoxemic signs.

The electrophoretic position and behavior of the native and activated forms of equine plasma alpha-2-macroglobulin (alpha 2M) were characterized and compared to human alpha 2M by nondenaturing polyacrylamide-gel electrophoresis (PAGE). Plasma alpha 2M was also compared between 6 normal horses and 6 horses with clinical signs of colic and endotoxemia due to volvulus or enteritis. Native and activated forms of alpha 2M were quantified by PAGE and densitometry. Binding of radio-labeled recombinant human tumour necrosis factor-alpha (125I-rhTNF-alpha) to native and activated forms of equine alpha 2M was also evaluated by autoradiography and densitometry of PAGE. Equine plasma alpha 2M migrated as a single band at a position equivalent to native human alpha 2M. Methylamine-reacted equine plasma samples resulted in faster migration of alpha 2M in a similar position to activated human alpha 2M. However, in methylamine-reacted equine plasma, an intermediate alpha 2M band was consistently present between the bands corresponding to native and activated alpha 2M. Amounts of plasma alpha 2M were similar in normal and endotoxemic horses, and remained in the electrophoretically slow or unreacted native form. The vast majority of 125I-rHuTNF-alpha did not bind to alpha 2M or other equine plasma proteins. 125I-rHuTNF-alpha bound weakly to both native and fast methylamine-reacted equine forms of alpha 2M, although binding was better to the activated form. This study indicates that: (1) equine plasma alpha 2M behaves similarly to human alpha 2M on PAGE, (2) plasma alpha 2M of horses can be activated to electrophoretically fast forms, but it is neither activated nor depleted during endotoxemia, and (3) the binding interactions between equine alpha 2M and TNF-alpha are too low to implicate equine alpha 2M as a regulator of TNF-alpha during endotoxemia in horses.