Papillomavirus infection of equids

Papillomavirus

Papillomavirus infection of equids

Current authors:
C G KNIGHT - Department of Veterinary Clinical and Diagnostic Sciences, Faculty of Veterinary Medicine, University of Calgary, Canada
J S MUNDAY - School of Veterinary Science, Massey University, Palmerston North, New Zealand

Introduction

Seven types of equine papillomavirus (Equus caballus papillomavirus; EcPV) infect horses.18, 31, 52 These are subclassified within the Zetapapillomavirus, Dyoiotapapillomavirus, or Dyorhopapillomavirus genera.49 Three distinct clinical syndromes caused by EcPV infection are recognized: cutaneous papillomas, aural plaques, and genital lesions including squamous cell carcinomas (SCCs). In addition, infection with bovine deltapapillomaviruses is associated with a fourth clinical syndrome, the development of fibroblastic sarcoids in horses and other equids, including donkeys, mules and zebras. A single donkey papillomavirus (Equus asinus papillomavirus type 1; EaPV-1) has been described, although this virus is not currently associated with any clinical disease.33

Cutaneous papillomas (viral warts, classical viral papillomatosis, “grass warts”)

Cutaneous papillomas are caused by Equus caballus papillomavirus type 1 (EcPV-1).18, 46 Papillomas affect horses throughout the world and, along with sarcoids, SCCs and melanomas, are one of the four most commonly reported skin tumors of horses.54, 63 Surveys of diagnostic laboratory submissions probably underestimate the prevalence of cutaneous papillomas because they are easily recognized by clinicians and so are seldom submitted for histologic diagnosis.54 It is likely that, similar to other species, most horses develop papillomas during their lifetime.

Equus caballus papillomavirus type 1 is transmitted directly and indirectly through small skin abrasions, as is typical for papillomaviruses.10, 54 The incubation period after experimental inoculation ranges from two weeks to two months and lesions typically regress spontaneously after several months.10, 22 Congenital papillomas have been reported in foals; however, these are probably hamartomas (epidermal nevi) and are unlikely to be caused by papillomavirus infection.

Cutaneous papillomas usually develop on horses that are less than three years old, although they may occur in older horses, particularly in immunocompromised animals.54 They develop most frequently on the muzzle and lips (Figure 1), less frequently on the distal limbs, eyelids, or paragenital region, and are uncommon in other body locations.54 Papillomas are typically multiple, and as many as 100 may be present. When numerous papillomas develop they may coalesce into a single large mass. Lesions begin as 1 mm diameter, well-demarcated, smooth, greyish-white papules. Over a one to two-month period they proliferate and progress to larger (5-20 mm diameter), broad-based or pedunculated masses that are pink, grey or white and have roughened or frond-like surfaces composed of thickened keratin. Cutaneous papillomas are typically neither pruritic nor painful.

Histologically, cutaneous papillomas have three phases: growth, development and regression.22 Papillomas initially progress from basal cell hyperplasia and mild hyperkeratosis to marked, papillary epidermal hyperplasia with cellular features typical of papillomavirus infection such as enlargement of cytoplasm by increased quantities of blue-grey material, enlarged, darkened nuclei surround by a perinuclear clear space (koilocytosis), and intranuclear viral inclusion bodies. The regression phase is characterized by infiltration of lymphocytes and mild proliferation of fibroblasts.

Diagnosis is often straightforward as the gross appearance and location of lesions and the signalment of affected horses are characteristic. Biopsy and histologic examination are diagnostic but rarely performed. However, these are indicated when lesions are present in atypical body locations, as sarcoids and SCCs can appear clinically similar to cutaneous papillomas.54

Cutaneous papillomas typically resolve without treatment in two to three months. However, as in humans, the time taken for resolution can be variable with a small proportion of papillomas persisting for up to a year prior to spontaneous resolution. If resolution has not occurred after 12 months, immunosuppression should be considered and investigated.54 Immunity post-resolution is lifelong. If removal is required for cosmetic or medical reasons, surgical excision and cryosurgery are effective. There is no proof that surgical removal of a proportion of lesions accelerates the regression of remaining lesions.56 Because cutaneous papillomas regress spontaneously without treatment it is difficult to interpret the results of therapeutic intervention. Suggested but unproven treatments include topical antiviral and “wart” creams, injection of intralesional or intravenous immunostimulants, injection of intralesional cisplatin or interleukin-2 (IL-2), and administration of autogenous vaccines.54

Cutaneous papillomas are contagious and their spread may be reduced by isolating affected horses, reducing exposure of naive horses to infected premises and pastures, and disinfecting feeding, grooming and tack equipment to preclude fomite transmission.

Aural plaques (equine ear papillomas, pinnal acanthosis, “ear fungus”)

Aural plaques are greyish flattened or proliferative hyperplastic lesions that develop on the concave surface of the ear.  

Four novel types of Equus caballus papillomavirus (EcPV-3, -4, -5 and -6) have been detected in aural plaques.3, 58 However, the specific contribution to lesion development of each particular PV type is currently unknown.19 The EcPVs in aural plaques are believed to be transmitted among horses by species of black flies (Simulium spp.) although this is not proven.12 The absence of flies that are able to transmit the papillomavirus is hypothesized to be the reason that horses in some countries (for example New Zealand) do not develop aural plaques. The prevalence of aural plaques is not known; as with cutaneous papillomas, their easy recognition by clinicians means that they are rarely submitted to diagnostic laboratories. These lesions are restricted to horses and have not been reported in other equine species.

Horses of any age can develop aural plaques, although they are rare in horses less than one year of age. There is no known breed or sex predilection. Lesions develop in the inner (concave) surface of the pinna, and consist of multiple discrete grey-white, roughened papules that can coalesce to form plaques up to several cm in diameter (Figures 2 and 3). Aural plaques may be unilateral or bilateral. Aural plaques are a cosmetic concern but are rarely bothersome to affected horses, although there are reports that some horses became ‘head shy’ because of them. Unlike cutaneous papillomas caused by EcPV-1 infection, aural plaques do not regress spontaneously. However, aural plaques have not been reported to progress to SCC.

The diagnosis of aural plaques is straightforward as the gross appearance and location of lesions are characteristic. Histologically, aural plaques resemble flat cutaneous papillomas. Relative to adjacent normal skin they have a characteristic reduction in melanin within the basilar epidermis (hypomelanosis).54

Aural plaques are rarely treated except for cosmetic reasons. Debulking or “shaving off” lesions may cause a reduction in size, or even apparent surgical cure of the aural plaques, although lesions tend to recur within 12 month.69 Repeated topical application of imiquimod cream (Aldara) over several months permanently resolved a high proportion of aural plaques, but treatment caused irritation, crusting and inflammation and horses usually required sedation to permit treatment.61

Genital lesions, including squamous cell carcinoma

Proliferative lesions of the external genitalia of horses associated with EcPV infections include SCCs, precancerous plaques (SCC in situ), papillomatosis (extensive, coalescing warts), and squamous papillomas (discrete warts). All are associated with EcPV-2 infection. Because these four syndromes may represent a continuum rather than distinct clinical entities, any EcPV-2-induced non-SCC genital lesion should be considered premalignant. EcPV-2-associated genital lesions are more common in stallions and geldings than in mares. EcPV-2 has not been reported to infect other equine species and these lesions are currently restricted to horses.

The external genitalia are predilection sites for SCC development in horses. Of equine cutaneous SCCs overall, 25-50 per cent affect the male external genitalia and approximately 10 per cent affect the female external genitalia.1, 35 Squamous cell carcinomas are the most frequent neoplasms of the penis and vulva40, 64, 65 and affect older horses, with an average age at diagnosis of 10-18 years for males and 12-19 years for females.16, 25, 26, 36, 63 They are reported anecdotally to be most common in breeds with non-pigmented genitalia, such as Appaloosas, Pintos and American paint horses, the assumption being that these breeds lack the protection to exposure to ultraviolet light afforded by the melanin of pigmented breeds.1, 6, 53 However, there is currently no strong evidence that genital SCCs are more common in breeds of horses with non-pigmented genitalia.

In 2010 an association between EcPV-2 infection and equine genital SCC was first recognized.52 This finding has been supported since by numerous studies, and EcPV-2 is now accepted to be the cause of a large proportion of precancerous plaques and SCCs of the genitals of horses.27, 28, 45, 57 In addition, EcPV-2 has been shown to be the cause of penile squamous papillomas and penile papillomatosis.29, 52, 67 It is not known how EcPV-2 infection is transferred among horses or when horses become infected. As with some human papillomaviruses, venereal transmission is possible. However, as horses with no breeding history or contact with other horses can be infected this is unlikely to be the exclusive mode of transmission. Mechanical transmission via sheath cleaning or insects has been proposed but not proven. Little is known about the natural biology of EcPV-2 infection, including prevalence in the overall horse population, incidence of infection, the time from infection to development of lesions, whether infection may be cleared over time, or oncogenic mechanisms.14, 27, 67

EcPV-2-induced lesions may develop anywhere on the male external genitalia but are more common on the free part (including the glans) of the penis than the prepuce or urethra.25, 26, 36 The majority of males with genital SCCs present with a malodorous, blood-stained and purulent preputial discharge,25 although other reported clinical signs include preputial oedema, dysuria, haematuria, weight loss and urinary incontinence.35 In mares, SCCs appear to be more common on the vulva than  the clitoral fossa.

Genital SCCs begin as small, plaque-like or papillary lesions that progress to larger cauliflower-like or pedunculated masses with areas of necrosis and ulceration (Figure 4).35 Histologically, they are usually well differentiated and keratinization is almost always present. Infiltration of inflammatory cells into the tumor is common, and foci of necrosis and mineralization are frequent.16 Penile squamous papillomas and precancerous plaques are recognized as potential precursors of penile SCCs, although the proportion of penile SCCs that develop from these versus the proportion that develop de novo is unknown. Squamous papillomas are keratinized exophytic epithelial tumors with a sparse, branching fibrous stroma. Approximately one third of equine male genital squamous papillomas occur in close proximity to a penile SCC, suggesting that these papillomas have the potential for neoplastic transformation.16, 25 Precancerous plaques are unique to horses and are intermediate in appearance between squamous papillomas and SCCs and cannot be reliably distinguished from either grossly (Figure 5). They frequently progress to invasive SCC.16, 25 Histologically, precancerous plaques show features consistent with SCC in situ at other locations including a hyperplastic to dysplastic epithelium, with or without koilocytes, in which cells of the basal layer exhibit an increased mitotic rate and loss of polarity but no invasion of the basement membrane.25, 68

Genital SCCs are typically slowly progressive with a low metastatic potential. However, the lack of specific clinical signs and difficulty in examining the external genitalia mean that penile SCCs may be advanced at the time of diagnosis, increasing the risk of local or metastatic spread by the time that treatment is started.36 Metastasis is usually first to local lymph nodes, with pulmonary metastasis rarely also reported.64, 65 There is little published information on the behavior of equine female genital SCCs, but they likely behave the same as male genital SCCs.

Definitive diagnosis of equine genital lesions is only possible by biopsy and histologic examination. Several lesions including sarcoids, habronemiasis, granulomas, and non-specific ulcerative balanoposthitis may mimic the gross lesions of EcPV-2-induced disease. Because even small precursor lesions may progress to SCCs it is important to diagnose genital lesions accurately. The addition of specific testing of biopsy samples for the presence of EcPV-2 by PCR or its distribution by RNA in situ hybridization may be useful.

Treatment options for penile SCCs range from minimally invasive (e.g. topical chemotherapy) to radical surgical procedures (e.g. penile amputation and removal of inguinal lymph nodes), with the choice depending primarily on the size and site of the lesion, the presence or absence of metastasis and the owner’s willingness for surgery.66 There is no standardized approach to treatment of penile SCCs and reported outcomes vary. Tumors may recur after incomplete excision and the long-term prognosis for non-resectable penile SCCs is poor.64 The largest study to date comparing different surgical treatment modalities reported an overall success rate, as determined by absence of tumor recurrence, of just over 50 per cent.64 Precursor lesions of penile SCC (squamous papillomas and precancerous plaques) are typically removed by surgical excision or cryotherapy, followed by adjuvant therapy such as topical 5-Fluorouracil or intralesional injection of cisplatin-impregnated beads, although it is currently uncertain whether not adjuvant therapy decreases the recurrence rate of these lesions.15, 60, 66

Because it is not known how EcPV-2 is transmitted, there are no proven guidelines for prevention of virus spread among horses. Attention to hygiene when cleaning the external genitalia of horses, particularly any with proliferative lesions, is advisable. Development and testing of a virus-like particle (VLP)-based vaccine against EcPV-2 are underway currently. It is possible that a prophylactic vaccine against development of EcPV-2-induced genital lesions will be available in the future.20

Equine sarcoids

Sarcoids are locally invasive fibroblastic neoplasms affecting horses, donkeys, mules and zebras. They are the most common skin tumor of equids worldwide.24, 59, 63 Although they rarely metastasize, they are not benign tumors and can be progressive, invasive lesions. The propensity of sarcoids to increase in size after treatment or to recur after excision make them a therapeutic challenge.

Sarcoids are most common in horses between three and six years of age, and are uncommon in horses less than one or greater than seven years old.54 Quarter horses, Appaloosas and Arabians are significantly more likely to develop sarcoids than Thoroughbreds, while Standardbreds are significantly less likely, suggesting a differing genetic susceptibility.2, 42 The concept of genetic predisposition to sarcoid is further supported by the proposed existence of a “sarcoid susceptibility gene” linked to the major histocompatibility complex class II region.9, 32, 41, 55 Geldings may be more susceptible to sarcoid development than either stallions or mares, but this predisposition has not been confirmed.42 An association between coat color and sarcoid development has not been reported.

Sarcoids are associated with infection with bovine papillomaviruses (BPVs) within the genus Deltapapillomavirus. BPV-1 and/or BPV-2 DNA sequences or RNA transcripts have been detected by PCR in up to 100 per cent of sarcoids.8, 39, 47, 70 In vitro experiments using BPV-transformed equine fibroblast cell lines74 have revealed several molecular mechanisms for tumor formation.71, 72, 73 According to the majority of studies BPV-1 is most frequently present in sarcoids, although two surveys of horses in the western USA and Canada found BPV-2 to be the most common type.8, 70 Phylogenetic analysis of BPV-1 from bovine warts and equine sarcoids has shown substantial genetic variation in the virus from different geographic locations, with 21 sequence variants (SVs) recognized across both species.62 One sequence variant (SV20) has been shown to be present in the majority of equine sarcoid samples but no bovine wart samples, suggesting the possibility of an “equine-specific” BPV-1 variant.43, 62 However, the existence of an “equine-specific” BPV-1 variant was not supported in a recent study and whether such a strain exists is currently unresolved.30 BPV-13 DNA was recently detected by PCR in sarcoids in horses in Brazil, but little is currently known about this virus.34

It is not known how horses become infected with BPVs, and sarcoids cannot be induced experimentally. Intradermal inoculation of horses with BPV-1 virions from bovine warts causes transient, spontaneously regressing fibroblastic proliferations termed “pseudosarcoids,” but these differ histologically and behaviorally from naturally occurring sarcoids.23, 48 It is also not clear whether BPVs are transmitted from infected cattle, from infected horses, or from both. Several studies support the possibility of direct BPV transmission between equids. In one, two sarcoid-affected donkeys with different variants of BPV-1 were each housed with a sarcoid-free donkey. In both pairs the sarcoid-free donkey developed a sarcoid containing the same BPV-1 variant as its stable-mate, providing strong evidence for equid to equid transmission.43 Another study reported that sarcoid-free donkeys in close proximity to sarcoid-affected donkeys are at increased risk for sarcoid development,50 and an outbreak of sarcoids in an isolated population of zebras has been described, again supporting equid to equid transmission of BPV.37 However, many equids also develop sarcoids with no history of close contact with sarcoid-affected animals, and the possibility of insect transmission of BPVs has been proposed. BPV-1 DNA has been detected by PCR in biting and non-biting flies trapped in the vicinity of sarcoid-affected horses, and BPV sequence variants in the insects were the same as those commonly found in sarcoids in that geographic location.13 Further work would be necessary to demonstrate that insects can actually transmit BPVs from horse to horse.

Horses have historically been considered dead-end hosts of BPV infection, with early studies detecting only BPV DNA in dermal fibroblasts, but never infectious BPV virions (surrounded by a protein capsid) in the epidermis.1, 7 However, several more recent studies have challenged the assumption that horses are dead-end hosts, with transcripts of the BPV-1 L1 gene, required for viral capsid assembly, detected in sarcoids, suggesting that infection may be productive in some cases.44 Additionally, full-length BPV-1 genomes complexed with L1 capsid proteins, possibly representing virions, have been detected in two equine sarcoids.4 Microdissected sarcoid epidermis has also been shown to contain BPV-1 DNA, regulatory proteins and capsid proteins, suggesting productive infection.3, 5 BPV capsid protein have been detected in epithelial cells of the sebaceous glands, hair follicles and epidermis of sarcoid samples using RNA in situ hybridization, suggesting both productive infection and a possible mechanism for viral dispersal.17 However, none of these studies definitively showed the presence of infectious BPV virions, and further research into whether BPV infection in horses can be productive is needed.

Sarcoids may occur anywhere on horses but are more common on the head (especially ears, lip margins, and periocular skin), neck, limbs and the ventral body surface (especially inguinal and perineal regions).54 They are frequently reported to occur at sites of previous (3-6 months) skin wounds or surgery, although many also develop at sites with no history of trauma. Affected horses may have one or several sarcoids, and it is common for an original lesion to be surrounded by several smaller “satellite” sarcoids. Lesions range in diameter from several mm to tens of cm, with the largest lesions typically being located on the distal limbs.

Six clinical ‘types’ of sarcoid have been described.

Occult sarcoids appear as roughly circular, plaque-like, non-elevated areas of scaly alopecia (Figure 6). They are reported to have the most benign clinical behavior and may remain static for years or grow only slowly, with rare progression to verrucous or fibroblastic sarcoids.

Verrucous sarcoids are hairless to scaly, irregular, and wart-like (Figure 7). Lesions may be pedunculated (on narrow stalks) or broad-based and flat. They can resemble benign cutaneous papillomas and clinical differentiation between these two entities can be difficult. Verrucous sarcoids typically grow slowly but may become more aggressive if traumatized.

Nodular sarcoids are firm, well circumscribed subcutaneous or dermal masses covered by intact, normal skin, although larger lesions may become alopecic. They can be solitary but are often multiple, sometimes with dozens to hundreds of variably sized nodules appearing in clusters. They may become more aggressive when traumatized or ulcerated and progress to fibroblastic forms.

Fibroblastic sarcoids are raised, fleshy, ulcerated and exudative. They may resemble proud flesh (exuberant granulation tissue), ulcerated SCCs or granulomas, making accurate diagnosis essential. Fibroblastic sarcoids can develop de novo, from other traumatized types of sarcoid (occult, verrucous or nodular), or at sites of previous wounds anywhere on the body. They are more locally invasive than the first three types of sarcoid.

Malevolent sarcoids are deeply invasive, often extending along local lymphatic vessels and causing cord-like thickening that is palpable and resembles lymphangitis. Superficially they appear as several to numerous ulcerated nodules that, like fibroblastic sarcoids, resemble proud flesh, ulcerated SCCs and granulomas.  Malevolent sarcoids are often reported to occur at sites of repeated trauma such as those caused by multiple attempts at surgical excision.

Mixed sarcoids share features of two or more of the previous five sarcoid types, and commonly develop from chronic or repeatedly traumatized sarcoids of other types.

It should be noted that these ‘types’ reflect different stages of tumor progression and the relative predominance of the dermal and epidermal components, rather than distinct neoplasms.59 A simpler, and possibly more clinically relevant classification system divides sarcoids into indolent (occult and verrucous) and invasive forms, and recognizes that invasive forms may arise de novo or from progression of an indolent lesion.59

The differential diagnosis for sarcoids includes: simple cutaneous papillomas for the verrucous type; proud flesh, SCCs, or ulcerated granulomas for the fibroblastic and malevolent types; and skin neoplasms (especially melanoma and mast cell tumor) or granulomas for the nodular type. Definitive diagnosis of sarcoids is only possible by biopsy and histologic examination; however, this should be done with caution as manipulation and biopsy are reported to accelerate the growth of the remaining sarcoid tissue.54, 59

Histologically, equine sarcoids consist of proliferating dermal fibroblasts and overlying epidermal hyperplasia. There may be characteristic dermoepidermal junction changes such as formation of long epidermal rete pegs or a perpendicular (“picket fence”) arrangement of fibroblasts relative to the epidermal basement membrane. Because of the variable histologic appearance of different types of sarcoid, a lesion may be misdiagnosed as proud flesh or as a mesenchymal neoplasm such as fibroma, fibrosarcoma or peripheral nerve sheath tumor. One study showed that the only features shared by all types of sarcoid was the presence of BPV DNA and increased density of dermal fibroblasts.38 Therefore, PCR testing for the presence or absence of BPV DNA may help distinguish sarcoids from other mesenchymal neoplasms and from proud flesh.

Early and complete surgical resection followed by histologic confirmation of adequate tumor-free margins is the most effective treatment for sarcoids.59 Incomplete resection or inadequate surgical margins frequently result in aggressive regrowth and conversion to a more aggressive type of sarcoid, and a previous unsuccessful attempt at removal is a strongly negative prognostic factor. Sarcoids at certain anatomic locations such as the distal limbs and periocular skin may be difficult to remove fully and require adjuvant therapy. Suggested therapies include radiation, laser or cryotherapy; intralesional injection of cisplatin-impregnated beads; and topical treatments such as acyclovir, 5-fluorouracil, imiquimod, zinc chloride, or bloodroot paste (Sanguinaria canadensis). Autologous vaccination using sarcoid tissue frozen in liquid nitrogen and implanted into subcutaneous sites along the neck has been described, but reports on clinical efficacy are limited.11, 51 Prevention of sarcoids is currently not possible given that the mechanism of transmission of BPVs to or among horses is unknown. Owners should be made aware that sarcoids are an infectious disease, albeit probably not a highly contagious one. It appears that precautions such as reducing sharing of feeding, grooming and tack equipment between affected and healthy horses may decrease the spread of sarcoids between horses.21 However, there is no evidence that sarcoid-affected horses should be removed from properties or isolated from other horses. Development and testing of a virus-like particle (VLP)-based vaccine against BPVs-1 and -2 are underway, and it is possible that a prophylactic vaccine against sarcoid development will be available in the future.20

Figure 1 Multiple cutaneous papillomas on the muzzle of a horse. This is a typical location and distribution. Lesions may coalesce when numerous. Photograph courtesy of Dr. Ashley Whitehead, University of Calgary, Canada.

Figure 2 Aural plaques within the concave surface of the pinna of a horse. This is a typical location and distribution for lesions, which appear as grey-white wart-like masses. Photograph courtesy of Dr. Ashley Whitehead, University of Calgary, Canada.

Figure 3 Numerous aural plaques within the concave surface of the pinna of a horse.  Aural plaques typically coalesce into larger masses when numerous. Photograph courtesy of Dr. Ashley Whitehead, University of Calgary, Canada.

Figure 4 Extensive squamous cell carcinoma affecting the glans penis and distal penile shaft of a horse. Photograph courtesy of Dr. Ashley Whitehead, University of Calgary, Canada.

Figure 5 Precancerous plaque (carcinoma in situ) at the junction of the glans penis and distal penile shaft of a horse. Precancerous plaques are intermediate in appearance between squamous papillomas and squamous cell carcinomas. They typically progress to invasive squamous cell carcinomas. Photograph courtesy of Dr. Ashley Whitehead, University of Calgary, Canada.

Figure 6 Occult sarcoid ventral to the eye of a horse. Occult sarcoids appear as plaque-like areas of scaly alopecia. Photograph courtesy of Dr. Ashley Whitehead, University of Calgary, Canada.

Figure 7 Large verrucous sarcoid on the neck of a horse. Verrucous sarcoids are hairless to scaly and wart-like. They may be pedunculated or broad-based. Image courtesy of Dr. Ashley Whitehead, Department of Veterinary Clinical and Diagnostic Sciences, University of Calgary, Alberta, Canada.

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