Equine Papillomavirus

Overview and Taxonomy of Equine Papillomavirus

The family Papillomaviridae encompasses a remarkably diverse and ancient group of small, non-enveloped, double-stranded DNA viruses that exhibit a profound degree of host specificity and epithelial tropism. Within this expansive family, the equine papillomaviruses (EcPVs) represent a phylogenetically and clinically significant cluster of pathogens that infect domestic horses (Equus caballus) and other equid species. The study of EcPVs has undergone a dramatic transformation over the past two decades, evolving from the characterization of a single, benign viral type associated with classical cutaneous warts to the discovery of a rapidly expanding genus of viruses implicated in a spectrum of diseases ranging from self-limiting papillomas to invasive, life-threatening squamous cell carcinomas (SCCs). This section provides a comprehensive overview of the taxonomy, genomic organization, phylogenetic relationships, and biological context of the known equine papillomaviruses, drawing upon the most recent molecular and epidemiological evidence.

Taxonomic Classification and Nomenclatural Framework

The taxonomic classification of papillomaviruses is governed by the International Committee on Taxonomy of Viruses (ICTV), which employs a hierarchical system based on pairwise nucleotide sequence identity of the highly conserved L1 capsid gene. A novel papillomavirus is considered a new type if its L1 gene shares less than 90% identity with any previously characterized type. Genera are defined by less than 60% L1 nucleotide identity, while species within a genus share between 60% and 70% identity. The equine papillomaviruses are distributed across several genera within the Papillomaviridae family, reflecting their substantial genetic divergence and likely ancient co-evolution with their equid hosts.

To date, at least nine distinct Equus caballus papillomavirus types (EcPV1 through EcPV9) have been formally described, with evidence for additional putative novel types emerging from metagenomic surveys [1, 2, 3]. The taxonomy of these viruses is not static; recent discoveries have necessitated the creation of new genera and the re-evaluation of existing phylogenetic relationships. EcPV1, the first equine papillomavirus to be characterized, is the type species of the genus Zetapapillomavirus and is the etiological agent of classical cutaneous papillomatosis, commonly known as viral warts [4, 5, 6]. EcPV2, EcPV4, and EcPV5 are classified within the genus Dyoiotapapillomavirus, while EcPV3 resides in the genus Dyorhopapillomavirus [1, 2]. EcPV6 is classified within the genus Dyochipapillomavirus, and EcPV7, EcPV8, and EcPV9 are more recently assigned members whose precise generic placement continues to be refined as whole-genome sequences become available [2, 7]. The discovery of EcPV9 in the semen of a Thoroughbred stallion with a penile lesion, for instance, revealed a virus that clusters with EcPV2, EcPV4, and EcPV5 but is sufficiently divergent to represent a new species within Dyoiotapapillomavirus [2]. This ongoing taxonomic expansion underscores the power of high-throughput sequencing technologies in uncovering the hidden viral diversity within equine populations.

Genomic Architecture and Conserved Features

The genome of all known EcPVs conforms to the canonical papillomavirus organization, consisting of a circular double-stranded DNA molecule of approximately 7.6 to 7.8 kilobase pairs [1, 2, 5]. The genome is functionally divided into three major regions: an early (E) region encoding proteins involved in viral replication, transcription, and cellular transformation; a late (L) region encoding the structural capsid proteins L1 and L2; and a non-coding long control region (LCR) that contains the origin of replication and regulatory elements for transcription. The early region typically comprises open reading frames (ORFs) for E1, E2, E4, E5, E6, and E7, although the presence and functionality of E5 can vary between genera. For example, EcPV3 and EcPV6, which belong to different genera, share only approximately 52% nucleotide sequence similarity across their entire genomes, yet both retain the core early and late gene architecture [1].

The E6 and E7 oncoproteins are of paramount importance in the context of EcPV-associated carcinogenesis. These proteins are consistently expressed in EcPV2-positive genital SCCs and their precursor lesions, and their transcriptional activity is a hallmark of virally driven malignancy [8, 9, 10]. In EcPV2, the E6 and E7 genes are transcribed as polycistronic messages, and RNA-seq analyses of SCC tissues have revealed complex splicing patterns that regulate the expression of these oncogenes, mirroring the strategies employed by high-risk human papillomaviruses (HPVs) [9]. The E2 protein, in addition to its role in viral replication and transcription, is often disrupted during viral integration into the host genome, an event that has been documented in EcPV2-associated SCCs and which likely contributes to the deregulation of E6/E7 expression [9]. The L1 gene, being the most conserved region of the genome, serves as the primary target for phylogenetic classification and for the design of consensus PCR primers used in diagnostic and epidemiological studies [5, 11, 12].

Phylogenetic Diversity and Evolutionary Relationships

Phylogenetic analyses based on concatenated amino acid sequences of the E1, E2, L1, and L2 genes have consistently resolved the equine papillomaviruses into distinct clades that correlate with their tissue tropism and pathogenic potential. The most clinically significant clade is that containing EcPV2, EcPV4, EcPV5, and EcPV9, all members of the genus Dyoiotapapillomavirus [2]. This clade is strongly associated with anogenital and oropharyngeal SCCs, with EcPV2 being the most prevalent and best-characterized oncogenic type. EcPV2 DNA has been detected in a high proportion of penile, vulvar, gastric, oral, and laryngeal SCCs, with detection rates ranging from 29% to 100% depending on the anatomical site and geographic region [13, 14, 15, 10, 16, 17]. The consistent association of EcPV2 with malignant lesions, coupled with the detection of viral oncogene transcripts and the demonstration of transforming activity in vitro, provides robust evidence for its causal role in equine carcinogenesis [18, 8, 9].

In contrast, EcPV1, the sole member of the Zetapapillomavirus genus, is strictly associated with benign, self-limiting cutaneous papillomas that typically affect young horses, particularly on the muzzle and lips [4, 6]. The lesions are characterized by exophytic, verrucous growths that exhibit acanthosis, hyperkeratosis, and the pathognomonic presence of koilocytes, cells with perinuclear halos indicative of viral cytopathic effect [4, 6]. EcPV1-induced papillomas rarely undergo malignant transformation, and the virus is not considered an oncogenic risk. EcPV3, EcPV4, EcPV5, and EcPV6 are predominantly associated with aural plaques, which are benign, hyperkeratotic lesions confined to the inner surface of the pinnae [19, 20, 21]. These viruses are frequently detected as co-infections, with EcPV4 being the most prevalent type in aural plaque samples, followed by EcPV3, EcPV6, and EcPV5 [19, 20]. Interestingly, EcPV2, EcPV7, EcPV8, and EcPV9 are not typically found in aural plaques, suggesting a strict tissue tropism that restricts these types to mucosal or genital epithelia [20].

The recent identification of a highly divergent EcPV from a horse in Denmark, which clusters with EcPV3 and EcPV6 but occupies a distinct phylogenetic branch, highlights the likelihood that many more equine papillomavirus types remain to be discovered [1]. This novel virus exhibited only 58.4% and 60.0% L1 nucleotide identity to EcPV3 and EcPV6, respectively, placing it at the threshold of a new genus [1]. Such discoveries are not merely taxonomic curiosities; they have profound implications for understanding the full spectrum of EcPV-associated disease and for the development of comprehensive diagnostic and prophylactic strategies.

Cross-Species Infections and the Bovine Papillomavirus Connection

A unique and clinically critical aspect of equine papillomavirus biology is the phenomenon of cross-species infection by bovine papillomaviruses (BPVs). Unlike the strictly host-specific EcPVs, BPV types 1, 2, and possibly 13 are capable of infecting equids, where they induce the formation of sarcoids, the most common skin tumor of horses worldwide [22, 23, 11, 24]. BPVs belong to the genus Deltapapillomavirus and are distinct from the EcPVs in their genomic organization and pathogenic mechanisms. In their natural bovine host, BPV1 and BPV2 cause benign cutaneous papillomas that typically regress spontaneously. However, when transmitted to horses, these viruses establish a non-productive, persistent infection in dermal fibroblasts, leading to the formation of locally aggressive, fibroblastic tumors that rarely, if ever, regress [23, 24, 25].

The molecular basis for this host-dependent difference in pathogenesis is an area of active investigation. Horses are considered dead-end hosts for BPV, as the virus does not complete its life cycle to produce infectious virions; viral DNA is maintained as episomes in the nucleus of transformed fibroblasts, and late gene expression is absent or severely restricted [23, 26]. The BPV E5 oncoprotein, which is a potent transforming protein in horses, plays a central role in sarcoid pathogenesis by constitutively activating the platelet-derived growth factor β receptor (PDGFβR) and by downregulating major histocompatibility complex class I (MHC-I) expression, thereby facilitating immune evasion [27, 28]. Recent genomic analyses of BPV1 and BPV2 isolates from equine sarcoids and bovine papillomas have revealed that the viral sequences are not segregated by host species, indicating that cross-species transmission is an ongoing, dynamic process rather than a single historical event that gave rise to horse-adapted variants [29, 30]. This finding has significant implications for the epidemiology of sarcoids, as it suggests that horses are continuously exposed to BPV from infected cattle, and that geographic variations in BPV type prevalence (e.g., BPV2 predominance in New Zealand versus BPV1 in Europe) reflect the local bovine reservoir [11, 12].

Intriguingly, ovine papillomavirus (OaPV) DNA has also been detected in a subset of equine sarcoids using highly sensitive digital droplet PCR, raising the possibility that other ungulate papillomaviruses may occasionally contribute to sarcoid disease [22]. The detection of OaPV types 1, 3, and 4 in 34.9% of sarcoid samples from Austria suggests that the etiological landscape of equine sarcoids may be broader than previously appreciated, although the clinical significance of these findings requires further validation [22].

Epidemiological Context and Clinical Implications

The taxonomy and phylogeny of EcPVs are not merely academic exercises; they provide the framework for understanding the epidemiology, pathogenesis, and potential control of equine papillomavirus-associated diseases. The recognition that EcPV2 is the primary oncogenic type in horses has spurred efforts to develop prophylactic and therapeutic vaccines based on virus-like particles (VLPs) derived from the L1 capsid protein [31, 32]. However, the recent discovery that EcPV7 can also cause penile SCCs, either alone or as a co-infection with EcPV2, complicates vaccine development, as a monovalent EcPV2 vaccine may not provide complete protection against all genital SCCs [7]. Similarly, the diversity of EcPV types associated with aural plaques (EcPV3, 4, 5, and 6) suggests that any vaccine aimed at preventing this common condition would need to be multivalent [19, 20].

The high genoprevalence of EcPV2 in clinically healthy horses, 30.3% in one Italian study of 234 asymptomatic animals, indicates that infection is widespread and that most horses are able to control the virus without developing malignant disease [33]. This parallels the situation in humans, where high-risk HPV infections are common but only a small fraction progress to cancer. The factors that determine whether an EcPV2 infection remains latent, causes benign papillomas, or progresses to invasive SCC are likely multifactorial, involving viral genotype, host genetics, immune status, and environmental cofactors such as UV radiation and trauma [34, 18, 15]. The detection of EcPV2 DNA in the semen of a stallion with a penile lesion [2] and in the oral cavity of healthy donkeys [35] further underscores the potential for sexual and horizontal transmission, and highlights the need for comprehensive screening programs in breeding populations.

In conclusion, the taxonomy of equine papillomaviruses is a rapidly evolving field that reflects the remarkable genetic diversity and biological complexity of these pathogens. From the benign, self-limiting warts caused by EcPV1 to the aggressive, metastatic SCCs driven by EcPV2 and EcPV7, and the enigmatic cross-species sarcoids induced by BPVs, the equine papillomaviruses offer a rich model for studying virus-host interactions, oncogenesis, and the evolutionary dynamics of host-switching events. Continued genomic surveillance, particularly through metagenomic approaches, will undoubtedly reveal additional novel types and deepen our understanding of the viral determinants of pathogenicity, ultimately informing the development of effective vaccines and therapeutic interventions.

Molecular Pathogenesis of EcPV1 and EcPV2

The molecular pathogenesis of equine papillomaviruses, particularly EcPV1 and EcPV2, represents a complex interplay between viral oncogene expression, host cellular signaling disruption, immune evasion, and progressive neoplastic transformation. While EcPV1 is primarily associated with benign, self-limiting cutaneous papillomas, EcPV2 has emerged as a significant oncogenic driver in the development of equine squamous cell carcinomas (SCCs) across multiple anatomical sites, including the genitalia, oropharynx, stomach, and ocular regions [13, 14, 10, 36]. Understanding the discrete molecular mechanisms underpinning these divergent clinical outcomes is critical for developing targeted therapeutic and prophylactic interventions.

Viral Genome Organization and Oncogene Function

Both EcPV1 and EcPV2 possess circular double-stranded DNA genomes of approximately 7.6–7.7 kb, organized into early (E) and late (L) coding regions flanked by a long control region (LCR) that contains regulatory elements for viral replication and transcription [4, 5]. The early region encodes the oncoproteins E6 and E7, which are central to viral-mediated cellular transformation, along with E1, E2, and E4, which are involved in genome replication, transcriptional regulation, and viral egress. The late region encodes the capsid proteins L1 and L2, which facilitate virion assembly and host cell entry [37]. For EcPV2, transcriptional profiling of naturally occurring lesions has demonstrated that the majority of viral reads map to the non-structural early genes, particularly E6, E7, and E2/E4, with a distinct pattern of alternative splicing events essential for the expression of functionally diverse gene products [9]. This splicing repertoire is a hallmark of papillomavirus biology, allowing a single genomic region to generate multiple protein isoforms that differentially modulate the host cellular environment.

The E6 and E7 oncoproteins of EcPV2 are the primary drivers of cellular immortalization and malignant progression. In human papillomavirus (HPV) biology, E6 targets the tumor suppressor p53 for ubiquitin-mediated proteasomal degradation, while E7 inactivates the retinoblastoma protein (pRb), leading to deregulated cell cycle progression. Although the precise biochemical interactions of EcPV2 E6 and E7 with equine homologs remain incompletely characterized, accumulating evidence supports functional conservation. In EcPV2-positive penile SCCs, immunohistochemical analysis has revealed non-basal p53 positivity that is significantly associated with malignancy, suggesting that p53 dysregulation, whether through degradation, stabilization of mutant forms, or disruption of downstream signaling, is a critical event in EcPV2-driven carcinogenesis [36]. Furthermore, the proliferation marker Ki67 demonstrates progressively higher expression from benign papillomas through carcinoma in situ (CIS) to invasive SCCs, directly correlating with the level of EcPV2 oncogene transcription [8, 36]. Three-dimensional organotypic raft cultures derived from EcPV2-positive penile lesions recapitulate this progression, with levels of E6/E7 transcription and p53, Ki67, and MCM7 expression increasing in parallel with the severity of dysplasia, thereby providing a robust ex vivo model for dissecting these molecular events [8].

Epithelial-to-Mesenchymal Transition and Tumor Cell Plasticity

A hallmark of EcPV2-associated malignant progression is the induction of epithelial-to-mesenchymal transition (EMT), a process by which polarized epithelial cells lose cell-cell adhesion and acquire migratory, invasive, and stem cell-like properties. In EcPV2-positive penile SCCs, immunohistochemical characterization has demonstrated a significant downregulation of epithelial markers, including E-cadherin, β-catenin, and pan-cytokeratin, at the tumor invasive front, coupled with the upregulation of mesenchymal markers such as N-cadherin and vimentin [18]. This phenotypic switch is orchestrated by EMT-related transcription factors, including TWIST-1 and ZEB-1, which are expressed in neoplastic cells at the leading edge of invasion [18]. Critically, the transcriptional profiling of EcPV2-positive SCCs has revealed the upregulation of genes within the canonical Wnt signaling pathway, including BCATN1 (β-catenin), LEF1, and FOSL1, alongside RANKL, a downstream target of Wnt activation [18]. This pathway activation is strikingly similar to that observed in human penile SCCs, reinforcing the utility of the equine model for comparative oncology.

Beyond EMT, EcPV2-positive SCCs exhibit broader tumor cell plasticity, including the adoption of stem cell-like and endothelial-like phenotypes. Immunohistochemical and immunofluorescence analyses of EcPV2-positive head and neck SCCs (HNSCCs) have identified CD44 and CD271 double-positive tumor cell subsets, indicative of a stem cell-like population that may drive tumor recurrence and therapeutic resistance [34]. These cells are particularly enriched at infiltrative tumor fronts, and their presence is independent of EcPV2 infection status, suggesting that once EMT is initiated, the resulting plasticity becomes self-sustaining [34]. More recently, vasculogenic mimicry (VM), the formation of perfused, vessel-like channels by tumor cells themselves, independent of endothelial cells, has been documented in equine SCCs. In EcPV2-positive genital and oronasal lesions, periodic acid-Schiff (PAS)-positive, CD31-negative lumens containing erythrocytes were identified, and triple immunofluorescence confirmed that these channels are lined by cytokeratin-positive tumor cells that also express type IV collagen and alpha-smooth muscle actin [38]. The recruitment of pericytes to these pseudo-vessels suggests that VM-forming cancer cells actively stabilize their own vascular network, a phenomenon that may contribute to the aggressive clinical behavior and metastatic potential of EcPV2-associated SCCs.

Immune Evasion and the Tumor Microenvironment

The progression from EcPV2 infection to invasive carcinoma is critically dependent on the virus’s ability to subvert host immune surveillance. Comparative analysis of the immune cell infiltrate (ICI) in EcPV2-positive versus EcPV2-negative penile neoplasms has revealed a distinct immunological landscape. While the overall densities of CD3+ T cells, CD20+ B cells, and IBA-1+ macrophages do not differ significantly according to EcPV2 status, the number of FoxP3+ regulatory T cells (Tregs) is significantly elevated in both the intraepithelial and stromal compartments of EcPV2-positive tumors [36]. Tregs are potent suppressors of anti-tumor immunity, and their enrichment in EcPV2-associated lesions suggests that the virus actively promotes an immunosuppressive microenvironment that facilitates persistent infection and neoplastic progression. This finding is consistent with the observation that EcPV2 DNA can be detected in the genital mucosa of 30% of clinically healthy horses, yet only a minority develop SCCs, implying that immune competence is a critical determinant of disease outcome [33]. In asymptomatic EcPV2-positive horses, the expression of the viral L1 gene is associated with increased transcription of pro-inflammatory cytokines IL1B and IL12p40, alongside decreased expression of immunosuppressive TGFB and RANKL, indicating that an effective innate immune response can control viral replication and prevent malignant transformation [33].

The role of macrophages in EcPV2-driven carcinogenesis is nuanced. IBA-1+ macrophage densities are significantly higher in invasive SCCs compared to papillomas or CIS, suggesting that tumor-associated macrophages (TAMs) are recruited during malignant progression [36]. In human cancers, TAMs can adopt either anti-tumor (M1) or pro-tumor (M2) polarization states, and the balance between these phenotypes influences tumor growth, angiogenesis, and metastasis. The functional polarization of macrophages in equine SCCs remains to be fully characterized, but the observed increase in macrophage density, coupled with the immunosuppressive Treg infiltrate, is consistent with a shift toward an M2-dominant, tumor-promoting microenvironment.

Host Transcriptome Dysregulation and Genomic Instability

RNA-seq analysis of EcPV2-positive genital SCCs compared to healthy control tissue has identified 1,957 differentially expressed host genes, with the most significantly affected pathways related to DNA replication, cell cycle regulation, extracellular matrix (ECM)-receptor interaction, and focal adhesion [9]. The upregulation of matrix metalloproteinase 1 (MMP1) and interleukin-8 (IL8) in SCCs suggests that these genes may serve as potential biomarkers for malignant progression, as they are implicated in ECM degradation, angiogenesis, and neutrophil recruitment [9]. The dysregulation of cell cycle-related genes, including multiple cyclins and cyclin-dependent kinases, directly reflects the activity of the EcPV2 E7 oncoprotein in overriding the G1/S checkpoint.

Genomic instability is a hallmark of EcPV2-associated carcinogenesis. While papillomavirus genomes typically persist as episomes in benign lesions and many cancers, integration into the host genome can occur and is associated with increased oncogene expression and genomic rearrangements. In one EcPV2-positive genital SCC, DNA sequencing followed by PCR confirmation revealed the integration of viral DNA into the host genome, a finding that parallels the integration events commonly observed in HPV-associated human cancers [9]. The consequences of integration include the disruption of host genes, the stabilization of viral oncogene transcripts, and the potential for insertional mutagenesis. Although integration appears to be a rare event in EcPV2-associated disease, its occurrence underscores the potential for severe genomic consequences in a subset of cases.

Comparative Pathogenesis of EcPV1

In contrast to the oncogenic potential of EcPV2, EcPV1 is primarily associated with benign, self-limiting cutaneous papillomas, typically affecting the muzzle, lips, and periocular region of young horses [4, 6]. The molecular pathogenesis of EcPV1 is characterized by productive viral replication within differentiated keratinocytes, leading to the classical histopathological features of acanthosis, hyperkeratosis, and koilocytosis, the latter representing a cytopathic effect of viral infection on the stratum spinosum and granulosum [4]. EcPV1 induces a robust host immune response that typically results in spontaneous regression of lesions within weeks to months, a process that is thought to be mediated by cell-mediated immunity targeting viral antigens. The autovaccination of horses with formalin-inactivated EcPV1 papilloma tissue has been shown to accelerate lesion regression, further supporting the role of adaptive immunity in controlling EcPV1 infection [6]. The molecular basis for the divergent pathogenicity of EcPV1 versus EcPV2 likely resides in differences in the E6 and E7 oncoproteins, which may have evolved distinct affinities for host tumor suppressor proteins, as well as differences in the LCR that govern tissue-specific and differentiation-dependent viral gene expression.

The Role of Coinfections and Epigenetic Regulation

Emerging evidence suggests that EcPV2 does not act in isolation but may synergize with other pathogens and host factors to drive carcinogenesis. EcPV2 DNA has been detected alongside gamma-herpesviruses, including equine herpesvirus 2 (EHV2) and EHV5, in a substantial proportion of SCCs from the head and neck, ocular, and genital regions [17]. While the functional significance of these coinfections remains to be established, herpesviruses are known to possess immunomodulatory and oncogenic properties, and their presence may contribute to the immunosuppressive microenvironment that facilitates EcPV2 persistence and progression. Furthermore, the detection of EcPV7 DNA in a subset of penile and oropharyngeal SCCs, either alone or as a coinfection with EcPV2, expands the repertoire of potentially oncogenic equine papillomaviruses and suggests that multiple viral types may contribute to SCC pathogenesis [7].

Epigenetic dysregulation is an emerging theme in EcPV2-associated disease. In equine sarcoids, which are caused by bovine papillomavirus (BPV) rather than EcPV, transcriptome and methylome sequencing has revealed altered expression of long non-coding RNAs (lncRNAs) and aberrant DNA methylation patterns that affect genes involved in ECM disassembly and cancer pathways [39]. Although these studies focus on BPV-induced sarcoids, the principles of epigenetic reprogramming are likely applicable to EcPV2-induced SCCs, as both viruses manipulate host gene expression through similar oncogenic pathways. The integration of transcriptomic and epigenomic data in future studies of EcPV2-associated lesions will be essential for a comprehensive understanding of the molecular pathogenesis.

Implications for Disease Prevention and Therapy

The detailed molecular characterization of EcPV2 pathogenesis has direct translational implications. The consistent association of EcPV2 with genital, oral, and gastric SCCs, coupled with the detection of viral DNA and RNA in precursor lesions and metastases, provides a strong rationale for the development of prophylactic vaccines targeting the L1 capsid protein, similar to the highly successful HPV vaccines used in human medicine [31]. Virus-like particle (VLP)-based vaccines have been shown to be immunogenic in horses and represent a promising strategy for preventing EcPV2 infection and its associated malignancies [31]. Additionally, the identification of FoxP3+ Tregs as a key component of the immunosuppressive microenvironment in EcPV2-positive tumors suggests that immunotherapies aimed at depleting Tregs or blocking their suppressive functions could enhance anti-tumor immunity and improve clinical outcomes [36]. The demonstration that EcPV2 E6 and E7 are consistently expressed in SCCs also opens the door for therapeutic vaccines targeting these oncoproteins, an approach that has shown promise in HPV-associated human cancers and is being actively explored in equine medicine using influenza virus vectors expressing BPV E6 and E7 antigens for sarcoid immunotherapy [32].

Epidemiology and Global Distribution of Equine Papillomavirus

The epidemiological landscape of equine papillomaviruses (EcPVs) is characterized by remarkable viral diversity, distinct tissue tropisms, and profound geographic heterogeneity in the prevalence of specific viral types and their associated clinical manifestations. Unlike human papillomaviruses, which have been extensively catalogued through decades of population-based surveillance, the global distribution of EcPVs remains incompletely mapped, with substantial gaps in knowledge across entire continents and among diverse equid populations. The emerging picture, drawn from a growing but still fragmented body of molecular epidemiological investigations, reveals a complex interplay between viral phylogeny, host genetics, environmental cofactors, and clinical outcomes that demands rigorous, systematic investigation.

Diversity and Phylogenetic Distribution of EcPV Types

The family Papillomaviridae encompasses an expanding roster of equine-associated viruses, with at least nine recognized EcPV types (EcPV1 through EcPV9) distributed across multiple genera, alongside cross-species infections by bovine papillomaviruses (BPV1, BPV2, and potentially BPV13) and, more recently, the detection of ovine papillomaviruses (OaPV1, OaPV3, OaPV4) in equine tissues [22]. This taxonomic diversity reflects millions of years of co-evolution with equid hosts, but the geographic boundaries of each viral type remain poorly defined. EcPV1, the classical agent of cutaneous papillomatosis, has been molecularly confirmed across disparate regions including Japan, Turkey, Saudi Arabia, and Brazil, suggesting a near-cosmopolitan distribution consistent with its long-recognized clinical phenotype [4, 5, 6, 20]. Whole-genome characterization of Japanese EcPV1 isolates revealed a 7,613 bp genome with conserved organization, while Arabian horse isolates from Saudi Arabia yielded 384 bp amplicons corresponding to the E4 and L2 genes, confirming that EcPV1 circulates widely even among genetically distinct horse populations [4, 5].

The most extensively studied type, EcPV2, exhibits a particularly complex epidemiological profile. Originally identified in association with genital squamous cell carcinomas (SCCs), EcPV2 has since been detected in a widening spectrum of anatomical sites, including the oropharynx, stomach, ocular tissues, and larynx, raising fundamental questions about its transmission dynamics and tissue-specific oncogenic mechanisms [13, 14, 10, 17]. A comprehensive survey of Western Canadian horses identified EcPV2 DNA in 18% of 101 papillomas, carcinomas in situ, and SCCs, with 29% of genital SCCs testing positive, while SCCs from non-genital locations were uniformly negative [15]. In stark contrast, studies from Europe and Scandinavia have reported substantially higher detection rates. Tuomisto et al. (2024) found EcPV2 nucleic acids in 100% of genital lesions (12/12), 100% of gastric SCCs (2/2), 33% of ocular SCCs (2/6), and the single laryngeal SCC examined from Finnish horses [10]. Similarly, a large Italian survey of 234 clinically healthy horses revealed an EcPV2 genoprevalence of 30.3% based on detection of L1 DNA in genital swabs, with 48% of those positive samples demonstrating active L1 gene expression, indicating ongoing viral transcription in the absence of clinical disease [33]. This striking discordance, between high subclinical carriage rates in some populations and variable detection in neoplastic tissues, suggests that EcPV2 infection is far more prevalent than SCC incidence would predict, implicating host immune status, genetic susceptibility, and environmental cofactors in malignant progression.

Geographic Patterns in Sarcoid-Associated BPV Distribution

Equine sarcoids, the most common cutaneous neoplasms of equids globally, present a unique epidemiological scenario involving cross-species infection by bovine papillomaviruses. The distribution of sarcoid-associated BPV types exhibits marked geographic structuring that challenges simple explanations based on viral biology alone. In New Zealand, a comprehensive analysis of 104 sarcoids using both consensus and type-specific PCR primers revealed that 88.3% of PV-positive lesions contained only BPV2 DNA, 9.6% harbored both BPV1 and BPV2, and merely 2.1% contained BPV1 alone [11]. This BPV2 predominance aligns with patterns observed in North American sarcoids but diverges substantially from European and Australian studies, where BPV1 typically dominates [11, 12]. Polish sarcoid isolates, for instance, demonstrated that variants of BPV-1 isolate EqSarc1 constituted the most prevalent viral type, with 66.7% of sequenced isolates sharing 100% homology with this specific lineage [12]. The biological basis for these geographic differences remains obscure but may reflect regional variations in cattle herd BPV prevalence, vector ecology, or yet-unidentified host genetic factors.

Yamashita-Kawanishi et al. (2020) provided critical phylogenetic evidence from Japan that BPV1 sequence variability correlates more strongly with geographic origin than with host species, suggesting that BPV1 circulates continuously between local bovine and equid populations rather than having undergone an ancient host-switch followed by independent equine adaptation [29]. This finding was substantially reinforced by Gysens et al. (2023), who analyzed 98 complete BPV1 and BPV2 genomes using Bayesian phylogenetic methods and found no significant phylogeny-host correlation, supporting an ongoing, dynamic process of cross-species transmission rather than the circulation of horse-adapted variants [30]. Intriguingly, these authors identified extensive deletions in the L1/L2 region (up to 1.5 kb) exclusively in horse-derived samples, a finding of unknown functional significance that warrants further investigation [30]. The epidemiological implication is profound: equine sarcoid burden in any given region is likely a function of local bovine BPV prevalence, management practices that facilitate equid-bovid contact, and individual horse susceptibility, rather than the result of stable, horse-adapted viral lineages.

Prevalence Dynamics in Anatomically and Demographically Defined Subpopulations

The prevalence of EcPV infection varies not only by geographic region but also by anatomical site, age, breed, sex, and reproductive history, reflecting complex transmission dynamics and host-virus interactions. Aural plaques, the most prevalent EcPV-associated dermatological condition, demonstrate exceptionally high viral detection rates. Zakia et al. (2019) employed quantitative real-time PCR to screen 103 aural plaque samples and detected at least one viral type in 90.29% of specimens, with EcPV4 present in 82.52%, EcPV3 in 36.89%, EcPV6 in 10.68%, and EcPV5 in just 0.97% [19]. Subsequent Brazilian work by Bromberger et al. (2023) confirmed these patterns, finding EcPV6 most prevalent at 81%, followed by EcPV3 (72%), EcPV4 (63%), and EcPV5 (47%), while EcPV2, EcPV7, EcPV8, and EcPV9 were entirely absent from aural plaque samples [20]. The near-ubiquity of multiple EcPV types in aural plaques suggests either that these lesions represent a particularly permissive environment for PV infection or that the pinna serves as a reservoir for viral persistence and potential transmission.

In the context of genital disease, breed and management factors emerge as significant epidemiological variables. Cappelli et al. (2022) demonstrated that among Italian horses, Thoroughbreds carried the highest risk of EcPV2 infection, while in mares, pluriparity and natural breeding history were significantly associated with increased positivity (p = 0.0111 and p = 0.0037, respectively) [33]. These findings suggest that mechanical trauma during coitus and parturition may facilitate viral entry into the genital epithelium, analogous to the role of mucosal disruption in human PV transmission. The equine penile and vulvar epithelia, particularly at sites prone to frictional injury, likely represent primary portals of EcPV2 entry, with subsequent establishment of persistent infection that may remain subclinical for years or decades.

Emerging Viral Types and Expanding Host Range

The application of high-throughput sequencing technologies has dramatically accelerated the discovery of novel EcPV types, revealing an unsuspected diversity that challenges existing taxonomic frameworks. Blomström et al. (2025) identified a divergent EcPV from a Danish horse via viral metagenomics, assembling a complete 7,767 bp genome that shared only 58.4% and 60.0% L1 gene sequence identity with EcPV3 and EcPV6, respectively, suggesting this isolate may represent a novel species or even genus within the Papillomaviridae [1]. Importantly, the index horse presented with neurological signs, and the detection of this divergent PV through unbiased sequencing underscores the potential for EcPVs to be identified in clinical contexts far removed from classical papillomatosis. Similarly, Li et al. (2019) employed meta-transcriptomics on semen from a Thoroughbred stallion with a genital lesion to identify EcPV9, which clustered with EcPV2, EcPV4, and EcPV5 but exhibited sufficient divergence to constitute a new viral species [2].

The detection of EcPV7 in penile SCCs by Munday et al. (2024) carries profound epidemiological and clinical implications. In a series of 20 archived penile SCCs, EcPV7 was the sole PV detected in one case, co-infected with EcPV2 in five cases, and absent in the remaining 14, which harbored only EcPV2 [7]. This demonstration that PV types other than EcPV2 can cause penile SCCs immediately complicates vaccine development strategies, as vaccines targeting EcPV2 alone would likely fail to prevent a subset of these neoplasms. Furthermore, EcPV7 was co-detected with EcPV2 in 3 of 10 oropharyngeal SCCs, indicating that this novel type may contribute to malignant transformation at multiple anatomical sites [7]. The absence of distinguishing clinical or histological features between EcPV2- and EcPV7-associated lesions means that molecular typing is essential for accurate epidemiological surveillance.

Co-infections and the Complexity of the Equine PV Virome

The epidemiological picture is further complicated by the frequent detection of mixed infections involving multiple PV types within individual lesions and even within individual animals. Miglinci et al. (2023) screened a series of 43 equine SCCs from head-and-neck, ocular, and genital regions, detecting EcPV2 in 100% of genital tumors, 45.5% of head-and-neck lesions, and 8.3% of ocular SCCs, while also identifying EcPV5 in two head-and-neck SCCs and EcPV4 in an ocular lesion [17]. Concurrently, herpesvirus DNA, primarily equine herpesvirus 2 (EHV2), EHV5, and asinine herpesvirus 5 (AsHV5), was detected in 63.6% of head-and-neck, 66.6% of ocular, and 47.2% of penile SCC cases, raising the possibility of synergistic interactions between PVs and gamma-herpesviruses in oncogenesis [17]. The detection of ovine PV DNA in 34.92% of 63 equine sarcoid samples using droplet digital PCR further expands the known virome of equine neoplasia, with OaPV1, OaPV3, and OaPV4 identified either alone or as multiple co-infections [22]. These findings challenge the traditional single-pathogen paradigm of PV-induced disease and suggest that the equine PV epidemiological unit may be better conceptualized as a complex viral community.

Asymptomatic Carriage and the Reservoir Hypothesis

A critical epidemiological insight emerging from recent studies is the recognition that EcPV infection far exceeds clinical disease, necessitating a reevaluation of transmission dynamics and disease risk. The Italian study by Cappelli et al. (2022) demonstrated that 30.3% of 234 clinically healthy horses harbored EcPV2 DNA on genital mucosa, with nearly half of these showing active L1 transcription [33]. Importantly, mares expressing L1 exhibited increased IL1B and IL12p40 expression and decreased RANKL and TGFB expression, suggesting that an effective innate immune response may control viral replication and prevent neoplastic progression in most infected individuals [33]. This high subclinical carriage rate, analogous to human HPV infection where most sexually active adults clear the virus without ever developing cancer, implies that EcPV2 transmission is likely far more efficient than SCC incidence would suggest. The vaginal and preputial mucosa, along with the pinna, may serve as primary viral reservoirs from which transmission occurs through direct contact, fomites, or environmental contamination. The World Organisation for Animal Health (WOAH) has recognized the economic importance of equine sarcoids, but comprehensive surveillance programs for EcPV carriage comparable to those implemented for high-risk HPV types in humans do not yet exist for equine populations.

Clinical Manifestations and Histopathological Features

The clinical and pathological landscape of equine papillomavirus (EcPV) infection is remarkably diverse, reflecting the broad tropism of these viruses for stratified squamous epithelium and, in the case of bovine papillomavirus (BPV)-induced sarcoids, dermal fibroblasts. The manifestations range from benign, self-limiting cutaneous papillomas to aggressive, life-threatening squamous cell carcinomas (SCCs) and the enigmatic, fibroblast-derived sarcoid. A comprehensive understanding of these features is critical for accurate diagnosis, prognostication, and the development of targeted therapeutic strategies. The clinical presentation is heavily influenced by the specific viral type, the anatomical site of infection, and the host’s immune status, with a growing body of evidence highlighting the role of viral oncoproteins in driving neoplastic progression and modulating the tumor microenvironment.

Benign Proliferative Lesions: Classical Papillomatosis and Aural Plaques

Classical Cutaneous Papillomatosis (EcPV-1) The most archetypal manifestation of EcPV infection is classical cutaneous papillomatosis, primarily associated with Equus caballus papillomavirus type 1 (EcPV-1) [4, 5, 6]. Clinically, these lesions present as multiple, raised, verrucous (warty) growths, typically ranging from 2 to 8 mm in diameter, though they can coalesce into larger, cauliflower-like masses [4]. They are most frequently observed on the muzzle, lips, and distal limbs of young horses, often resolving spontaneously within weeks to months as the host mounts an effective immune response [6]. The lesions are non-painful and non-pruritic, and their distribution often suggests direct or fomite-mediated transmission through contact with contaminated equipment [4, 37]. Histopathologically, these papillomas are characterized by marked thickening and hyperplasia of all epidermal layers. Key diagnostic features include acanthosis (thickening of the stratum spinosum), hyperkeratosis (thickening of the stratum corneum, often of the orthokeratotic type), and the pathognomonic presence of koilocytes [4, 6]. Koilocytes are large, vacuolated keratinocytes with shrunken, pyknotic nuclei, representing the cytopathic effect of active viral replication and assembly within the granular and spinous layers. Occasional intranuclear viral inclusions may also be observed [6]. The dermis typically shows a mild to moderate perivascular inflammatory infiltrate, composed primarily of lymphocytes and macrophages, reflecting a host immune response that is ultimately responsible for lesion regression.

Aural Plaques (EcPV-3, -4, -5, -6) Aural plaques represent a distinct clinical entity, presenting as well-demarcated, hypochromic (depigmented), hyperkeratotic plaques on the inner surface of the pinnae [19, 20]. These lesions are typically small (1–2 cm), flat to slightly raised, and may coalesce to form larger, irregular patches. They are often bilaterally symmetrical and are considered a cosmetic issue, though they can be associated with sensitivity or head-shaking in some horses. Unlike classical papillomas, aural plaques are persistent and do not typically undergo spontaneous regression [19]. The etiology is strongly linked to multiple EcPV types, with EcPV-4 being the most prevalent, followed by EcPV-3, EcPV-6, and EcPV-5 [19, 20]. Histologically, aural plaques exhibit moderate epidermal hyperplasia with hyperkeratosis and parakeratosis. The presence of koilocytes is a consistent feature, though often less pronounced than in classical papillomas. A key differentiating feature is the presence of a superficial, perivascular to lichenoid inflammatory infiltrate, often rich in lymphocytes and plasma cells, which contributes to the depigmentation seen clinically. The chronicity of these lesions and the persistent viral presence suggest a degree of immune evasion, a hallmark of papillomavirus biology.

Premalignant and Malignant Lesions: The EcPV-2 Carcinogenic Spectrum

The most clinically significant association is between EcPV-2 and the development of squamous cell carcinoma (SCC), particularly of the external genitalia, but also involving the ocular, oronasal, and gastric mucosa [13, 14, 15, 10, 16, 36]. This relationship is now considered etiological, with EcPV-2 DNA and RNA consistently detected in a high proportion of these tumors [18, 9, 10]. The progression from benign papilloma to invasive carcinoma is a multi-step process, with distinct clinical and histopathological correlates.

Penile and Vulvar Lesions In male horses, the disease often begins as benign penile papillomas or plaques, which are flat or slightly raised, hyperkeratotic lesions on the glans penis, shaft, or prepuce [36, 40]. These precursor lesions may be single or multiple and can persist for months to years. The transition to carcinoma in situ (CIS) is marked by increasing epithelial atypia, loss of normal stratification, and the presence of atypical mitotic figures within the full thickness of the epithelium, without breaching the basement membrane [36]. Clinically, CIS may appear as a roughened, erythematous, or leukoplakic patch. Progression to invasive squamous cell carcinoma (SCC) is characterized by the penetration of neoplastic keratinocytes through the basement membrane into the underlying dermis. These tumors often present as exophytic, cauliflower-like masses that are friable, ulcerated, and prone to secondary bacterial infection and hemorrhage [40]. In severe cases, they can cause phimosis, paraphimosis, and dysuria. A recent study by Bacci et al. (2025) demonstrated that EcPV-2-positive penile tumors harbor a significantly different immune microenvironment compared to EcPV-2-negative tumors, characterized by a higher density of FoxP3+ regulatory T-cells (Tregs) in both intraepithelial and stromal compartments [36]. This suggests that EcPV-2 actively promotes an immunosuppressive milieu, facilitating immune evasion and tumor progression. Furthermore, the same study found that non-basal p53 expression was associated with malignancy, and Ki67 proliferation indices progressively increased from papilloma to CIS to SCC, confirming the biological aggressiveness of these lesions [36].

Histopathologically, EcPV-2-associated SCCs are typically well- to moderately-differentiated, with prominent keratinization and the formation of keratin pearls. A hallmark feature is the presence of koilocytotic atypia in the adjacent or overlying epithelium, a remnant of the viral cytopathic effect [7]. The invasive front of these tumors often shows evidence of epithelial-to-mesenchymal transition (EMT), a process whereby neoplastic cells lose their epithelial characteristics (e.g., E-cadherin expression) and acquire a mesenchymal phenotype (e.g., vimentin expression), enabling them to invade and metastasize [34, 18]. Armando et al. (2021) demonstrated that EcPV-2-positive penile SCCs exhibit significant upregulation of EMT-related transcription factors like TWIST-1 and ZEB-1 at the invasive front, along with activation of the canonical Wnt pathway, mirroring findings in human penile SCC [18]. In female horses, vulvar and clitoral SCCs present similarly, often arising from the clitoral fossa or labia, and are also strongly associated with EcPV-2 [15, 17].

Ocular, Oronasal, and Gastric SCC While the genital tract is the most common site for EcPV-2-associated SCC, the virus has been implicated in a subset of SCCs at other mucosal sites. Ocular SCCs, particularly those affecting the nictitating membrane and conjunctiva, have shown variable EcPV-2 detection rates, with some studies finding a low prevalence (8.3%) [17], while others have detected viral nucleic acids in a subset of cases [10, 38]. Oronasal SCCs, including those of the tongue, gingiva, and nasal cavity, have been associated with EcPV-2 in approximately 32-45% of cases [34, 13, 17]. These tumors are often locally invasive and can be highly destructive. A particularly intriguing finding is the association of EcPV-2 with gastric SCC, the most common neoplasm of the equine stomach. Alloway et al. (2020) detected EcPV-2 DNA and RNA in 64% and 45% of gastric SCCs, respectively, including in distant metastases, providing strong evidence for a causal role [14]. This is a critical finding, as gastric SCC carries a grave prognosis due to its late diagnosis and aggressive behavior. The histopathology of these non-genital SCCs is similar to their genital counterparts, with koilocytosis, keratin pearl formation, and varying degrees of differentiation. The presence of vasculogenic mimicry (VM), the formation of vessel-like channels by tumor cells themselves, independent of endothelial cells, has been recently documented in equine ocular, oronasal, and genital SCCs [38]. This phenomenon, characterized by PAS-positive, CD31-negative channels containing erythrocytes, is associated with aggressive tumor behavior and poor prognosis in human cancers, and its discovery in equine SCC opens new avenues for understanding metastasis.

Equine Sarcoids: A Unique Fibroblastic Neoplasm

Equine sarcoids represent a unique and enigmatic entity in veterinary oncology. Unlike other papillomavirus-induced lesions, sarcoids are mesenchymal tumors of dermal fibroblasts, not epithelial cells, and are caused by cross-species infection with bovine papillomavirus (BPV) types 1, 2, and possibly 13 [22, 23, 11, 24]. This is the only known natural cross-species infection by a papillomavirus, making it a fascinating model for viral oncogenesis [27].

Clinical Presentation and Subtypes Sarcoids are the most common skin tumor of horses worldwide, with a reported incidence of 12-67% of all skin neoplasms [41]. They are non-metastasizing but are locally invasive, highly recurrent, and can be severely debilitating. The clinical presentation is remarkably pleomorphic, and six distinct subtypes are recognized: occult, verrucous, nodular, fibroblastic, mixed, and malevolent [42, 41]. The occult sarcoid is the earliest form, presenting as a poorly defined area of alopecia and mild skin thickening, often with a roughened texture. Verrucous sarcoids are wart-like, hyperkeratotic lesions that can be easily confused with viral papillomas. Nodular sarcoids are firm, spherical, subcutaneous nodules with an intact overlying epidermis. The fibroblastic subtype is the most aggressive, presenting as a rapidly growing, fleshy, ulcerated, and highly vascularized mass that bleeds easily. Mixed sarcoids combine features of verrucous, nodular, and fibroblastic types. The malevolent form is rare but devastating, characterized by the rapid spread of tumor nodules along lymphatic vessels, leading to extensive local invasion and ulceration [41]. Sarcoids have a predilection for sites of trauma, including the groin, axilla, inner thighs, eyelids, and the base of the ears [41].

Histopathological and Molecular Features Histologically, sarcoids are characterized by a proliferation of spindle-shaped fibroblasts within the dermis, often arranged in interlacing bundles or a whorled pattern (storiform). The overlying epidermis may be normal, hyperplastic, or ulcerated. A key feature is the presence of hyperplastic rete pegs that extend into the underlying dermis, creating a characteristic "picket fence" appearance. The dermal-epidermal junction is often indistinct. The tumor cells exhibit variable degrees of atypia, and mitotic figures are common, particularly in the fibroblastic subtype. The presence of BPV DNA, particularly the E5 and E6 oncogenes, is a defining feature [23, 26, 42]. The BPV genome persists as a stable, high-copy-number episome within the nucleus of the transformed fibroblasts, and recent evidence suggests that it is not integrated into the host genome [26]. The viral E5 oncoprotein is a potent transforming agent, driving cell proliferation and inhibiting apoptosis. It also plays a key role in immune evasion by downregulating MHC class I expression on the surface of infected fibroblasts, allowing them to escape cytotoxic T-cell recognition [27, 28]. This immune evasion is further supported by the observation that sarcoids are associated with an impaired immune response to BPV infection, and spontaneous regression is an exceptional event [25]. The host immune response is characterized by a mixed inflammatory infiltrate, with elevated expression of both pro-inflammatory (IL-6, IL-1β) and anti-inflammatory (IL-10) cytokines, suggesting a complex and often ineffective immune reaction [42]. The tumor microenvironment is further shaped by the expression of BAG3, a pro-survival protein that protects BPV-transformed fibroblasts from cell death signals, and aberrant expression of p53, a key tumor suppressor [43, 44]. Recent transcriptomic studies have also revealed significant alterations in long non-coding RNA (lncRNA) expression and DNA methylation patterns in sarcoids, highlighting the complex epigenetic reprogramming that underlies this disease [39].

Diagnostic Approaches: Molecular, Histological, and Immunological Methods

The accurate diagnosis of equine papillomavirus (EcPV) infections and their associated neoplastic sequelae requires a sophisticated, multi-modal diagnostic paradigm that integrates molecular virology, classical histopathology, and advanced immunological profiling. The diagnostic landscape has evolved considerably from simple clinical inspection, now encompassing high-throughput genomic sequencing, quantitative nucleic acid detection, in situ localization of viral transcripts, and detailed characterization of the host immune microenvironment. Given the economic significance of equine papillomavirus-associated diseases, particularly sarcoids and squamous cell carcinomas (SCCs), and the potential for these conditions to compromise animal welfare and athletic performance, the World Organisation for Animal Health (WOAH) recognizes the importance of standardized diagnostic protocols for these transmissible neoplasms. The diagnostic approaches must be tailored to the specific clinical presentation, the suspected viral type, and the stage of disease, as the biological behavior of EcPV-induced lesions ranges from benign, self-limiting papillomas to invasive, life-threatening malignancies.

Molecular Detection and Genotyping: From Conventional PCR to High-Throughput Sequencing

Polymerase chain reaction (PCR)-based methods remain the cornerstone of molecular diagnosis for equine papillomaviruses, offering exceptional sensitivity and the capacity for genotyping. Conventional PCR targeting conserved regions of the viral genome, such as the L1 capsid gene or the E2 open reading frame, has been widely employed for initial detection. The use of consensus primers, such as the FAP59/FAP64 system targeting the L1 gene, allows for the amplification of a broad spectrum of papillomavirus types, including novel or uncharacterized variants [12]. This approach has been instrumental in identifying the diversity of EcPVs, including the detection of EcPV1 in Arabian horses in Saudi Arabia, where a 384-bp amplicon corresponding to the E4 and L2 genes confirmed infection in all affected animals [4]. Similarly, a 334-bp fragment spanning the E2 and L2 genes has been used for molecular typing of EcPV1 in Turkey, demonstrating 98.78% to 98.97% identity to the reference sequence [6].

However, the diagnostic sensitivity of conventional PCR can be suboptimal, particularly in samples with low viral copy numbers or in formalin-fixed, paraffin-embedded (FFPE) tissues where nucleic acid degradation is a concern. Quantitative real-time PCR (qPCR) has largely supplanted conventional methods for many applications, offering not only detection but also precise quantification of viral load. This is particularly critical for understanding the pathogenesis of equine sarcoids, where the relationship between bovine papillomavirus (BPV) copy number and clinical phenotype has been a subject of debate. Studies utilizing qPCR have demonstrated that BPV DNA copy number does not differ significantly between sarcoid subtypes, but is significantly higher in animals with fewer tumors, suggesting a threshold effect for disease manifestation [42]. The standardization of qPCR protocols for EcPV types 3, 4, 5, and 6 has enabled the high-throughput screening of aural plaque samples, revealing a prevalence of at least one viral type in 90.29% of lesions, with EcPV4 being the most common (82.52%) [19].

The advent of digital droplet PCR (ddPCR) represents a significant leap forward in diagnostic precision. ddPCR partitions the sample into thousands of nanoliter-sized droplets, allowing for absolute quantification of target DNA without the need for standard curves. This technology has proven superior to qPCR for detecting low-abundance viral targets, as demonstrated in a study of equine sarcoids where ddPCR detected ovine papillomavirus (OaPV) DNA in 34.92% of samples, compared to only 22.72% by qPCR [22]. Furthermore, ddPCR was able to detect multiple OaPV types in seven cases where qPCR failed, highlighting its enhanced resolution for mixed infections [22]. This capability is clinically relevant, as co-infections with multiple papillomavirus types, such as EcPV2 and EcPV7 in penile SCCs, are increasingly recognized and may influence disease progression and response to therapy [7].

For comprehensive viral characterization, next-generation sequencing (NGS) and metagenomic approaches have become indispensable. Whole-genome sequencing using platforms such as Nanopore and Illumina has enabled the discovery of novel EcPV types, including EcPV9 from stallion semen [2] and a divergent EcPV from a Danish horse exhibiting neurological signs [1]. These techniques allow for the assembly of complete viral genomes, facilitating phylogenetic classification and the identification of genetic determinants of pathogenicity. RNA sequencing (RNA-seq) has further advanced our understanding by enabling simultaneous analysis of host and viral transcriptomes. In EcPV2-positive genital SCCs, RNA-seq revealed 1,957 differentially expressed host genes, with upregulation of MMP1 and IL8 identified as potential biomarkers for malignant transformation [9]. Moreover, RNA-seq can detect viral splicing events and integration sites, with one study identifying EcPV2 DNA integration into the host genome, a finding with profound implications for understanding viral oncogenesis [9].

Histopathological and In Situ Hybridization Techniques

Histopathological examination remains the gold standard for definitive diagnosis of papillomavirus-associated lesions, providing critical information on tissue architecture, cellular morphology, and the presence of viral cytopathic effects. Classical features of EcPV-induced papillomas include epidermal hyperplasia, acanthosis, hyperkeratosis, and the pathognomonic presence of koilocytes, enlarged keratinocytes with perinuclear halos and pyknotic nuclei [4, 6]. These changes reflect the virus's ability to disrupt normal keratinocyte differentiation and induce cellular proliferation. In malignant lesions such as SCCs, histopathological evaluation assesses the degree of dysplasia, invasion depth, and the presence of keratin pearls, which are essential for grading and prognostication. A recent comprehensive study of 185 equine SCCs identified six histopathological subtypes according to WHO criteria, usual/invasive, verrucous, pseudoglandular, papillary, warty, and basaloid, each with distinct prognostic implications [16]. This classification system, previously applied only in human medicine, represents a significant advance in equine oncological pathology.

Immunohistochemistry (IHC) extends the diagnostic power of histopathology by enabling the detection of specific cellular and viral proteins. The proliferation marker Ki67 is routinely used to assess tumor growth fraction, with studies demonstrating progressively higher Ki67 indices from benign papillomas to carcinomas in situ (CIS) and invasive SCCs [36]. The tumor suppressor protein p53 is another critical marker; aberrant p53 expression, particularly non-basal nuclear positivity, is associated with malignancy in equine penile lesions [36]. Notably, p53 immunohistochemistry can be correlated with TP53 mutational status, as determined by next-generation sequencing, although the immune cell infiltrate does not appear to vary according to TP53 status [36]. In the context of equine sarcoids, IHC for BAG3, a pro-survival protein, has shown strong positivity in tumor samples compared to normal dermal fibroblasts, suggesting a role in BPV-induced carcinogenesis [43].

In situ hybridization (ISH) provides spatial resolution of viral nucleic acids within tissue sections, allowing for the direct visualization of EcPV DNA or RNA in specific cell types. Chromogenic ISH (CISH) for EcPV2 E6/E7 oncogenes has been instrumental in establishing the etiological link between viral infection and SCC development across multiple anatomical sites. In gastric SCCs, intense hybridization signals were detected within neoplastic epithelial cells in 45% of cases, while adjacent normal mucosa remained negative [14]. Similarly, ISH has confirmed the presence of EcPV2 nucleic acids in oral SCCs (26% of cases) [13] and in a series of penile lesions, where all 12 genital lesions tested positive by both PCR and ISH [10]. The specificity of ISH is particularly valuable for distinguishing true viral association from incidental detection, as PCR alone may amplify viral DNA from contaminating or non-lesional tissue. RNA-ISH, targeting viral transcripts rather than DNA, provides evidence of active viral transcription and is considered the gold standard for demonstrating a causal role of papillomaviruses in oncogenesis [15].

Immunological Profiling and Serological Assays

The host immune response to papillomavirus infection is a critical determinant of disease outcome, and immunological methods are increasingly integrated into diagnostic workflows. Multiplex immunohistochemistry (mIHC) enables the simultaneous detection of multiple immune cell markers within a single tissue section, providing a comprehensive picture of the tumor immune microenvironment. In equine penile tumors, mIHC for CD3 (T cells), CD20 (B cells), and IBA-1 (macrophages), combined with IHC for FoxP3 (regulatory T cells), has revealed that EcPV2-positive tumors harbor significantly higher numbers of FoxP3+ regulatory T cells in both intraepithelial and stromal compartments compared to EcPV2-negative lesions [36]. This finding suggests that EcPV2 infection actively shapes the immune landscape, potentially promoting immune evasion through the recruitment of immunosuppressive regulatory T cells. The density of IBA-1+ macrophages was also higher in SCCs than in papillomas or CIS, indicating a progressive shift in the inflammatory milieu with malignant progression [36].

The study of epithelial-mesenchymal transition (EMT) markers has provided insights into tumor cell plasticity and metastatic potential. Immunohistochemical analysis of EcPV2-positive penile SCCs has demonstrated loss of epithelial markers (E-cadherin, β-catenin, pan-cytokeratin) and gain of mesenchymal markers (N-cadherin, vimentin) at the invasive front, indicative of EMT [18]. Double immunofluorescence for keratins and vimentin has confirmed the presence of hybrid epithelial/mesenchymal phenotypes, while CD44 and CD271 staining has identified stem-cell-like tumor cell subsets that may drive disease progression and therapy resistance [34]. These markers, while not diagnostic per se, provide valuable prognostic information and may guide therapeutic decision-making.

Serological assays offer a non-invasive approach to assessing immune responses to papillomavirus infection. An enzyme-linked immunosorbent assay (ELISA) targeting the C-terminal peptide of the BPV E5 oncoprotein has demonstrated the presence of IgG antibodies in horses with sarcoids, with antibody levels above a certain threshold being specific to sarcoid-positive animals [27]. This finding confirms that a humoral immune response to viral oncoproteins can be elicited, and serological testing may have utility for screening at-risk populations or monitoring response to immunotherapy. The development of virus-like particle (VLP)-based serological assays, analogous to those used in human papillomavirus diagnostics, represents a promising avenue for future research [31].

Comparative Performance and Clinical Integration

The choice of diagnostic method depends on the clinical context, the type of lesion, and the specific diagnostic question. For routine clinical diagnosis of cutaneous papillomas, histopathology combined with PCR for EcPV1 is often sufficient. However, for suspected malignant lesions or when evaluating response to therapy, a more comprehensive approach is warranted. The performance of sampling methods is critical; for equine sarcoids, fine-needle aspirates (FNA) have been shown to provide a significantly better approximation of actual BPV viral load compared to superficial swabs, with a strong correlation to postoperative tissue biopsies (r = 0.59 for occult sarcoids) [45]. This finding has direct implications for clinical trial design and monitoring of therapeutic interventions.

The integration of multiple diagnostic modalities is essential for accurate diagnosis and prognostication. For example, in a study of 115 archived equine tissue samples, the combination of broad-spectrum PCR, EcPV2-specific PCR, and RNA-ISH was necessary to accurately determine EcPV2 status, with 18% of lesions testing positive overall [15]. Importantly, EcPV2 status was not associated with overall survival in genital SCCs, whereas lack of treatment and post-treatment recurrence were strong negative predictors [15]. This underscores the importance of integrating virological data with clinical parameters for meaningful prognostication.

The detection of vasculogenic mimicry (VM), the formation of vessel-like structures by tumor cells, represents a novel diagnostic and prognostic dimension in equine oncology. Using a combination of Periodic Acid-Schiff (PAS) reaction and CD31 immunohistochemistry, followed by triple immunofluorescence for CD31, pan-cytokeratin, and type 4 collagen, VM was identified in 13 of 43 equine SCCs, including ocular, genital, and oronasal lesions [38]. The presence of VM is associated with aggressive tumor behavior and may represent a therapeutic target, highlighting the value of advanced immunohistochemical techniques in characterizing tumor biology.

Immune Response and Tumor Microenvironment in EcPV-Associated Lesions

The interplay between equine papillomaviruses (EcPVs) and the host immune system is a critical determinant of whether an infection resolves spontaneously, establishes a persistent benign lesion, or progresses to invasive squamous cell carcinoma (SCC). The tumor microenvironment (TME) in EcPV-associated lesions is a complex ecosystem where viral oncoproteins, recruited immune cells, stromal fibroblasts, and neoplastic keratinocytes engage in a dynamic cross-talk that ultimately dictates the trajectory of disease. While research into the immunobiology of EcPV infections lags behind the human papillomavirus (HPV) field, the convergence of high-resolution transcriptomics, multiplex immunohistochemistry, and functional in vitro models is now revealing a remarkably nuanced landscape that shares both parallels and unique features with HPV-driven carcinogenesis.

The Cellular Composition and EcPV-2 Status: A Dichotomy in Regulatory T-Cell Recruitment

A seminal study employing multiplex immunohistochemistry (mIHC) for CD3, CD20, and IBA-1, alongside FoxP3 immunohistochemistry, has provided the most granular characterization of the immune cell infiltrate (ICI) across the spectrum of penile lesions, papillomas, carcinoma in situ (CIS), and invasive SCCs [36]. The most striking finding was the significant enrichment of FoxP3+ regulatory T lymphocytes (Tregs) in EcPV-2-positive tumors compared to their EcPV-2-negative counterparts, observed in both intraepithelial and stromal compartments [36]. This Treg accumulation was independent of the overall density of T-cells, B-cells, or macrophages, which did not significantly differ between virus-positive and virus-negative lesions [36]. This suggests a specific, virus-driven mechanism for Treg recruitment or expansion rather than a generalized increase in inflammation.

The functional implications of this Treg enrichment are profound. In human HPV-associated cancers, Tregs are known to suppress effector T-cell responses, particularly cytotoxic CD8+ T cells, thereby facilitating immune evasion and tumor progression. The presence of elevated FoxP3+ cells in EcPV-2+ lesions implies that the virus actively sculpts the local immune milieu to be tolerogenic. This is further supported by transcriptomic data from EcPV-2-positive SCCs, which reveals a marked dysregulation of immune-related pathways. RNA sequencing of these lesions identified 1,957 differentially expressed host genes compared to normal tissue, with significant enrichment in pathways related to extracellular matrix (ECM)-receptor interaction, focal adhesion, and, critically, immune signaling, including the upregulation of IL8 [9]. IL8 (CXCL8) is a potent chemoattractant for neutrophils and can also promote angiogenesis and tumor cell proliferation. Its overexpression in EcPV-2-positive SCCs is consistent with a TME that harbors both active inflammatory signals and potent immunosuppressive elements. The presence of both pro-inflammatory (IL8) and anti-inflammatory (Treg) signals indicates a TME that is not simply "hot" or "cold," but rather a carefully orchestrated inflammatory milieu that the virus manipulates to its advantage.

Macrophage Polarization, Tumor-Associated Macrophages, and the Cytokine Milieu

While overall macrophage densities (IBA-1+ cells) were higher in invasive SCCs than in papillomas or CIS, the functional polarization of these macrophages remains a critical unresolved question [36]. In human cancers, tumor-associated macrophages (TAMs) typically adopt an M2-like, immunosuppressive phenotype that promotes tumor growth, angiogenesis, and metastasis. The transcriptomic landscape of EcPV-2-positive SCCs provides indirect evidence for this. The upregulation of extracellular matrix remodeling genes and the enrichment of focal adhesion pathways [9] are consistent with the activity of M2 macrophages, which are key producers of matrix metalloproteinases (MMPs). Indeed, MMP1 was identified as a potential marker gene for equine SCC development [9]. The interplay between macrophages and the ECM is likely a central feature of the EcPV-2 TME.

Further insights into the cytokine microenvironment come from studies of asymptomatic, EcPV-2-positive horses. Healthy horses harboring EcPV-2 DNA with active L1 gene expression showed a distinct immune signature: increased expression of IL1B and IL12p40, and decreased expression of RANKL and TGFB [33]. This profile suggests a robust, effective Th1-biased immune response capable of controlling viral replication and preventing neoplastic progression. The elevated IL12p40 is particularly telling, as it indicates a drive toward a cell-mediated immune response. In stark contrast, horses that develop sarcoids in response to BPV infection, a cross-species infection model, display a different pattern. In BPV-positive sarcoids, both pro-inflammatory (IL6, IL1B) and anti-inflammatory (IL10) pathways are upregulated, with IL10 and CCL5 being elevated in all lesion types compared to normal skin [42]. IL10 is a quintessential immunosuppressive cytokine that can inhibit antigen presentation and T-cell effector functions. The presence of this dual activation, pro- and anti-inflammatory, suggests that the TME in sarcoids, and likely EcPV-associated SCCs, is in a state of "smoldering inflammation," where the immune system is activated but is actively counterbalanced by viral evasion mechanisms and host regulatory pathways. Notably, TGFB1 expression was specifically elevated in occult sarcoids, hinting at its early role in establishing an immunosuppressive niche [42]. The absence of significant changes in TLR9, ATR, VEGFA, and PTGS2 expression in BPV sarcoids compared to normal skin [42] marks a difference from HPV-driven cancers and suggests that BPV-mediated tumorigenesis may rely on different pathways.

Evasion of Immune Surveillance: The E5 Oncoprotein and BPV-Associated Sarcoids

The immune evasion strategies deployed by EcPVs are best characterized in the context of BPV-induced sarcoids, which serve as a powerful comparative model. The BPV E5 oncoprotein is a primary driver of immune escape. E5 has been shown to downregulate major histocompatibility complex class I (MHC I) on the surface of infected fibroblasts, a critical mechanism that prevents recognition and lysis by cytotoxic CD8+ T cells [28, 24]. This is a fundamental barrier to effective anti-viral immunity. The development of an ELISA to detect antibodies against the C-terminal peptide of the BPV E5 protein in horses has provided direct serological evidence that a humoral immune response to this oncoprotein can be mounted [27]. Antibodies against E5 were found at significantly higher levels in horses with sarcoids compared to healthy controls, indicating that the immune system can recognize this target, but the antibody response is evidently insufficient to clear the infection or prevent tumor growth [27]. This underscores the concept that the failure of immunity in sarcoids is not a lack of recognition, but rather an ineffective response, likely due to the combined effects of MHC I downregulation and the local immunosuppressive TME.

The BPV E6 and E7 oncoproteins also play roles in subverting cellular defenses. They can interfere with p53 function and the retinoblastoma (Rb) pathway, respectively, promoting cellular proliferation and inhibiting apoptosis. While not directly immune evasion, this creates a permissive environment for the virus. In sarcoid cell lines, BPV-1 has been shown to increase p53 expression, though often in a cytoplasmic, non-functional localization [44]. This aberrant p53 expression could contribute to resistance to apoptosis, allowing infected cells to persist. The Cas9-mediated editing of BPV-1 oncogenes E5, E6, and the long control region (LCR) has been shown to reduce viral load in equine fibroblasts [26], confirming the essential role of these early region transcripts in maintaining the transformed state.

The Role of Epithelial-Mesenchymal Transition and Vasculogenic Mimicry in the TME

The TME in EcPV-positive SCCs is not a passive scaffold but an active participant in disease progression. Epithelial-mesenchymal transition (EMT), a process where epithelial cells lose their polarity and acquire a mesenchymal, migratory phenotype, is a hallmark of aggressive cancers and is a key factor in the TME. EcPV-2-positive penile SCCs exhibit clear evidence of EMT at the invasive front, characterized by the downregulation of epithelial markers (E-cadherin, β-catenin) and the upregulation of mesenchymal markers (N-cadherin, vimentin), alongside the activation of EMT-related transcription factors TWIST-1 and ZEB-1 [18]. This phenotypic switching is associated with the canonical Wnt pathway, as evidenced by the significant upregulation of RANKL, BCATN1, LEF1, and FOSL1 genes [18]. The activation of EMT in EcPV-2-positive SCCs mirrors findings in HPV-positive human penile SCCs, positioning the equine disease as a valuable spontaneous model.

Further complicating the TME is the phenomenon of vasculogenic mimicry (VM), identified in a subset of equine genital, oronasal, and ocular SCCs [38]. VM refers to the formation of fluid-conducting channels by tumor cells themselves, independent of endothelial cells. In these equine SCCs, VM was confirmed by the presence of PAS-positive, CD31-negative vessel-like structures containing erythrocytes [38]. The detection of collagen type IV (Col4) and alpha-smooth muscle actin (αSMA) in the lining of these channels suggests that the tumor cells are recruiting pericytes to stabilize the pseudovascular networks [38]. While VM was observed in both EcPV-2-positive and -negative genital lesions, its presence provides an alternative mechanism for tumor perfusion and metastasis that is independent of angiogenesis, and it represents a significant challenge for anti-angiogenic therapies. In the context of the TME, VM is a dramatic manifestation of tumor cell plasticity. This plasticity is further highlighted by the detection of CD44+ and CD271+ stem-cell-like tumor cell subsets in both EcPV-2-positive and -negative head and neck SCCs, indicating that these hybrid epithelial/mesenchymal phenotypes are a common feature of the advanced TME, regardless of viral etiology [34].

Therapeutic Implications and Immunomodulation of the TME

Understanding the immune landscape of EcPV-associated lesions is not merely academic; it has direct therapeutic implications. The presence of a dense, yet suppressed, immune infiltrate suggests that immunotherapeutic strategies aimed at reactivating this response could be effective. The use of a live influenza virus vector (iNS1) expressing shuffled BPV1 E6 and E7 antigens has shown remarkable success in a clinical trial for equine sarcoids, inducing complete regression of both injected and non-injected lesions in a significant proportion of horses [32]. The induction of a systemic anti-tumor response underscores that a robust, T-cell-mediated immunity can overcome the local immunosuppressive TME.

Similarly, the historical use of intratumoral BCG immunotherapy for sarcoids, while yielding variable results, is thought to act by activating innate immunity via TLR2/4 signaling and promoting a local Th1 response, thereby breaking the immunosuppressive state of the TME [46]. The variable efficacy of BCG may be due to the robustness of the Treg-mediated suppression present in individual lesions. The development of a therapeutic vaccine for EcPV-2-induced SCCs would likely need to include strategies to deplete or inhibit Tregs, given their key role in the EcPV-2-positive TME [36]. The concept of prime-boost spacing, as studied in HPV vaccine models, suggests that longer intervals between prime and boost can enhance the durability of memory and anti-tumor responses [47]. These principles, derived from basic immunology, are directly translatable to designing protocols for equine cancer vaccines.

Notably, the discovery that EcPV-2 DNA can persist in an episomal state, with the potential for integration into the host genome [9], represents a further challenge, as integrated viral DNA may alter the way viral antigens are processed and presented. The Staggering complexity of the TME in EcPV-associated lesions, from Treg recruitment and macrophage polarization to EMT and VM, underscores the need for multi-modal therapeutic approaches that combine direct anti-viral strategies with immunomodulation to overcome the sophisticated evasion tactics employed by these viruses.

Disease Management, Prognosis, and Preventive Strategies

The management of equine papillomavirus (EcPV)-associated diseases and bovine papillomavirus (BPV)-induced sarcoids presents a formidable clinical challenge, rooted in the fundamentally different biology of these two viral families and the variable immune competence of the equine host. A comprehensive approach must distinguish between benign, self-limiting conditions such as classical cutaneous papillomatosis (EcPV-1) and the potentially life-threatening, treatment-resistant neoplasms like EcPV-2-associated squamous cell carcinoma (SCC) and BPV-driven sarcoids. The therapeutic landscape has evolved from purely surgical extirpation to encompass immunomodulatory, topical chemotherapeutic, and, most recently, molecularly targeted strategies, yet significant gaps remain in achieving durable remission and preventing recurrence.

Therapeutic Strategies for EcPV-Associated Lesions

Benign Papillomatosis (EcPV-1): Classical viral papillomatosis in young horses is frequently self-limiting, with spontaneous regression typically occurring within 1–3 months as the host mounts an effective cell-mediated immune response. However, in cases where lesions are numerous, cosmetically unacceptable, or located in areas prone to trauma or secondary infection, intervention may be warranted. Surgical excision, cryotherapy, or laser ablation are effective for individual lesions but do not address the underlying viral infection or prevent recurrence. A historically noteworthy approach is the use of formalin-inactivated autogenous vaccines. Onen [6] demonstrated in a small case series that two doses of a formalin-inactivated autovaccine, derived from the horse's own papillomatous tissue and administered at 7-day intervals, led to gradual lesion regression within 2–3 weeks in three treated horses, while an untreated control showed no change. This preparation, confirmed to contain EcPV-1 DNA, presumably works by exposing the immune system to a concentrated dose of viral antigens, thereby accelerating the natural adaptive immune response. Despite its efficacy in this report, autovaccination has not been standardized, carries risks of injection-site reactions and potential transmission of other pathogens, and is not commercially available. In a separate case, topical 5-fluorouracil (5-FU) applied for 5 months to a penile papilloma with cellular atypia (EcPV-2-positive) failed to prevent regrowth, ultimately leading to euthanasia [40], underscoring the limitations of topical monotherapy for lesions with malignant potential.

Squamous Cell Carcinoma (EcPV-2, EcPV-7, EcPV-5): The management of EcPV-related SCC, particularly of the penis, vulva, and oronasal and gastric regions, is considerably more complex and often requires multimodal intervention. Surgical excision remains the cornerstone of treatment for localized disease. Complete surgical margins are strongly associated with improved outcomes, although recurrence rates remain high, up to 30–50% in some series, due to the field cancerization effect where adjacent mucosa may harbor subclinical viral infection. In the Western Canadian retrospective study by Greenwood et al. [15], the strongest negative predictors of overall survival (OS) were a lack of treatment (p < 0.01) and recurrence post-treatment (p < 0.01), while completeness of surgical margins did not significantly influence OS (p > 0.1). This counterintuitive finding may reflect the difficulty in achieving true histological clearance in a virally driven disease where the oncogenic process extends beyond the visible tumor margin. For non-resectable or recurrent tumors, intralesional chemotherapy, particularly cisplatin-based protocols, has demonstrated efficacy. Cisplatin has been shown to induce apoptosis in BPV-transformed fibroblasts [44], and extrapolation to EcPV-driven SCC is reasonable, though formal trials are lacking. The use of topical imiquimod, an immune response modifier that activates Toll-like receptor 7, has been reported anecdotally for early or in situ SCC lesions, but rigorous equine-specific data are absent. The detection of vasculogenic mimicry (VM) in 13 of 43 equine SCCs, including genital and oronasal lesions [38], has profound therapeutic implications. VM provides an alternative blood supply independent of endothelial-dependent angiogenesis, rendering anti-angiogenic therapies (e.g., targeting VEGF) potentially ineffective. This finding suggests that treatment strategies must also target the tumor cells themselves or the pericyte recruitment (Col4, αSMA expression) that stabilizes these pseudo-vessels.

Therapeutic Strategies for BPV-Induced Sarcoids

Equine sarcoids represent the most common and therapeutically challenging neoplasm in horses worldwide. Unlike EcPV-induced lesions, sarcoids are fibroblastic tumors caused by cross-species infection with BPV types 1, 2, and possibly 13 [11, 30, 24], where the equid is a dead-end host. The failure of the equine immune system to clear the infection, coupled with the episomal persistence of BPV DNA in transformed fibroblasts, underpins the high recurrence rate and therapeutic resistance.

Conventional and Surgical Approaches: Complete surgical excision is the standard of care, yet recurrence rates of 50–80% are reported, likely due to incomplete removal of virally infected fibroblasts in the wound bed and the activation of latent infection by the trauma of surgery itself. Cryotherapy, laser ablation, and hyperthermia have been used as monotherapies or adjuncts, with variable success. The clinical subtype of sarcoid (occult, verrucose, nodular, fibroblastic, mixed, malevolent) profoundly influences management decisions. Fibroblastic and malevolent subtypes are the most aggressive, with rapid growth and extensive infiltration, often necessitating radical excision or even euthanasia [41]. Intriguingly, viral load does not appear to correlate with sarcoid subtype [42, 45], suggesting that host factors, including genetic predisposition and immune status, are more critical determinants of clinical behavior than the quantity of viral DNA.

Immunotherapy: Immunotherapy has emerged as a rational and increasingly successful strategy for sarcoid management, given that the disease fundamentally represents a failure of antiviral immunity.

• BCG Immunotherapy: Bacillus Calmette–Guérin (BCG) has been used for decades, particularly for periocular sarcoids, with reported success rates of 50–80% for complete regression. The mechanism involves TLR2/4 signaling, macrophage polarization, and enhanced CD8+ T-cell responses [46]. However, efficacy is highly variable and dependent on protocol heterogeneity (dose, strain, adjuvant use). Modern comparative analyses suggest that BCG is now rarely considered first-line therapy in many settings due to the availability of more effective alternatives (cryotherapy, cisplatin, imiquimod) with fewer adverse inflammatory reactions. Nonetheless, BCG remains a relevant, cost-effective option in resource-limited regions, such as the Amazon Biome, where access to advanced therapies is restricted [46].
• Therapeutic Vaccines: The development of rationally designed therapeutic vaccines targeting BPV oncoproteins represents a paradigm shift. Jindra et al. [32] reported the use of influenza virus vectors (iNS1) expressing shuffled BPV-1 E6 and E7 antigens in a patient trial involving 29 horses. Intratumoral injection and boosting induced a systemic antitumour response, with complete regression in 10/29 horses and ongoing regression in another 10 at the time of reporting. Crucially, scrapings from former tumour sites in two patients tested negative for BPV1 DNA, suggesting durable viral clearance. Nine severely affected horses with a history of unsuccessful previous treatments did not respond, indicating that the disease burden and prior therapeutic failures may compromise immune competence. A subsequent review by the same group [28] consolidates these findings and highlights the potential of VLP-based vaccines [31] for prophylactic use, though no equine VLP vaccine is currently commercially available.
• Autogenous Vaccines and Immune Modulation: While Onen’s autovaccination approach [6] targeted EcPV-1, similar concepts could theoretically be applied to BPV-induced sarcoids, though the fibroblastic nature of sarcoids (as opposed to the epithelial origin of papillomas) may limit antigen presentation. The detection of antibodies to the BPV E5 oncoprotein in sarcoid-affected horses [27] confirms that a humoral immune response to this key transforming protein is possible, opening the door for E5-targeted immunotherapeutic strategies.

Topical and Intralesional Chemotherapy: Topical 5-fluorouracil (5-FU) has been used with some success for small, superficial sarcoids, but as noted above, it failed to prevent regrowth in a case of EcPV-2-positive penile papilloma with atypia [40]. Intralesional cisplatin, often combined with electrochemotherapy to enhance drug uptake, is one of the most effective treatments for nodular and fibroblastic sarcoids, with reported response rates exceeding 80%. The mechanism involves cisplatin-induced apoptosis, to which BPV-1-transformed fibroblasts are sensitized [44], though increased clonogenic survival after repeated treatments may limit long-term efficacy.

Emerging Molecular Therapies: The advent of CRISPR/Cas9 technology has opened a new frontier. Monod et al. [26] demonstrated that targeting BPV-1 episomal E5 and E6 oncogenes, as well as the long control region (LCR), resulted in a pronounced reduction of BPV-1 load in primary equine sarcoid fibroblasts. Furthermore, deletion of the equine Vimentin (VIM) gene, which is highly expressed in sarcoids, also decreased the number of BPV-1 episomes. This proof-of-concept study suggests that gene editing could be used to directly eliminate the viral reservoir, though delivery challenges, off-target effects, and ethical considerations remain substantial barriers to clinical application. BAG3, a protein that sustains cell survival and is highly expressed in sarcoids, has also been identified as a potential therapeutic target; silencing BAG3 in sarcoid cells promoted apoptosis and sensitized them to chemotherapeutic agents [43].

Prognostic Indicators and Factors Influencing Outcome

Prognostication in EcPV-associated disease is nuanced and must account for viral type, lesion site, and host immune microenvironment.

Viral Status and Tumor Site: For genital SCC, the presence of EcPV-2 DNA is widely accepted as a causal factor, yet its prognostic utility is limited. In the large Western Canadian study [15], EcPV-2 status of genital SCCs was not associated with overall survival (p = 0.76). This finding is corroborated by O’Brien et al. [16], who found no association between EcPV-2 status and prognosis in a series of 185 equine SCCs. However, the pattern of infection matters: EcPV-2 nucleic acids have been detected in a high proportion of genital (100%), gastric (45–64%), and a subset of ocular (33%) and laryngeal SCCs [14, 10, 17], suggesting that the virus can drive carcinogenesis at multiple mucosal sites. The detection of EcPV-7 in a subset of penile SCCs, both alone and as a co-infection with EcPV-2 [7], further complicates the picture and implies that vaccine strategies targeting only EcPV-2 may be insufficient.

Immune Microenvironment: The host immune response is a critical determinant of disease progression. Bacci et al. [36] found that FoxP3+ regulatory T-cells (Tregs) were significantly more abundant in EcPV-2-positive tumors compared to EcPV-2-negative lesions, both in intraepithelial and stromal compartments. This enrichment of immunosuppressive Tregs likely contributes to immune evasion and tumor progression, suggesting that Treg depletion strategies could be a therapeutic avenue. In contrast, EcPV-2-positive tumors did not show significant differences in overall B-cell, T-cell, or macrophage densities [36], indicating that the qualitative nature of the immune infiltrate, specifically the regulatory component, is more important than the total number of immune cells.

Molecular and Histologic Markers: Several molecular features correlate with malignant progression and may inform prognosis. Non-basal p53 expression is associated with malignancy [36], and p53 variants were detected in 4/21 SCC cases, though the immune infiltrate did not vary according to TP53 status. Ki67 proliferation index increases progressively from papilloma to carcinoma in situ to invasive SCC [8, 36]. The presence of epithelial-to-mesenchymal transition (EMT), characterized by loss of E-cadherin and gain of vimentin and N-cadherin at the invasive front, is a hallmark of aggressive SCC and is associated with EcPV-2 infection [34, 18]. The upregulation of MMP1 and IL8 has been proposed as potential marker genes for SCC development [9]. Additionally, the demonstration of stem-cell-like tumor cell phenotypes (CD44+/CD271+ double-positive cells) and vasculogenic mimicry [38] in equine SCCs suggests that tumors have multiple mechanisms to resist therapy and metastasize. HER-2 expression is widespread in equine SCCs and, while not prognostic, could represent a therapeutic target [16].

Clinical and Treatment-Related Factors: The strongest predictors of poor outcome remain clinical. Lack of treatment and recurrence post-treatment are consistently associated with decreased overall survival [15]. Older age at diagnosis is a weaker predictor, likely reflecting cumulative exposure to environmental carcinogens (e.g., UV radiation) and accumulated viral damage. Anatomic location (anogenital vs. other) does not independently influence OS when treatment is accounted for [15]. Histologic subtype of SCC (usual invasive, verrucous, pseudoglandular, papillary, warty, basaloid) may have prognostic significance, as different subtypes show varying clinical behavior [16].

Preventive Strategies: Vaccination and Biosecurity

Prophylactic Vaccination: The development of an effective prophylactic vaccine against EcPV infection is a major unmet need. The success of human papillomavirus (HPV) VLP-based vaccines provides a strong precedent. EcPV L1 proteins self-assemble into highly immunogenic VLPs, and such vaccines have been shown to protect against experimental infection in other animal models [31]. However, the diversity of EcPV types, at least nine described, with new types continuously emerging (e.g., EcPV-9 in semen, a novel EcPV in Danish horses) [1, 2], presents a significant challenge. A vaccine containing only EcPV-2 L1 VLPs may not protect against EcPV-7-induced penile SCCs [7] or against the multiple EcPV types associated with aural plaques (EcPV-1, 3, 4, 5, 6) [19, 20]. A multivalent vaccine, analogous to the 9-valent HPV vaccine, would be ideal but poses substantial economic and manufacturing hurdles. The finding that EcPV-2 DNA is present in 30.3% of asymptomatic Italian horses, with L1 expression in 48% of those [33], indicates that subclinical infection is common and that a prophylactic vaccine would need to be administered to a large population to prevent the small fraction of infections that progress to cancer.

For sarcoid prevention, vaccination against BPV is a theoretically attractive strategy, as BPV is a cross-species infection. BPV-1/2 are enzootic in cattle populations worldwide, and horses are repeatedly exposed through contact with contaminated fomites (e.g., halters, grooming tools) or vectors (flies). A prophylactic BPV vaccine could reduce the incidence of sarcoids, but it would need to cover the predominant viral types in a given geographic region. In New Zealand, BPV-2 is responsible for 88% of sarcoids, while BPV-1 accounts for only 2% [11], suggesting a BPV-2-focused vaccine would be most impactful there. In contrast, in Polynesia, BPV-1 predominates. The ongoing cross-species transmission of BPV between cattle and horses [30] means that eradication of the virus from the equine population is implausible without also addressing it in cattle.

Biosecurity and Management: Given the absence of a licensed vaccine, prevention relies on biosecurity and management practices. For EcPV-associated genital disease, the identification of subclinical shedders, 30.3% of healthy horses harbor EcPV-2 DNA [33], highlights the need for routine screening of breeding stock. Mares that are pluriparous and those bred naturally have a higher risk of infection [33], suggesting that artificial insemination using EcPV-free semen could reduce transmission. The detection of a novel equine papillomavirus (EcPV-9) in the semen of a Thoroughbred stallion with a penile lesion [2] underscores the potential for venereal transmission and the importance of testing semen before use. Genomic characterization of a novel EcPV from a horse in Denmark [1] shows that new viral types capable of causing neurological signs (though likely an incidental finding) continue to emerge, necessitating ongoing surveillance.

For sarcoids, the role of fomites and vectors in BPV transmission is critical. BPV DNA has been detected in flies, and the virus is stable in the environment. The use of shared equipment between BPV-infected and naïve horses should be avoided. Trauma is a known cofactor, so prompt management of wounds to reduce exposure to contaminated environments is prudent. The genetic predisposition to sarcoid development, likely inherited as a polygenic trait [24], means that breeding of affected individuals, particularly those with multiple or severe lesions, should be discouraged. The statement from Koch [24] that "limited recognition of sarcoids as a significant health problem has hindered the implementation of breeding-based prevention strategies" is a call to action for the veterinary community. International bodies such as WOAH (World Organisation for Animal Health) should be engaged to establish reporting standards for sarcoid and EcPV-associated SCC, facilitating global epidemiological tracking. The WHO's recognition of papillomaviruses as oncogenic agents in humans provides a framework that could be adapted for equine medicine, emphasizing the need for a One Health approach to oncogenic virus management.

References

[1] Blomström A, Hansen S, Riihimäki M. Identification and whole-genome characterization of a novel equine papillomavirus. Virus genes. 2025. DOI: https://doi.org/10.1007/s11262-025-02190-y

[2] Li C, Chang W, Mitsakos K, Rodger J, Holmes E, Hudson B. Identification of a Novel Equine Papillomavirus in Semen from a Thoroughbred Stallion with a Penile Lesion. Viruses. 2019. DOI: https://doi.org/10.3390/v11080713

[3] Munday J, Grant K, Orbell G, Vaatstra B. Cutaneous plaques associated with a putative novel papillomavirus type in a horse. New Zealand Veterinary Journal. 2022. DOI: https://doi.org/10.1080/00480169.2022.2157347

[4] Al-Hammadi M. First report on equine papillomavirus type 1 in Arabian horses in Saudi Arabia: Clinical, histopathological, and molecular characterization. Open Veterinary Journal. 2025. DOI: https://doi.org/10.5455/OVJ.2025.v15.i4.32

[5] Dong J, Zhu W, Yamashita N, Chambers J, Uchida K, Kuwano A, et al.. Isolation of equine papillomavirus type 1 from racing horse in Japan. Journal of Veterinary Medical Science. 2017. DOI: https://doi.org/10.1292/jvms.17-0322

[6] Onen EA. Molecular typing of equine papillomavirus and autovaccination to treat horses with cutaneous papillomatosis.. Australian Veterinary Journal. 2020. DOI: https://doi.org/10.1111/avj.12954

[7] Munday J, Knight CG, Bodaan CJ, Codaccioni C, Hardcastle MR. Equus Caballus Papillomavirus Type 7 is A Rare Cause Of Equine Penile Squamous Cell Carcinomas.. The Veterinary Journal. 2024. DOI: https://doi.org/10.1016/j.tvjl.2024.106155

[8] Ramsauer A, Wachoski-Dark G, Fraefel C, Ackermann M, Brandt S, Grest P, et al.. Establishment of a Three-Dimensional In Vitro Model of Equine Papillomavirus Type 2 Infection. Viruses. 2021. DOI: https://doi.org/10.3390/v13071404

[9] Ramsauer A, Kubacki J, Favrot C, Ackermann M, Fraefel C, Tobler K. RNA-seq analysis in equine papillomavirus type 2-positive carcinomas identifies affected pathways and potential cancer markers as well as viral gene expression and splicing events.. Journal of General Virology. 2019. DOI: https://doi.org/10.1099/jgv.0.001267

[10] Tuomisto L, Virtanen J, Kegler K, Levanov L, Sukura A, Sironen T, et al.. Equus caballus papillomavirus type 2 (EcPV2)‐associated benign penile lesions and squamous cell carcinomas. Veterinary Medicine and Science. 2024. DOI: https://doi.org/10.1002/vms3.1342

[11] Munday J, Orbell G, Fairley R, Hardcastle M, Vaatstra B. Evidence from a Series of 104 Equine Sarcoids Suggests That Most Sarcoids in New Zealand Are Caused by Bovine Papillomavirus Type 2, although Both BPV1 and BPV2 DNA Are Detectable in around 10% of Sarcoids. Animals. 2021. DOI: https://doi.org/10.3390/ani11113093

[12] Szczerba-Turek A, Siemionek J, Raś A, Bancerz-Kisiel A, Platt-Samoraj A, Lipczyńska-Ilczuk K, et al.. Genetic evaluation of bovine papillomavirus types detected in equine sarcoids in Poland.. Polish journal of veterinary sciences. 2019. DOI: https://doi.org/10.24425/pjvs.2018.125602

[13] Luff J, Weingart S, May S, Murphy B. A subset of equine oral squamous cell carcinomas are associated with Equus caballus papillomavirus 2 infection. Journal of Comparative Pathology. 2023. DOI: https://doi.org/10.1016/j.jcpa.2023.06.003

[14] Alloway E, Linder K, May S, Rose T, Delay J, Bender S, et al.. A subset of equine gastric squamous cell carcinomas is associated with Equus caballus papillomavirus-2. Veterinary Pathology-Supplement. 2020. DOI: https://doi.org/10.1177/0300985820908797

[15] Greenwood S, Chow-Lockerbie B, Epp T, Knight C, Wachoski-Dark G, MacDonald-Dickinson V, et al.. Prevalence and Prognostic Impact of Equus caballus Papillomavirus Type 2 Infection in Equine Squamous Cell Carcinomas in Western Canadian Horses. Veterinary Pathology-Supplement. 2020. DOI: https://doi.org/10.1177/0300985820941266

[16] O'Brien K, Mair T, Mudhar H, Pesavento P, Miller H, Priestnall S, et al.. Equine genital and ocular squamous cell carcinomas: clinical, histopathological, molecular and viral characterization with proposed histopathological classification system. Veterinary Quarterly. 2026. DOI: https://doi.org/10.1080/01652176.2026.2648939

[17] Miglinci L, Reicher P, Nell B, Koch M, Jindra C, Brandt S. Detection of Equine Papillomaviruses and Gamma-Herpesviruses in Equine Squamous Cell Carcinoma. Pathogens. 2023. DOI: https://doi.org/10.3390/pathogens12020179

[18] Armando F, Mecocci S, Orlandi V, Porcellato I, Cappelli K, Mechelli L, et al.. Investigation of the Epithelial to Mesenchymal Transition (EMT) Process in Equine Papillomavirus-2 (EcPV-2)-Positive Penile Squamous Cell Carcinomas. International Journal of Molecular Sciences. 2021. DOI: https://doi.org/10.3390/ijms221910588

[19] Zakia LS, Herman M, Basso RM, Hernadez J, Araújo J, Borges AS, et al.. Equine papillomavirus detection in aural plaques by qPCR. Brazilian Journal of Veterinary Pathology. 2019. DOI: https://doi.org/10.24070/BJVP.1983-0246.V12I1P1-4

[20] Bromberger CR, Costa JR, Herman M, Hernandez J, Albertino L, Alves C, et al.. Detection of Equus caballus papillomavirus in equine aural plaque samples.. Journal of Equine Veterinary Science. 2023. DOI: https://doi.org/10.2139/ssrn.4393742

[21] Zakia LS, Basso RM, Olivo G, Herman M, Araújo J, Borges AS, et al.. Detection of papillomavirus DNA in formalin-fixed paraffin-embedded equine aural plaque samples. Arquivo Brasileiro De Medicina Veterinaria E Zootecnia. 2015. DOI: https://doi.org/10.1590/1678-4162-8077

[22] Falco FD, Cutarelli A, Pellicanò R, Brandt S, Roperto S. Molecular Detection and Quantification of Ovine Papillomavirus DNA in Equine Sarcoid. Transboundary and Emerging Diseases. 2024. DOI: https://doi.org/10.1155/2024/6453158

[23] Hainisch E, Jindra C, Reicher P, Miglinci L, Brodesser DM, Brandt S. Bovine Papillomavirus Type 1 or 2 Virion-Infected Primary Fibroblasts Constitute a Near-Natural Equine Sarcoid Model. Viruses. 2022. DOI: https://doi.org/10.3390/v14122658

[24] Koch C. Equine sarcoids. Part 1: the aetiopathogenesis of equine sarcoid disease. UK-Vet Equine. 2026. DOI: https://doi.org/10.12968/ukve.2025.0027

[25] Brandt S. Spontaneous regression of equine sarcoids is an exceptional event.. Equine Veterinary Journal. 2026. DOI: https://doi.org/10.1002/evj.70158

[26] Monod A, Koch C, Jindra C, Haspeslagh M, Howald D, Wenker C, et al.. CRISPR/Cas9-Mediated Targeting of BPV-1-Transformed Primary Equine Sarcoid Fibroblasts. Viruses. 2023. DOI: https://doi.org/10.3390/v15091942

[27] Hoikhman R, Molínková D, Pillarova D, Linhart P, Kopecká A, Jahn P. The serological detection of Bovine papillomavirus's E5 oncoprotein antibodies in horses.. Veterinary Immunology and Immunopathology. 2023. DOI: https://doi.org/10.1016/j.vetimm.2023.110633

[28] Jindra C, Hainisch E, Brandt S. Immunotherapy of Equine Sarcoids, From Early Approaches to Innovative Vaccines. Vaccines. 2023. DOI: https://doi.org/10.3390/vaccines11040769

[29] Yamashita-Kawanishi N, Chambers J, Uchida K, Tobari Y, Yoshimura H, Yamamoto M, et al.. Genomic characterisation of bovine papillomavirus types 1 and 2 identified in equine sarcoids in Japan.. Equine Veterinary Journal. 2020. DOI: https://doi.org/10.1111/evj.13398

[30] Gysens L, Vanmechelen B, Maes P, Martens A, Haspeslagh M. Complete genomic characterization of bovine papillomavirus type 1 and 2 strains infers ongoing cross-species transmission between cattle and horses.. The Veterinary Journal. 2023. DOI: https://doi.org/10.1016/j.tvjl.2023.106011

[31] Hainisch E, Jindra C, Kirnbauer R, Brandt S. Papillomavirus-like Particles in Equine Medicine. Viruses. 2023. DOI: https://doi.org/10.3390/v15020345

[32] Jindra C, Hainisch E, Rümmele A, Wolschek M, Muster T, Brandt S. Influenza virus vector iNS1 expressing bovine papillomavirus 1 (BPV1) antigens efficiently induces tumour regression in equine sarcoid patients. PLoS ONE. 2021. DOI: https://doi.org/10.1371/journal.pone.0260155

[33] Cappelli K, Ciucis CD, Mecocci S, Nervo T, Crescio MI, Pepe M, et al.. Detection of Equus Caballus Papillomavirus Type-2 in Asymptomatic Italian Horses. Viruses. 2022. DOI: https://doi.org/10.3390/v14081696

[34] Strohmayer C, Klang A, Kummer S, Walter I, Jindra C, Weissenbacher-Lang C, et al.. Tumor Cell Plasticity in Equine Papillomavirus-Positive Versus-Negative Squamous Cell Carcinoma of the Head and Neck. Pathogens. 2022. DOI: https://doi.org/10.3390/pathogens11020266

[35] Ikechukwu C, Qin K, Zhang H, Pan J, Zhang W. Novel equid papillomavirus from domestic donkey.. Equine Veterinary Journal. 2023. DOI: https://doi.org/10.1111/evj.13957

[36] Bacci B, Martinoli G, Gallina L, Avallone G, Brunetti B, Franceschini T, et al.. Immune cell analysis in equine penile papilloma, in situ squamous cell carcinoma and invasive squamous cell carcinoma: FoxP3+ T regulatory lymphocytes differ according to equine papillomavirus 2 status. Veterinary Pathology-Supplement. 2025. DOI: https://doi.org/10.1177/03009858251341544

[37] Peletto S. Papillomavirus infection in equids. UK-Vet Equine. 2025. DOI: https://doi.org/10.12968/ukve.2025.0015

[38] Schwarz S, Kummer S, Klang A, Walter I, Nell B, Brandt S. Detection of vasculogenic mimicry in equine ocular, oronasal, and genital squamous cell carcinoma. bioRxiv. 2025. DOI: https://doi.org/10.1371/journal.pone.0328584

[39] Semik-Gurgul E, Gurgul A, Szmatoła T. Transcriptome and methylome sequencing reveals altered long non-coding RNA genes expression and their aberrant DNA methylation in equine sarcoids. Functional & Integrative Genomics. 2023. DOI: https://doi.org/10.1007/s10142-023-01200-2

[40] Lee S, Yoon J, Kim Y, Lee I. Penile neoplasm associated with Equus caballus papillomavirus type 2 infection in a miniature Appaloosa. Korean Journal of Veterinary Research. 2024. DOI: https://doi.org/10.14405/kjvr.20240011

[41] Portenko M, Shchebentovska O. Clinic and anatomic aspects of verification and monitoring of various types of equine sarcoid in the western regions of Ukraine. Scientific Messenger of LNU of Veterinary Medicine and Biotechnologies. 2023. DOI: https://doi.org/10.32718/nvlvet10918

[42] Parkinson N, Ward A, Malbon AJ, Reardon R, Kelly P. Bovine papillomavirus gene expression and inflammatory pathway activation vary between equine sarcoid tumour subtypes.. Veterinary Immunology and Immunopathology. 2024. DOI: https://doi.org/10.1016/j.vetimm.2024.110838

[43] Cotugno R, Gallotta D, d’Avenia M, Corteggio A, Altamura G, Roperto F, et al.. BAG3 protects Bovine Papillomavirus type 1-transformed equine fibroblasts against pro-death signals. Veterinary Research. 2013. DOI: https://doi.org/10.1186/1297-9716-44-61

[44] Finlay MA, Yuan Z, Morgan I, Campo M, Nasir L. Equine sarcoids: Bovine Papillomavirus type 1 transformed fibroblasts are sensitive to cisplatin and UVB induced apoptosis and show aberrant expression of p53. Veterinary Research. 2012. DOI: https://doi.org/10.1186/1297-9716-43-81

[45] Gysens L, Martens A, Haspeslagh M. Performance of fine-needle aspirate testing compared with superficial swab testing for quantification of BPV-1/-2 viral load in equine sarcoids.. Research in Veterinary Science. 2023. DOI: https://doi.org/10.1016/j.rvsc.2023.04.014

[46] Monteiro MM, Castro ELAd, Pereira AJM, Thiesen R, Thiesen RMC, Salvarani FM. BCG Immunotherapy in Equine Sarcoid Treatment: Mechanisms, Clinical Efficacy, and Challenges in Veterinary Oncology. Viruses. 2025. DOI: https://doi.org/10.3390/v17101322

[47] Silva DDD, Martínez E, Bogaert L, Kast W. Investigation of the Optimal Prime Boost Spacing Regimen for a Cancer Therapeutic Vaccine Targeting Human Papillomavirus. Cancers. 2022. DOI: https://doi.org/10.3390/cancers14174339 *** Disclaimer: This article is for educational and informational purposes only. It is not intended to substitute for professional veterinary advice, diagnosis, treatment, or regulatory guidance. Always consult a licensed veterinarian or qualified specialist regarding animal health, disease diagnosis, and therapeutic decisions.