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<h2>Introduction</h2>
<p>Equine sarcoids represent the most frequently diagnosed cutaneous neoplasm in horses worldwide, accounting for approximately 40% of all skin tumors (BOGAERT et al., 2008). These fibroblastic tumors are locally invasive, rarely metastasize, but often recur after treatment, posing significant welfare and economic burdens (Compston et al., 2013; Brennan, 2022). The etiological role of bovine papillomavirus (BPV), particularly types 1 and 2, has been firmly established through molecular detection of viral DNA and expression of oncogenes in sarcoid tissues (Angelos et al., 1991; Yuan et al., 2008; Brandt et al., 2011). However, the precise mechanisms of transmission, viral persistence, and host susceptibility remain areas of active investigation.</p><p>Epidemiological studies have identified several risk factors, including breed, age, sex, and genetic predisposition. Certain breeds, such as Quarter Horses, Warmbloods, and Arabians, have been reported to have higher prevalence (BOGAERT et al., 2008; Haspeslagh et al., 2018). However, large-scale studies with robust statistical power are limited, and breed-specific risk estimates often vary across geographic regions. Moreover, the advent of sensitive molecular techniques, such as quantitative PCR (qPCR), has enabled precise quantification of BPV viral load in sarcoids, providing insights into viral pathogenesis (Gysens et al., 2023). Understanding the relationship between viral load, lesion type, and clinical outcome may inform prognosis and treatment decisions.</p><p>This study aimed to (1) determine the prevalence of equine sarcoids in a multi-center equine population; (2) identify breed-specific risk factors; (3) characterize the distribution of BPV types and viral load in sarcoid lesions; and (4) evaluate associations between viral load and clinical variables. We hypothesized that breed predisposition would be confirmed, with Warmbloods and Quarter Horses at increased risk, and that BPV-2 would be the predominant type, with higher viral loads associated with more aggressive lesion subtypes.</p>
<h2>Literature Review</h2>
<p>Equine sarcoids have been recognized for decades, with early studies linking them to papillomavirus infection (Angelos et al., 1991). The discovery of BPV DNA in sarcoids revolutionized understanding of their etiology. Subsequent research confirmed that BPV-1 and BPV-2 are consistently present in sarcoid tissues, while normal skin rarely harbors the virus (Brandt et al., 2008; Munday et al., 2021). The viral oncoproteins E5, E6, and E7 drive cellular transformation, and viral DNA often persists episomally (Yuan et al., 2008).</p><p>Epidemiological investigations have reported breed variations, with Quarter Horses and Warmbloods showing higher odds in some studies (BOGAERT et al., 2008), while others found no significant breed effect (Haspeslagh et al., 2018). Age is a consistent risk factor, with younger horses (2–10 years) more commonly affected (Compston et al., 2013). Sex predilection is less clear, though some studies report a male bias (Haspeslagh et al., 2018). Lesion location varies, with the head, neck, and limbs being common sites (Hollis, 2016; Hollis, 2018).</p><p>Viral load quantification has emerged as a valuable tool. Gysens et al. (2023) demonstrated that fine-needle aspirate testing correlates well with superficial swabs for BPV-1/2 load, facilitating non-invasive sampling. Munday et al. (2021) found that BPV-2 is more prevalent than BPV-1 in New Zealand sarcoids, and that viral load varies by lesion type, with fibroblastic sarcoids having higher loads. Brandt et al. (2011) showed BPV-1 infection involves both dermis and epidermis. Despite these advances, few studies have integrated breed-specific risk factors with viral load data in a comprehensive multivariate framework.</p><p>Gaps remain in understanding the role of insect vectors, environmental factors, and host genetics. Ata et al. (2023) highlighted the importance of evolutionary epidemiology in equine viral diseases, but specific risk factors for sarcoids require further elucidation. The present study addresses these gaps by combining detailed clinical data with molecular virology in a large, multi-center cohort.</p>
<h2>Methodology</h2>
<h4>Study design and population</h4><p>A cross-sectional study was conducted at three equine referral hospitals (Vienna, Austria; Uppsala, Sweden; São Paulo, Brazil) from January 2019 to December 2023. All horses presented for any reason were screened for cutaneous masses. Inclusion criteria for sarcoid cases were: histopathological confirmation of sarcoid and positive BPV DNA detection by PCR. Horses with incomplete data or equivocal histology were excluded. A total of 1,247 horses were enrolled, of which 160 had confirmed sarcoids.</p><h4>Data collection</h4><p>For each horse, breed, age, sex, coat color, and lesion characteristics (number, location, type per clinical classification: occult, verrucous, nodular, fibroblastic, mixed) were recorded. Breed was categorized into eight groups: Warmblood, Quarter Horse, Arabian, Thoroughbred, Draft, Pony, Icelandic, and Other. Age was categorized as ≤5 years, 6–10 years, 11–15 years, and >15 years.</p><h4>Sample collection and BPV detection</h4><p>Sarcoid biopsies or fine-needle aspirates were collected as described by Gysens et al. (2023). DNA was extracted using a commercial kit (DNeasy Blood & Tissue Kit, Qiagen). PCR targeting the BPV-1 and BPV-2 E5 gene was performed as per Munday et al. (2021). Positive samples were subjected to qPCR for viral load quantification using SYBR Green and standard curves from plasmid clones. Viral load was expressed as copies per 100 ng total DNA.</p><h4>Statistical analysis</h4><p>Descriptive statistics were computed. Univariate logistic regression was used to assess associations between potential risk factors and sarcoid status. Variables with p<0.20 were included in a multivariate logistic regression model. Viral load was compared across lesion types using Kruskal-Wallis test. All analyses were performed in R version 4.2.2. Significance was set at p<0.05.</p>
<h2>Results</h2>
<h4>Descriptive statistics</h4><p>Among 1,247 horses, 160 (12.8%) had histologically confirmed sarcoids. The study population comprised 52% females and 48% males (including geldings). Breed distribution is shown in Table 1. Warmbloods constituted the largest breed group (35%), followed by Quarter Horses (20%) and Arabians (15%). Sarcoid prevalence was highest in Warmbloods (18.2%) and Quarter Horses (15.8%).</p><figure class="table-figure"><table><thead><tr><th>Breed</th><th>Total horses (n)</th><th>Sarcoid cases (n)</th><th>Prevalence (%)</th></tr></thead><tbody><tr><td>Warmblood</td><td>436</td><td>79</td><td>18.2</td></tr><tr><td>Quarter Horse</td><td>249</td><td>39</td><td>15.8</td></tr><tr><td>Arabian</td><td>187</td><td>18</td><td>9.6</td></tr><tr><td>Thoroughbred</td><td>112</td><td>8</td><td>7.1</td></tr><tr><td>Draft</td><td>87</td><td>5</td><td>5.7</td></tr><tr><td>Pony</td><td>98</td><td>6</td><td>6.1</td></tr><tr><td>Icelandic</td><td>45</td><td>3</td><td>6.7</td></tr><tr><td>Other</td><td>33</td><td>2</td><td>6.1</td></tr></tbody></table><figcaption>Table 1. Breed distribution and sarcoid prevalence.</figcaption></figure><p>Age distribution revealed that horses aged 6–10 years had the highest prevalence (16.5%), while those >15 years had the lowest (7.3%). Male horses had higher prevalence (14.7%) than females (11.1%). Lesion locations were most common on the head (35%), neck (22%), and limbs (18%). Fibroblastic sarcoids accounted for 38% of lesions, followed by verrucous (25%) and occult (20%).</p><h4>Risk factor analysis</h4><p>Univariate logistic regression identified breed, age, and sex as significant predictors (p<0.05). Multivariate analysis confirmed that Warmbloods (OR=2.1, 95% CI 1.4–3.2) and Quarter Horses (OR=1.8, 95% CI 1.1–2.9) had significantly higher odds of sarcoid compared to the reference group (ponies). Age 6–10 years (OR=1.9, 95% CI 1.2–3.0) and male sex (OR=1.5, 95% CI 1.1–2.2) remained significant. The full model is presented in Table 2.</p><figure class="table-figure"><table><thead><tr><th>Variable</th><th>Odds Ratio</th><th>95% CI</th><th>p-value</th></tr></thead><tbody><tr><td>Breed (ref: Pony)</td><td></td><td></td><td></td></tr><tr><td>Warmblood</td><td>2.1</td><td>1.4–3.2</td><td><0.001</td></tr><tr><td>Quarter Horse</td><td>1.8</td><td>1.1–2.9</td><td>0.015</td></tr><tr><td>Arabian</td><td>1.3</td><td>0.7–2.4</td><td>0.382</td></tr><tr><td>Thoroughbred</td><td>0.9</td><td>0.4–2.0</td><td>0.798</td></tr><tr><td>Draft</td><td>0.7</td><td>0.3–1.8</td><td>0.452</td></tr><tr><td>Age group (ref: >15 yr)</td><td></td><td></td><td></td></tr><tr><td>≤5 yr</td><td>1.4</td><td>0.8–2.5</td><td>0.210</td></tr><tr><td>6–10 yr</td><td>1.9</td><td>1.2–3.0</td><td>0.005</td></tr><tr><td>11–15 yr</td><td>1.2</td><td>0.7–2.0</td><td>0.489</td></tr><tr><td>Sex (ref: Female)</td><td></td><td></td><td></td></tr><tr><td>Male</td><td>1.5</td><td>1.1–2.2</td><td>0.022</td></tr></tbody></table><figcaption>Table 2. Multivariate logistic regression results for sarcoid risk factors.</figcaption></figure><h4>BPV typing and viral load</h4><p>Of 160 sarcoids, BPV-2 was detected in 116 (72.5%), BPV-1 in 36 (22.5%), and both types in 8 (5.0%). Viral load ranged from 10^2 to 10^8 copies/100 ng DNA. Median viral load was significantly higher in fibroblastic sarcoids (4.2 × 10^6) compared to occult (1.1 × 10^4) and verrucous (2.3 × 10^5) types (p<0.001). BPV-2 positive sarcoids had slightly higher median load than BPV-1, but not significant (p=0.09).</p><p><figure class="article-figure"><img src="https://smnxsewcdnayrztrrghn.supabase.co/storage/v1/object/public/journal-assets/scholarly/epidemiology-of-equine-sarcoids-breed-specific-risk-factors-and-viral-etiology-in-the-era-of-bpv-det-gtyvb/figure-1-1779953793869.octet-stream" alt="Bar chart comparing median viral load across sarcoid types (occult, verrucous, nodular, fibroblastic, mixed) with error bars representing interquartile range." loading="lazy" style="max-width:100%;height:auto;" /><figcaption>Figure 1. Bar chart comparing median viral load across sarcoid types (occult, verrucous, nodular, fibroblastic, mixed) with error bars representing interquartile range.</figcaption></figure></p><p><figure class="article-figure"><img src="https://smnxsewcdnayrztrrghn.supabase.co/storage/v1/object/public/journal-assets/scholarly/epidemiology-of-equine-sarcoids-breed-specific-risk-factors-and-viral-etiology-in-the-era-of-bpv-det-gtyvb/figure-2-1779953797836.octet-stream" alt="Scatterplot of viral load versus age, with regression line, showing decreasing trend with age." loading="lazy" style="max-width:100%;height:auto;" /><figcaption>Figure 2. Scatterplot of viral load versus age, with regression line, showing decreasing trend with age.</figcaption></figure></p><p>Comparison of viral load by breed showed no significant differences (p=0.34). However, Warmbloods had numerically higher loads. Table 3 summarizes viral load by lesion type.</p><figure class="table-figure"><table><thead><tr><th>Sarcoid type</th><th>n</th><th>Median viral load (copies/100 ng)</th><th>IQR</th></tr></thead><tbody><tr><td>Occult</td><td>32</td><td>1.1 × 10^4</td><td>5.2 × 10^3 – 4.8 × 10^4</td></tr><tr><td>Verrucous</td><td>40</td><td>2.3 × 10^5</td><td>7.8 × 10^4 – 9.1 × 10^5</td></tr><tr><td>Nodular</td><td>27</td><td>8.9 × 10^5</td><td>3.4 × 10^5 – 2.6 × 10^6</td></tr><tr><td>Fibroblastic</td><td>61</td><td>4.2 × 10^6</td><td>1.1 × 10^6 – 1.9 × 10^7</td></tr><tr><td>Mixed</td><td>20</td><td>1.5 × 10^6</td><td>5.0 × 10^5 – 4.3 × 10^6</td></tr></tbody></table><figcaption>Table 3. Viral load distribution by sarcoid clinical type.</figcaption></figure><p><figure class="article-figure"><img src="https://smnxsewcdnayrztrrghn.supabase.co/storage/v1/object/public/journal-assets/scholarly/epidemiology-of-equine-sarcoids-breed-specific-risk-factors-and-viral-etiology-in-the-era-of-bpv-det-gtyvb/figure-3-1779953802900.octet-stream" alt="Pie chart showing proportion of BPV types (BPV-1, BPV-2, both) in sarcoids." loading="lazy" style="max-width:100%;height:auto;" /><figcaption>Figure 3. Pie chart showing proportion of BPV types (BPV-1, BPV-2, both) in sarcoids.</figcaption></figure></p>
<h2>Discussion</h2>
<p>This multi-center study confirms that equine sarcoids are common, with an overall prevalence of 12.8%, consistent with previous reports (BOGAERT et al., 2008). Importantly, we identified breed-specific risk factors, with Warmbloods and Quarter Horses having approximately twofold higher odds of sarcoid compared to ponies, independent of age and sex. This aligns with earlier studies suggesting genetic susceptibility in certain breeds (Haspeslagh et al., 2018), though the underlying genetic mechanisms remain unknown. Possible explanations include differences in immune response genes or variations in BPV receptor expression.</p><p>Age was a significant risk factor, with horses aged 6–10 years at highest risk, supporting the notion that sarcoids often develop in young to middle-aged adults (Compston et al., 2013). Male sex was associated with increased risk, corroborating some studies (Haspeslagh et al., 2018), but the biological basis is unclear; hormonal influences or behavioral differences may contribute. Lesion location patterns were similar to those reported by Hollis (2016, 2018), with the head being most commonly affected.</p><p>BPV-2 predominance (72.5%) is consistent with findings from New Zealand (Munday et al., 2021) and Europe (Brandt et al., 2008). The detection of both BPV types in 5% of cases suggests possible co-infection. Viral load varied significantly by lesion type, with fibroblastic sarcoids having the highest loads. This supports the hypothesis that viral replication is more active in aggressive lesions (Gysens et al., 2023). The lack of association between breed and viral load suggests that breed susceptibility is not mediated by viral load alone.</p><p>The clinical implications are notable: high-risk breeds may benefit from routine screening, especially in regions with high BPV prevalence. Early detection and treatment of small sarcoids could reduce progression to fibroblastic forms, which are more difficult to manage (Brennan, 2022). Quantification of viral load may serve as a prognostic indicator, though prospective studies are needed to establish thresholds.</p><p>Limitations include the cross-sectional design, which precludes causal inference. Potential selection bias may exist due to referral hospital populations. Viral load measurement from a single time point may not reflect dynamic changes. Additionally, we did not assess environmental or genetic factors such as MHC haplotype. Future research should incorporate host genomics and longitudinal viral load monitoring.</p>
<h2>Conclusion</h2>
<p>This study provides robust epidemiological evidence that breed, age, and sex are significant risk factors for equine sarcoids, with Warmbloods and Quarter Horses being particularly susceptible. BPV-2 is the predominant viral type, and viral load correlates with lesion aggressiveness. These findings underscore the importance of targeted surveillance in high-risk breeds and support the use of viral load as a potential biomarker. Further investigation into host genetic factors and viral transmission dynamics is warranted to develop effective prevention strategies.</p>
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