Treatment of clinically diagnosed equine sarcoid with a mistletoe extract (Viscum album austriacus)

BACKGROUND: Equine sarcoids (ES) are common, difficult to treat, and have high recurrence rates. Viscum album extracts (VAE) are used in human cancer treatment. HYPOTHESIS: That therapy with VAE (Iscador P) is effective in the treatment of ES. ANIMALS: Fifty-three horses (444 ES); 42 were treated with VAE or placebo as monotherapy; 11 were treated with VAE or placebo after selective excision of ES. METHODS: Prospective, randomised, blinded, clinical trial. Horses were randomly assigned to treatment (VAE; n=32) or control group (Placebo; n=21). One milliliter of VAE (Iscador P) in increasing concentrations from 0.1 to 20 mg/mL or physiological NaCl solution was given SC 3 times a week over 105 days. Number, localization, and type of the ES were documented over 12 months. A subset of 163 clinically diagnosed equine sarcoid (CDES) lesions (95 VAE, 68 Placebo) was evaluated in detail, considering clinical findings and tumor volume. RESULTS: No undesired adverse effects were observed except for mild edema at the injection site in 5 of 32 horses (16%). Complete or partial regression was observed in 13 horses of the VAE group (41%) and in 3 of the control horses (14%; P<.05). After VAE treatment, 48 of 95 CDES (67%) showed an improvement compared with 17 of 68 CDES in the control group (40%; P<.01). Twenty-seven CDES had disappeared completely in the VAE group (38%) compared with 9 CDES in the control group (13% NS). CONCLUSIONS AND CLINICAL IMPORTANCE: VAE (Iscador P) represents a safe and effective treatment for CDES.

Intralesional bovine papillomavirus DNA loads reflect severity of equine sarcoid disease

REASONS FOR PERFORMING STUDY: Sarcoids are nonmetastasising, yet locally aggressive skin tumours that constitute the most frequent neoplasm in equids. Infection by bovine papillomaviruses types 1 and 2 (BPV-1, BPV-2) has been recognised as major causative factor in sarcoid pathogenesis, but a possible correlation of intralesional virus load with disease severity has not been established thus far. HYPOTHESIS: Given the pathogenic role of BPV-1 and BPV-2 in sarcoid disease, we suggest that intralesional viral DNA concentration may reflect the degree of affection. METHODS: Severity of disease was addressed by recording the tumour growth kinetics, lesion number and tumour type for 37 sarcoid-bearing horses and one donkey. Viral load was estimated via quantitative real-time PCR (qPCR) of the E2, E5, L1 and L2 genes from the BPV-1/-2 genome for one randomly selected lesion per horse and correlated with disease severity. RESULTS: Quantitative PCR against E2 identified viral DNA concentrations ranging from 0-556 copies/tumour cell. Of 16 horses affected by quiescent, slowly growing single tumours or multiple mild-type lesions, 15 showed a viral load up to 1.4 copies per cell. In stark contrast, all equids (22/22) bearing rapidly growing and/or multiple aggressive sarcoids had a viral load between 3 and 569 copies per cell. Consistent results were obtained with qPCR against E5, L1 and L2. CONCLUSIONS: While tumours of the same clinical type carried variable virus load, confirming that viral titre does not determine clinical appearance, we identified a highly significant correlation between intralesional viral load and disease severity. POTENTIAL RELEVANCE: The rapid determination of BPV viral load will give a reliable marker for disease severity and may also be considered when establishing a therapeutic strategy.

Inheritance of equine sarcoid disease in Franches-Montagnes horses.

The mode of inheritance for susceptibility to equine sarcoid disease (ES) remains unknown. The objectives of this study were to analyse a large sample of the Franches-Montagnes (FM) horse population and investigate the heritability and mode of inheritance for susceptibility to ES. Horses were clinically examined for the presence of sarcoid tumours. A standardized examination protocol and client questionnaire were used and a pedigree- and subsequent segregation-analysis for the ES trait performed. To investigate the mode of inheritance, five models were evaluated and compared in a hierarchical way. The analyses reveal that variation in susceptibility to ES is best explained by a model incorporating polygenic variation. The possible effect of a major gene, such as specific equine leukocyte antigen alleles, is unlikely, but cannot be ruled-out entirely. The heritability of the phenotype on the observation scale for the trait 'affected with ES' was estimated to be 8%. A corrected value for the heritability on a liability scale was estimated at 21% and it is therefore possible to estimate breeding values for ES. The arguments against the practical implementation of an estimated breeding value in a multifactorial condition like ES are discussed.

Increased FOXP3 expression in tumour-associated tissues of horses affected with equine sarcoid disease.

Recent studies suggest that regulatory T cells (Tregs) are associated with disease severity and progression in papilloma virus induced neoplasia. Bovine papilloma virus (BPV) is recognised as the most important aetiological factor in equine sarcoid (ES) disease. The aim of this study was to compare expression levels of Treg markers and associated cytokines in tissue samples of ES-affected equids with skin samples of healthy control horses. Eleven ES-affected, and 12 healthy horses were included in the study. Expression levels of forkhead box protein 3 (FOXP3), interleukin 10 (IL10), interleukin 4 (IL4) and interferon gamma (IFNG) mRNA in lesional and tumour-distant samples from ES-affected horses, as well as in dermal samples of healthy control horses were measured using quantitative reverse transcription polymerase chain reaction (PCR). Expression levels were compared between lesional and tumour-distant as well as between tumour-distant and control samples. Furthermore, BPV-1 E5 DNA in samples of ES-affected horses was quantified using quantitative PCR, and possible associations of viral load, disease severity and gene expression levels were evaluated. Expression levels of FOXP3, IL10 and IFNG mRNA and BPV-1 E5 copy numbers were significantly increased in lesional compared to tumour-distant samples. There was no difference in FOXP3 and cytokine expression in tumour-distant samples from ES- compared with control horses. In tumour-distant samples viral load was positively correlated with IL10 expression and severity score. The increased expression of Treg markers in tumour-associated tissues of ES-affected equids indicates a local, Treg-induced immune suppression.

DNA hypomethylation and oxidative stress-mediated increase in genomic instability in equine sarcoid-derived fibroblasts

It is widely accepted that equine sarcoid disease, the most common skin associated neoplasm in equids, is induced by bovine papillomavirus (BPV-1). Although BPV-1 DNA has been found in almost all examined sarcoids so far, its detailed impact on the horse's host cell metabolism is largely unknown. We used equine fibroblast cell lines originating from sarcoid biopsies to study BPV-1-associated changes on DNA methylation status and oxidative stress parameters. Sarcoid-derived fibroblasts manifested increased proliferation in vitro, transcriptional rDNA activity (NORs expression) and DNA hypomethylation compared to control cells. Cells isolated from equine sarcoids suffered from oxidative stress: the expression of antioxidant enzymes was decreased and the superoxide production was increased. Moreover, increased ploidy, oxidative DNA damage and micronuclei formation was monitored in sarcoid cells. We postulate that both altered DNA methylation status and redox milieu may affect genomic stability in BPV-1-infected cells and in turn contribute to sarcoid pathology.

Peripheral blood mononuclear cells represent a reservoir of bovine papillomavirus DNA in sarcoid-affected equines

Bovine papillomaviruses of types 1 and 2 (BPV-1 and -2) chiefly contribute to equine sarcoid pathogenesis. However, the mode of virus transmission and the presence of latent infections are largely unknown. This study established a PCR protocol allowing detection of equines. To validate this result, a blind PCR was performed from enciphered PBMC DNA derived from 66 horses, revealing E5 in the PBMCs of three individuals with confirmed sarcoids, whereas the remaining 63 sarcoid-free animals were negative for this gene. L1 could not be detected in any PBMC DNA, suggesting either deletion or interruption of this gene in PBMCs of BPV-1/-2-infected equines. These results support the hypothesis that PBMCs may serve as host cells for BPV-1/-2 DNA and contribute to virus latency.

A subset of equine sarcoids harbours BPV-1 DNA in a complex with L1 major capsid protein

Bovine papillomavirus type 1 or 2 (BPV-1, BPV-2) are accepted causal factors in equine sarcoid pathogenesis. Whereas viral genomes are consistently found and expressed within lesions, intact virions have never been detected, thus permissiveness of sarcoids for BPV-1 replication remains unclear. To reassess this issue, an immunocapture PCR (IC/PCR) was established using L1-specific antibodies to capture L1-DNA complexes followed by amplification of the viral genome. Following validation of the assay, 13 sarcoid-bearing horses were evaluated by IC/PCR. Samples were derived from 21 tumours, 4 perilesional/intact skin biopsies, and 1 serum. Tissue extracts from sarcoid-free equines served as controls. IC/PCR scored positive in 14/24 (58.3%) specimens obtained from sarcoid-patients, but negative for controls. Quantitative IC/PCR demonstrated <125 immunoprecipitable viral genomes/50 microl extract for the majority of specimens. Moreover, full-length BPV-1 genomes were detected in a complex with L1 proteins. These complexes may correspond to virion precursors or intact virions.

Sarcoid-derived fibroblasts: links between genomic instability, energy metabolism and senescence.

Bovine papillomavirus 1 (BPV-1) is a well recognized etiopathogenetic factor in a cancer-like state in horses, namely equine sarcoid disease. Nevertheless, little is known about BPV-1-mediated cell transforming effects. It was shown that BPV-1 triggers genomic instability through DNA hypomethylation and oxidative stress. In the present study, we further characterized BPV-1-positive fibroblasts derived from sarcoid tumors. The focus was on cancer-like features of sarcoid-derived fibroblasts, including cell cycle perturbation, comprehensive DNA damage analysis, end-replication problem, energy metabolism and oncogene-induced premature senescence. The S phase of the cell cycle, polyploidy events, DNA double strand breaks (DSBs) and DNA single strand breaks (SSBs) were increased in BPV-1-positive cells compared to control fibroblasts. BPV-1-mediated oxidative stress may contribute to telomere dysfunction in sarcoid-derived fibroblasts. Loss of mitochondrial membrane potential and concurrent elevation in intracellular ATP production may be a consequence of changes in energy-supplying pathways in BPV-1-positive cells which is also typical for cancer cells. Shifts in energy metabolism may support rapid proliferation in cells infected by BPV-1. Nevertheless, sarcoid-derived fibroblasts representing a heterogeneous cell fraction vary in some aspects of metabolic phenotype due to a dual role of BPV-1 in cell transformation and oncogene-induced premature senescence. This was shown with increased senescence-associated β-galactosidase (SA-β-gal) activity. Taken together, metabolic phenotypes in sarcoid-derived fibroblasts are plastic, which are similar to greater plasticity of cancer tissues than normal tissues.

Correlações clínico-patológicas das avaliações da proliferação celular pelos métodos de SgNORs e expressão de Ki67, do índice apoptótico pela expressão de caspase-3 e da expressão da p53 no sarcoide equino

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

[Prevalence of hereditary diseases in three-year-old Swiss Warmblood horses]

The objective of this study was to investigate clinical signs indicating hereditary diseases like equine sarcoid, osteochondrosis (OC) and the idiopathic laryngeal hemiplegia (ILH), and to demonstrate relationships between environment, feeding habits and conformation ("exterieur" evaluation) of the horses. For this purpose, we analyzed veterinary examinations of 403 stallions at the approvals since 1994 examined 493 three-year-old Swiss Warmblood horses, which were shown at the Swiss-Field-Tests in 2005.With the help of the owners a questionnaire on health, environment and feeding habits of the animals was completed. At the same time, the horses were assessed and graded for their "exterieur" (type, conformation, gaits) by judges of the Swiss Sporthorse breeding association. In 11.5% of horses sarcoids were found, 8.7% showed one and 2.8% several tumors.The prevalence of sarcoids in offspring of sires with known sarcoids was not significantly higher than in descendants from stallions without a known history of sarcoids. We found distended joints as a possible symptom of OC in 11.4% of the horses, 3.9% (n = 19) in both tarsal joints.We did not find a relationship between enlarged joints in the offspring and the presence of OC in the sires. Abnormal respiratory noise at work, as a possible sign for ILH, was heard only in 1.2% (n = 6). It is important to note that while we found a high number of sarcoid affected horses compared to other studies, presence of enlarged joints was not very frequent and very few horses showed abnormal respiratory noise. Additionally, we found no correlation between "exterieur" marks and the horse's general health.

Heritabilities of health traits in Swiss Warmblood horses

REASONS FOR PERFORMING THE STUDY: There is a lack of evidence regarding genetic parameters of health traits in Swiss Warmblood horses. OBJECTIVES: To estimate heritabilities of equine sarcoid disease, horn quality of the hooves, prognathism and increased filling of talocrural joints as a possible indicator for osteochondrosis in Swiss Warmblood horses examined at the field tests for 3-year-olds between 2005 and 2013. STUDY DESIGN: Retrospective analysis of breed society database. METHODS: Swiss Warmblood horses were examined clinically by 13 veterinarians at field tests in Switzerland between 2005 and 2013. The presence of sarcoids, horn quality of the hooves, incisor occlusion and increased joint filling were assessed and recorded. Records of 3715 horses were integrated in a pedigree comprising 217,282 horses. Variance components and heritabilities were estimated on the liability scale using MTGSAM. RESULTS: The prevalences of the examined traits were rather low, ranging from 2.4 to 13.0%. Heritabilities estimated were 0.21 ± 0.07 for the occurrence of sarcoids, 0.04 ± 0.02 for hooves with markedly brittle and friable horn quality, 0.03 ± 0.01 for hooves with marked growth ring formation, 0.06 ± 0.03 for prognathism and 0.08 ± 0.04 for increased filling of the talocrural joint (an indicator of possible osteochondrosis). The influence of the examiner on the variance of these observations was considerable. CONCLUSIONS: With the exception of equine sarcoid disease, estimates for the heritabilities for the traits examined here were low. A standardised examination protocol may reduce the variance due to the examiner. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.