Agreement among equine veterinarians and between equine veterinarians and inertial sensor system during clinical examination of hindlimb lameness in horses.

BACKGROUND: Hindlimb lameness evaluation is known to be challenging. Experience is essential for the ability of equine veterinarians to detect lameness. Nevertheless, even an experienced veterinarian is still subject to bias. Objective lameness detecting methods have been established to aid veterinarians. OBJECTIVES: 1) To estimate the effect of experience on the interobserver agreement and the agreement between a body-mounted inertial sensor system (BMISS) and veterinarians on detecting hindlimb lameness, and 2) to estimate the agreement between the BMISS and highly experienced veterinarians on change in lameness after diagnostic analgesia. STUDY DESIGN: Cross-sectional study. METHODS: Twenty-six horses with hindlimb lameness were evaluated in clinical conditions by clinicians and simultaneously measured by the BMISS. Videos of their lameness examination were recorded and shown to 13 veterinarians from three groups of varying experience for evaluation. The interobserver agreement and the agreement between veterinarians and the BMISS were calculated. RESULTS: Interobserver agreement from all three groups was recorded as 'fair'. The strength of agreement between veterinarians and BMISS was 'fair' for the highly experienced group, 'slight to fair' for the moderately experienced group and 'slight' in the inexperienced group. The BMISS and the highly experienced veterinarians declared a 'strong' agreement in assigning an improvement in lameness after diagnostic analgesia. MAIN LIMITATIONS: Lameness evaluation through video viewing might be more challenging for some evaluators than live situations. CONCLUSIONS: Given the task of evaluating videos of horses trotting in a straight line, the more experienced veterinarians did not show more reliability than those with less experience. Due to 1) the moderate agreement between the BMISS and clinicians (highly experienced and moderately experienced) in the live clinical evaluation in determining hindlimb lameness, and 2) the strong association between the BMISS and highly experienced veterinarians in determining improvement of lameness after anaesthesia, therefore the use of the BMISS as a supporting tool for veterinarians is encouraged.

DNA methylation patterns of the S100A14, POU2F3 and SFN genes in equine sarcoid tissues.

Genetic and epigenetic alterations in the equine sarcoid, a locally invasive skin tumour of equids, are still poorly characterized. Numerous studies have provided reliable evidence for the relationship between the development of cancer and the loss of function of a number of tumour suppressor genes. In the present study, we assessed methylation levels in the promoter region of SFN, S100A14 and POU2F3 genes in sarcoid samples to clarify whether DNA methylation may be associated with previously identified changes in the expression level of these genes during the course of tumour progression. Using bisulfite sequencing and clone sequencing, we detected that lesional samples had a significantly higher rate of DNA methylation in the analyzed S100A14A region than the corresponding normal skin tissue. A frequent methylation of the SFN and POU2F3 promoter sequences were observed in both the tumour samples and the control skin tissues. Further studies are needed to evaluate the role of aberrant methylation in sarcoid progression and to understand the mechanisms involved in reduced expression of SFN, S100A14 and POU2F3 genes in the lesional tissues.

Comparative analysis of DNA methylation patterns of equine sarcoid and healthy skin samples.

OBJECTIVE: In this study, for the first time we report the genome-wide DNA methylation profile of skin tumour in horses and describe differentially methylated genomic regions (DMRs) with respect to healthy skin. MATERIALS & METHODS: The comparative analysis of DNA methylation patterns detected using Reduced Representation Bisulfite Sequencing (RRBS) technique, allowed identification of 136 regions showing differential methylation between sarcoid and normal skin tissue. RESULTS: Most of the identified DMRs were short fragments, less than 1 kb in size, located in the intergenic regions. Among identified DMRs there were also regions located within genes directly or indirectly related with oncogenesis. We additionally validated 9 CpG sites showing hypomethylation and 9 CpG sites that were hypermethylated in lesional sample, confirming the identified changes in the DNA methylation. CONCLUSION: Knowledge on the changes taking place in the process of DNA methylation may provide a basis for the development of new alternative diagnostic or therapeutic approaches to equine sarcoids.

Transcriptome analysis of equine sarcoids.

Equine sarcoids are the most commonly detected skin tumours in Equidae. In the present research, a comparative transcriptomic analysis was performed which aimed at looking inside a tumour biology and identification of the expression profile as a potential source of cancer specific genes useful as biomarkers. We have used Horse Gene Expression Microarray data from matched equine sarcoids and tumour-distant skin samples. In total, 901 significantly differentially expressed genes (DEGs) between lesional and healthy skin samples have been identified (fold change ≥ 2; P < 0.05). The large subset of DEGs, with decreased expression, was associated with a suppression of malignant transformation, whereas several overexpressed genes were involved in the processes associated with growth and progression of a tumour or immune system activity. Our results, as a first to date, showed comprehensive transcriptome analysis of skin tumour in horses and pinpointed significant pathways and genes related with oncogenesis processes.

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.