A multicentric intermediate-size B-cell lymphoma with epitheliotropism in a Freiberger mare.

This report describes a multicentric intermediate-size B-cell lymphoma with epitheliotropism in a Freiberger mare affecting multiple mucous membranes, skin and internal organs. The clonal neoplastic B-cell population was accompanied by numerous reactive polyclonal small T cells. Differential diagnoses for these unusual findings are discussed.

An outbreak of Arthroderma vanbreuseghemii dermatophytosis at a veterinary school associated with an infected horse.

We report a case of an outbreak of inflammatory dermatophytoses caused by Arthroderma vanbreuseghemii (formally Trichophyton mentagrophytes pro parte) that involved an infected horse, the owner and at least 20 students, staff and stablemen at a veterinary school in Bern (Switzerland) that presented highly inflammatory dermatitis of the body and the face. Transmission from human to human was also recorded as one patient was the partner of an infected person. Both the phenotypic characteristics and ITS sequence of the dermatophytes isolated from the horse and patients were identical, consistent with the conclusion that the fungus originated from the horse. Three infected persons had not been in direct contact with the horse. Although direct transmission from human to human cannot be ruled out, fomites were most likely the source of infection for these three patients. Inspection of the literature at the end of the nineteenth and beginning of the twentieth century revealed that this dermatophyte was frequently transmitted from horses to humans in contact with horses (stablemen, coachmen, carters and artillery soldiers). The rarity of the present case report at the present time is likely related to the transformation of civilisation from the nineteenth century to nowadays in Europe with the change of horse husbandry. In addition, the inadequate immune response of the horse and the high number of people in contact with it at the equine clinic may explain the exceptional aspect of this case report.

Evaluation of the conjunctival fungal flora and its susceptibility to antifungal agents in healthy horses in Switzerland.

OBJECTIVE: To characterize the conjunctival fungal flora and to determine the susceptibility of 2 isolated molds to antifungal drugs in samples of 64 healthy horses from The National Stud in Switzerland. PROCEDURE: Conjunctival cytobrush samples were collected from both eyes of 64 ophthalmologically normal horses in August 2012 and subsequently cultured on Sabouraud's agar medium. Growing fungi were identified and counted. Etests or broth microdilution tests for Aspergillus fumigatus and Eurotium amstelodami were carried out to determine antifungal drug sensitivity. These species had previously been detected in samples from eyes with keratomycosis in Switzerland. Minimal inhibitory concentrations (MICs) for voriconazole, fluconazole, itraconazole, amphotericin B, and miconazole were recorded. RESULTS: Fifty-nine of the horses were tested positive for fungal growth from at least one eye (92%). Eleven genera of fungi were identified. The most common fungal genera were Alternaria, Eurotium, Rhizopus, and Cladosporium. Aspergillus spp. and Penicillium spp. were isolated frequently, while no Fusarium spp. was found. In only 2 cases, yeasts were identified as Candida guilliermondii. For certain fungal species, the type of bedding and housing appeared to influence their prevalence. Susceptibility testing of A. fumigatus showed lowest MICs for voriconazole, E. amstelodami for voriconazole and itraconazole. High MICs for fluconazole were detected for all tested fungi while MICs for amphotericin B and miconazole were variable. CONCLUSIONS: A large range of fungal mold species was identified including A. fumigatus and E. amstelodami, which have been causative agents of keratomycosis in Switzerland. Best in vitro susceptibility results for these two species were obtained for voriconazole.

A randomized placebo-controlled double-blinded study comparing oral and subcutaneous administration of mistletoe extract for the treatment of equine sarcoid disease.

BACKGROUND: Equine sarcoids (ES) are the most common cutaneous tumors in equids. Systemic treatment options are sparse. Subcutaneous (SC) injections of Viscum album extract (VAE) demonstrate efficacy as a systemic treatment directed against ES. OBJECTIVES/AIM: To critically assess the therapeutic efficacy of orally administered VAE. ANIMALS: Forty-five ES-affected, privately owned, 3-12 year-old horses. METHODS: A 3-armed randomized placebo-controlled, double-blinded study was conducted in a double-dummy design. Horses were subjected to oral administration and SC injections of either VAE or placebo (VAE oral/placebo SC, VAE SC/placebo oral, placebo oral/placebo SC) over a 7-month treatment period. Primary endpoint was the change of baseline of a composite index of ES number and ES area after 14 months. Second endpoint was the clinical response. RESULTS: No statistically significant difference in the composite endpoint between the 3 study arms was found. The primary endpoint showed 4 (27%) horses in the VAE oral group with complete ES regression, 3 (21%) in the VAE SC injection group, and 2 (13%) in the placebo group. The clinical response revealed complete or partial regression in 6 horses of the oral VAE group (40%), 4 of the SC injection group (29%), and 4 of the placebo group (25%). Direct comparison of oral VAE and placebo showed an odds ratio, stratified for prognosis of 2.16 (95%-CI: 0.45-10.42) and a P-value of 0.336. CONCLUSION AND CLINICAL IMPORTANCE: Oral administration of VAE is well tolerated. No statistically significant difference in the effectiveness of systemic VAE versus placebo against ES was found.

Outbreak of equine coronavirus disease in adult horses, Switzerland 2021.

Coronaviruses are causing severe respiratory and enteric diseases in humans and animals. Here, we report an outbreak of equine coronavirus disease in adult horses, detected by a voluntary syndromic surveillance scheme for equine diseases in Switzerland. This scheme allowed a rapid concerted action to diagnose and contain the disease.

Diagnostic potential of three serum microRNAs as biomarkers for equine sarcoid disease in horses and donkeys.

BACKGROUND: MicroRNAs (miRNAs) are potential biomarkers for equine sarcoids (ES). OBJECTIVES: To assess eca-miR-331, eca-miR-100, and eca-miR-1 as serum biomarkers for ES disease. ANIMALS: Sixty-eight ES cases (56 horses, 12 donkeys), 69 tumor-free controls (60 horses, 9 donkeys), and 20 horses with other skin tumors. METHODS: For this case-control study, expression of serum eca-miR-331, eca-miR-100, and eca-miR-1 in ES-affected equids was compared to tumor-free age-, sex-, and breed-matched control horses and donkeys with other skin tumors using reverse transcription quantitative PCR (polymerase chain reaction) for relative miRNA quantification. Biological, preanalytical, and clinical variable influences on miRNA expression were examined. Receiver operator characteristic (ROC) curve analyses were used to determine differences in miRNA expression between groups. RESULTS: The expression of eca-miR-100 was affected by age (P = .003) and expression of eca-miR-100 and eca-miR-1 were affected by hemolysis (both P < .001). Eca-miR-331 was unaffected by biological variation, hemolysis, ES type, and disease severity. Eca-miR-331 concentrations were higher in ES-affected compared to tumor-free controls (P = .002). The ROC curve analysis indicated an area under the curve of 0.65 (P = .002) with a sensitivity of 60%, specificity of 71%, and positive and negative likelihood ratios of 2.1 and 0.56, respectively, to diagnose ES. Eca-miR-331 expression did not discriminate between horses with ES and other skin tumors. Expression of eca-miR-100 and eca-miR-1 was not different between groups. CONCLUSIONS AND CLINICAL IMPORTANCE: Serum eca-miR-331 expression is neither sensitive nor specific enough as a single ES biomarker. If combined with other miRNAs, it may be helpful for ES diagnosis.

Molecular characterization of equine thymidine kinase 1 and preliminary evaluation of its suitability as a serum biomarker for equine lymphoma.

BACKGROUND: Thymidine kinase 1 (TK1) plays a key role in the synthesis of deoxythymidine triphosphate (dTTP) and is thus important for DNA replication and cell proliferation. The expression of TK1 is highest during S-phase, and it is rapidly degraded after mitosis. In cancer cells, TK1 is upregulated, resulting in leakage of excess TK1 into the blood. Consequently, serum TK1 has been used as a diagnostic and prognostic cancer biomarker, mainly in human medicine. The aims of this work were to characterize equine TK1 and to evaluate its suitability as a serum biomarker for equine lymphoma. RESULTS: Equine TK1 was cloned, expressed in E. coli and affinity purified. The purified recombinant horse TK1 showed broad substrate specificity, phosphorylating pyrimidine deoxyribo- and ribonucleosides and, to some extent, purine deoxynucleosides, including anticancer and antiviral nucleoside analogues. ATP was the preferred phosphate donor. Serum TK1 activity was measured in samples collected from horses with confirmed or suspected lymphoma and control horses with and without concurrent diseases. Serum TK1 activity levels were significantly higher in horses with lymphoma (p <  0.0005) and suspected lymphoma (p <  0.02) and in tumour-free groups with diverse diseases (p <  0.03) than in controls without concurrent diseases. There was a significant difference between the lymphoma group and the tumour-free group with diverse diseases (p <  0.0006). Furthermore, receiver operating characteristic analysis revealed a sensitivity of 0.86, a specificity of 0.95 and an AUC (area under the curve) of 0.92 compared to the controls without concurrent diseases, with a sensitivity of 0.97, a specificity of 0.71 and an AUC of 0.88 when compared with the tumour-free group with diverse diseases. CONCLUSION: Equine TK1 showed high specific activity and broader substrate specificity than human TK1. Anticancer and antiviral thymidine analogues were efficiently phosphorylated by horse TK1, suggesting that these analogues might be good candidates for chemotherapy in horses. Serum TK1 activity was significantly higher in horses with lymphoma than in controls. ROC analysis indicated that serum TK1 could serve as a promising cancer biomarker in horses.

Diagnostic and prognostic potential of eight whole blood microRNAs for equine sarcoid disease.

MicroRNAs have been proposed as biomarkers for equine sarcoids, the most prevalent equine skin tumors globally. This study served to validate the diagnostic and prognostic potential of whole blood microRNAs identified in a previous study for long-term equine sarcoid diagnosis and outcome prediction. Based on findings of a clinical examination at the age of 3 years and a follow-up following a further 5-12 years, 32 Franches-Montagnes and 45 Swiss Warmblood horses were assigned to four groups: horses with regression (n = 19), progression (n = 9), new occurrences of sarcoid lesions (n = 19) and tumor-free control horses (n = 30). The expression levels for eight microRNAs (eca-miR-127, eca-miR-432, eca-miR-24, eca-miR-125a-5p, eca-miR-134, eca-miR-379, eca-miR-381, eca-miR-382) were analyzed through reverse transcription quantitative polymerase chain reaction in whole blood samples collected on initial examination. Associations of sex, breed, diagnosis, and prognosis with microRNA expression levels were examined using multivariable analysis of variance. Sex and breed influenced the expression level of five and two microRNAs, respectively. Eca-miR-127 allowed discrimination between sarcoid-affected and tumor-free horses. No variation in microRNA expression was found when comparing horses with sarcoid regression and progression. Expression levels of eca-miR-125a-5p and eca-miR-432 varied in male horses that developed sarcoids throughout the study period in comparison to male control horses. While none of the investigated miRNAs was validated for predicting the prognosis of sarcoid regression / progression within young horses with this condition, two miRNAs demonstrated potential to predict if young male (though not female) tumor-free horse can develop sarcoids within the following years. Sex- and breed- biased miRNAs exist within the equine species and have an impact on biomarker discovery.

Differentially expressed microRNAs, including a large microRNA cluster on chromosome 24, are associated with equine sarcoid and squamous cell carcinoma.

The aim of this study was to investigate microRNA (miRNA) differential expression in the two most common equine skin tumours, equine sarcoid (ES) and squamous cell carcinoma (SCC), and its potential influence on the tumour microenvironment at post-transcriptional level. We investigated miRNA fingerprints in four subgroups: mild (ESM) and aggressive (ESA) ES and ocular SCC (oSCC) and genital SCC (gSCC). Three tumours and three control samples were included in each of the four subgroups. Following next generation sequencing, miRNA differential expression analysis using DESeq2 was carried out. Pathways associated with the human mature homologues of identified dysregulated miRNAs were predicted using DIANA- miRPath v3.0. When comparing tumour vs control tissue, 57 miRNAs in ESM, six in ESA, 47 in oSCC and zero in gSCC were found to be differentially expressed and may thus serve as potential diagnostic tissue biomarkers. Whereas, ES lesions in general were associated with downregulation of the miR-200 family, which may trigger epithelial-mesenchymal transition, ESM lesions were associated with upregulation of the proposed tumour-suppressive miRNA cluster on equine chromosome 24. In contrast, the oSCC tumours showed downregulation of this cluster as well as downregulation of the miR-34 family, which may favour oSCC tumour cell metabolism. To further validate the proposed diagnostic miRNA fingerprints and their suggested biological effects, further miRNA studies need to be carried out in larger study cohorts.

MicroRNA fingerprints in serum and whole blood of sarcoid-affected horses as potential non-invasive diagnostic biomarkers.

Serum and whole blood microRNA (miRNA) fingerprints have been proposed as a new class of non-invasive human cancer biomarkers. In this study, we compared equine sarcoid (ES) disease-specific serum and whole blood miRNA fingerprints and correlated them to miRNA expression in sarcoid tissue. After high throughput sequencing, miRNA differential expression analysis between six ES-affected and five control horses was carried out in serum and whole blood using a DESeq algorithm, accounting for the influence of hemolysis and the white blood cell count. Target gene, pathway prediction and enrichment analyses were conducted using TarBase, mirPath and GeneCodis. After exclusion of 4 hemolyzed out of a total of 11 serum samples, 9 miRNAs were found to be differentially expressed in serum of ES vs control horses. In whole blood, all 11 samples showed normal white blood cell counts and 19 miRNAs were found to be differentially expressed. A total of 2/9 serum and 7/19 whole blood differentially expressed miRNAs were also highly expressed at the tissue level and their predicted target genes were associated with cancer pathways. Serum and whole blood miRNA expression allowed discrimination between ES and control horses and merits further validation in a larger study cohort. The use of whole blood might be superior because it has higher miRNA content and is less influenced by pre-analytical variables compared to serum. Concurrent dysregulation of single miRNAs in tissue and blood suggests a possible biological function of circulating miRNAs.

Differences in miRNA differential expression in whole blood between horses with sarcoid regression and progression.

BACKGROUND: Currently no methods are available to predict the clinical outcome of individual horses with equine sarcoid (ES) disease. OBJECTIVE: To investigate if whole blood microRNA (miRNA) profiles can predict the long-term development of ES tumors. ANIMALS: Five horses with regression and 5 with progression of ES lesions monitored over 5-7 years and 5 control horses free of ES for at least 5 years. METHODS: For this cohort study, RNA extracted from whole blood samples from the regression, progression, and control groups was used for high throughput sequencing. Known and novel miRNAs were identified using miRDeep2 and differential expression analysis was carried out by the DESeq2 algorithm. Target gene and pathway prediction as well as enrichment and network analyses were conducted using TarBase, mirPath, and metaCore from GeneGo. RESULTS: Fourteen miRNAs were differentially expressed between regression and progression groups after accounting for the control condition: 4 miRNAs (28.6%) were upregulated and 10 miRNAs (71.4%) were downregulated with >2-fold change. Seven of the 10 downregulated miRNAs are encoded in an miRNA cluster on equine chromosome 24, homologous to the well-known 14q32 cluster in humans. Their target genes show enrichment for pathways involved in viral carcinogenesis. CONCLUSIONS AND CLINICAL IMPORTANCE: Whole blood miRNA expression profiles are associated with long-term ES growth in horses and warrant further validation as prognostic biomarkers in a larger study cohort. Deregulation of miRNAs on equine chromosome 24 might represent a trigger for ES development.

Optimized methods for extracting circulating small RNAs from long-term stored equine samples.

Circulating miRNAs in body fluids, particularly serum, are promising candidates for future routine biomarker profiling in various pathologic conditions in human and veterinary medicine. However, reliable standardized methods for miRNA extraction from equine serum and fresh or archived whole blood are sorely lacking. We systematically compared various miRNA extraction methods from serum and whole blood after short and long-term storage without addition of RNA stabilizing additives prior to freezing. Time of storage at room temperature prior to freezing did not affect miRNA quality in serum. Furthermore, we showed that miRNA of NGS-sufficient quality can be recovered from blood samples after >10 years of storage at -80 °C. This allows retrospective analyses of miRNAs from archived samples.

European outbreak of atypical myopathy in the autumn 2009.

BACKGROUND: Atypical myopathy is an acute, severe rhabdomyolysis occurring in grazing horses. In the beginning of October 2009, a new outbreak occurred in several European countries. Geographic, demographic and clinical data of the reported cases in the month October 2009 are described. KEY FINDINGS: The survival rate in this outbreak was 25%. The most frequently observed clinical signs were congested mucous membranes, dyspnea, tachycardia, depression, weakness, stiffness, recumbency, trembling, sweating, and myoglobinuria. Nonsurvivors were significantly more likely to be recumbent than survivors. Prognostic factors, symptomatic treatment, and preventive measures are discussed. SIGNIFICANCE: Differences were encountered during the described outbreak of atypical myopathy in October 2009 compared with previous outbreaks reported. Equine practitioners should be aware that previous epidemiological studies have shown that after a high prevalence in the autumn, new cases are likely to occur in the following spring.

Immune response of horses to vaccination with the recombinant Hc domain of botulinum neurotoxin types C and D.

Botulinum neurotoxins, predominantly serotypes C and D, cause equine botulism through forage poisoning. The C-terminal part of the heavy chain of botulinum neurotoxin types C and D (HcBoNT/C and D) was expressed in Escherichia coli and evaluated as a recombinant mono- and bivalent vaccine in twelve horses in comparison to a commercially available toxoid vaccine. A three-dose subcutaneous immunization of adult horses elicited robust serum antibody response in an ELISA using the immunogen as a capture antigen. Immune sera showed dose-dependent high potency in neutralizing specifically the active BoNT/C and D in the mouse protection assay. The aluminium hydroxide based mono- and bivalent recombinant HcBoNT/C and D vaccines were characterized by good compatibility and the ability to elicit protective antibody titers similar or superior to the commercially available toxoid vaccine.