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Background: Quality of life (QoL) provides a comprehensive concept underpinning veterinary decision-making that encompasses factors beyond physical health. It becomes particularly pertinent when seeking responsible choices for chronically ill or old horses that emphasise their well-being and a good QoL over the extension of life. How different stakeholders use the concept of QoL is highly relevant when considering the complexity of these decisions in real-life situations., Methods: Seven focus group discussions (N = 39) were conducted to gain insights into how stakeholders assess and use equine QoL in veterinary care decisions for chronically ill and/or old horses. The discussions included horse owners (n = 17), equine veterinarians (n = 7), veterinary officers (n = 6), farriers (n = 4), and horse caregivers (n = 5). The combination of deductive and inductive qualitative content analysis of the group discussions focused on identifying both similarities and differences in the views of these groups regarding QoL for old and/or chronically ill horses., Results: Findings show agreement about two issues: the importance of the individuality of the horse for assessing QoL and the relevance of QoL in making decisions about veterinary interventions. We identified differences between the groups with respect to three issues: the time required to assess QoL, stakeholders' contributions to QoL assessments, and challenges resulting from those contributions. While owners and caregivers of horses emphasised their knowledge of a horse and the relevance of the time they spend with their horse, the veterinarians in the study focused on the differences between their own QoL assessments and those of horse owners. In response to challenges regarding QoL assessments and decision-making, stakeholders described different strategies such as drawing comparisons to human experiences., Conclusions: Differences between stakeholders regarding equine QoL assessments contribute to challenges when making decisions about the care of chronically ill or old horses. The results of this study suggest that individual and collaborative reflection about a horse's QoL should be encouraged, for example by developing practicable QoL assessment tools that support relevant stakeholders in this process., (© 2024. The Author(s).)

Background: Equine parvovirus hepatitis (EqPV-H) can cause Theiler's disease and subclinical hepatitis in horses., Objectives: Assess the frequency of subclinical EqPV-H infection in hospitalized horses and to study viral transmission by investigating potential shedding routes., Animals: One hundred sixteen equids, that presented to the University Equine Hospital of the University of Veterinary Medicine Vienna between February 2021 and March 2022, for causes other than hepatopathy., Methods: In this cross-sectional study, samples (serum, feces, nasal, and buccal swabs) of hospitalized horses were collected. Sera were screened for the presence of anti-EqPV-H antibodies by a luciferase immunoprecipitation system assay. Quantitative PCR was used for the detection of EqPV-H DNA in the samples and a nested PCR was used for further validation., Results: Seroprevalence was 10.3% (12/116) and viremia occurred in 12.9% (15/116) of the serologically positive horses. The detected viral load in serum varied from non-quantifiable amount to 1.3 × 10 6 genome equivalents per milliliter of serum. A low viral load of EqPV-H DNA was detected in 2 nasal swabs and 1 fecal sample., Conclusion and Clinical Importance: EqPV-H DNA was detected in nasal secretions and feces of viremic horses, which could pose a risk to naive hospitalized horses. It is advisable to screen hospitalized horses that are potential donors of blood or plasma to reduce the risk of iatrogenic EqPV-H transmission., (© 2024 The Author(s). Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

Background: To determine whether sensory nerve conduction stimulus threshold measurements of the infraorbital nerve are able to differentiate horses with idiopathic trigeminal-mediated headshaking (i-TMHS) from healthy horses and from horses with secondary trigeminal-mediated headshaking (s-TMHS). In a prospective trial, headshaking horses were examined using a standardized diagnostic protocol, including advanced diagnostics such as computed tomography and 3-Tesla-magnetic resonance imaging (MRI), to differentiate s-TMHS from i-TMHS. Clinically healthy horses served as controls. Within this process, patients underwent general anesthesia, and the minimal sensory nerve conduction stimulus threshold (SNCT) of the infraorbital nerve was measured using a bipolar concentric needle electrode. Sensory nerve action potentials (SNAP) were assessed in 2.5-5 mA intervals. Minimal SNCT as well as additional measurements were calculated., Results: In 60 horses, SNAP could be recorded, of which 43 horses had i-TMHS, six had suspected s-TMHS, three horses had non-facial headshaking, and eight healthy horses served as controls. Controls had a minimal SNCT ≥ 15 mA, whereas 14/43 horses with i-TMHS and 2/6 horses with s-TMHS showed a minimal SNCT ≤ 10 mA. Minimal SNCT ≤ 10 mA showed 100% specificity to distinguish TMHS from controls, but the sensitivity was only 41%., Conclusion: A minimal SNCT of the infraorbital nerve ≤ 10 mA was able to differentiate healthy horses from horses with TMHS. Nevertheless, a higher minimal SNCT did not exclude i-TMHS or s-TMHS and minimal SNCT does not distinguish s-TMHS from i-TMHS., (© 2024. The Author(s).)

Background: Gray horses are predisposed to equine malignant melanoma (EMM) with advancing age. Depending on the tumor's location and size, they can cause severe problems (e.g., defaecation, urination, feeding). A feasible therapy for EMM has not yet been established and surgical excision can be difficult depending on the location of the melanoma. Thus, an effective and safe therapy is needed. Naturally occurring betulinic acid (BA), a pentacyclic triterpene and its synthetic derivate, NVX-207 (3-acetyl-betulinic acid-2-amino-3-hydroxy-2-hydroxymethyl-propanoate) are known for their cytotoxic properties against melanomas and other tumors and have already shown good safety and tolerability in vivo. In this study, BA and NVX-207 were tested for their permeation potential into equine skin in vitro in Franz-type diffusion cell (FDC) experiments after incubation of 5 min, 30 min and 24 h, aiming to use these formulations for prospective in vivo studies as a treatment for early melanoma stages. Potent permeation was defined as reaching or exceeding the half maximal inhibitory concentrations (IC 50 ) of BA or NVX-207 for equine melanoma cells in equine skin samples. The active ingredients were either dissolved in a microemulsion (ME) or in a microemulsion gel (MEG). All of the formulations were transdermally applied but the oil-in-water microemulsion was administered with a novel oxygen flow-assisted (OFA) applicator (DERMADROP TDA)., Results: All tested formulations exceeded the IC 50 values for equine melanoma cells for BA and NVX-207 in equine skin samples, independently of the incubation time NVX-207 applied with the OFA applicator showed a significant time-dependent accumulation and depot-effect in the skin after 30 min and 24 h (P < 0.05)., Conclusions: All tested substances showed promising results. Additionally, OFA administration showed a significant accumulation of NVX-207 after 30 min and 24 h of incubation. Further in vivo trials with OFA application are recommended., (© 2024. The Author(s).)

Malignant melanomas (MMs) are the abnormal proliferation of melanocytes and are one of the lethal skin cancers in humans, equines, and canines. Accordingly, MMs in companion animals can serve as naturally occurring animal models, completing conventional cancer models. The common constitutive activation of the MAPK and PI3K pathways in MMs has been described in all three species. Targeting the related pathways is considered a potential option in comparative oncologic approaches. Herein, we present a cross-species comparative analysis exposing a set of ten melanoma cell lines (one human, three equine, and six canine) derived from primary tumors or metastasis to a pan-RAF and RAF dimer inhibitor (LY3009120). Cellular response (proliferation, biomass, metabolism, early and late apoptosis/necrosis, and morphology) and the presence of pathogenic single-nucleotide variants (SNVs) within the mutational hotspot genes BRAF exon 11 and 15, NRAS exon 2 and 3, KRAS exon 2, and KIT exon 11 were analyzed. This study showed that equine malignant melanoma (EMM) cells (MelDuWi) harbor the KRAS p.Q61H mutation, while canine malignant melanoma (CMM) cells (cRGO1 and cRGO1.2) carry NRAS p.G13R. Except for EMM metastasis cells eRGO6 (wild type of the above-mentioned hotspot genes), all melanoma cell lines exhibited a decrease in dose dependence after 48 and 72 h of exposure to LY3009120, independent of the mutation hotspot landscape. Furthermore, LY3009120 caused significant early apoptosis and late apoptosis/necrosis in all melanoma cell lines except for eRGO6. The anti-tumor effects of LY3009120 were observed in nine melanoma cell lines, indicating the potential feasibility of experimental trials with LY3009120. The present study reveals that the irradiation-resistant canine metastasis cells (cRGO1.2) harboring the NRAS p.G13R mutation are significantly LY3009120-sensitive, while the equine metastases-derived eRGO6 cells show significant resistance to LY3009120, which make them both valuable tools for studying resistance mechanisms in comparative oncology.

Acute abdominal pain (colic) is one of the major equine health threats worldwide and often necessitates intensive veterinary medical care and surgical intervention. Equine coronavirus (ECoV) infections can cause colic in horses but are rarely considered as a differential diagnosis. To determine the frequency of otherwise undetected ECoV infections in horses with acute colic, fresh fecal samples of 105 horses with acute colic and 36 healthy control horses were screened for viruses belonging to the Betacoronavirus 1 species by RT-PCR as well as for gastrointestinal helminths and bacteria commonly associated with colic. Horses with colic excreted significantly fewer strongyle eggs than horses without colic. The prevalence of anaerobic, spore-forming, gram-positive bacteria ( Clostridium perfringens and Clostridioides difficile ) was significantly higher in the feces of horses with colic. Six horses with colic (5.7%) and one horse from the control group (2.8%) tested positive for Betacoronaviruses. Coronavirus-positive samples were sequenced to classify the virus by molecular phylogeny (N gene). Interestingly, in three out of six coronavirus-positive horses with colic, sequences closely related to bovine coronaviruses (BCoV) were found. The pathogenic potential of BCoV in horses remains unclear and warrants further investigation.

In equine stables and their surroundings, a large number of insects are present that can be a nuisance to their equine hosts. Previous studies about dipterans transmitting infectious agents to Equidae have largely focused on Nematocera. For the preparation of this systematic review, the existing literature (until February 2022) was systematically screened for various infectious agents transmitted to Equidae via insects of the suborder Brachycera, including Tabanidae, Muscidae, Glossinidae and Hippoboscidae, acting as pests or potential vectors. The PRISMA statement 2020 (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines for systematic reviews were followed. The two concepts, Brachycera and Equidae, were combined for the search that was carried out in three languages (English, German and French) using four different search engines. In total, 38 articles investigating Brachycera as vectors for viral, bacterial and parasitic infections or as pests of equids were identified. Only 7 of the 14 investigated pathogens in the 38 reports extracted from the literature were shown to be transmitted by Brachycera. This review clearly shows that further studies are needed to investigate the role of Brachycera as vectors for pathogens relevant to equine health.

Horses and other equids can be infected with several viruses of the family Flaviviridae, belonging to the genus Flavivirus and Hepacivirus. This consensus statement focuses on viruses with known occurrence in Europe, with the objective to summarize the current literature and formulate clinically relevant evidence-based recommendations regarding clinical disease, diagnosis, treatment, and prevention. The viruses circulating in Europe include West Nile virus, tick-borne encephalitis virus, Usutu virus, Louping ill virus and the equine hepacivirus. West Nile virus and Usutu virus are mosquito-borne, while tick-borne encephalitis virus and Louping ill virus are tick-borne. The natural route of transmission for equine hepacivirus remains speculative. West Nile virus and tick-borne encephalitis virus can induce encephalitis in infected horses. In the British Isle, rare equine cases of encephalitis associated with Louping ill virus are reported. In contrast, equine hepacivirus infections are associated with mild acute hepatitis and possibly chronic hepatitis. Diagnosis of flavivirus infections is made primarily by serology, although cross-reactivity occurs. Virus neutralization testing is considered the gold standard to differentiate between flavivirus infections in horses. Hepacivirus infection is detected by serum or liver RT-PCR. No direct antiviral treatment against flavi- or hepacivirus infections in horses is currently available and thus, treatment is supportive. Three vaccines against West Nile virus are licensed in the European Union. Geographic expansion of flaviviruses pathogenic for equids should always be considered a realistic threat, and it would be beneficial if their detection was included in surveillance programs., (© 2022 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

Equine hepacivirus (EqHV) is the closest known genetic homologue of hepatitis C virus. An effective prophylactic vaccine is currently not available for either of these hepaciviruses. The equine as potential surrogate model for hepacivirus vaccine studies was investigated, while equine host responses following vaccination with EqHV E2 recombinant protein and subsequent EqHV inoculation were elucidated. Four ponies received prime and booster vaccinations (recombinant protein, adjuvant) four weeks apart (day -55 and -27). Two control ponies received adjuvant only. Ponies were inoculated with EqHV RNA-positive plasma on day 0. Blood samples and liver biopsies were collected over 26 weeks (day -70 to +112). Serum analyses included detection of EqHV RNA, isotypes of E2-specific immunoglobulin G (IgG), nonstructural protein 3-specific IgG, haematology, serum biochemistry, and metabolomics. Liver tissue analyses included EqHV RNA detection, RNA sequencing, histopathology, immunohistochemistry, and fluorescent in situ hybridization. Al-though vaccination did not result in complete protective immunity against experimental EqHV inoculation, the majority of vaccinated ponies cleared the serum EqHV RNA earlier than the control ponies. The majority of vaccinated ponies appeared to recover from the EqHV-associated liver insult earlier than the control ponies. The equine model shows promise as a surrogate model for future hepacivirus vaccine research.

Introduction: Equine granulocytic anaplasmosis (EGA) and equine piroplasmosis (EP) are triggered by tick-borne pathogens - the intracellular bacterium Anaplasma phagocytophilum and the intracellular protozoa Babesia caballi and Theileria equi. These pathogens attack cells in the blood stream and cause similar clinical symptoms and changes in laboratory values. Although the treatment principles are naturally different, similarities in prophylaxis exists due to the transmission route. Tick transmitted pathogens can play a greater role in equine medicine in the future due to various factors, such as the tendency of relevant tick species to spread, but also the increasing import and travel activities of and with pets (both in the context of sporting events and as a leisure activity). While EGA is endemic in Central Europe, EP is a sporadic disease in Switzerland, Austria and Germany. However, EP must be viewed as underdiagnosed, as horses persistently infected with T. equi are also repeatedly detected in Central Europe. These diseases should be considered in horses with a fever and corresponding laboratory changes. Available diagnostic tests are direct pathogen detection by blood smear or PCR, and, indirect antibody detection, which is considered to be highly sensitive and (as a competitive ELISA) also very specific. Acute infections can be detected with PCR, serology is more suitable for chronic infections. A pathogen-free condition after treatment can be demonstrated with decreasing antibody titers in combination with repeated PCR tests. In addition, clinically healthy horses infected with T. equi should be identified by antibody detection and appropriate preventative transmission measures must be initiated. The prophylaxis of tick bites in horses is difficult due to the high exposure, and long-term tick bite prevention can hardly be guaranteed. Monitoring of tick activity and strict measures to prevent the spread of the pathogen within the tick population are therefore of great importance.

The naturally occurring betulinic acid (BA) and its derivative NVX-207 induce apoptosis in equine melanoma cells in vitro. After topical application, high concentrations of the substances can be reached in healthy equine skin. With the aim to investigate the effect and safety of topically applied BA and NVX-207 in horses with melanocytic tumors, the longitudinal, prospective, randomized, double-blind, placebo-controlled study protocol included eighteen Lipizzaner mares with early-stage cutaneous melanoma assigned to three groups. Melanocytic lesions were topically treated either with a placebo, 1% BA or 1% NVX-207 twice a day for 91 days. Caliper measurements, clinical examinations and blood tests were performed to assess the effects and safety of the medication. The topical treatment was convenient and safe. The volumes of tumors treated with BA were significantly reduced over time as compared to tumors treated with the placebo from day 80 of the study. Although treatment with NVX-207 seemed to decrease tumor volume, these results did not reach statistical significance. The findings must be regarded as preliminary due to the limited group size and need to be replicated in a larger cohort with modified pharmaceutical test formulations. Accordingly, the treatment protocol cannot yet be recommended in its current form.

The emergence of West Nile virus (WNV) and Usutu virus (USUV) in addition to the autochthonous tick-borne encephalitis virus (TBEV) in Europe causes rising concern for public and animal health. The first equine case of West Nile neuroinvasive disease in Austria was diagnosed in 2016. As a consequence, a cross-sectional seroprevalence study was conducted in 2017, including 348 equids from eastern Austria. Serum samples reactive by ELISA for either flavivirus immunoglobulin G or M were further analyzed with the plaque reduction neutralization test (PRNT-80) to identify the specific etiologic agent. Neutralizing antibody prevalences excluding vaccinated equids were found to be 5.3% for WNV, 15.5% for TBEV, 0% for USUV, and 1.2% for WNV from autochthonous origin. Additionally, reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to detect WNV nucleic acid in horse sera and was found to be negative in all cases. Risk factor analysis did not identify any factors significantly associated with seropositivity.

Equine parvovirus hepatitis (EqPV-H) was first described in 2018 in a fatal case of Theiler's disease which followed the administration of an equine-origin biological product. The virus has since been frequently identified in serum and liver tissue of horses affected by Theiler's disease-an acute, severe hepatitis characterised by fulminant hepatic necrosis with a fatal outcome in most cases. EqPV-H is hepatotropic, appears to be associated with subclinical to severe hepatitis in horses, and is a likely cause of Theiler's disease. Although this disease is most frequently reported following the administration of equine-origin biological products, it can also occur among in-contact horses. Horizontal transmission may be iatrogenic, via contaminated equine-origin biological products such as equine serum, botulism or tetanus antitoxin, and mesenchymal stem cells or by means of the oral route of infection. Other horizontal transmission routes, for example, arthropod vectors, warrant further investigation. A worldwide prevalence of EqPV-H antibodies and DNA has been reported in asymptomatic horses. EqPV-H-positive horses suffering from acute, severe hepatitis have reportedly developed clinical signs including icterus, lethargy, inappetence, and neurological abnormalities and have had increased liver-associated biochemistry parameters recorded. The most common histopathological abnormalities of the liver have been hepatocellular necrosis, collapse of the lobular architecture, and lymphocytic infiltration. Most horses infected experimentally with EqPV-H have developed subclinical hepatitis, and close temporal associations between peak viraemia, seroconversion, and the onset of hepatitis have been observed. Based on strong evidence indicating that EqPV-H causes hepatitis in horses, veterinarians should consider this virus an important differential diagnosis in such cases. Potential risks associated with the administration of equine-origin biological products must be emphasised., (© 2021 The Authors. Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.)

There is strong evidence that equine parvovirus-hepatitis (EqPV-H) is associated with the onset of Theiler's disease, an acute hepatic necrosis, in horses. However, the impact of this virus on other hepatopathies remains unknown. The objective of this retrospective study was to evaluate the prevalence and quantify the viral loads of EqPV-H in formalin-fixed, paraffin-embedded equine and donkey livers with various histopathologic abnormalities. The pathologies included cirrhosis, circulatory disorders of the liver, toxic and metabolic hepatic diseases as well as neoplastic and inflammatory diseases ( n = 84). Eight normal liver samples were included for comparison as controls. EqPV-H DNA was qualitatively and quantitatively measured by real-time PCR and digital PCR, respectively. The virus was detected in two livers originating from horses diagnosed with abdominal neoplasia and liver metastasis (loads of 5 × 10 3 and 9.5 × 10 3 genome equivalents per million cells). The amount of viral nucleic acids measured indicates chronic infection or persistence of EqPV-H, which might have been facilitated by the neoplastic disease. In summary, this study did not provide evidence for EqPV-H being involved in hepatopathies other than Theiler's disease.

Since its first discovery by Arnold Theiler in 1918, serum hepatitis also known as Theiler's disease has been reported worldwide, causing idiopathic acute hepatitis and liver failure in horses. Recent studies have suggested a novel parvovirus, named equine parvovirus hepatitis (EqPV-H), to be associated with Theiler's disease. Despite the severity and potential fatality of EqPV-H infection, little is known about the possibility of developing chronic infections and putative cross-species infection of equine sister species. In the present longitudinal study, we employed qPCR analysis, serology, and biochemical testing as well as pathology examination of liver biopsies and sequence analysis to investigate potential chronic EqPV-H infection in an isolated study cohort of in total 124 horses from Germany over five years (2013-2018). Importantly, our data suggest that EqPV-H viremia can become chronic in infected horses that do not show biochemical and pathological signs of liver disease. Phylogenetic analysis by maximum likelihood model also confirms high sequence similarity and nucleotide conservation of the multidomain nuclear phosphoprotein NS1 sequences from equine serum samples collected between 2013-2018. Moreover, by examining human, zebra, and donkey sera for the presence of EqPV-H DNA and VP1 capsid protein antibodies, we found evidence for cross-species infection in donkey, but not to human and zebra. In conclusion, this study provides proof for the occurrence of persistent EqPV-H infection in asymptomatic horses and cross-species EqPV-H detection in donkeys.

The naturally occurring betulinic acid (BA) and its derivative NVX-207 show anticancer effects against equine malignant melanoma (EMM) cells and a potent permeation in isolated equine skin in vitro. The aim of the study was to determine the in vivo concentration profiles of BA and NVX-207 in equine skin and assess the compounds' local and systemic tolerability with the intent of developing a topical therapy against EMM. Eight horses were treated percutaneously in a crossover design with 1% BA, 1% NVX-207 or a placebo in a respective vehicle twice a day for seven consecutive days with a seven-day washout period between each formulation. Horses were treated at the neck and underneath the tail. Concentration profiles of the compounds were assessed by high-performance liquid chromatography in the cervical skin. Clinical and histopathological examinations and blood analyses were performed. Higher concentrations of NVX-207 were found in the skin compared to BA. Good systemic tolerability and only mild local adverse effects were observed in all three groups. This study substantiates the topical application of BA and NVX-207 in further clinical trials with horses suffering from EMM; however, penetration and permeation of the compounds may be altered in skin affected by tumors., (© 2020 The Authors. Journal of Veterinary Pharmacology and Therapeutics published by John Wiley & Sons Ltd.)

Equine sarcoid (ES) is the most prevalent skin tumor in equids worldwide. Additionally, aging grey horses frequently suffer from equine malignant melanoma (EMM). Current local therapies targeting these skin tumors remain challenging. Therefore, more feasible topical treatment options should be considered. In order to develop a topical therapy against ES and EMM, betulinyl-bis-sulfamate and NVX-207, derivatives of the naturally occurring betulin and betulinic acid, respectively, were evaluated for their antiproliferative (crystal violet staining assay), cytotoxic (MTS assay) and apoptotic (AnnexinV staining, cell cycle investigations) effects on primary ES cells, EMM cells and equine dermal fibroblasts in vitro. The more potent derivative was assessed for its in vitro penetration and permeation on isolated equine skin within 30 min and 24 h using Franz-type diffusion cells and HPLC analysis. Betulinyl-bis-sulfamate and NVX-207 inhibited the proliferation and metabolism in ES cells, EMM cells and fibroblasts significantly (p < 0.001) in a time- and dose-dependent manner. NVX-207 had superior anticancer effects compared to betulinyl-bis-sulfamate. Both compounds led to the externalization of phosphatidylserines on the cell membrane and DNA fragmentation, demonstrating that the effective mode of action was apoptosis. After 48 h of treatment with NVX-207, the number of necrotic cells was less than 2% in all cell types. Detected amounts of NVX-207 in the different skin layers exceeded the half-maximal inhibitory concentrations calculated by far. Even though data obtained in vitro are auspicious, the results are not unconditionally applicable to the clinical situation. Consequently, in vivo studies are required to address the antitumoral effects of topically applied NVX-207 in ES and EMM patients., Competing Interests: The authors declare that no competing interests exist. The affiliation "BioSolutions Halle GmbH” of Dr. Jutta Kalbitz does not alter our adherence to PLOS ONE policies on sharing data and materials. As described in the Funding Statement, BioSolutions Halle GmbH is not a commercial funder, but an equal partner in the TopiDrugHorse project.

We report details of the first seven equine cases of confirmed West Nile neuroinvasive disease in Austria. The cases presented during summer and autumn of 2016 (n = 2), 2017 (n = 3) and 2018 (n = 2). All horses showed gait abnormalities and 6 of 7 horses exhibited fasciculations and/or tremors, and we provide video recordings of these. Three horses also showed cranial nerve involvement. Following rapid improvement, three horses were discharged. Four horses were euthanized due to the severity of clinical signs and subjected to neuropathological examination. West Nile virus (WNV) lineage 2 nucleic acid was detected in 5 of 7 horses, and WNV-specific neutralizing antibodies in all 7 horses. In addition, serologic evidence of WNV infection was found in two out of fourteen in-contact horses. Horses may be considered a sentinel species for human WNV infections, integrating human and veterinary medicine and thus contributing to the one health concept., (© 2019 The Authors. Transboundary and Emerging Diseases published by Blackwell Verlag GmbH.)

Background: Equine malignant melanoma (EMM) is a frequently occurring dermoepidermal tumor in grey horses. Currently available therapies are either challenging or inefficient. Betulinic acid (BA), a naturally occurring triterpenoid, is a promising compound for cancer treatment. To evaluate the potential of BA as a topical therapy for EMM, its anticancer effects on primary equine melanoma cells and dermal fibroblasts and its percutaneous permeation through isolated equine skin were assessed in vitro., Results: BA showed antiproliferative and cytotoxic effects on both primary equine melanoma cells and fibroblasts in a time- and dose-dependent manner. The lowest half-maximal inhibitory concentrations were obtained 96 h after the beginning of drug exposure (12.7 μmol/L and 23.6 μmol/L for melanoma cells eRGO1 and MelDuWi, respectively, in cytotoxicity assay). High concentrations of the compound were reached in the required skin layers in vitro., Conclusion: BA is a promising substance for topical EMM treatment. Further clinical studies in horses are necessary to assess safety and antitumoral effects in vivo.

Prevalence studies have demonstrated a global distribution of equine hepacivirus (EqHV), a member of the family Flaviviridae. However, apart from a single case of vertical transmission, natural routes of EqHV transmission remain elusive. Many known flaviviruses are horizontally transmitted between hematophagous arthropods and vertebrate hosts. This study represents the first investigation of potential EqHV transmission by mosquitoes. More than 5000 mosquitoes were collected across Austria and analyzed for EqHV ribonucleic acid (RNA) by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Concurrently, 386 serum samples from horses in eastern Austria were analyzed for EqHV-specific antibodies by luciferase immunoprecipitation system (LIPS) and for EqHV RNA by RT-qPCR. Additionally, liver-specific biochemistry parameters were compared between EqHV RNA-positive horses and EqHV RNA-negative horses. Phylogenetic analysis was conducted in comparison to previously published sequences from various origins. No EqHV RNA was detected in mosquito pools. Serum samples yielded an EqHV antibody prevalence of 45.9% (177/386) and RNA prevalence of 4.15% (16/386). EqHV RNA-positive horses had significantly higher glutamate dehydrogenase (GLDH) levels ( p = 0.013) than control horses. Phylogenetic analysis showed high similarity between nucleotide sequences of EqHV in Austrian horses and EqHV circulating in other regions. Despite frequently detected evidence of EqHV infection in Austrian horses, no viral RNA was found in mosquitoes. It is therefore unlikely that mosquitoes are vectors of this flavivirus.

An equine parvovirus-hepatitis (EqPV-H) has been recently identified in association with equine serum hepatitis, also known as Theiler's disease. The disease was first described by Arnold Theiler in 1918 and is often observed with parenteral use of blood products in equines. However, natural ways of viral circulation and potential risk factors for transmission still remain unknown. In this study, we investigated the occurrence of EqPV-H infections in Thoroughbred horses in northern and western Germany and aimed to identify potential risk factors associated with viral infections. A total of 392 Thoroughbreds broodmares and stallions were evaluated cross-sectionally for the presence of anti-EqPV-H antibodies and EqPV-H DNA using a luciferase immunoprecipitation assay (LIPS) and a quantitative PCR, respectively. In addition, data regarding age, stud farm, breeding history, and international transportation history of each horse were collected and analysed. An occurrence of 7% EqPV-H DNA positive and 35% seropositive horses was observed in this study cohort. The systematic analysis of risk factors revealed that age, especially in the group of 11-15-year-old horses, and breeding history were potential risk factors that can influence the rate of EqPV-H infections. Subsequent phylogenetic analysis showed a high similarity on nucleotide level within the sequenced Thoroughbred samples. In conclusion, this study demonstrates circulating EqPV-H infections in Thoroughbred horses from central Europe and revealed age and breeding history as risk factors for EqPV-H infections., Competing Interests: The authors declare no conflict of interest.

An equine parvovirus-hepatitis (EqPV-H) has been recently identified in association with equine serum hepatitis, also known as Theiler's disease. This disease was first described by Arnold Theiler in 1918 and is often observed after applications with blood products in equines. So far, the virus has only been described in the USA and China. In this study, we evaluated the presence of EqPV-H in several commercial serum samples to assess the potential risk of virus transmission by equine serum-based products for medical and research applications. In 11 out of 18 commercial serum samples, EqPV-H DNA was detectable with a viral load up to 10 5 copies/mL. The same serum batches as well as three additional samples were also positive for antibodies against the EqPV-H VP1 protein. The countries of origin with detectable viral genomes included the USA, Canada, New Zealand, Italy, and Germany, suggesting a worldwide distribution of EqPV-H. Phylogenetic analysis of the EqPV-H NS1 sequence in commercial serum samples revealed high similarities in viral sequences from different geographical areas. As horse sera are commonly used for the production of anti-sera, which are included in human and veterinary medical products, these results implicate the requirement for diagnostic tests to prevent EqPV-H transmission.

Atypical myopathy (AM) in horses is caused by ingestion of seeds of the Acer species (Sapindaceae family). Methylenecyclopropylacetyl-CoA (MCPA-CoA), derived from hypoglycin A (HGA), is currently the only active toxin in Acer pseudoplatanus or Acer negundo seeds related to AM outbreaks. However, seeds or arils of various Sapindaceae (e.g., ackee, lychee, mamoncillo, longan fruit) also contain methylenecyclopropylglycine (MCPG), which is a structural analogue of HGA that can cause hypoglycaemic encephalopathy in humans. The active poison formed from MCPG is methylenecyclopropylformyl-CoA (MCPF-CoA). MCPF-CoA and MCPA-CoA strongly inhibit enzymes that participate in β-oxidation and energy production from fat. The aim of our study was to investigate if MCPG is involved in Acer seed poisoning in horses. MCPG, as well as glycine and carnitine conjugates (MCPF-glycine, MCPF-carnitine), were quantified using high-performance liquid chromatography-tandem mass spectrometry of serum and urine from horses that had ingested Acer pseudoplatanus seeds and developed typical AM symptoms. The results were compared to those of healthy control horses. For comparison, HGA and its glycine and carnitine derivatives were also measured. Additionally, to assess the degree of enzyme inhibition of β-oxidation, several acyl glycines and acyl carnitines were included in the analysis. In addition to HGA and the specific toxic metabolites (MCPA-carnitine and MCPA-glycine), MCPG, MCPF-glycine and MCPF-carnitine were detected in the serum and urine of affected horses. Strong inhibition of β-oxidation was demonstrated by elevated concentrations of all acyl glycines and carnitines, but the highest correlations were observed between MCPF-carnitine and isobutyryl-carnitine (r = 0.93) as well as between MCPA- (and MCPF-) glycine and valeryl-glycine with r = 0.96 (and r = 0.87). As shown here, for biochemical analysis of atypical myopathy of horses, it is necessary to take MCPG and the corresponding metabolites into consideration., Competing Interests: After the present study was completed, Aleksandra Zuraw became employed by Definiens AG. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Non-primate hepacivirus (NPHV), a recently discovered hepatotropic virus infecting horses, is phylogenetically the closest known homologue of hepatitis C virus (HCV). The main route for acquiring HCV infection in childhood is vertical transmission. However, nothing is known about the natural mode of transmission for NPHV. To investigate the possibility of vertically transmitted NPHV infection in horses, 20 Thoroughbred broodmares and their foals were monitored during foaling season 2015 until 6 months post-partum. Prepartal serum was taken from the mares, and during foaling umbilical cord blood and colostrum samples were collected. Postnatal serum samples were taken from the foals after delivery. In addition, serum was taken at 3 and 6 months after foaling from all mares and foals. Samples were analysed for the presence of NPHV RNA by quantitative real-time PCR and for the presence of anti-NPHV NS3 antibodies by luciferase immunoprecipitation system. Identified NPHV isolates were sequenced and phylogenetic analysis of the viral glycoproteins was used to track the course of naturally occurring infections and the circulation of distinct isolates within the herd. At parturition, 16 mares were seropositive, including four viraemic mares. Vertical transmission occurred in one of these four mare-foal pairs. Interestingly, NPHV isolates of newly infected foals and mares after 3 and 6 months cluster in their respective pasture herds suggesting another horizontal route of transmission.

The recent discovery of hepatitis C virus (HCV)-related viruses in different animal species has raised new speculations regarding the origin of HCV and the possibility of a zoonotic source responsible for the endemic HCV transmission. As a consequence, these new findings prompt questions regarding the potential for cross-species transmissions of hepaciviruses. The closest relatives to HCV discovered to date are the non-primate hepaciviruses (NPHVs), which have been described to infect horses. To evaluate the risk of a potential zoonotic transmission, we analysed NPHV RNA and antibodies in humans with occupational exposure to horses in comparison with a low-risk group. Both groups were negative for NPHV RNA, even though low seroreactivities against various NPHV antigens could be detected irrespective of the group. In conclusion, we did not observe evidence of NPHV transmission between horses and humans.