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Transdermal drug administration is an elegant method to overcome various side effects of oral or parenteral drug administration. Nevertheless, due to an effective skin barrier, which is provided by the stratum corneum , transdermal drug delivery is sometimes very slow and ineffective. Thus, the effect of a medical device (DERMADROP TDA) for transdermal penetration of drugs in conjunction with a special vehicle emulsion on percutaneous permeation of several substances (with different physicochemical properties) was investigated in Franz-type diffusion cells with porcine skin over 28 h. This medical device disperses pharmaceutical agents via oxygen flow through an application system, which is used in conjunction with specially developed vehicle substances. Substance permeation of various substances with different physicochemical properties (diclofenac, enrofloxacin, flufenamic acid, indomethacin, and salicylic acid) was examined after application with a pipette and with the medical device. Therefore, acceptor media samples were collected up to 28 h after drug administration. Drug concentration in the acceptor medium was determined via high-performance liquid chromatography. Enhanced permeation was observed for diclofenac, enrofloxacin, flufenamic acid, indomethacin, and salicylic acid after oxygen-based administration. This correlates negatively with the molecular weight. Thus, drug administration can effectively be enhanced by a medical device using oxygen.

Hepatitis C virus (HCV) displays a restricted host species tropism and only humans and chimpanzees are susceptible to infection. A robust immunocompetent animal model is still lacking, hampering mechanistic analysis of virus pathogenesis, immune control, and prophylactic vaccine development. The closest homolog of HCV is the equine nonprimate hepacivirus (NPHV), which shares similar features with HCV and thus represents an animal model to study hepacivirus infections in their natural hosts. We aimed to dissect equine immune responses after experimental NPHV infection and conducted challenge experiments to investigate immune protection against secondary NPHV infections. Horses were i.v. injected with NPHV containing plasma. Flow cytometric analysis was used to monitor immune cell frequencies and activation status. All infected horses became viremic after 1 or 2 wk and viremia could be detected in two horses for several weeks followed by a delayed seroconversion and viral clearance. Histopathological examinations of liver biopsies revealed mild, periportally accentuated infiltrations of lymphocytes, macrophages, and plasma cells with some horses displaying subclinical signs of hepatitis. Following viral challenge, an activation of equine immune responses was observed. Importantly, after a primary NPHV infection, horses were protected against rechallenge with the homologous as well as a distinct isolate with only minute amounts of circulating virus being detectable.

The recently discovered nonprimate hepacivirus (NPHV) naturally infects horses and is the closest known homolog of hepatitis C virus to date. Within a follow-up study acute field infections were monitored in four young Thoroughbred horses until the ages of 12-13 months. Serum samples were analyzed for the presence of NPHV RNA and anti-NPHV NS3 antibodies and liver specific parameters were evaluated. The four young horses were not able to clear infection, but remained chronically infected for the entire monitored time period despite the presence of NPHV specific antibodies.

Hypoglycin A (2-amino-3-(2-methylidenecyclopropyl)propanoic acid) is the plant toxin shown to cause atypical myopathy in horses. It is converted in vivo to methylenecyclopropyl acetic acid, which is transformed to a coenzyme A ester that subsequently blocks beta oxidation of fatty acids. Methylenecyclopropyl acetic acid is also conjugated with carnitine and glycine. Acute atypical myopathy may be diagnosed by quantifying the conjugates of methylenecyclopropyl acetic acid plus a selection of acyl conjugates in urine and serum. We describe a new mass spectrometric method for sample volumes of <0.5 mL. Samples were extracted with methanol containing 5 different internal standards. Extracts were analyzed by ultra-high-performance liquid chromatography-tandem mass spectrometry focusing on 11 metabolites. The total preparation time for a series of 20 samples was 100 min. Instrument run time was 14 min per sample. For the quantification of carnitine and glycine conjugates of methylenecyclopropyl acetic acid in urine, the coefficients of variation for intraday quantification were 2.9% and 3.0%, respectively. The respective values for interday were 9.3% and 8.0%. Methylenecyclopropyl acetyl carnitine was detected as high as 1.18 µmol/L in serum (median: 0.46 µmol/L) and 1.98 mmol/mol creatinine in urine (median: 0.79 mmol/mol creatinine) of diseased horses, while the glycine derivative accumulated up to 1.97 mmol/mol creatinine in urine but was undetectable in most serum samples. In serum samples from horses with atypical myopathy, the intraday coefficients of variation for C4-C8 carnitines and glycines were ≤4.5%. Measured concentrations exceeded those in healthy horses by ~10 to 1,400 times., (© 2015 The Author(s).)

Betulinic acid, a pentacyclic triterpene, and its derivatives are promising compounds for cancer treatment in humans. Melanoma is not only a problem for humans but also for grey horses as they have a high potential of developing melanoma lesions coupled to the mutation causing their phenotype. Current chemotherapeutic treatment carries the risk of adverse health effects for the horse owner or the treating veterinarian by exposure to antineoplastic compounds. Most treatments have low prospects for systemic tumor regression. Thus, a new therapy is needed. In this in vitro study, Betulinic acid and its two derivatives B10 and NVX-207, both with an improved water solubility compared to Betulinic acid, were tested on two equine melanoma cell lines (MelDuWi and MellJess/HoMelZh) and human melanoma (A375) cell line. We could demonstrate that all three compounds especially NVX-207 show high cytotoxicity on both equine melanoma cell lines. The treatment with these compounds lead to externalization of phosphatidylserines on the cell membrane (AnnexinV-staining), DNA-fragmentation (cell cycle analysis) and activation of initiator and effector caspases (Caspase assays). Our results indicate that the apoptosis is induced in the equine melanoma cells by all three compounds. Furthermore, we succeed in encapsulating the most active compound NVX-207 in 2-Hydroxyprolyl-β-cyclodextrine without a loss of its activity. This formulation can be used as a promising antitumor agent for treating grey horse melanoma. In a first tolerability evaluation in vivo the formulation was administered every one week for 19 consecutive weeks and well tolerated in two adult melanoma affected horses., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Novel viruses belonging to the genera Hepacivirus and Pegivirus have recently been discovered in horses and other animal species. Viral genomes of non-primate hepaciviruses (NPHV), equine pegivirus 1 (EPgV 1) and Theiler's disease associated virus (TDAV) were detected in a horse serum routinely used for cell culture propagation in our laboratory. Therefore, a study was carried out to further investigate the presence of these human Hepatitis C virus (HCV) related viruses in equine serum based products used in veterinary medicine and for research and to characterize the viral genomes. Without exception all commercially available equine sera purchased for cell culture propagation (n=6) were tested positive for NPHV, EPgV 1 or TDAV genomes by qRT-PCR. Molecular analyses of one single commercial horse serum from Europe confirmed multiple viral genomes, including two TDAV genomes significantly different from the only published TDAV sequence. Furthermore, multiple batches of horse serum pools (n=35) collected for manufacturing of biological products turned out to be positive for NPHV and EPgV 1 genomes. Nevertheless, the final commercial products (n=9) were exclusively tested qRT-PCR negative. Field samples (n=119) obtained from two premises located in the same region as the donor horses were analyzed, demonstrating the frequent presence of NPHV and EPgV 1, but the absence of TDAV genomes. The presence of NPHV, EPgV 1 and TDAV in commercial equine sera and serum based products could have considerable consequences for biosecurity and may also bias the outcome of research studies conducted with related viruses., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Deoxyribonucleic acid (DNA) vaccines are used for experimental immunotherapy of equine melanoma. The injection of complexed linear DNA encoding interleukin (IL)-12/IL-18 induced partial tumour remission in a clinical study including 27 grey horses. To date, the detailed mechanism of the anti-tumour effect of this treatment is unknown., Results: In the present study, the clinical and cellular responses of 24 healthy horses were monitored over 72 h after simultaneous intradermal and intramuscular application of equine IL-12/IL-18 DNA (complexed with a transfection reagent) or comparative substances (transfection reagent only, nonsense DNA, nonsense DNA depleted of CG). Although the strongest effect was observed in horses treated with expressing DNA, horses in all groups treated with DNA showed systemic responses. In these horses treated with DNA, rectal temperatures were elevated after treatment and serum amyloid A increased. Total leukocyte and neutrophil counts increased, while lymphocyte numbers decreased. The secretion of tumour necrosis factor alpha (TNFα) and interferon gamma (IFNγ) from peripheral mononuclear blood cells ex vivo increased after treatments with DNA, while IL-10 secretion decreased. Horses treated with DNA had significantly higher myeloid cell numbers and chemokine (C-X-C motif) ligand (CXCL)-10 expression in skin samples at the intradermal injection sites compared to horses treated with transfection reagent only, suggesting an inflammatory response to DNA treatment. In horses treated with expressing DNA, however, local CXCL-10 expression was highest and immunohistochemistry revealed more intradermal IL-12-positive cells when compared to the other treatment groups. In contrast to non-grey horses, grey horses showed fewer effects of DNA treatments on blood lymphocyte counts, TNFα secretion and myeloid cell infiltration in the dermis., Conclusion: Treatment with complexed linear DNA constructs induced an inflammatory response independent of the coding sequence and of CG motif content. Expressing IL-12/IL-18 DNA locally induces expression of the downstream mediator CXCL-10. The grey horses included appeared to display an attenuated immune response to DNA treatment, although grey horses bearing melanoma responded to this treatment with moderate tumour remission in a preceding study. Whether the different immunological reactivity compared to other horses may contributes to the melanoma susceptibility of grey horses remains to be elucidated.

Background: Equine melanoma has a high incidence in grey horses. Xenogenic DNA vaccination may represent a promising therapeutic approach against equine melanoma as it successfully induced an immunological response in other species suffering from melanoma and in healthy horses. In a clinical study, twenty-seven, grey, melanoma-bearing, horses were assigned to three groups (n = 9) and vaccinated on days 1, 22, and 78 with DNA vectors encoding for equine (eq) IL-12 and IL-18 alone or in combination with either human glycoprotein (hgp) 100 or human tyrosinase (htyr). Horses were vaccinated intramuscularly, and one selected melanoma was locally treated by intradermal peritumoral injection. Prior to each injection and on day 120, the sizes of up to nine melanoma lesions per horse were measured by caliper and ultrasound. Specific serum antibodies against hgp100 and htyr were measured using cell based flow-cytometric assays. An Analysis of Variance (ANOVA) for repeated measurements was performed to identify statistically significant influences on the relative tumor volume. For post-hoc testing a Tukey-Kramer Multiple-Comparison Test was performed to compare the relative volumes on the different examination days. An ANOVA for repeated measurements was performed to analyse changes in body temperature over time. A one-way ANOVA was used to evaluate differences in body temperature between the groups. A p-value < 0.05 was considered significant for all statistical tests applied., Results: In all groups, the relative tumor volume decreased significantly to 79.1 ± 26.91% by day 120 (p < 0.0001, Tukey-Kramer Multiple-Comparison Test). Affiliation to treatment group, local treatment and examination modality had no significant influence on the results (ANOVA for repeated measurements). Neither a cellular nor a humoral immune response directed against htyr or hgp100 was detected. Horses had an increased body temperature on the day after vaccination., Conclusions: This is the first clinical report on a systemic effect against equine melanoma following treatment with DNA vectors encoding eqIL12 and eqIL18 and formulated with a transfection reagent. Addition of DNA vectors encoding hgp100 respectively htyr did not potentiate this effect.

Leukocytes and their functional capacities are used extensively as biomarkers in immunological research. Commonly employed indicators concerning leukocytes are as follows: number, composition in blood, response to discrete stimuli, cytokine release, and morphometric characteristics. In order to employ leukocytes as biomarkers for disease and therapeutic monitoring, physiological variations and influencing factors on the parameters measured have to be considered. The aim of this report was to describe the ranges of selected leukocyte parameters in a sample of healthy horses and to analyse whether age, sex, breed, and sampling time point (time of day) influence peripheral blood leukocyte composition, cell morphology and release of cytokines ex vivo. Flow cytometric comparative characterisation of cell size and complexity in 24 healthy horses revealed significant variance. Similarly, basal release of selected cytokines by blood mononuclear cells also showed high variability [TNFα (65-16,624pg/ml), IFNγ (4-80U/ml), IL-4 (0-5069pg/ml), IL-10 (49-1862pg/ml), and IL-17 (4-1244U/ml)]. Each animal's age influenced leukocyte composition, cell morphology and cytokine release (TNFα, IL-4, IL-10) ex vivo. Geldings showed smaller monocytes and higher spontaneous production of IL-10 when compared to the mares included. The stimulation to spontaneous release ratios of TNFα, IL-4 and IL-17 differed in Warmblood and Thoroughbred types. Sampling time influenced leukocyte composition and cell morphology. In summary, many animal factors - age being the dominant one - should be considered for studies involving the analysis of equine leukocytes. In addition, high inter-individual variances argue for individual baseline measurements., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Unlabelled: Hepatitis C virus (HCV) has a very narrow species and tissue tropism and efficiently replicates only in humans and the chimpanzee. Recently, several studies identified close relatives to HCV in different animal species. Among these novel viruses, the nonprimate hepaciviruses (NPHV) that infect horses are the closest relatives of HCV described to date. In this study, we analyzed the NPHV prevalence in northern Germany and characterized the clinical course of infection and viral tissue tropism to explore the relevance of HCV-related horse viruses as a model for HCV infection. We found that approximately 31.4% of 433 horses were seropositive for antibodies (Abs) against NPHV and approximately 2.5% carried viral RNA. Liver function analyses revealed no indication for hepatic impairment in 7 of 11 horses. However, serum gamma-glutamyl transferase (GGT) concentrations were mildly elevated in 3 horses, and 1 horse displayed even highly elevated GGT levels. Furthermore, we observed that NPHV infection could be cleared in individual horses with a simultaneous emergence of nonstructural (NS)3-specific Abs and transient elevation of serum levels of liver-specific enzymes indicative for a hepatic inflammation. In other individual horses, chronic infections could be observed with the copresence of viral RNA and NS3-specific Abs for over 6 months. For the determination of viral tissue tropism, we analyzed different organs and tissues of 1 NPHV-positive horse using quantitative real-time polymerase chain reaction and fluorescent in situ hydridization and detected NPHV RNA mainly in the liver and at lower amounts in other organs., Conclusion: Similar to HCV infections in humans, this work demonstrates acute and chronic stages of NPHV infection in horses with viral RNA detectable predominantly within the liver., (© 2014 by the American Association for the Study of Liver Diseases.)

Research on equine cytokines is often performed by analyses of mRNA. For many equine cytokines an analysis on the actual protein level is limited by the availability of antibodies against the targeted cytokines. Generation of new antibodies is ongoing but time consuming. Thus, testing the reactivity of commercially available antibodies for cross-reactivity with equine cytokines is of particular interest. Fifteen monoclonal antibodies against IL-1β, IL-6, IL-8, IL-12, IL-18 and Granulocyte Macrophage Colony stimulating factor (GM-CSF) of different species were evaluated for reactivity with their corresponding equine cytokines. Dot Blot (DB) and Western Blot (WB) analyses were performed using recombinant equine cytokines as positive controls. Immunohistochemistry (IHC) was carried out on equine tissue and flow cytometry on equine PBMC as positive controls. As expected, three equine IL-1β antibodies detected equine IL-1β in DB, WB and IHC. For these, reactivity in IHC has not been described before. One of them was also found to be suitable for intracellular staining of equine PBMC and flow cytometric analysis. Two antibodies raised against ovine GM-CSF cross-reacted with equine GM-CSF in DB, WB and IHC. For these anti-GM-CSF mAbs this is the first experimental description of cross-reactivity with equine GM-CSF (one mAb was predicted to be cross-reactive in WB in the respective data sheet). The other clone additionally proved to be appropriate in flow cytometric analysis. Two mAbs targeting porcine IL-18 cross-reacted in IHC, but did not show specificity in the other applications. No reactivity was shown for the remaining five antibodies in DB, although cross-reactivity of two of the antibodies was described previously. The results obtained in this study can provide beneficial information for choosing of antibodies for immunological tests on equine cytokines., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Cerebellar abiotrophy (CA) is a rare but significant disease in Arabian horses caused by progressive death of the Purkinje cells resulting in cerebellar ataxia characterized by a typical head tremor, jerky head movements and lack of menace response. The specific role of magnetic resonance imaging (MRI) to support clinical diagnosis has been discussed. However, as yet MR imaging has only been described in one equine CA case. The role of MR morphometry in this regard is currently unknown. Due to the hereditary nature of the disease, genetic testing can support the diagnosis of CA. Therefore, the objective of this study was to perform MR morphometric analysis and genetic testing in four CA-affected Arabian horses and one German Riding Pony with purebred Arabian bloodlines in the third generation., Results: CA was diagnosed pathohistologically in the five affected horses (2 months - 3 years) supported by clinical signs, necropsy, and genetic testing which confirmed the TOE1:g.2171G>A SNP genotype A/A in all CA-affected horses. On MR images morphometric analysis of the relative cerebellar size and relative cerebellar cerebrospinal fluid (CSF) space were compared to control images of 15 unaffected horses. It was demonstrated that in MR morphometric analyses, CA affected horses displayed a relatively smaller cerebellum compared to the entire brain mass than control animals (P = 0.0088). The relative cerebellar CSF space was larger in affected horses (P = 0.0017). Using a cut off value of 11.0% for relative cerebellar CSF space, the parameter differentiated between CA-affected horses and controls with a sensitivity of 100% and a specificity of 93.3%., Conclusions: In conclusion, morphometric MRI and genetic analysis could be helpful to support the diagnosis of CA in vivo.

The article summarizes the relevant clinical pathological assessment of horses with acute colic. A minimal laboratory evaluation should include the patient's haematocrit (or packed cell volume), total protein, and lactate concentration in the blood. Haematocrit and total protein provide an indication of the severity of dehydration (haematocrit < 0.45 l/l is evidence of no to mild dehydration whereas > 0.5 l/l points to a severe dehydration). The degree of dehydration is often associated with the severity of the colic. Additionally, the blood lactate concentration rises with increasing intestinal compromise with a concentration of > 4 mmol/l indicating a guarded prognosis. However, it is crucial to assess laboratory values only in the context of the clinical findings. If an abdominocentesis is performed, the leukocyte count and the protein and lactate concentrations offer valuable information regarding the type of colic, the severity of the lesion, further therapy, and prognosis of the colic. Reddish discolouration of peritoneal fluid may be a sign of a strangulating obstruction. Transcutaneous abdominal ultrasonography may provide a crucial insight into the colic cause and severity in a relatively short time, even for inexperienced examiners. In regards to small intestinal lesions, dilated small intestinal loops can often be imaged ultrasonographically before they can be palpated transrectally. The occurrence of free peritoneal fluid and dilated small intestine as well as the evaluation of the intestinal wall and the extent of the gastric wall, allow a better management of the acute colic patient. In ponies and foals, radiography as a further diagnostic imaging modality of the abdomen is of great value. It can help to visualise sand impactions, meconium impactions, or gastrointestinal atresia in the neonate.