Isolation, establishment, and characterization of ex vivo equine melanoma cell cultures

Gray horses spontaneously develop metastatic melanomas that resemble human disease, and this is often accompanied with metastasis to other organs. Unlike in other species, the establishment of primary equine melanoma cultures that could be used to develop new therapeutic approaches has remained a major challenge. The purpose of the study was to develop a protocol for routine isolation and cultivation of primary equine melanocytes. Melanoma tissues were excised from 13 horses under local anesthesia, mainly from the perianal area. The melanoma cells were isolated from the melanoma tissue by serial enzymatic digestion using dispase and collagenase. Out of the 13 excised melanomas, cell cultures from eight melanomas were established, which corresponded to a success rate 62%. These cells showed different degrees of melanin pigmentation. Characterization of these cells using confocal microscopy, FACs analysis and western blotting showed that they expressed melanoma-associated antigens; Melan-A, MAGE-1, and MAGE-3, and PCNA expression was higher in fast-proliferating isolates. The protocol we developed and established proved successful for routine isolation and cultivation of primary equine melanoma cells. This method provided a large number of primary equine melanoma cells that could be used to study new therapeutic approaches for treatment of equine melanoma

Comparison of nanoparticle-mediated transfection methods for DNA expression plasmids: efficiency and cytotoxicity

<p>Abstract</p> <p>Background</p> <p>Reproducibly high transfection rates with low methodology-induced cytotoxic side effects are essential to attain the required effect on targeted cells when exogenous DNA is transfected. Different approaches and modifications such as the use of nanoparticles (NPs) are being evaluated to increase transfection efficiencies. Several studies have focused on the attained transfection efficiency after NP-mediated approaches. However, data comparing toxicity of these novel approaches with conventional methods is still rare.</p> <p>Transfection efficiency and methodology-induced cytotoxicity were analysed after transfection with different NP-mediated and conventional approaches. Two eukaryotic DNA-expression-plasmids were used to transfect the mammalian cell line MTH53A applying six different transfection protocols: conventional transfection reagent (FuGENE HD, FHD), FHD in combination with two different sizes of stabilizer-free laser-generated AuNPs (PLAL-AuNPs_S1,_S2), FHD and commercially available AuNPs (Plano-AuNP), and two magnetic transfection protocols. 24 h post transfection efficiency of each protocol was analysed using fluorescence microscopy and GFP-based flow cytometry. Toxicity was assessed measuring cell proliferation and percentage of propidium iodide (PI%) positive cells. Expression of the respective recombinant proteins was evaluated by immunofluorescence.</p> <p>Results</p> <p>The addition of AuNPs to the transfection protocols significantly increased transfection efficiency in the pIRES-hrGFPII-<it>eIL-12 </it>transfections (FHD: 16%; AuNPs mean: 28%), whereas the magnet-assisted protocols did not increase efficiency. Ligand-free PLAL-AuNPs had no significant cytotoxic effect, while the ligand-stabilized Plano-AuNPs induced a significant increase in the PI% and lower cell proliferation. For pIRES-hrGFPII-<it>rHMGB1 </it>transfections significantly higher transfection efficiency was observed with PLAL-AuNPs (FHD: 31%; PLAL-AuNPs_S1: 46%; PLAL-AuNPs_S2: 50%), while the magnet-assisted transfection led to significantly lower efficiencies than the FHD protocol. With PLAL-AuNPs_S1 and _S2 the PI% was significantly higher, yet no consistent effect of these NPs on cell proliferation was observed. The magnet-assisted protocols were least effective, but did result in the lowest cytotoxic effect.</p> <p>Conclusions</p> <p>This study demonstrated that transfection efficiency of DNA-expression-plasmids was significantly improved by the addition of AuNPs. In some combinations the respective cytotoxicity was increased depending on the type of the applied AuNPs and the transfected DNA construct. Consequently, our results indicate that for routine use of these AuNPs the specific nanoparticle formulation and DNA construct combination has to be considered.</p

First detection and frequent occurrence of Equine Hepacivirus in horses on the African continent

Since the discovery of equine hepacivirus (EqHV) in 2011, the virus has been detected in horse populations from more than twelve countries across five continents. EqHV seroprevalence has been reported to be as high as 61.8% and EqHV ribonucleic acid (RNA) prevalence to range between 0.9% and 34.1%. Molecular and serological indications of EqHV infection have never been reported in equids on the African continent. Therefore, investigation of EqHV prevalence in South African horses and subsequent viral genetic characterization contribute to a better understanding of the global epidemiology of this virus. In a cross-sectional study, serum samples from 454 Thoroughbred foals (aged 58–183 days) were analysed for anti-EqHV non-structural protein 3 (NS3)-specific antibodies (abs) with a luciferase immunoprecipitation system (LIPS) and for EqHV RNA by quantitative real-time polymerase chain reaction (qRT-PCR). Farms of origin (n = 26) were situated in South Africa’s Western Cape Province. The associations between EqHV infection state and farm of origin, foal gender and foal age were subsequently described. Furthermore, nested PCRs were performed on parts of the 5′UTR, NS3 and NS5B genes of 17 samples. Samples were sequenced and phylogenetic analyses were conducted. The population’s seroprevalence was 83.70% and RNA was detected in 7.93% of samples. Increasing foal age was associated with decreasing ab prevalence and increasing prevalence of EqHV RNA. Sequences from South African EqHV strains did not show in-depth clustering with published sequences of EqHV isolates from particular continents. In conclusion, EqHV is present in the South African Thoroughbred population and appears more prevalent than reported in other horse populations worldwide.M.B. was supported by the Deutscher Akademischer Austauschdienst (DAAD). B.T. was supported by the Hannover Medical School. E.S. was supported by the Helmholtz Centre for Infection Research.http://www.elsevier.com/locate/vetmic2019-09-01hj2018Companion Animal Clinical StudiesEquine Research Centr

Local and systemic effect of transfection-reagent formulated DNA vectors on equine melanoma

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

Isolation, establishment, and characterization of ex vivo equine melanoma cell cultures

Gray horses spontaneously develop metastatic melanomas that resemble human disease, and this is often accompanied with metastasis to other organs. Unlike in other species, the establishment of primary equine melanoma cultures that could be used to develop new therapeutic approaches has remained a major challenge. The purpose of the study was to develop a protocol for routine isolation and cultivation of primary equine melanocytes. Melanoma tissues were excised from 13 horses under local anesthesia, mainly from the perianal area. The melanoma cells were isolated from the melanoma tissue by serial enzymatic digestion using dispase and collagenase. Out of the 13 excised melanomas, cell cultures from eight melanomas were established, which corresponded to a success rate 62%. These cells showed different degrees of melanin pigmentation. Characterization of these cells using confocal microscopy, FACs analysis and western blotting showed that they expressed melanoma-associated antigens; Melan-A, MAGE-1, and MAGE-3, and PCNA expression was higher in fast-proliferating isolates. The protocol we developed and established proved successful for routine isolation and cultivation of primary equine melanoma cells. This method provided a large number of primary equine melanoma cells that could be used to study new therapeutic approaches for treatment of equine melanomas

Einführung und Evaluierung des praktischen Jahres an der Tierärztlichen Hochschule Hannover [Introduction and evaluation of final-year practice-oriented education at the University of Veterinary Medicine in Hanover, Germany]

[english] The introduction of a year of practice-oriented education should provide students of the University of Veterinary Medicine in Hanover greater integration in the clinical and scientific setting in order to gain “real-life” experience for their later profession before the end of their studies. This year replaces the old 9th and 10th semesters and builds a bridge between the well-organized and structured studies and the later profession. It includes internships outside the university in private practice, private clinics, slaughterhouses, etc., and a practical semester that can be completely at any time of the year in clinics and other institutes of the university. The online evaluation of this year has been positive. [german] Mit der Einführung des praktischen Jahres sollen die Studierenden der Tierärztlichen Hochschule Hannover (TiHo) stärker in den klinischen und wissenschaftlichen Alltag eingebunden werden, um schon vor Ende des Studiums praxisnahe Erfahrungen zu sammeln. Das praktische Jahr ersetzt das bisherige 9. und 10. Semester und bildet so die Überleitung zwischen dem eng organisierten Studium und der anschließenden beruflichen Laufbahn. Es beinhaltet die Praktika außerhalb der TiHo in Tierarztpraxen, Privatkliniken, Schlachthöfen, etc. und ein internes praktisches Semester, welches das gesamte Jahr über in den Einrichtungen der TiHo abgeleistet werden kann. Die Online-Evaluierung dieses Jahres verlief positiv

Auswirkungen von Haltung, Fütterung und Nutzung auf die Pferdegesundheit unter besonderer Berücksichtigung respiratorischer und gastrointestinaler Krankheiten

In einer zufällig ausgewählten Pferdepopulation in der Schweiz leiden 54% aller Pferde an einer COPD. Ursache der COPD ist eine Hypersensitivität des Respirationstraktes gegenüber im Heu enthaltenen Pilzsporen und thermophilen Aktinomyceten. Ebenfalls ursächlich im Zusammenhang mit der Fütterung entstehen Zahnprobleme, die dritthäufigsten von praktischen Tierärzten behandelten Krankheiten. Nutzungsbedingt entwickeln sich Magenulzera am häufigsten bei Rennpferden, die je nach Untersuchung eine Magenulkus-Prävalenz von ca. 63 bis 90% aufweisen. Bei einer Untersuchung in der Schweiz konnten bei einer aus Ausbildungs-, Schul-, Sport- und Fahrpferden bestehenden Population bei 57.1% der Pferde Magenulzera festgestellt werden. Überhöhte Kraftfuttergaben, zu seltene und mengenmässig zu geringe Heufütterung pro Tag sowie der Einsatz von hygienisch nicht einwandfreien Futtermitteln und mangelhafte Bewegung sind wesentliche Gründe, die zur Entstehung von Koliken beitragen können