Evaluation of Stress Response under a Standard Euthanasia Protocol in Horses Using Analysis of Heart Rate Variability

The effects of a standard protocol for euthanasia on heart rate variability (HRV) as a consequence of stress response were analyzed in this prospective clinical study. The HRV was determined in 40 horses undergoing euthanasia due to various reasons, at different locations, and with/without owner presence. For euthanasia, horses were sedated with xylazine or a combination of xylazine and butorphanol. General anesthesia was induced using diazepam and ketamine. Afterwards, horses were euthanized with pentobarbital. The ECG data were taken by a Telemetric ECG at three time points (sedation, anesthesia, anesthesia until death). The HRV was analyzed including the low (LF) and high frequency (HF) components of HRV and the sympathovagal balance (LF/HF ratio). Significant differences in the LF, HF and LF/HF ratio were found between the three time points of euthanasia (p < 0.001). The HRV analysis showed dominating sympathetic activity in the preparation phase of euthanasia and during the injection of pentobarbital. The location of euthanasia, presence of owner and type of primary diseases had no influence on stress parameters. Horses showing excitations or groaning during euthanasia did not differ in HRV. Horse with colic were however more likely to show reoccurrence of breathing during euthanasia. In conclusion, HRV is a sensitive, noninvasive parameter to obtain sympathovagal stimulations during euthanasia and adapted protocols for euthanasia in horse with colic should be studied

Treatment of equine sarcoids using recombinant poxviruses expressing feline interleukin-2

Background Interleukin (IL)‐2 stimulates antitumour immunity and is successfully used for the treatment of different neoplasias. Hypothesis/Objectives Canarypox virus locally expressing feline IL‐2 is safe and can be used to treat equine sarcoids. Animals Twenty horses of different breeds with a median age of eight years (interquartile range 6.0–13.3 years) and a total number of 59 sarcoids were included in the study. Methods In this prospective clinical trial, sarcoids were injected twice seven days apart, with a recombinant canarypox virus expressing feline IL‐2. Complete blood counts (CBC) and fibrinogen levels were measured before treatment and on days 1, 2, 7 and 8. Results Complete regression was achieved in eight horses (40%) and partial regression in two horses (10%). No change in sarcoid size was observed in two horses (10%) and the disease progressed in five horses (25%). Sarcoids of three horses (15%) showed initial response followed by tumour growth. There were no significant changes in CBC and fibrinogen levels after either injection. One horse developed a mild fever the day after each injection, which subsided without treatment the following day. Conclusions Treatment of equine sarcoids with recombinant canarypox virus expressing feline IL‐2 seems to be a safe therapy option. Although the expression of IL‐2 after vector injection and its biological activity in horses were not proven in this study, the treatment resulted in regression and partial regression in 50% of the cases. Further studies are necessary to verify these findings and to establish a treatment protocol

Treatment of equine sarcoids with interleukin 2

Das Equine Sarkoid ist der am häufigsten diagnostizierte Hauttumor der Pferde. Die Pathogenese ist multifaktoriell und Viren, genetische Prädisposition, Mikrotraumata und Vektoren scheinen eine Rolle zu spielen. Pferde jeden Alters und Geschlechts können Sarkoide entwickeln. Makroskopisch lassen sich 6 Formen (okkult, verrukös, nodulär, fibroblastisch, maligne und gemischt) unterscheiden. Im ersten Teil dieser Arbeit wurde die aktuelle Literatur zur Therapie Equiner Sarkoide untersucht. Die Literaturstudie zeigt die aktuellen Erkenntnisse zu Equinen Sarkoiden, insbesondere die verschiedenen Therapiemöglichkeiten mit ihren Erfolgsraten sowie ihrer Verfügbarkeit. Alle hier erwähnten Therapieoptionen bergen jedoch die Gefahr von Rezidiven, welche häufig aggressiver sind als das ursprüngliche Sarkoid. Eine gründliche Besitzeraufklärung ist unbedingt erforderlich und die Prognose ist in jedem Fall vorsichtig zu stellen. Die beschriebenen Therapieoptionen beinhalten chirurgische Therapie, Thermotherapie, Strahlentherapie, lokale Chemotherapie, antivirale Therapie, Immuntherapie, Phototherapie und Phytotherapie. Die publizierten Studien über Therapiemöglichkeiten bei Equinen Sarkoiden haben unterschiedliche Rahmenbedingungen bezüglich Beobachtungzeitraum oder Definition von Therapieerfolg. Keine der bisher beschriebenen Therapieoptionen kann eine Heilung garantieren. Im zweiten und dritten Teil der Arbeit wurde ein rekombinanter Kanarienpockenvirus, das lokal felines Interleukin 2 (IL-2) (Oncept IL-2, Merial, Zulassungsnummer: EU/2/13/150/001) exprimiert, auf Sicherheit und Wirksamkeit als Therapieoption bei Equinen Sarkoiden untersucht. IL-2 soll durch Aktivierung von natürlichen Killerzellen und T-Helferzellen sowie über die vermehrte Ausschüttung von weiteren Zytokinen die spezifische Tumorimmunität stimulieren (Gansbacher et al. 1990). Die publizierten Sequenzen von felinem und equinem IL-2 zeigen eine hohe Übereinstimmung (Dunham et al. 1995). Die Sicherheit des Präparats „Oncept“ wurde bei vier gesunden Pferden getestet. Es wurde zweimal im Abstand von einer Woche subkutan injiziert werden. Durch wiederholte Allgemeinuntersuchungen, dermatologische Untersuchungen inklusive Messung der Hautdicke und labordiagnostische Untersuchungen sowie Biopsien der Injektionsstellen wurden eine rein lokale Wirkung des Medikaments und nur geringgradige Nebenwirkungen wie lokale Schwellung nachgewiesen. Die histologische Untersuchung zeigte eine mittel- bis hochgradige, diffuse, vorrangig perivaskuläre, lympho-plasmazelluläre Dermatitis / Pannikulitis in den IL-2 behandelten Hautbiopsien aller Tiere. Aufgrund des Fehlens ähnlicher Befunde in den Kontrollproben erscheint somit eine Einschätzung als reaktive, behandlungsassoziierte Veränderung wahrscheinlich. Im dritten Teil der Arbeit wurde die Wirksamkeit des rekombinanten Kanarienpockenvirus, das lokal felines Interleukin 2 (IL-2) (Oncept IL-2, Merial, Zulassungsnummer: EU/2/13/150/001) exprimiert, an 20 Pferden mit insgesamt 59 Sarkoiden überprüft. Bei acht Pferden (40%) konnte eine vollständige Regression, bei zwei Pferden (10%) eine partielle Regression erzielt werden. Bei zwei Patienten (10%) zeigte sich keine Veränderung der Tumoren, während es bei fünf Patienten (25%) zu weiterem Wachstum der Tumore kam. Bei drei Pferden (15%) kam es zwar initial zur Regression, jedoch gefolgt von rezidivem Wachstum. Die untersuchten Blutparameter lagen vor und nach der Behandlung im Referenzbereich. Ein Pferd zeigte am Tag nach der Behandlung leichtes Fieber. Die Behandlung Equiner Sarkoide mit rekombinantem Kanarienpockenvirus, das lokal felines Interleukin 2 exprimiert, scheint eine für Patient und Anwender sichere und vielversprechende Therapieoption zu sein. Es sind jedoch weitere Studien nötig, um einerseits die Wirkung des Vektors und des felinen IL-2 zu überprüfen und um andererseits ein optimales Behandlungsschema zu finden sowie die Ergebnisse bei einer größeren Population zu überprüfen.The first part of this study is a literature review to asses current knowledge about equine sarcoids, its therapy options and their availability. The equine sarcoid is a locally invasive growing, non-metastatic tumor of the equine skin. It is with 90% the most common skin tumor in the horse. The pathogenesis is poorly understood and many factors seem to play a role. There is a strong association between the bovine papilloma virus and equine sarcoids. There also seems to be a genetic component to the establishment of disease as well as the participation of vectors. There are six different types of sarcoids (occult, verrucous, nodular, fibroblastic, malevolent and mixed). Many treatment options are available but there is always a high risk of recurrence. Failure of treatment is often followed by a more aggressive tumor. The owners should be advised carefully before any treatment and prognosis is always guarded. There various, available treatment options include surgical removal, cryrosurgery, radiation therapy, chemotherapy, antiviral therapy, immunotherapy, phototherapy and phytotherapy. The study designs investigating these different therapy options differ in their study designs. Different observation periods and different definitions of treatment response make a comparison infeasible. The second and third part of this study asses the safety and efficiency of poxviruses expressing feline interleukin 2 (IL-2) for the treatment of equine sarcoids. Injections into the tumor bed of feline fibrosarcomas with poxviruses expressing feline interleukin 2 (IL-2) reduced the recurrence rate after surgical resection, due to a stimulation of antitumor immunity. The published sequences of feline and equine IL-2 show a high homology. To examine the safety of the drug four healthy horses were injected subcutaneously with the recombinant poxviruses expressing feline IL-2. One horse developed temporary mild swelling at the injection side. In one horse there was a mild leukocytosis the day after the first treatment. CBC and fibrinogen levels were within the reference range in all horses after the second injection. Main findings in the histopathologic examination were a moderate to severe, diffuse perivascular lymphoplasmacellular dermatitis and panniculitis with mild edema, while no pathological changes were found in the untreated skin. Treatment of equine sarcoids with recombinant poxviruses expressing feline IL-2 seems to be a safe therapy option. In the third part the efficacy of recombinant poxviruses expressing feline IL-2 for the treatment of equine sarcoids was examined. 20 horses of different breeds and age with a total number of 59 sarcoids were included in the study. Sarcoids were injected twice at intervals of seven days with recombinant poxviruses expressing feline IL-2. A clinical examination was performed daily. Complete blood count (CBC) and fibrinogen concentration were measured before treatment and on days 1, 2, 7 and 8. Complete regression of all sarcoids, including those that had not been treated, was achieved in eight horses (40 %) CR and partial response in two horses (10 %). Sarcoids did not change in size in two horses (10 %), and the disease progressed in five horses (25 %). Sarcoids showed response in three horses (15 %) initially but then progressed again. All measured blood parameters were within the reference ranges before and one day after treatment. One of the horses developed mild fever the day after injection, which subsided the following day without treatment. Treatment of equine sarcoids with canarypox virus locally expressing IL-2 was easy and safe for both user and horse. Equine sarcoids injected with canarypox virus expressing feline IL-2 showed a reduction in tumor size or a complete remission of the tumor in 50 % of cases. Further studies are necessary to research vector and feline IL-2 effects in horses, establish an optimal treatment regime for equines and verify the results in a larger population of patients