bufadienolide and Plant-Poisoning

Intoxication by Moraea pallida Bak. (yellow tulp) in livestock is of great importance in South Africa, ranking top among all plant-induced cardiac glycoside toxicosis. The toxic principle, a bufadienolide, is 1α, 2α-epoxyscillirosidine. Treatment of poisoning is challenging and affected livestock often succumbs due to the stress of handling. Manipulating animals to resist poisoning is a potential management strategy. The goal of this study was to explore the potential to develop a vaccine against epoxyscillirosidine by raising antibodies against epoxyscillirosidine in sheep and to assess the neutralization ability of the antibodies in vitro. Epoxyscillirosidine was successfully conjugated to keyhole limpet haemocyanin (KLH) and bovine serum albumin (BSA) rendering them immunogenic. The sheep, vaccinated with epoxyscillirosidine-KLH conjugate (n = 4) and KLH (n = 2) with Montanide, developed antibodies as determined with an indirect enzyme linked immunosorbent assay (ELISA). Total immunoglobulins from sera of vaccinated and control sheep that were purified and concentrated using ammonium sulphate precipitation were 11,940 and 7850 μg, respectively. The in vitro neutralization assay using the methyl blue tetrazolium bromide (MTT) cell viability assay indicated no significant difference (p > 0.05) between anti-epoxyscillirosidine-KLH and KLH antibodies. Rather, the antibodies seemed to enhance the cytotoxicity of epoxyscillirosidine in H9c2 cells. Thus, it is necessary to develop improved vaccination methods to generate antibodies capable of neutralizing the functional group responsible for epoxyscillirosidine toxicity.

Topics: Animals; Antibodies, Neutralizing; Bufanolides; Cell Line; Enzyme-Linked Immunosorbent Assay; Hemocyanins; Immunoglobulins; Iridaceae; Male; Neutralization Tests; Plant Poisoning; Rats; Serum Albumin, Bovine; Sheep, Domestic; Vaccination; Vaccines, Conjugate

Two anaesthetized sheep were intoxicated with epoxyscillirosidin, the main cardio-active bufadienolide, extracted from Homeria pallida (Natal yellow tulp). The epoxyscillirosidin was injected intravenously as a bolus of 50 micrograms/kg, followed 30 min later by a continuous infusion in a normal saline drip (0.9% NaCl) at 25 micrograms/kg/h. In addition, another two conscious sheep were poisoned by intraruminal dosing of 1,25 g/kg of dried H. pallida plant material. Electrocardiograms, heart and respiratory rates and venous-acid-base levels were recorded prior to and at approximately 30-60 min intervals during the course of the experiment. Additional recordings were made when animals showed signs of intoxication. R56865 (Janssen Pharmaceutica, Pty Ltd), a novel Ca++ antagonist, was administered at the first distinct signs of cardiac disturbances in the sheep given epoxyscillirosidin and after development of tachycardia and dyspnoea in those that received plant material. Activated charcoal was drenched at 3 g/kg to both sheep that received H. pallida about 1 h after the initial administration of R56865. All H. pallida sheep and one of the epoxyscillirosidin sheep survived. The signs of intoxication with H. pallida, namely groaning and tachypnoea, abated within minutes of treatment with R56865, but returned c. 30 min later in both animals. The treatment apparently had little effect on heart rate and EKG changes. One of the epoxyscillirosidin sheep was treated while exhibiting paroxysmal ventricular tachycardia. Although a transient improvement in conduction disturbance was recorded, the animal died soon afterwards. The results of this study indicate that the in vivo response of R56865 against induced bufadienolide cardiac disturbance in sheep is not as evident as that observed with R56865 against similar cardiac disturbance in vitro. The potential use of R56865 together with activated charcoal is discussed.

Topics: Animals; Benzothiazoles; Bufanolides; Calcium Channel Blockers; Cholenes; In Vitro Techniques; Piperidines; Plant Extracts; Plant Poisoning; Sheep; Sheep Diseases; Thiazoles; Toxins, Biological