Towards a yellow tulp vaccine : preliminary studies exploiting the potential for cross-reactivity with related bufadienolides

Abstract

Poisoning by Moraea pallida Bak. (yellow tulp) is the most important of all cardiac glycoside induced toxicoses which collectively account for 33% and 10% of deaths in large and small stock due to plant poisoning, respectively, in the Republic of South Africa. This study was conducted to investigate the potential for developing a vaccine against epoxyscillirosidine, the toxin contained by yellow tulp. Epoxyscillirosidine was extracted, isolated, purified and confirmed using 13C NMR spectroscopy. Bufalin and proscillaridine were purchased and together with epoxyscillirosidine were coupled to BSA and KLH to render them immunogenic. The immunogens (4 mg/ml) were emulsified with an equal volume of Montanide ISA, as an adjuvant. Adult male New Zealand White rabbits (n=15) were randomly assigned to 5 equal groups. Rabbits in groups I, II, III and IV were immunized with proscillaridine-BSA, bufalin-BSA, epoxyscillirosidine-KLH and epoxyscillirosidine-BSA conjugates, respectively. Group V served as control where animals were administered BSA only. The rabbits were immunized on Days 0, 21 and 42 by intradermal injection of 0.1ml of the vaccine at four sites on the dorsum. Blood was collected prior to each vaccination and on Day 67. An ELISA was performed to determine antibody response. Antibodies were raised against proscillaridine, bufalin and epoxyscillirosidine. Furthermore, the antibodies synthesized by Group I and II rabbits also cross-reacted with epoxyscillirosidine. However, the degree of cross-reactivity was low. This may be enhanced by optimizing the vaccine to induce stronger antibody response. The antibodies will be evaluated to determine the neutralization efficacy against epoxyscillirosidine.