The aim of this study was to investigate if a vaccine can be developed against
epoxyscillirosidine, to prevent yellow tulp poisoning in livestock. Moraea pallida Bak. (yellow
tulp) poisoning is the most important cardiac glycoside toxicosis in ruminants in South Africa.
Cardiac glycoside poisonings collectively account for about 33 and 10% mortalities due to
plants, in large and small ruminants, respectively. The toxic principle contained by yellow tulp
1α, 2α-epoxyscillirosidine, is a bufadienolide.
Epoxyscillirosidine, proscillaridin and bufalin, were conjugated to [hen ovalbumin (OVA),
bovine serum albumin (BSA) and keyhole limpet haemocyanin (KLH)]. Adult male New
Zealand White rabbits were vaccinated in 3 trials. In trial 1 (T1) and 2 (T2), experimental (n=7)
and control (n=5) animals were vaccinated with epoxyscillirosidine-OVA (0.4 mg/rabbit) and
OVA (0.8 mg/rabbit), respectively. In T1 Freund’s (complete and incomplete) and in T2
Montanide was used as adjuvant, respectively. In Trial 3 (T3), five equal groups of 3 animals
each, were vaccinated with proscillaridin-BSA (group 1), bufalin-BSA (group 2), epoxyscillirosidine-KLH (group 3), epoxyscillirosidine-BSA (group 4) and BSA (group 5), with
Montanide as adjuvant, on days (D) 0, 21 and 42 (0.8 mg/rabbit, intradermally). Blood was
collected before each vaccination and at 3 weeks after the last vaccination. Antibody response
was determined using an indirect ELISA. There was a poor immune response associated with the
dose and/or adjuvant in T1. However, after increasing the dose of the immunogen to 0.8 mg (per
rabbit) and changing the adjuvant to Montanide, in T2 and T3, antibodies against the conjugates
were successfully raised. In T3, epoxyscillirosidine-KLH (group 3) induced the highest immune
response. Furthermore, proscillaridin and bufalin antibodies cross-reacted with
epoxyscillirosidine and its OVA conjugate in the ELISAs.
Preparatory to in vitro studies to assess the efficacy of the raised antibodies to neutralize
epoxyscillirosidine, a rat embryonic cardiomyocyte (H9c2) cell line was established and the
cytotoxic effect of epoxyscillirosidine was determined. Cells (10 000/well) exposed to
epoxyscillirosidine (10–200 μM) for 24, 48 and 72 h were evaluated using 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the lactate dehydrogenase
(LDH) assays. Cells (100 000/well) exposed to epoxyscillirosidine (40–200 μM, for 24, 48 and
72 h), were processed and viewed with a transmission electron microscope (TEM). Cell viability
indicated a hormetic dose/concentration response, characterized by higher viability (relative to
control) at low doses (10–40, 10 and 10–20 μM for 24, 48 and 72 h, respectively) and decreased
viability at higher doses. The cytotoxic effect and ultra-structural changes were dose and time
dependent. Numerous cytoplasmic vacuoles, karyolysis and damage to the cell membrane,
indicative of necrosis, were observed.
The animal vaccination trial was scaled up, to generate more antibodies, for the in vitro
neutralization studies. Six, adult Mutton Merino whethers were vaccinated with epoxyscillirosidine-KLH (2 mg subcutaneously), on D0, 21 and 42. Immune response was
determined with an indirect ELISA. Antibodies were concentrated and purified using ammonium
sulphate precipitation, before evaluation of in vitro neutralization efficacy. There was no
significant (p > 0.05) difference in viability, between cells exposed to a pre-incubated solution of
antibodies and epoxyscillirosidine and the epoxyscillirosidine exposed control cells. The
antibodies failed to neutralize the toxic effect of epoxyscillirosidine.
In conclusion, conjugated epoxyscillirosidine was an effective immunogen following
conjugation to carrier proteins and antibodies were raised in vaccinated animals. Although
antibodies against epoxyscillirosidine-KLH were raised in sheep, they failed to neutralize the
toxin in the in vitro H9c2 cell model. This is possibly because higher ratios of antibodies to toxin
are needed to effectively neutralize epoxyscillirosidine than those used in this study. Since
antibodies failed to neutralize epoxyscillirosidine in the current study, further studies could
optimize the vaccine to produce more specific antibodies with stronger affinity and avidity to be
able to neutralize epoxyscillirosidine. Furthermore, the antibody purification method could be
adjusted or changed for optimal results in the future. Antibodies against the related commercially
available bufadienolides, namely proscillaridin and bufalin, cross-reacted with
epoxyscillirosidine and could be investigated in future studies to prevent yellow tulp poisoning
by vaccination. / Thesis (PhD)--University of Pretoria, 2018. / Paraclinical Sciences / PhD / Unrestricted
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:up/oai:repository.up.ac.za:2263/70595 |
| Date | January 2018 |
| Creators | Isa, Hamza Ibrahim |
| Contributors | Botha, C.J. (Christoffel Jacobus), u16286431@tuks.co.za, Crafford, Jan Ernst |
| Publisher | University of Pretoria |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Rights | © 2019 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
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