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Development and evaluation of immunogens for a yellow tulp (Moraea pallida) vaccine
Thesis (PhD)--University of Pretoria, 2018.
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University of Pretoria
2019
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| _version_ | 1867613705240313856 |
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| access_status_str | Open Access |
| author2 | Botha, C.J. (Christoffel Jacobus) |
| author_browse | Botha, C.J. (Christoffel Jacobus) |
| author_facet | Botha, C.J. (Christoffel Jacobus) |
| collection | Thesis |
| dc_rights_str_mv | © 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. |
| description | Thesis (PhD)--University of Pretoria, 2018. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/70595 |
| institution | University of Pretoria (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:40:23.116Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UPSpace — University of Pretoria Institutional Repository |
| publishDate | 2019 |
| publishDateRange | 2019 |
| publishDateSort | 2019 |
| publisher | University of Pretoria |
| publisherStr | University of Pretoria |
| record_format | dspace |
| source_str | UPSpace — University of Pretoria Institutional Repository |
| spelling | oai:repository.up.ac.za:2263/70595 Development and evaluation of immunogens for a yellow tulp (Moraea pallida) vaccine Botha, C.J. (Christoffel Jacobus) u16286431@tuks.co.za Crafford, Jan Ernst Isa, Hamza Ibrahim UCTD Veterinary science theses SDG-01 SDG-01: No poverty Thesis (PhD)--University of Pretoria, 2018. 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. Paraclinical Sciences PhD Unrestricted 2019-07-08T09:46:58Z 2019-07-08T09:46:58Z 2019/04/04 2018 Thesis Isa, HI 2018, Development and evaluation of immunogens for a yellow tulp (Moraea pallida) vaccine, PhD Thesis, University of Pretoria, Pretoria, viewed yymmdd <http://hdl.handle.net/2263/70595> A2019 http://hdl.handle.net/2263/70595 en © 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. application/pdf University of Pretoria |
| spellingShingle | UCTD Veterinary science theses SDG-01 SDG-01: No poverty Development and evaluation of immunogens for a yellow tulp (Moraea pallida) vaccine |
| title | Development and evaluation of immunogens for a yellow tulp (Moraea pallida) vaccine |
| title_full | Development and evaluation of immunogens for a yellow tulp (Moraea pallida) vaccine |
| title_fullStr | Development and evaluation of immunogens for a yellow tulp (Moraea pallida) vaccine |
| title_full_unstemmed | Development and evaluation of immunogens for a yellow tulp (Moraea pallida) vaccine |
| title_short | Development and evaluation of immunogens for a yellow tulp (Moraea pallida) vaccine |
| title_sort | development and evaluation of immunogens for a yellow tulp moraea pallida vaccine |
| topic | UCTD Veterinary science theses SDG-01 SDG-01: No poverty |
| url | http://hdl.handle.net/2263/70595 |