Department of Biochemistry

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Antibody production against Staphylococcus aureus CoA biosynthesis enzymes and their application in protein level quantification
(Stellenbosch : Stellenbosch University, 2021-04) Bothma, Karli; De Villiers, Marianne; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Antimicrobial resistance has become an increased burden worldwide as more and more human pathogens are becoming resistant to current antimicrobials. Therefore, the identification of novel drug targets and development of new antimicrobial drugs are currently of high priority. A drug target that has gained increased attention is the coenzyme A (CoA) biosynthesis pathway. CoA is an essential cofactor that is necessary for life in all organisms, including human pathogens, making it an attractive target for the development of new antimicrobial drugs. The CoA biosynthesis pathway of Staphylococcus aureus, which is the leading cause of hospital-associated infections, was the focus of this study. Although various studies have investigated this pathway in S. aureus as a possible drug target, there is still a lot that needs to be elucidated in this regard. One gap in our knowledge is that the levels of the CoA biosynthesis enzymes (PanK, CoaBC, PPAT and DPCK) under physiological conditions are currently unknown. This study therefore aimed to develop immunological techniques which could be implemented as tools to quantify the levels of these enzymes at different growth phases of S. aureus cultivated under physiological growth conditions. To achieve this aim, the four CoA biosynthesis enzymes of S. aureus were recombinantly expressed and purified using established methods. Polyclonal antibodies were raised in rabbits by immunising the animals with the respective enzymes adsorbed to acid-treated, naked Salmonella minnesota R595. This method has been used to successfully produce antibodies to a wide variety of antigens, especially in cases where only small amounts of the antigen were available. With these antibodies, indirect competition enzyme-linked immunosorbent assays (ELISAs) with excellent standard curves were obtained for the quantification of each of the respective enzymes. Furthermore, cross-reactivity studies performed with ELISA and western blot revealed that the anti-SaPanK, anti-SaPPAT and anti-SaDPCK antibodies showed limited cross-reactivity. In an attempt to quantify the amount of cross-reactivity of each antibody-antigen pair, however, it was found that only the cross-reactivity of the anti-SaPPAT antibodies had an effect on the final optimised assay. In this study an original, highly sensitive ELISA method for the detection and quantification of each of the enzymes of the CoA biosynthesis pathway of S. aureus was developed. These assays provide a cost-effective method for enzyme level quantification that have the potential to provide better insight on the levels of the different enzymes under physiological conditions and ultimately aid in the development of new antimicrobial drugs.
An assessment of the diversity and pathogenicity of Potato leafroll virus in South Africa
(Stellenbosch : Stellenbosch University, 2018-03) Roos, Wiets Gideon; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Potato production in South Africa has steadily intensified through improved pivot irrigation. Since potatoes are vegetatively propagated the potato industry is continuously threatened by Potato leafroll virus (PLRV) which is responsible for increasing yield losses in South Africa. Effective management of PLRV is dependent on its accurate detection in seed potato stocks. PLRV is a small spherical plant virus consisting of a protein capsid and an RNA genome of approximately 5900 bases. This virus is phloem restricted and is vectored by, most notably, by the green peach aphid. Plant RNA viruses pose a threat due to their high mutation rate and the ability to adapt rapidly to a changing environment. To effectively manage PLRV infection, detection of virus infection in seed potatoes is paramount. In this study, five field trials were carried out in potato production fields, to compare the commonly used DAS-ELISA with RT-PCR for PLRV detection. From the results obtained it was concluded that DAS-ELISA detection greatly underestimates the number of infected samples when compared to RT-PCR. The hotter climate of the Sandveld region appears to inhibit PLRV accumulation in infected plants and these infections then remain undetected by DAS-ELISA. PLRV isolates were sequenced and phylogenetic and bioinformatic analyses were performed to identify and characterise local variants of PLRV. PLRV isolates found in the Sandveld region were closely related to PLRV isolates from Australia. Some of these isolates had recombined with variants commonly found in Europe, Asia and the Americas as well as with those similar to PLRV isolates from Peru and Germany. Three locally produced cultivars were graft-inoculated with two PLRV isolates that represent the two main variant groups found to assess symptom development and yield reduction. Symptom development in locally produced cultivars was typical for PLRV. A yield loss resulted from this infection with no difference between the Australian type and the European/Australian recombinant type. The proteins produced by the newly sequenced isolates were further analysed in comparison to other isolates found worldwide. The variation in the proteins produced by the newly sequenced isolates was mainly due to recombination between distinct groups of PLRV in the 5’ half of the genome and through mutation in the 3’region of the genome. A differential RT-PCR was designed to distinguish, in a single reaction, between the Australian type and the European/Australian recombinant type of PLRV. This revealed that simultaneous infections with both types occurred commonly, and could explain why recombination has occurred.
An assessment of the mutation patterns in South African isolates of Potato leafroll virus and the expression of recombinant viral coat protein genes in Escherichia coli
(Stellenbosch : Stellenbosch University, 2007-03) Rothmann, Adri Hilda; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Presently, the observed variation in symptoms of Potato leafroll virus (PLRV) infection in potato cultivars in South Africa cannot be reconciled with PLRV symptoms obtained 10-15 years ago, even if the different interactions between the pathogen and the cultivar are taken into account. In an effort to analyze this variation, mutations in the coat protein (CP) gene of South African isolates of PLRV were assessed. The CP gene of PLRV isolates from different areas within South Africa was amplified by reverse transcription-polymerase chain reaction (RT-PCR), cloned and sequenced. Significant sequence variation in the CP gene was found within the analyzed South African isolates of PLRV. Phylogenetic analysis revealed two major clades with most South African isolates and an Australian and North American isolate grouped together and the remainder grouped with isolates from diverse countries worldwide. The deduced amino acid sequences from representatives of these two clades indicated differences in CP threedimensional structure. In an effort to produce recombinant PLRV CP for the production of antibodies specific for South African isolates of PLRV for use in enzyme-linked immunosorbent assay (ELISA), the CP gene of a South African isolate of PLRV was subcloned into a bacterial expression vector (pET14-b). Expression of full length recombinant PLRV CP was attempted in Escherichia coli strains BL21(DE3)pLysS, Rosetta-gami B(DE3)pLysS and Rosetta-2(DE3)pLysS. As this was not successful, the PLRV CP gene was subcloned in another expression vector (pGEX) for expression as an N-terminal fusion protein with glutathione-S-transferase (GST) in E. coli strains BL21(DE3)pLysS and Rosetta-2(DE3)pLysS. The recombinant GST-PLRV CP fusion protein was purified and used for antibody production in rabbits. Using western blots, the effectiveness of antibodies produced to recombinant GST-PLRV CP fusion protein was assessed for PLRV recognition. It was found that antibodies to the recombinant GST-PLRV CP fusion protein were more effective for the detection of GST than PLRV CP and that production of antibodies to the cleaved PLRV CP product would be necessary if antibodies are required for ELISA applications.
Biochemical and genetic characterization of bacteria isolated from diseased rainbow trout (Oncorhynchus mykiss) farmed in Lesotho and Mpumalanga province of South Africa
(Stellenbosch : Stellenbosch University, 2017-12) Kutu, Vuyokazi; Bellstedt, D. U.; Macey, B. M.; Mouton, A.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Rainbow trout farms in Mpumalanga Province, South Africa and Lesotho, have periodically suffered significant losses from infections caused by Gram-positive bacteria. Such outbreaks have hampered the development of this industry in both South Africa and Lesotho. A total of 55 bacterial strains had been isolated between 2006-2012 from infected trout farmed in Lesotho and Mpumalanga Province and had been stored for long term by freeze drying. Some isolate identification had been performed and a few were used for vaccine development. Vaccines were however only effective for one or two seasons, highlighting the need to properly characterize these Gram-positive bacteria. The aims of the study were therefore to: (i) investigate the genetic diversity of these bacterial isolates by their phenotype; antimicrobial susceptibility and 16S ribosomal RNA (rRNA) sequencing and phylogenetic analysis, (ii) investigate the different antigenic epitopes that exist within this group of bacterial isolates by development of an enzyme-linked immunosorbent assay (ELISA) utilizing six rabbit produced polyclonal antibodies, produced against six selected bacterial isolates from the 55 isolates investigated in this study. Phenotypic analysis showed that fifty of the isolates were Gram-positive cocci and five were Gram-positive rods. Their growth characterists and antimicrobial susceptibility were extensively characterized. The 16S rRNA analysis indicated the following isolate composition: 49 Lactococcus garvieae, one Lactococcus lactis, three Carnobacterium maltaromaticum and two Weissella species, which is the first report of Weissella from diseased trout from South Africa. Antigenicity analysis showed that there were highly specific epitopes that were limited to very few isolates, but also common epitopes that were shared between isolates of the same genus, but even some epitopes that were shared between different bacterial genera. The patterns of epitope sharing broadly correlated with the 16S rRNA phylogeny, but not entirely which was not unexpected as phylogeny does not indicate the presence or absence of bacterial epitopes. These results address the importance and accuracy of molecular identification of disease causing species and the need to investigate the antigenic differences expressed by these pathogenic bacteria to assist in generating correct information needed for the development of vaccines of high efficacy.
A biochemical and immunochemical study of ovine adrenal cytochrome P-450 reductase
(Stellenbosch : Stellenbosch University, 1992-03) Petersen, Carmen J.; Swart, P.; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: This study describes: (a) the isolation · of ovine adrenal cytochrome P-450 reductase, (b) the preparation of , antibodies against this enzyme and (c) comparitive immunochemical studies with ovine' liver and .bovine. adrenal cytochrome P-450 'reductases
A biochemical investigation into the compounds involved in pigmentation of apple and pear skin and the manipulation thereof
(Stellenbosch : Stellenbosch University, 1998) Arends, Arrie Paul; Bellstedt, D. U.; Swart, P.; Stellenbosch University. Faculty of Science. Department of Biochemistry.
ENGLISH ABSTRACT: This study describes (a) Identification of the main anthocyanin pigment in the skin of the 'Fuji' apple cultivar and in the pear cultivars 'Bon Rouge' 'Forelle', Red d'Anjou', 'Rosemarie' and 'Flamingo', (b) an investigation of the effect of on-tree bagging on anthocyanin pigment accumulation in the skin of the 'Fuji' apple cultivar by means of high performance liquid chromatography (HPLC) technology. (c) an investigation of the influence of cold storage and ripening on anthocyanin concentration and accumulation in the skin of the pear cultivars, 'Forelle', 'Rosemarie', 'Flamingo', 'Bon Rouge' and Red d'Anjou' pear cultivars by means of HPLC technology and (d) an investigation of the level of induction of dihydroflavonol 4-reductase (DFR) gene in 'Fuji' apple cultivar during bagging trial, through mRNA studies.
A biochemical study of budbreak and plant growth regulators in table grapes
(Stellenbosch : Stellenbosch University, 2002-03) Lombard, Petrus Johannes; Bellstedt, D. U.; Cook, N. C.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: The cultivation of table grapes in the warmer areas of South Africa, indeed worldwide, is complicated by rest breaking problems in spring due to delayed budbreak. In order to overcome these problems rest breaking agents, mainly hydrogen cyanamide, are applied. However, instead of alleviating the problem, additional problems such as uneven budbreak and reduced production are often induced. This study was initiated to further understand the physiological processes occurring during budbreak and how the application of hydrogen cyanamide influences these processes. The following aspects were investigated in this study: a. The effect of hydrogen cyanamide on tissue cytokinin (specifically zeatin riboside) levels of Sultanina table grape vines after application at different times before natural budbreak was studied over two seasons. In 1997, hydrogen cyanamide was applied at three weeks before induced budbreak and in 1998 at six weeks before induced budbreak. One year-old canes were sampled weekly after hydrogen cyanamide application, divided into distal and proximal sections, then further divided into buds, bark and wood tissues and the zeatin riboside (ZR) levels determined. A relatively high amount of chilling coupled to late hydrogen cyanamide application in 1997 led to a large effect on ZR release, but did not lead to significant shifting of the budbreak pattern. Zeatin riboside peaks were observed in buds, internode wood and bark of treated vines compared to control vines. The peaks were higher in distal portions compared to proximal portions in all tissues. The relatively lower chilling and earlier application of hydrogen cyanamide in 1998 had a larger effect on the budbreak pattern while the bud ZR peak was shifted earlier. The distal portion bud ZR . peak was again higher than the proximal portion bud ZR peak. In 1997, as sampling was not initiated early enough, bud ZR peaks were only observed after budbreak, while in 1998 bud ZR peaks were observed before and after budbreak. The effect of these ZR increases on the development of inflorescence primordia, subsequent bunch development and ultimately production, are discussed. b. Free xylem sap was sampled at cane and spur pruned lengths from unpruned canes of Sultanina from budswell until after budbreak in 1999 and from three table grape cultivars, i.e Sultanina, Alphonse Lavalleé and Sunred Seedless, in 2001 and ZR levels determined. The ZR levels in the buds of these three table grape cultivars, pruned to different cane lengths were also determined. One year old canes of these cultivars, were each pruned to long canes (14 buds) and short spurs (2 buds). The ZR content in buds of these canes at distal and proximal positions were determined weekly from budswell until after budbreak in 1999. Xylary ZR peaks occurred before 50% budbreak. Spur xylary ZR levels of all three cultivars followed a similar pattern, although at lower ZR levels than that of the canes. This is similar to previous studies on xylary ZR levels of apple shoots. The high levels of free ZR found in xylem sap at the distal portions of canes support the hypothesis of a cumulative ZR build-up effect as cane length increases. Spur pruning resulted in earlier budbreak and a higher final budbreak than cane pruning. The proximal portions of shoots, whether spur pruned or the proximal portions of canes, showed elevated ZR levels in all cultivars. This difference in ZR levels in bud tissue of different portions of the cane would suggest a difference in ZR consumption or turnover. The results of this study have important management implications for the cultivation of vines in warmer areas in which hydrogen cyanamide is used to alleviate budbreak problems.
Cloning and identification of genes involved in the interaction between the bacterial stone fruit pathogen Pseudomonas syringae pv. syringae strain NV and plum trees
(Stellenbosch : Stellenbosch University, 2001-03) Appel, Maryke; Bellstedt, D. U.; Mansvelt, E. L.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Bacterial canker of stone fruit, caused by Pseudomonas syringae pv. syringae, is one of the most destructive crop diseases in South Africa. Chemical control has failed completely and effective long-term management strategies will have to rely on the breeding of resistant host trees. To assist in such breeding programmes, investigations into the molecular basis of the interaction between P. s. pv. syringae and stone fruit trees have been undertaken in collaboration with the ARC-Fruit, Wine and Vine Research Institute in Stellenbosch. The aim of this dissertation was to clone and identify genes that are involved in interaction between the bacterial canker pathogen and stone fruit trees. In the first part of the study, the harpin encoding gene of a local strain of the pathogen, P. s. pv. syringae NV, was amplified in a polymerase chain reaction (PCR) strategy with primers based on the hrpAZB sequences of the bean pathogen, P. s. pv. syringae 61. Sequencing of this hrpZpssNvgene revealed a high degree of homology (96%) between the harpin encoding genes and harpin proteins of the two strains. The hrpZpssNvgene was subsequently cloned into the pMAL-c2 expression vector and expressed in Escherichia co/i. This system was used for the production of purified, biologically active, recombinant HrpZpSSNV protein. In the second part of the study, differential display (DD) technology was used to identify genes that are induced in stone fruit trees in response to P. s. pv. syringae and/or its harpin elicitor. For this purpose, actively growing shoots of two Prunus sa/icina cultivars, the moderately resistant cv. 'Laetitia' and the highly susceptible cv. 'Songold' were treated with recombinant harpinpssNvprotein or live P. s. pv. syringae NV bacteria. An untreated control and wounding control was included in the experiment. Total RNA was isolated for comparative mRNA analysis 24 hours after treatment. DD profiles were generated with fifteen primer combinations. Eight candidate bands were re-amplified, cloned and sequenced. Reverse transcription PCR was employed to verify the expression patterns of the cloned bands in the original RNA sample set. Two bands, DDc and DD4 were shown to be differentially expressed between treatments and/or cultivars, while no differences in the expression levels of the remaining six bands (DDa, DDe, DD3, DD5, DD6 and DD7) were observed. BLAST similarity searches yielded significant matches for DDe, DD4 and DD7 with plant defense-related genes.
The development of a DNA vaccine against Mycoplasma nasistruthionis sp. nov. for use in ostriches
(Stellenbosch : Stellenbosch University, 2015-12) Wium, Martha; Botes, Annelise; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Mycoplasma nasistruthionis sp. nov. str. Ms03 (Ms03) is one of three Mycoplasma species that were identified from ostriches. Mycoplasmas infections have been implicated in ostrich chick mortalities, growth retardation and downgrading of ostrich carcasses. Currently there is no vaccine available for the treatment of mycoplasmosis in ostriches. This study investigated the development of DNA vaccines against Ms03 infections in ostriches. To this end, the Ms03 genome was sequenced and annotated. The vaccine candidate gene, oppA, was identified within the genome sequence and characterized before DNA vaccines containing the oppA were developed and tested. The genome of Ms03 was sequenced and the resulting 172 contigs were annotated. This dissertation presents the first Ms03 draft genome and annotation which contributed to the understanding of Ms03 as a miniature genetically independent organism. In Ms03, genome replication, cell division, RNA transcription, protein translation and glycolysis resemble that of the closely related Mycoplasma synoviae 53. Purine and pyrimidine metabolism was incomplete and de novo synthesis thereof was not possible. Amino acid synthesis in Ms03 was mostly absent and only the genes that convert aspartate to asparagine and glycine to serine were found. More importers than exporters were annotated owing to the lack of synthesis pathways in Ms03, which is typical for mycoplasmas that have parasitic life styles. Two oligopeptide permease (opp) operons were annotated within the Ms03 genome. The potential of the oppA as a vaccine candidate gene was evaluated by investigating the need for a substrate-binding domain (OppA) as part of the OppBCDF transporter within Mycoplasma species. An oppA homologue could be identified for each oppBCDF operon in all species and therefore must play an essential role in oligopeptide transport. All mycoplasmas (except for hemoplasma) had one, two or three opp operons that could be divided into three types (Type A, B and C). Each type had unique InterPro and MEME domains and motifs which together with the phylogenetic analysis suggest unique roles in their survival under different conditions. Ms03 had a Type A and a Type B opp operon, the Type A oppA was used as vaccine candidate gene. The Type A oppA was cloned and site-directed mutagenesis was used for codon correction before the mutated gene was sub-cloned into three DNA vaccine vectors. The three DNA vaccines (pCI-neo_oppA, VR1012_oppA and VR1020_oppA) were used to vaccinate ostriches and the OppA-antibody response was analysed by ELISA. The VR1020_oppA and pCI-neo_oppA constructs elicited a primary immune response in ostriches, indicating that the OppA protein was expressed in vivo and was immunogenic. This can therefore be viewed as the first step in the development of a DNA vaccine for the control of mycoplasma infections in ostriches.
Development of an oral vaccine against the ostrich-specific mycoplasma, Mycoplasma struthionis
(Stellenbosch : Stellenbosch University, 2013-03) Van Tonder, Amanda; Bellstedt, D. U.; Botes, Annelise; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: The ostrich-specific mycoplasmas Mycoplasma struthionis, Ms02 and Mycoplasma nasistruthionis, are associated with respiratory disease in ostriches, which is threatening the South African ostrich industry. Antibiotics are available to manage Mycoplasma infections, but a need to prevent infections led to an investigation into the development of vaccines against the ostrich-specific mycoplasma. After commercially available poultry mycoplasma vaccines proved to be unsuccessful in protecting ostriches against ostrich mycoplasma infections, the genome of M. struthionis was analysed and the OppA gene identified as a good vaccine candidate. The gene was isolated and used to develop the pCI-neo, VR1012 and VR1020 naked DNA vaccines. M. struthionis infects the respiratory tract of ostriches, therefore a vaccine that results in mucosal immunity is required. The use of a bacterial vector for DNA vaccines has been shown to elicit both a humoral and mucosal immune response. Salmonella enterica serovar typhimurium SL3261 was used to develop mucosal pCI-neo, VR1012 and VR1020 DNA vaccines. The pCI-neo mucosal DNA vaccine was found to be unstable in vivo and the stable mucosal VR1020 and VR1012 DNA vaccines were considered for subsequent vaccine trials. The tissue plasminogen activator (TSA) signal peptide found in the VR1020 plasmid to direct the secretion of the membrane protein, OppA, makes it a good candidate vaccine to compare against the naked DNA vaccine. A preliminary vaccine trial conducted with this vaccine was influenced by various factors including avian influenza and the statistical results proved to be invalid, but enzyme-linked immunosorbent assays (ELISAs) were developed for the successful measurement of the immune response of the ostriches. The dose of the mucosal vaccine administered in the preliminary trial might not have been enough to elicit an effective immune response in the ostriches. Different doses of the mucosal VR1012 and VR1020 DNA vaccines were therefore used in a second trial, but the trial was also influenced by a variety of factors. Even though the results of the vaccine trials were not successful, a few observations were made that could be used to improve future trials and reduce the effect of the factors on the vaccine trials, such as the effect of prior infection, as well as the stress on the ostriches.
The development of enzyme-linked immunosorbent assays to detect potato virus Y and potato leaf roll virus using recombinant viral coat proteins as antigens
(Stellenbosch : University of Stellenbosch, 2005-04) Matzopoulos, Mark; Bellstedt, D. U.
ENGLISH ABSTRACT: Potato Virus Y (PVY) and Potato Leafroll Virus (PLRV) are two of the most destructive potato viruses capable of drastically diminishing crop yields by up to 80%. The presence of these viruses in planting material namely seed potato stocks are routinely diagnosed by enzyme-linked immunosorbent assay (ELISA) kits. The kits currently used by Potatoes South Africa are obtained from Europe. These kits have produced false positive and false negative results in the past. Potatoes South Africa required an ELISA that was reliable, cheap and specific for the detection of South African strains of the two respective viruses. In this study the viral coat protein genes were amplified by RT-PCR from a South African source of infected plant material. The PVY and PLRV coat protein genes were subsequently cloned into pGEM-T Easy vector and sequenced. The sequences of the two viruses were aligned and compared to corresponding viral coat protein gene sequences obtained from Genbank. Subsequently the two amplified and cloned coat protein genes of PVY and PLRV were sub-cloned into an expression system (pET-14b) to induce and express the respective recombinant viral coat proteins. The induction of the cloned coat protein genes yielded successful production of the recombinant PVY coat protein but the induction and expression of the recombinant PLRV coat protein was unsuccessful. The isolated recombinant PVY CP was then used to immunize a rabbit to produce highly specific anti-PVY CP immunoglobulins. The antiserum obtained from the rabbit was used to develop an ELISA to detect the presence of PVY in seed potato stocks in South Africa. The ELISA kit was subsequently used in preliminary trials to determine if the kit could detect PVY infected plant material. The initial results of the ELISA trials using PVY infected material obtained from Potatoes South Africa yielded positive results.
Evaluation of DNA vaccines developed against Mycoplasma struthionis sp. nov. str. Ms01 in ostriches
(Stellenbosch : Stellenbosch University, 2016-03) De Wet, Bertus; Botes, Annelise; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: The vast demand for ostrich meat has made South Africa the leader in not only the production of ostrich meat but also ostrich associated products such as feathers and leather. Ostrich specific mycoplasmas (referred to as Ms01, Ms02 and Ms03) cause respiratory tract infections with subsequent reduction in physical growth rate and therefore reduced production. To date no vaccines are available to combat these infections in ostriches. In this laboratory three DNA vaccines (pCI-neo, VR1012 and VR1020) have been developed with each containing the Ms01 oppA gene as antigen. The aim of this study was to evaluate these developed DNA vaccines in a mammalian cell culture based system as well as an ostrich vaccination trial. COS-1 cells were transfected with the three developed DNA vaccines. Transcription of the oppA gene was proven for all the plasmids. Translation into the OppA protein was shown to be limited to the VR1020_oppA plasmid. The protein was visualised by SDS-PAGE and detected by western blot using chemiluminescence. Two of the vaccines, VR1020_oppA and pCI-neo_oppA, were administered in three concentrations (100 μg/ml, 300 μg/ml and 600 μg/ml) to ostriches during a vaccination trial followed by a booster injection. The ability of the vaccines to elicit anti-OppA antibodies was measured using ELISA. The pCI-neo_oppA vaccine failed to induce an immune response against the antigen after both the first and booster vaccinations. The VR1020_oppA vaccine on the other hand was able to elicit an anti-OppA immune response.
Immunological and epidemiological investigations in South African ostriches and penguins
(Stellenbosch : Stellenbosch University, 2004-04) Botes, Annelise; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry
ENGLISH ABSTRACT: Newcastle disease (NO) and mycoplasma infections in ostriches have considerable economic implications for the South African ostrich industry in that NO is a limiting factor in the export of ostrich products to the European Union and mycoplasma infections cause stock losses, reduced production, reduced hatchability and downgrading of carcasses. In the first section of this dissertation, the role of passively acquired and mucosal immunity in protection of ostrich chicks against Newcastle disease virus (NOV) was investigated. Ostrich hen serum IgG and yolk IgY were isolated and characterized, and the transfer of maternal anti-NOV antibodies to the egg yolk was determined using an enzyme-linked immunosorbent assay (ELISA). Results indicated that anti-NOV antibodies were successfully transferred from the ostrich hen to the egg yolk. In addition, ostrich IgA was isolated, characterized and rabbit anti-ostrich IgA antibodies produced and used for measuring mucosal anti- NOV IgA antibodies produced in response to mucosal vaccination. Results indicated that the live La Sota vaccine stimulates IgA production and thus mucosal immunity in ostrich chicks. In the second section of this dissertation, ostrich mycoplasmas were isolated and identified using 16S rRNA gene sequencing. These sequences indicated that ostriches carry three unique mycoplasmas, which are phylogenetically quite divergent. The 16S rRNA gene sequences of the ostrich mycoplasmas were subsequently used for the development of specific primers for the detection and diagnosis of mycoplasma infections in ostriches by PCR. The last section of this dissertation focuses on avian malaria in African penguins and the management of this disease during rehabilitation. The Foundation for the Conservation of Coastal Birds (SANCCOB) is a seabird rescue and rehabilitation centre, which is largely dedicated to the rehabilitation of diseased, injured and oiled penguins. Significant mortalities due to avian malaria occur at this facility. The aim of this study was the development of an ELISA for the purpose of assessing the natural levels of anti-Plasmodium antibodies in African penguins on entry into the SANCCOB facility and during rehabilitation. Results indicated significant increases in anti- Plasmodium antibody levels after entry, which was not influenced by oiling. Infection with malaria and not parasite recrudescence was viewed to be the cause of this increase, indicating a possible role of the SANCCOB facility in exposing penguins to avian malaria.
Immunological and epidemiological investigations into avian malaria in the African penguin during rehabilitation and in breeding colonies
(Stellenbosch : University of Stellenbosch, 2005-04) Thiart, Hanlie; Bellstedt, D. U.; University of Stellenbosch. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: The African penguin, which occurs along the south-eastern and south-western shores of South-Africa and Namibia, has experienced a severe reduction in population numbers due to guano and egg collection in the first half of the 19th century, and oil pollution in the second half of the 19th century as a result of oil tankers rounding the Cape of Good Hope. The population would have been reduced by a further 19% had it not been for the rehabilitation of penguins at the South African National Council for the Conservation of Coastal Birds (SANCCOB) facility. Although this has been very successful, mortalities as a result of avian malaria infection have considerably reduced the efficiency of rehabilitation. In an effort to assess the role of immunity against malaria in combating the disease, an enzyme-linked immunosorbent assay (ELISA) for the detection of antibody levels to avian malaria was developed. The ELISA was used to detect antibody levels to avian malaria of penguins on entry and during rehabilitation from October 2001 to January 2003. The aim of this study was to continue the determination of antibody levels to avian malaria of penguins entering the SANCCOB facility, in order to allow an evaluation of the antibody levels to avian malaria for two full calendar years. This investigation was combined with a polymerase chain reaction (PCR)-based method, capable of detecting any Plasmodium species in penguin serum. These two methods were also used to investigate avian malaria in several breeding colonies in order to assess the role avian malaria may play in the survival of the African penguin in the wild. Results indicated that the ability of penguins to produce anti-Plasmodium antibodies was not influenced by oiling and that infection with malaria was not due to recrudescence but rather due to infection via mosquitoes. This indicated a possible role of the SANCCOB facility in exposing the penguins to avian malaria. However a large number of penguins arrived at the facility previously infected with malaria, indicating that malaria was present in the breeding colonies. Investigations in the breeding colonies revealed extremely high avian malaria prevalence even though no sick birds or mortalities were observed. This raised the question whether different types of malaria are responsible for infection in the SANCCOB facility and breeding colonies.
The indentification, contiguous sequence annotation, cloning and site-directed mutagenesis of the P100 vaccine candidate gene of the ostrich mycoplasma Ms02
(Stellenbosch : University of Stellenbosch, 2010-12) Steenmans, Shandre; Bellstedt, D. U.; Botes, Annelise; University of Stellenbosch. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: The ostrich industry in South Africa is currently threatened by respiratory disease in feedlot ostriches which causes a dramatic loss in production. Ms01, Ms02 and Ms03 were identified as the three ostrichspecific mycoplasmas to be associated with this respiratory disease in ostriches of South Africa. The ostrich-specific mycoplasmas have a major impact on ostrich production and for this reason there is a serious need for treatment for these infections. For this reason, the ostrich industry has undertaken an investigation into the development of vaccines against mycoplasma infections. In this study, an approach to DNA vaccine development will be investigated and applied, specifically for the ostrich mycoplasma Ms02. Firstly, the whole genome of Ms02 was sequenced using GS FLX sequencing technology. The contiguous sequences obtained from the whole-genome sequencing were analysed bioinformatically which included the annotation of the contiguous sequences and the subsequent search for a vaccine candidate gene for the development of a DNA vaccine. The P100 gene of Ms02, which showed a high degree of homology with the P100 gene of the human pathogen M. hominis, was chosen as a vaccine candidate gene for the development of a DNA vaccine. The P100 gene was successfully cloned and subsequently modified by means of site-directed mutagenesis to correct for alternative codon usage, where after the modified P100 gene was subcloned into the mammalian expression vector, pCI-neo for vaccination trials in the near future.
Investigating the developmental changes in the gut microbiome of naturally reared ostrich chicks.
(Stellenbosch : Stellenbosch University, 2021-03) Wells, Felicia; Botes, Annelise; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: The South African ostrich industry has suffered losses due to premature losses of intensively reared ostrich chicks because of gastrointestinal tract (GIT) infections. It is known that survival increases with less GIT infections observed if a production system is used where chicks are raised naturally with the hen. The purpose of this study was therefore to evaluate the change in gut bacteria of naturally reared ostrich chicks during the first three months post hatch. To this end, Ion Torrent 16S-fragment metagenomic sequencing was used in combination with the Ion Reporter pipeline for analysis of sequence data. Different genomic (gDNA) isolation protocols were evaluated as well as the use of a stabilising solution to allow for sampling in remote areas. Overall, the protocol using the PSP® Spin Stool DNA Plus Kit, outperformed the rest of the protocols regarding time, efficiency and extracting the most and most highly diverse bacteria. Although the stabilising solution improved the ease of sampling, repeated freezing and thawing reduced gDNA yield. Results also indicated that the targeting of multiple 16S V-regions with Ion Torrent improved the identification of microbial taxa. Analysis of samples at weeks 0, 2, 4, 6 and 12 after hatch from different sections of the ostrich chick GIT indicated that the chicks already have a bacterial microbiome present within their GIT at hatch. A substantial bacterial shift was observed between week 0 and week 2, and another from week 6-12. The changes from weeks 0-2 can be attributed to the change from yolk as main nutrient to feed whilst the gradual stabilization in bacterial population from week 6-12 is the result of the chick developing an adult-like hindgut fermenter. A comparison of microbiome of the different GIT sections with each other, showed that the stool sample, colon and caecum had the same dominant phyla (Figure 4.16: B), classes and families (Figure 4.19), however, at the genus level the differentiation between the stool sample, colon and caecum became more distinct (Figure 4.20: A). These changes between the different GIT sections are attributed to the unique physiological environments within each GIT section. Thus, the extrapolation of the ostrich chick GIT microbiome from a stool sample only must be done with caution. The microbiome data obtained in this study could in future studies be compared to that obtained for intensively reared ostrich chicks to shed some light on the impact of production systems on the intestinal health of ostrich chicks.
An investigation into the potato leafroll virus problem in the Sandveld region, South Africa
(Stellenbosch : Stellenbosch University, 2017-12) Van Wyk, Lezan; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Potato leafroll virus (PLRV) is responsible for significant yield losses in the South African (SA) potato industry. PLRV incidence in the Sandveld region has increased dramatically over the past 15 years. Enzyme-linked immunosorbent assay (ELISA) is used for routine testing by the SA Seed Potato Certification Scheme to diagnose PLRV infection, but many countries have changed to reverse transcription polymerase chain reaction (RT-PCR) for detection of PLRV because of its greater sensitivity. This project aimed to develop and validate a probe-based quantitative real-time reverse transcription PCR (RT-qPCR) to detect PLRV in potatoes and obtain an assessment of PLRV incidence in the Sandveld region, SA. This project also aimed to confirm infection in aphids and characterise aphid transmitted PLRV isolates by sequencing. Finally, this project aimed to apply a next-generation sequencing (NGS) technology to identify and characterise isolates, to compare non-coding 5’ and 3’ regions of the genome and lastly, to identify unknown viruses and other pathogens that possibly occur in potatoes in the Sandveld region. Suitable primers and a TaqMan probe were designed to develop a highly sensitive RT-qPCR detection method for PLRV. An amplified complementary DNA (cDNA) was cloned into a plasmid and used for assay quantification and validation. Thereafter, potato leaves were tested over a full calendar year and results were compared to vector pressure. Overall high infection levels were found, but in certain times of the year low infection levels were found due to low vector pressure. SA tubers were also tested with this method. This study indicates that the SA Potato Certification Scheme should reconsider the use of ELISA as the method for PLRV detection and replace it with the described RT-qPCR method. Secondly, the presence of PLRV in aphids was confirmed with RT-qPCR. A whole PLRV genome was amplified and sequenced after extraction from an infectious aphid. This generated whole PLRV genome was aligned in a data matrix with other whole genome sequences. Phylogenetic analysis of the whole genomes revealed that the aphid extracted PLRV isolate grouped with eight other SA isolates from the Sandveld region. Lastly, Ion Torrent was used to obtain information about further PLRV isolates present in the Sandveld region. Samples with low Cq values corresponded to a high number mapping, coverage and sequencing depth of small interfering RNAs (siRNAs). Three complete genomes were obtained by mapping siRNAs to the reference sequence, as de novo assembly could not obtain contigs longer than 700 nucleotides. Phylogenetic analysis of the whole genomes revealed that three of the samples grouped with an Australian isolate and seven SA isolates. The remaining isolate grouped with nine other SA isolates. Minor variation between upstream and downstream non-coding regions was seen. No other potato or unknown viruses were identified, but an unknown fungus was identified in all samples which needs further investigation.
An investigation of prevalance and the detection and race identification of South African potato viruses
(Stellenbosch : Stellenbosch University, 2013-03) Roos, Wiets Gideon; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Infection of potatoes by viral pathogens causes reduced crop yield and subsequent economic loss. In South Africa Potato virus Y (PVY) and Potato leafroll virus (PLRV) are the two most destructive viruses infecting potatoes. Several other viral pathogens exist, including Potato virus X (PVX), Potato virus M (PVM), Potato virus A (PVA), Potato virus S (PVS), Potato mop-top virus (PMTV), Tomato spotted wilt virus (TSWV) and Potato spindle tuber viroid (PSTVd). Although the aforementioned pathogens are found infecting potatoes around the world, there are no published information pertaining to the prevalence of these viral agents in South Africa. Currently, the occurrence of PLRV infection in potatoes of South Africa has reached epidemic proportions. A previous phylogenetic investigation undertaken in our laboratory of South African PLRV isolates, using coat protein (CP) gene sequences, found large variation between native South African PLRV isolates and most other isolates from elsewhere in the world; with their nearest relatives being single isolates from Australia and North America. In this study the incidence of PVX, PVM, PVA, PVS, PMTV, TSWV and PSTVd was investigated. A large number of potato plant and tuber samples was collected and infected samples were identified with reverse transcriptase polymerase chain reaction (RT-PCR) amplification of the CP gene or the whole genome in the case of PSTVd. The amplified nucleic acid segments were sequenced, aligned with international reference sequences and analysed phylogenetically to determine their relative relationships with these reference sequences. The CP genes of PLRV isolates were sequenced and phylogenetically investigated to determine how these new isolates compared relative to the previous findings from our laboratory. In addition, the complete genomes of two PLRV isolates were sequenced and phylogenetically investigated as a preliminary study to investigate the apparent increase of pathogenicity of certain variants of South African PLRV. Results obtained showed that only PVX and PVS were present in the samples collected and the incidences of these viruses were very low (2.0 and 1.1% respectively). The phylogenetic analyses of the CP genes, indicated that the PVX and PVS variants isolated in this study, were part of the dominant types of variants found worldwide. From the analyses of the PLRV CP and whole genome sequences, it was determined that many of the PLRV variants found in South Africa, are genetically distinctly different from those around the world. This warrants further investigation into the increased pathogenicity experienced with South African PLRV.
Investigations into the production of a harpin elicitor by Pseudomonas syringae pv. syringae isolated from a nectarine tree
(Stellenbosch : Stellenbosch University, 1996) Appel, Maryke; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Department of Biochemistry.
ENGLISH ABSTRACT: Bacterial canker of stone fruit trees, caused by Pseudomonas syringae pv. syringae, has become one of the most destructive crop diseases in South Africa Failure of chemical control of the disease has rendered the selection and breeding of resistant host trees an important aspect of future control strategies. To assist in such breeding programmes, investigations into the molecular basis of the host-pathogen interaction were initiated The fundamental ability of phytopathogenic pseudomonads, xanthomonads and non-soft rot erwinias to cause necrotic diseases in their hosts and hypersensitivity in non-host plants is controlled by their widely conserved hrp gene clusters. The only known secreted hrp gene product, dubbed "harpin", has been identified as the molecule ("elicitor"') both responsible and required for eliciting hypersensitivity or disease symptoms. In this study, the production of a harpin elicitor by a strain of Pseudomonas syringae pv. syringae, isolated locally from nectarine tree (P. s. pv. syringae NV) was investigated. The HR test in tobacco was used to assess the elicitor activity of bacterial fractions. It was established that the bacterium produces an extracellular protein elicitor similar to harpin Pss the harpin elicitor of the wheat and bean pathogen, Pseudomonas syringae pv. syringae 61. Antibodies were raised against harpin Pss and used to confirm homology between the elicitors of the two strains, using Western blot analysis. Homology between the two proteins was exploited on the gene level in the design of a polymerase chain reaction strategy for the amplification of the harpin encoding gene of P. s. pv. syringae NV from its genomic DNA. Partial sequencing of the single PCR product and Southern blot hybridization with a probe based on the P. s. pv. syringae 61 harpin encoding gene, confirmed its identity as the harpin encoding gene of P. s pv syringae NV.
Isolation and characterisation of a polygalacturonase-inhibiting protein (PGIP) and its encoding gene from Vitis vinifera L.
(Stellenbosch : Stellenbosch University, 2001-12) De Ascensao, Ana; Pretorius, I. S.; Vivier, Melane A.; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Polygalacturonase-inhibiting proteins (PGIPs) are present in the cell walls of a variety of plant species. These proteins have been shown to specifically inhibit endopolygalacturonases (endo-PGs) secreted by invading fungal pathogens as part of the induced disease resistance mechanism of plants. This is the first report on the isolation and characterisation of a pgip gene from Vitis vinifera L., designated grapevine pgip1. A single open reading frame encoding a deduced polypeptide of 333 amino acids with a predicted molecular mass of 37.1 kOa and a calculated isoelectric point of 8.61 was identified from a 5.6 kb subgenomic fragment of V. vinifera cv Pinotage. Nucleotide and derived amino acid sequence analysis of grapevine pgip1 showed significant homology with other characterised PGIP encoding genes and revealed features characteristic of PGIPs found in several other plant families. Genomic DNA analysis showed that grapevine pgip1 belongs to a small multigene family in Vitis cultivars. From Northern blot analysis it was evident that expression of the PGIP family is both tissue- and developmental stage specific. The grapevine pgip1 was transiently expressed in Nicotiana benthamiana L. with potato virus X (PVX) as a vector. Grapevine PGIP1 isolated from crude protein extracts of PVX-infected N. benthamiana were tested and showed inhibitory activity against polygalacturonases (PGs) from Botrytis cinerea. Grapevine PGIPs have not previously been purified and characterised. Molecular analyses have confirmed that PGIPs are typically encoded by multigene families and that the inhibitor specificities and kinetics of the isolated proteins differ within and among species. In this study, two PGIP isomers from V. vinifera berries were isolated. The one isomer, designated PGIP-A, was partially purified and had a molecular mass of 39 kOa, whereas the other PGIP, designated PGIP-B, was purified and had a molecular mass of 42 kOa as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SOS-PAGE) and Western blot analysis. Both proteins were cell wall-bound. Enzymatic deglycosylation confirmed that PGIP-B is a glycosylated protein. Grapevine PGIP-A showed strong inhibitory activity against a homogeneous PG from Aspergillus niger and to a lesser extent against PG from Fusarium moniliforme, but was unable to interact with a crude PG preparation from B. cinerea. Grapevine PGIP-B was able to strongly inhibit PGs from B. cinerea as well as from Colletotrichum gleosporoides, yet showed no inhibition towards PG from A. niger. The grapevine pgip1 gene was expressed under the control of the Cauliflower mosaic virus (CaMV) 35S promoter in tobacco plants via Agrobacterium tumefaciensmediated transformation. Transgenic tobacco plants expressing the grapevine PGIP (gPGIP1) were used to demonstrate the effectiveness of this inhibitor against fungal PGs and to investigate whether gPGIP1 influences disease development. Northern blot analysis identified 19 transgenic plants expressing pgip1 transcript levels. Crude PGIP extracts from the transgenic tobacco plants inhibited PGs from B. cinerea and C. gleosporoides, but not PG from A. niger. Leaves from untransformed tobacco plants, from transgenic tobacco lines showing high and low PG inhibition, and from transgenic plants that did not express pgip1, were inoculated with B. cinerea. Transgenic leaves showed a reduction in the size of necrotic lesions of macerated tissues of approximately 45% relative to control and non-expressing transgenic leaves. The results from the heterologous expression of gPGIP1, together with the results from the protein purifications and inhibition studies, indicate that the isolated grapevine pgip1 gene encodes the isolated PGIP-B isomer. This work has ; established a good model system to study certain aspects of plant-pathogen interactions in grapevine. Heterologous expression of gPGIP1 has demonstrated that PGIP inhibition of fungal PGs slows disease development of B. cinerea in planta.

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