Doctoral Degrees (Microbial, Biochemical and Food Biotechnology)
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Browsing Doctoral Degrees (Microbial, Biochemical and Food Biotechnology) by Author "Bragg, R. R."
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Item Open Access Development of a DNA vaccine for the prevention of Psittacine beak and feather disease(University of the Free State, 2008-05) Kondiah, Kulsum; Bragg, R. R.; Albertyn, J.Psittacine beak and feather disease (PBFD) is a readily recognisable dermatologic condition in wild and captive psittacines worldwide. It is caused by Beak and feather disease virus (BFDV) which is classified in the family Circoviridae and the genus Circovirus. BFDV has a circular ss-DNA genome consisting of seven open reading frames (ORFs), three being conserved in all BFDV isolates, ORF 1 which encodes the Rep protein, ORF 2 which encodes the coat or capsid protein (CP) and ORF 5 which encodes a protein whose function is as yet unknown. General symptoms of the disease include the symmetrical loss of feathers, feather abnormalities, beak and claw deformities, weight loss, anorexia and immunosuppression. The inability to grow BFDV in tissue culture or in embryonated eggs has hindered the routine diagnosis of PBFD affected birds and the development of reliable diagnostic tests and an effective vaccination program. PBFD is widespread in South Africa, leading to a loss of at least 10% of psittacine breeding stocks annually. The disease is also a major threat to the already endangered Cape Parrot (Poicephalus robustus) and the black-cheeked lovebird (Agapornis nigrigenis) and it is only a matter of time before we may see the extinction of these and other parrot species due to the lack of a preventative vaccine. The economical and natural implications of the attack by PBFD led to the aims of the present study which were to develop a potential DNA vaccine candidate, develop an expression system for production of recombinant CP as antigenic protein and establish an enzyme linked immunosorbent assay for the detection of BFDV-specific antibodies in parrots. The entire CP gene which has been suggested to encode for the epitopic protein of the virus was amplified by polymerase chain reaction (PCR) and ligated into a bacterial vector, pBAD/His B or a yeast vector, pKOV136 for expression of recombinant CP in Escherichia coli or Yarrowia lipolytica, respectively. Alternatively, CP gene PCR products were ligated into the mammalian expression vector pcDNA™3.1D/V5-His-TOPO® which was the vector of choice for DNA vaccine design and used to transiently transfect Chinese hamster ovary cells. Subsequently, the candidate DNA vaccine was used in a basic vaccine trial where budgerigars (Melopsittacus undulatus) were vaccinated either with the DNA vaccine candidate or a sub-unit vaccine consisting of purified recombinant CP. Expression of recombinant CP was monitored using polyacrylamide gel electrophoresis (PAGE), chemiluminescent and colorimetric detection on Western blots and ELISAs. While expression of the recombinant CP was unsuccessful in the yeast system using pKOV136, expression of recombinant CP was achieved in E. coli cells using the pBAD vector. Recombinant CP was partially purified and applied in both indirect and indirect competitive ELISAs as coating antigen for the detection of BFDV specific antibodies. Using the established ELISAs, BFDV specific antibodies could be detected in naturally infected parrots as well as in budgerigars vaccinated with the DNA vaccine and sub-unit vaccine. Comparable results were obtained when nonpurified recombinant CP was applied in the ELISAs in lieu of partially purified recombinant CP. Vaccinated budgerigars formed BFDV specific antibodies in response to the DNA vaccine and sub-unit vaccine that were detected using the indirect competitive ELISA established in the study. The antibody responses to the sub-unit vaccine were higher than those in response to vaccination with the DNA vaccine candidate. Although the indirect competitive ELISA could not provide an indication of whether these antibody responses are protective, the results obtained during the trial are a preliminary indication that both the DNA vaccine and sub-unit vaccine may be functional in parrots and safe to use as no adverse reactions were observed.Item Open Access Expression of avian pathogenic Escherichia coli (APEC) virulence factors, Iss and HlyF, as potential sub-unit vaccine candidates(University of the Free State, 2017-06) Van der Westhuizen, Wouter Andre; Bragg, R. R.; Boucher, C. E.; Theron, C. W.Avian pathogenic Escherichia coli (APEC) is the causative agent of colibacillosis in poultry and leads to economic losses in the poultry industry. Due to rising concerns of antibiotic resistance and antibiotic carry-over into food, bans have been implemented on antibiotic use in animal production. The process of discovering new antibiotics and having them registered and approved can take up to eight years, and their application will most likely be limited to human-use. Alternative therapies for the control of bacterial diseases in animals, including poultry, are therefore becoming increasingly important. Alternative treatment options could include the use of bacteriophages, bacteriophage enzymes, essential oils and vaccines. Bacterial vaccines are generally based on whole bacterial cells, and in the case of commensal bacteria such as E. coli, this can lead to poor gut health. Therefore, the need for highly specific bacterial vaccines are required, such as sub-unit vaccines containing only antigens found in pathogenic strains of E. coli. Various virulence genes have been found in APEC that contribute to the pathogenicity of the strains. Five of these genes have been found to be highly prevalent in most clinical cases of avian colibacillosis. In this study, two of these genes were selected as potential candidates for sub-unit vaccine development, namely increased serum survival (iss) and haemolysin F (hlyF). The increased serum survival gene, an outer-membrane protein of APEC, was successfully expressed in E. coli BL21 (DE3) using the pET28b(+) vector system, although the protein was mostly water-insoluble due to hydrophobic N -and C-terminals. The sequence of iss was then modified to create a truncated version of the gene encoding the hydrophilic region of the gene, also the potential epitope of the Iss antigen, to improve solubility during over-expression. Water-soluble truncated Iss was produced in conjunction with the full Iss protein, solubilised using a zwitterionic detergent, purified using the hexahistidine regions flanking the inserted gene in the pET28b(+) vector system. The purified proteins were mixed with adjuvant and injected into chickens raise antibodies against the two expressed proteins. The antibodies obtained from the chickens were used to perform western blotting and ELISA and both proteins were confirmed to be immunogenic. Furthermore, the obtained serum was cross-reacted with the full and truncated forms of the protein, indicating potentially similar epitopes, showing promise of using the highly water-soluble truncated Iss protein for potential future vaccine development. Yarrowia lipolytica heterologous protein expression was also attempted with the full iss gene. However, no expressed protein was detected using SDS-PAGE, so alternative methods of expression, purification and isolation were attempted. An enterokinase proteolytic cleavage site was introduced between the iss gene and the GPI-anchor sequence, the secretion vector pINA1317 and the C-terminus hexahistidine region bearing pINA1317 secretion vector were used as alternatives methods to express the full iss gene sequence. These methods failed to produce the desired results. Western blotting using antiserum raised against E. coli expressed protein could also not detect any expressed Iss protein. It was then concluded that a yet unknown issue is preventing adequate, if not any, expression of the iss gene in Y. lipolytica and further research is required. During the study, published literature indicated that the putative avian haemolysin F, was not a haemolysin but an enzyme involved in outer-membrane vesicle (OMV) biogenesis. This was validated by our failure to detect haemolytic activity after hlyF-overexpression and the presence of OMVs observed by TEM. As this was very recent research into the involvement of this gene in OMV biogenesis, the impact of the expression of the hlyF gene was investigated regarding the regulation of outer-membrane proteins which are scavenged during OMV release. Relative quantitative PCR was used to compare hlyF-expressing and non-expressing cells, and it was shown that ompA expression is increased during hlyF expression, while ompF expression remained nearly the same, which could lead to osmotic-stress susceptible cells during hlyF induction. It was concluded that a decrease in ompA expression is not involved in the mechanism of hlyF-induced OMV biogenesis, contrary to one of the biogenesis mechanisms described in literature. Sub-unit bacterial vaccines could be the future method of preventing bacterial diseases in the poultry industry. Alternative methods such as bacteriophage therapy might not be possible due to the non-linear pharmacokinetics observed, which will hinder registration of these products. Sub-unit vaccines should elicit highly specific immune responses to virulence-related antigens and not target commensal bacteria. Various antigens accessible to the immune system of the host are outer-membrane or cell wall-associated proteins, protein expression of these antigens could pose problems such as high costs for production and problems during the development of expression systems for these proteins, even complete failure of expression. Knowledge and correct characterisation of virulence-related proteins is also essential, as seen with the HlyF protein expressed in this study which is not directly suitable for vaccine production as previously thought. Future work is required in the optimisation of protein expression parameters, and chicken challenge studies with APEC will need to be conducted to determine if protective immunity is gained by vaccination with the E. coli-expressed Iss proteins. It will also be highly advantageous if the problems encountered during protein expression in Y. lipolytica could solved and the yeast-expressed Iss protein tested during challenge studies with APEC.Item Open Access An investigation of resistance to quaternary ammonium compound disinfectants in bacteria(University of the Free State, 2012) Jansen, Arina Corli; Bragg, R. R.The widespread and unrestricted use of antibiotics in animal production has led to a surge in antibiotic resistant bacterial strains. The poultry industry is steadily headed for a post antibiotic era, thus fuelling the search for alternative treatments for bacterial infections. One of these alternative treatments is the use of quaternary ammonium compound (QAC) based disinfectants. QACs are cationic surface active detergents widely used in the poultry industry because of their low relative toxicity and good antibacterial properties. Reports on QAC resistant bacteria have been on the increase in the food industry and thus studies on bacterial resistance to QACs are on the increase. In order to try and understand disinfectant resistance, it is important to gain a better understanding of the mode of action of QAC based disinfectants of bacterial cells, particularly in the light of a pending post antibiotic era. In order to do this, bacteria treated with DDAC were examined using Scanning electron microscopy (SEM) and Nano Scanning Auger Microscopy (NanoSAM). Staphylococcus aureus strain ATCC 2357 treated with DDAC revealed protuberances or “bleb” formations on their cell walls when observed with SEM. The DDAC treated cells were further investigated using NanoSAM. NanoSAM is the combination of Scanning Auger Microscopy (SAM) and etching with an Argon (Ar+) gun. SAM has the ability to perform semi-quantitative elemental analysis on extremely small volumes while visualizing the sample with SEM. Using NanoSAM technology we were able to visualize morphological changes caused by the disinfectant that SEM could not show. Clear evidence of a disruption of the cell membrane and the leaking out of cellular content was obtained. Resistance to QAC has been attributed to the presence of the qac resistance genes, smr, qacJ, qacG, qacH. During this study the presence of the qac resistance genes could be correlated to the degree of resistance QACs. The qac resistance genes were identified using conventional PCR in strains that displayed higher tolerance to the different QACs. No qac resistance genes where identified in the susceptible strain ATCC 25923 using conventional PCR even though this strain was resistant to one of the QACs, benzalkonium chloride. An increased resistance to the different QACs could not be attributed to the presence of one specific qac resistance gene.Real time PCR was introduced in this study since it is a technique known to be more sensitive than conventional PCR. Using real time PCR, it was revealed that all the bacterial strains contained more than one qac resistance gene. Interesting results were obtained with the susceptible strain ATCC 25923, where qac resistance genes were detected with real time PCR, while these genes were not detected using conventional PCR. Similar results were obtained with the Avian pathogenic Escherichia coli (APEC) strain isolated from poultry pens. After detecting the presence of the genes, the focus of the study changed to investigate the levels of expression of one of the qac resistance genes, smr. The expression study was performed using relative quantitative real time PCR. The hypothesis was that expression is increased when QACs are present in a culture medium. During the study it was revealed that there was no significant difference in the expression of the qac genes during cultivation in the presence of different QACs. There was, however a difference in the expression of the different strains tested where the smr was only expressed in the strain VB4-smr and not in the strains VB3-qacJ and ATCC 25923 during cultivation in the QAC didecyldimethylammonium chloride (DDAC). An additional hypothesis was subsequently formed. This hypothesis postulates that there is a difference in the expression of the smr gene over a time interval. During this study it was revealed that there was a significant difference in the expression of smr cultivated in different concentrations of DDAC, but there was no significant difference in the expression over a time interval. From this study, it has been established that qac resistance genes are present in various bacteria and that using the more sensitive real time PCR test, additional qac genes were found in most of the strains. From the expression studies, it can be concluded that the levels of resistance is not merely related to the presence or absence of a particular qac resistance gene. It was also established that resistance is also not always directly related to increased levels of expression of a particular qac resistance genes. From this study, it is evident that resistance to disinfectants is multi factorial and substantial additional research is required to fully understand resistance to disinfectants.Item Open Access Towards unravelling the genome of Avibacterium paragallinarum(University of the Free State, 2009) Roodt, Yolandi; Albertyn, J.; Bragg, R. R.Avibacterium paragallinarum is an avian pathogen and has the ability to cause vast economical losses. This bacterium forms part of the Pasteurellaceae family and factors contributing to pathogenicity, immunogenicity and serotyping are not clearly understood. One of the main questions that were addressed in this study was the identification of genetic tools are that is responsible for the NAD+- independence ability of this organism. NAD+ recycling genes were implicated for this bacterium and Av. paragallinarum seem to follow the recycling pathway as set out for the Pasteurellaceae family. Still the question regarding NAD+- independence remains unanswered as no complete pathway could be implicated for this trait. Furthermore, no plasmid(s) could be isolated that conferred this trait and no complete NAD+ synthesis pathway could be implicated. Only two genes were identified to form part of a NAD+-independent pathway which indicated that Av. paragallinarum may use genetic tools for NAD+-independence as set out for bacteria in general and not as the rest of the Pasteurellaceae family members. Plasmid isolation studies revealed only the plasmid p250 identical to the established plasmid p250 for Av. paragallinarum. Literature reports on two other native plasmids namely pYMH5 and pA14, however, these two plasmids could not be detected in any of the strains used in this study during plasmid screens. This study confirms and illustrates the integration of plasmid p250 within the genome of Av. paragallinarum by different serovars. In order to study this bacterium on a genomic level a whole genome sequencing project was launched. This project experienced vast amount of difficulties regarding the assembly of a complete genome for Av. paragallinarum. This bacterium contains numerous repeated regions within its genome which, with current sequencing technology, prevent the genome assembly into a single chromosome. A complete assembled genome can only be achieved when longer read lengths are available in high-throughput sequencing technologies and will thus enable clarification regarding the repeated regions. A pseudo-assembled molecule was sent to the JCVI for genome annotation. Annotated data revealed that Av. paragallinarum contains a high degree of complexity regarding its cell envelope which may shed light on pathogenicity and immunogenicity problems endured. This bacterium contains numerous mobile and extrachromosomal elements which contribute to the inability to close the genome of Av. paragallinarum. Nine site-specific integrases and 10 transposases were identified. These tools could allow for a high degree of genetic diversity within the Av. paragallinarum specie. Alongside these tools two putative prophages was identified for Av. paragallinarum. One prophage resembles the Mu-like prophages and was termed ФAvpmuC-2M, the other resembles the HP2-like prophages and was termed ФAvpC-2M-HP2.