Masters Degrees (Microbial, Biochemical and Food Biotechnology)
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Browsing Masters Degrees (Microbial, Biochemical and Food Biotechnology) by Author "Boucher, C. E."
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Item Open Access Antibody fragments as a possible therapeutic treatment for infectious bronchitis in poultry(University of the Free State, 2018-09) Coetzee, Janetta Magrieta; Bragg, R. R.; Boucher, C. E.; Van der Westhuizen, W. A.Infectious bronchitis virus (IBV), a coronavirus, is the etiological agent for infectious bronchitis (IB), an acute respiratory disease of poultry. Infectious bronchitis is a notifiable disease, and taking in consideration that poultry is the second most consumed meat within South Africa, it highlights the importance of monitoring IBV outbreaks. Recombinant single chain variable fragments (scFv) have been used in therapeutic treatments for various human and veterinarian viruses, including other coronaviruses, such as SARS-CoV and MERS-CoV. The study aims to select scFv against the IBV antigen, with the use of phage display technology and commercial ELISA plates coated with the most prevalent IBV strains namely, H120 and M41. It has been proven that the S1 protein induces the binding of neutralising antibodies which provides protection against lethal CoV infections, thus, indicating a possible application for a therapeutic treatment. In this study, phage clones were selected from a human domain (dAb) library (Source BioScience, Australia). Panning was repeated three times using commercial ELISA plates coated with M41 and H120 IBV strains. Positive monoclonal phage clones were retrieved from the polyclonal mix by a sandwich ELISA and sequenced. The selected scFvs were expressed by Isopropyl β-D-1 thiogalactopyranoside induction and purified by immunoprecipitation with the Pierce Anti-c-Myc Agarose kit (Thermo Scientific, USA). After purification, binding ability of the scFvs were determined by means of a direct competitive ELISA. The neutralising ability of the scFvs was then determined by a virus neutralisation assay in ovo with the Avipro IBV H120 strain (Lohmann Animal Health Gmbh, Germany). This was performed in 9-day old SPF eggs over a time-period of six days. One set of eggs were injected with a dilution range of the IBV H120 strain and another set by a mixture of 2 μg/ml scFv with the IBV H120 strain. The end-point titres were determined and compared by the Spearman-Karber method (Spearman, 1908). Statistical analysis was performed using the student t-test with a p-value of 0.05. Round 1 of panning resulted in a total of 1.2 x 106 phages/ml and after round 3 a total of 3.0 x 1010 phages/ml were obtained. A total of 96 phage clones were manually selected from which only 12.5% showed a positive result during the sandwich ELISA. The 12 positive clones were sequenced and analysed based on nucleotide and amino acid composition. A total of five scFvs contained a complete variable heavy (VH) chain sequence, two of which was identical. This resulted in four unique and complete scFv sequences. These sequences showed a high variance in the nucleotide composition through- out the sequence. However, variation in the amino acid composition was only observed in the third complementary determining region. The scFvs were expressed and resulted in concentrations ranging from 204.38 μg/ml to 265.07 μg/ml. Detection of IBV antigen binding ability of the purified scFvs was conducted by a direct competitive ELISA. However, no statistical difference in absorbance values were observed, indicating insufficient binding of the scFvs. The in ovo virus neutralisation assay resulted in a one log reduction of the end-point titres. Statistical analysis proved one of the reductions to be statistically significant with a p- value less than 0.05, resulting in a partial neutralisation effect from the scFv. In conclusion, the selection process showed a progressive enrichment of antigen specific clones from the dAb library. The scFvs were successfully expressed, purified and characterised in terms of binding ability.Item Open Access The development of a molecular serotyping system and an investigation into the presence of prophages in Avibacterium paragallinarum serogroups(University of the Free State, 2014-01) Coetsee, Elke; Bragg, R. R.; Boucher, C. E.Avibacterium paragallinarum is an avian pathogen that causes the upper respiratory disease Infectious coryza (IC) in chickens. This disease has the ability to cause vast economic losses due to a decrease in egg production. To date the factors contributing to pathogenicity, immunogenicity and serotyping are still not clearly understood. Vaccine failures are a major problem that occurs due to no or poor cross-protection occurring especially between the C-serovars of A. paragallinarum. This problem will be overcome by having a more accurate serotyping technique available for the diagnosis of IC. Therefore one of the aims of this study was the development of a molecular serotyping technique, where a serotyping PCR was developed which distinguished between the Modesto (C-2) and SA-3 (C-3) A. paragallinarum isolates which is the major cause of IC in South Africa. Reported in a recently published article was the presence of a HP2-like and Mu-like phage within the genome of the Modesto (C-2) strain of A. paragallinarum. Therefore another major question addressed during this study was whether there are prophage genes present in all of the reference isolates as well as in field isolates of A. paragallinarum and what the effect of these phages might be on the virulence and pathogenicity of these isolates. Phage genes that are important during lysogeny were selected and screened for by means of PCR. From the results it was able to determine that some of these genes are present in some of these isolates but no discernible patterns were detected in terms of the effect on pathogenicity. Therefore future studies will be conducted to mainly focus on the effect these phages might have on the virulence and pathogenicity as well as whether these phages are responsible for the occurrence of different A. paragallinarum serovars.Item Open Access Improving the current diagnostic strategy for beak and feather disease virus in parrots(University of the Free State, 2014) Munsamy, Yuri; Bragg, R. R.; Boucher, C. E.English: Beak and feather disease (BFD), caused by Beak and feather disease virus (BFDV) is a dermatological condition afflicting parrot species. It is becoming increasingly difficult to ignore not only the significant negative economic impact that the virus has on the parrot breeding industry but also the detrimental effect it has on the survival of the endemic Cape parrot (Poicephalus robustus). The virus, a member of the Circoviridae, is known to possess a non-enveloped, circular, single-stranded DNA genome. Two major open reading frames (ORFs) encode the replication associated protein (Rep) and the coat protein (CP). The study was set out to evaluate and improve the current diagnostic strategy for BFDV, with both molecular and serological techniques. The following objectives were attempted: 1. To evaluate polymerase chain reaction (PCR) and quantitative real-time polymerase chain reaction (qPCR) as diagnostic tools for BFDV. Detection of BFDV with conventional PCR is not always sensitive, especially in birds without clinical symptoms. Furthermore, genetic variance was suggested to have a detrimental effect on primer hybridisation. A real-time assay was designed to address these problems. It amplified a 115 bp fragment of ORF V1 and was able to quantify viral load. 2. To recombinantly express BFDV coat protein. A sustainable source of the main immunogen, coat protein, was needed for use in serological test development. Bacterial expression of BFDV CP was unsuccessful; however, BFDV CP from an alternative expression study was used as a serological diagnostic antigen. 3. To develop serological diagnostic tests for BFDV. A novel slide agglutination test was developed and will serve as an initial screening tool in serological diagnosis. Steps were made in the development of a competitive Enzyme Linked Immunosorbent Assay (ELISA) for a quantitative indication of immune response to BFDV. A significant proportion of asymptomatic BFDV infections exist. Using a combination approach of both molecular and serological tests increases the capacity to detect infections or exposure to virus. New techniques described should be used in conjunction with existing tests and should not completely replace conventional techniques for diagnosis of BFDV infection or detection of exposure to the virus.Item Open Access An investigation into the efflux mechanism against quaternary ammonium compounds as a contribution of bacterial resistance(University of the Free State, 2014-01) Coetzee, Marisa; Bragg, R. R.; Jansen, A. C.; Boucher, C. E.Bacterial infections, a major problem in the poultry industry, are controlled through the use of antibiotics. Due to the increase in antibiotic resistance and the restrictions placed on the use of antibiotics in animals, the poultry industry is slowly heading for a post-antibiotic era. The use of disinfectants, like quaternary ammonium compounds (QACs), could possibly be the last resort in the fight against bacterial infections. Resistance against QACs has been observed but needs to be investigated in order to prevent similar resistance problems. The overall aim of this study was to understand bacterial resistance to QACs, using the following objectives: To examine the presence of qac resistance genes in field isolates and determining if the number of genes present confer higher resistance. To study the expression of one of these qac resistance genes in the presence of increasing QAC concentration. To study the efflux system, to determine the uptake and efflux of disinfectant, and to determine if the bacteria causes structural changes of the disinfectant. Bacterial resistance is conferred through acquisition of resistance genes; therefore qac resistance genes were studied to understand resistance to QACs. Screening of field isolates using conventional PCR for qac resistance genes (smr, qacJ, qacH and qacG) showed that one strain could contain more than one resistance gene. This could not be correlated with the minimum inhibitory concentration (MIC) of the three QACs tested and possession of more genes did not necessarily make the strain more resistant. Staphylococcus aureus strains known to contain at least one of the resistance genes were also screened for these qac genes. Conventional PCR showed that all the genes could only be detected in the strain when it was exposed to QAC. Conversely, real-time PCR showed that the genes could be detected even in the absence of QAC, and was detected in susceptible strains as well. Therefore containing the genes does not necessarily confer resistance. Relative quantitative real-time PCR was used to determine the expression of the qacJ gene in the S. aureus strains VB3_qacJ and ATCC 25923. It was hypothesised that the expression of the qacJ gene would increase with increasing didecyldimethylammonium chloride (DDAC) concentration. However, in the VB3_qacJ strain there was no significant difference in expression when induced with different DDAC concentrations. Expression of known qac resistance genes needs to be investigated simultaneously to determine whether a relationship exists between the qac genes conferring resistance. The mechanism of resistance is mainly efflux of the disinfectants; thereby reducing the disinfectant concentration inside the cell. The efflux mechanism of S. aureus was also studied using liquid chromatography-mass spectrometry (LC-MS). The hypothesis was that the bacteria are able to alter the structure of QAC before extruding it from the cell. Samples were prepared for LC-MS using solid phase extraction (SPE). The SPE protocol was optimised for extraction DDAC from growth media. It was shown that the DDAC concentration in the growth media decreased after 18 hrs of growth. It was not determined if structural changes of DDAC occurred. Resistance to disinfectants is much more complex than originally thought, and therefore resistance is still not fully understood. Further research is required to understand the full mechanism of resistance against QACs, so that similar problems associated with antibiotic resistance can be prevented.Item Open Access A molecular study of Mycoplasma gallisepticum field isolates from poultry in southern Africa(University of the Free State, 2012-07) Moretti, Serena Angela; Bragg, R. R.; Boucher, C. E.English: Mycoplasma gallisepticum (MG) is an economically important pathogen of poultry worldwide, causing chronic respiratory disease in both chickens and turkeys. Little research has been done to characterize the MG field strains present in southern Africa. Field evidence however, suggested breaks in the vaccinations against MG, despite the proper cold chain and correct administration. Molecular methods were used to screen various poultry farms in South Africa and Zimbabwe for MG. Isolates were further characterized by gene-targeted sequencing (GTS). Portions of the cytadhesin pvpA, gapA, mgc2 genes and the uncharacterized surface lipoprotein gene designated MGA_0319 were sequenced and analysed. Three MG strains were identified in this study: a Ts-11-like strain; a strain showing closest percentage identity to the atypical MG RV-2 strain found in Israel, however with the lack of a large deletion within the pvpA region; and lastly an isolate from Zimbabwe, likely to be a novel MG strain. The latter contained a unique, large, in-frame nucleotide insertion in the mgc2 gene. Antigenically-significant variation (determined in silico) within the membrane surface proteins of this isolate was found compared to the MG Rlow strain used as an inactive vaccine on the poultry farm. It is thus postulated that by MG altering its antigenic profile, it allows effective avoidance of immune recognition and antibodies produced as a result of vaccination with inactivated and possibly live MG vaccines. Further research is however needed to substantiate this claim.Item Open Access Studying the regulation of immune signalling molecules related to immunity during Avibacterium paragallinarum infection(University of the Free State, 2019-06) Jawallapersand, Poojah; Boucher, C. E.; Janse van Rensburg, W. J.; Van der Westhuizen, W. A.Infectious coryza is a contagious and acute upper respiratory poultry disease caused by Avibacterium paragallinarum, that plagues predominantly layers but also affects broiler breeds. The chicken’s immune response to Av. paragallinarum serovar C-3 (SA-3) infection (reported to be most virulent in South Africa), and the underlying genetic mechanisms involved, are poorly understood and not well documented. The aim of the study is to understand the complexity of the regulation of immune functions by identifying the molecules that are expressed during Av. paragallinarum serovar C-3 (SA-3 strain) infection. In this study, chickens (control versus experimental groups) were directly challenged via infraorbital injection with Av. paragallinarum serovar C-3 (SA-3 strain) and the immune response was monitored. The mean disease score and mean daily egg production score were recorded and calculated. Blood and sera were obtained for blood microscopy, leukocyte population profiling by flow cytometry analysis, and antibody/cytokine screening with ELISA assays. Finally, control and experimental chickens were sacrificed based on the clinical scores obtained (0, 1, 2 or 3). Post-mortem examination was conducted, and organs were harvested for immunohistochemistry staining for identification of distinct immune cell populations. The in vivo results obtained from the experimental studies in combination with the in silico results obtained from bioinformatics tools for the generation of immune signalling pathway maps may provide insight and a birds-eye view into the immune mechanisms between host-pathogen interactions for this disease. Results from our study could potentially assist with diagnostic tests for serovar C-3 and provide insight towards more efficient vaccine development. Hence, if vaccine practices are improved this will limit importations of birds from huge global markets, thus preventing carry-over poultry diseases and zoonosis as well as maintaining a safe and sustainable economy.