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Von Willebrand disease is the most common inherited bleeding disorder caused by a deficiency or defect in von Willebrand factor. Quantitative defects of von Willebrand factor include, type 1 von Willebrand disease (partial deficiency of von Willebrand factor) and type 3 von Willebrand disease (complete deficiency of von Willebrand factor). Type 2 von Willebrand disease includes all qualitative defects of von Willebrand factor. Type 1 von Willebrand disease is either due to decreased synthesis and secretion or increased clearance of von Willebrand factor from plasma. It is essential to diagnose individuals with an increased clearance rate of von Willebrand factor, as the treatment of these patients with 1-deamino-8-D-arginine vasopressin is not effective. The ratio between the von Willebrand factor propeptide and the von Willebrand factor antigen is used to identify conditions of reduced half-life, such as type 1 von Willebrand disease with increased clearance. Currently, there is only one commercial assay available to measure von Willebrand factor propeptide levels. This assay is not only too expensive to be used in developing countries but is also very time consuming. The von Willebrand factor propeptide protein assay is an expensive test as it uses monoclonal antibodies. Mammalian cells are commonly used for the expression of monoclonal antibodies. The production of monoclonal antibodies is expensive. With this research an effort was made to develop more cost-effective and more rapid assays to determine the von Willebrand factor propeptide levels in patient’s plasma. The aim of this study was to therefore validate a von Willebrand factor propeptide assay. First, two single chain variable fragments that bind to the von Willebrand factor propeptide were expressed by yeast. The von Willebrand factor propeptide protein was also expressed, as it is not commercially available. However, the expression of the propeptide and the two single chain variable fragments were not

Since their discovery in the 1920s, antibiotics have saved generations of people from succumbing to bacterial infections. Antibiotic usage has resulted in increased antibiotic resistance which has become a problem in various fields and industries. Without effective antibiotics, even simple bacterial infections can be fatal and this has the potential to create a post-antibiotic era where fatalities from currently controlled bacterial infections will become normal. This also extends to the poultry industry where Avian Pathogenic Escherichia coli (APEC) are known to affect the market due to colibacillosis and resulting in poor quality meat and egg products. As bans against antibiotics used in the growth of food animals are established there is a necessity for alternative treatment options. One of the potential solutions is harnessing the power of bacteriophages which are viruses that infect bacteria and proliferate within them. Bacteriophage therapy has been developing and the diverse use of bacteriophages in treatment of bacterial infections ranges from using the whole bacteriophage within its packaged virion, to only parts of the bacteriophage such as proteins/enzymes. Some of the challenges of using bacteriophage cocktails for therapy, extended also to the treatment of APECs, are the myriad environmental conditions that bacteriophages require to proliferate in a host. In this thesis, the use of lysogenic bacteriophages and the heterologous expression of bacteriophage endolysins were investigated as alternative treatments against bacteria. Screening PCRs in combination with induction of prophages within lysogenic bacteria were used to determine whether temperate bacteriophages could be used and genetically manipulated to remain in lysis and therefore be used in therapy or treatment. Potential lysogens were induced using UV-, heat- and mitomycin C inducers. Heterologous expression of bacteriophage proteins was performed through both bacterial expression and yeast expr, National Research Foundation (NRF)

Mastitis is a common disease affecting dairy herds, with high occurance incidents and tremendous economic losses associated with it. Varieties of infectious agents are associated with mastitis, but bacteria are responsible for most of the cases. Apart from large numbers and heterogeneity between bacterial species associated with mastitis, it is an infectious disease and the process of milk production makes it easy for the disease to spread and difficult to control. Good milking hygiene, antibiotic therapy during lactation and dry off and chemical teat dips are some of the measures used in its control and treatment of mastitis. However, more cases of antibiotic therapy in treatment and control end up in failure and antimicrobial resistance is the reason attributed to this. The main goal of this study was to investigate the diversity of bacterial species causing mastitis in South African dairy farms, determine their antimicrobial susceptibility profile and isolate lytic bacteriophages for these species as potential alternative for the control of mastitis caused by bacterial pathogens. Milk samples from mastitis and normal cows were analysed using traditional isolation methods on non-selective and selective differential agar plates, identification by standard biochemical tests. The following bacterial species were predominantly found: Staphylococcus aureus and members of Coagulase-negative Staphylococcus group (Staphylococcus chromogenes, Staphylococcus haemolyticus, Staphylococcus xylosis, Staphylococcus epidermis, Staphylococcus hominis, Staphylococcus hycus, Staphylococcus capitis, Staphlococcus sciuri), Streptococcus dysgalactiae, Streptococcus uberis, Escherichia coli, Klebsiella spp., Pseudomonas spp., Enterococcus spp., Enterobacteriacea spp., Proteus spp., Citrobacter spp., Bacillus spp., Bacillus pumilus, Acinetobacter spp., Lactococcus lactis and Pasteurella spp. For their antimicrobial susceptibility evaluation, disk-diffusion and broth microdilution methods, National Research Foundation (NRF)

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 zwitteri, National Research Foundation (NRF)

Bacterial infections cause severe economic loss in the rapidly growing aquaculture industry. Lactococcus garvieae, a Gram-positive bacterium, causes lethal, hyperacute septicaemia in numerous fish species of importance to aquaculture worldwide. The virulence factors of this pathogen is not well understood, although the exopolysaccharide (EPS) capsule is considered the most important virulence determinant. A conserved metabolic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), may function as a virulence factor of L. garvieae via the phenomenon of “moonlighting”, a term that refers to the ability of a protein to perform more than one function. Previous investigations have also identified L. garvieae GAPDH as an antigen, suggesting that GAPDH is localised to the extracellular environment. The general aim of this study was to improve the current understanding of factors contributing to L. garvieae virulence by means of the following objectives:  Identify putative virulence factor genes of South African L. garvieae isolates by PCR and sequencing.  Identify extracellular proteins from South African L. garvieae isolates.  Clone, express and purify L. garvieae GAPDH.  Identify putative ligands to recombinant GAPDH using a commercially available random peptide phage display library. Isolates obtained from diseased rainbow trout (Oncorynchus mykiss) from locations in present day Gauteng and Mpumalanga, South Africa, were screened for the presence of a set of putative virulence factor genes by PCR. The presence of EPS capsules were tested by negative staining and amplification of EPS biosynthesis genes by PCR. All virulence factor genes were found in the isolates tested, including an avirulent strain NCFB 657. However, negative staining indicated the absence of EPS capsules in the virulent isolates and NCFB 657 and EPS biosynthesis genes could not be amplified. Extracellular proteins detected and identified by LC/MS-MS included L-lactate dehydrogenase, enolase, 60, University of the Free State (Department of Microbial, Biochemical and Food Biotechnology)

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.

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 combinatio, Afrikaans: Beak and feather disease (BFD), wat veroorsaak word deur Beak and feather disease virus (BFDV) is 'n dermatologiese toestand wat papegaai spesies versmag. Dit word toenemend moeilik om nie net die beduidende negatiewe ekonomiese impak wat die virus het op die papegaai teling bedryf nie, maar ook die nadelige uitwerking wat dit op die voortbestaan van die endemiese Kaapse papegaai (Poicephalus robustus) te ignoreer. Die virus, 'n lid van die Circoviridae, is bekend as 'n nie-omvou, sirkulêre, enkelstring DNA genoom in besit te neem. Twee groot oop lees rame (OLRe) enkodeer die replikasie verwante proteïene (Rep) en die kapsied proteïen (KP). Die studie was uiteengesit om die huidige diagnostiese strategie vir BFDV te evalueer en te verbeter, met beide molekulêre en serologiese tegnieke. Die volgende doelwitte is gepoog: 1. Om polimerase kettingreaksie (PKR) en kwantitatiewe werklike tyd polimerase kettingreaksie (qPCR) as diagnostiese instrumente vir BFDV te vergelyk. Opsporing van BFDV met konvensionele PCR is nie altyd sensitief nie, veral in voëls sonder kliniese simptome. Verder is genetiese afwyking voorgestel om 'n nadelige uitwerking op primer verbastering te hê. 'n qPCR toets is ontwerp om hierdie probleme aan te spreek. Dit versterk 'n 115 bp fragment van OLR V1 en was in staat om virale lading te kwantifiseer. 2. Om die BFDV kapsied proteïen in 'n bakteriële gasheer uit te druk. 'n Volhoubare bron van die hoof immunogeen, kapsied proteïen, is wat nodig is vir die gebruik in serologiese toets ontwikkeling. Bakteriële uitdrukking van BFDV KP was onsuksesvol, maar BFDV KP van 'n alternatiewe uitdrukking studie is gebruik as 'n serologiese diagnose antigeen. 3. Om serologiese diagnostiese toetse vir BFDV te ontwikkel. 'n Plaat agglutinasie toets is ontwikkel en sal as 'n aanvanklike keuring instrument in serologiese diagnose dien. Stappe is gedoen in die ontwikkeling van 'n mededingende Ensiem-Gekoppelde Immunosorbent toets (ELISA) vir 'n kwantitatie, National Research Foundation (NRF)

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

Introduction: Care farms, where all or part of the farm is used for therapeutic purposes, show much potential for improving the health and well-being of a range of disadvantaged groups. Studies to date have been qualitative or observational, with limited empirical evidence of the effectiveness of care farms in improving health and well-being. Understanding the underlying mechanisms that lead to improvements for different disadvantaged groups is a further gap in the evidence. Participants in this study are offenders serving community orders. Their low socioeconomic status and poor health outcomes relative to the general population exemplifies disadvantage. Methods and analysis: This paper describes the protocol of a study to understand the impacts of care farms and to pilot the design and tools for a study to assess cost-effectiveness of care farms in improving the quality of life of offenders. As a pilot study, no power calculation has been conducted. However, 150 offenders serving community sentences on care farms and 150 on other probation locations (eg, litter picking, painting) will be recruited over a 1-year period. Changes in quality of life, measured by Clinical Outcome in Routine Evaluation-Outcome Measure, health and reconvictions of offenders at care farms compared to other probation locations will be analysed to inform the sample size calculation for the follow on study. The feasibility of recruitment, retention, collecting cost data and modelling cost-effectiveness will also be assessed. The study will use qualitative methods to explore the experiences of offenders attending care farms and perceptions of probation and care farm staff on the processes and impacts of the intervention. Ethics and dissemination: Findings will be published and inform development of a natural experiment and will be disseminated to probation services, care farms and academics. University of Leeds Ethical Review Board approved: SoMREC/13/014. National Offender Management Service