Browsing by Author "Neveling, Deon Pieter"

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    Development of a ZnO nanowire-array biosensor for the detection and quantification of immunoglobulins
    (Stellenbosch : Stellenbosch University, 2013-12) Neveling, Deon Pieter; Dicks, Leon Milner Theodore; Perold, W. J.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: The aim of this study was to develop a ZnO nanowire-array biosensor that would detect immunoglobulins and record changes in the concentration of an antibody. Early detection of disease-causing agents is essential for an early response. In contrast to conventional methods, biosensors may detect disease-associated agents much faster and more accurate, which holds specific benefits to rural communities. The development of such a biosensor would be favourable for diagnostics in underprivileged communities without infrastructure. The hypothesis was that binding of antibodies to the surface of ZnO nanowires would result in the generation of a piezoelectric potential that, when channelled through a Schottky barrier, would produce a constant voltage reading. Piezoelectricty would be generated due to the bending of the nanowires, or tensile stress applied to the nanowires due to binding of the antibodies. The performance of such a device largely depends on the methods used to construct the ZnO nanowires and methods used to funtionalize the sensor surface. The biggest challenge was thus to chemically modify the self-assembled monolayers (SAMs) and create intermediate monolayers that would react to primary amino groups of lysozyme and form a covalent amide bond. Lysozyme was selected as model antigen, since its structure and reaction with antibodies has been well studied. Alkanethiol and dialkyl disulphides were used to form SAMs. Different SAMs were compared to select the absorbate that would bind the highest concentration of lysozyme. Lysozyme was best immobilized onto Au film layers in the presence of SAM 3-mercaptopropionic acid. Weakest immobilization was in the presence of combined SAM 11-mercaptoundecanoic acid/1-nonanethiol. The sensitivity of the constructed ZnO nanowire biosensor was tested in vitro, in the presence of different concentrations of lysozyme antibodies. An increase in the dimension of the ZnO seed layer led to an increase in the mean diameter of the ZnO seed grains, and subsequently an increase in the mean diameter of the synthesized ZnO nanowires. Deposition of the ZnO seed layer, using the RF cylindrical magnetron sputtering technique, improved the c-axis alignment of the nanowires and produced nanowires with similar dimensions. However, deposition of the ZnO seed layer using the sol-gel spin coating technique, produced nanowires with irregular c-axis alignments and irregular diameters. An increase in the Au film thickness led to a decrease in the mean diameter of the synthesized ZnO nanowires and worsening of the c-axis alignment. In contrast to single crystalline Au (111) film layers, polycrystalline Au layers increased the mean diameter of the synthesized nanowires. The crystal orientation of the Au film layer had no effect on the c-axis alignment. Increased voltage readings were recorded with an increase in antibody binding, indicating that the ZnO nanosensor may be used to record changes in immunoglobulin levels. Antibody concentrations ranging from 10 ng/ml to 20 μg/ml were sensed. This is the first study showing that ZnO nanowires, conformed into piezoelectric transducers, may be used in the detection of antibodies. The current size of the chip with ZnO nanowires is approximately 1 cm², which is too big to incorporate into a compact monitoring device. Apart from the challenge to produce smaller nanowire-arrays, highly sensitive sensors and miniature amplifiers will have to be developed to increase the strength of the signals generated by the nanowires. The biosensor will also have to be optimised to detect a variety of immunoglobulins.
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    Safety of antibiotic and probiotic feed additives for Gallus gallus domesticus
    (Stellenbosch : Stellenbosch University, 2018-09-05) Neveling, Deon Pieter; Dicks, Leon Milner Theodore; Smith, Carine; Pieterse, Elsje; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: The inclusion of antibiotics in broiler feed is of great concern, as many resistant pathogenic bacteria may spread to other farming animals and humans. Alternative methods are thus required to improve broiler health and performance without detrimental consequences. The objective of this study was to evaluate the effect of a multi-species probiotic on the health and growth performance of Gallus gallus domesticus. Bacteria from different segments of the gastrointestinal tract (GIT) of healthy free-range broilers were isolated, identified to species level by amplifying the genes encoding 16S rDNA, recA and gyrB, and comparing the sequences with those listed in GenBank. A select few isolates were screened for probiotic characteristics. Among the 609 isolates sampled from the GIT, Lactobacillus johnsonii DPN184, Lactobacillus salivarius DPN164, Lactobacillus crispatus DPN167, Lactobacillus gallinarum DPN164, Enterococcus faecalis DPN94 and Bacillus amyloliquefaciens DPN123 tolerated acidic conditions (pH 2 to 3), were resistant to bile salts (0.2 to 2.0 % w/v) and produced exopolysaccharides. Bacillus amyloliquefaciens DPN123, isolated from the duodenum, produced extracellular amylase, phytase and antimicrobial lipopeptides (surfactin and iturinA1). Enterococcus faecalis DPN94, isolated from the jejunum and ileum, produced phytase and bile salt hydrolase. The genome of E. faecalis DPN94 contained several genes that may encode virulence, but not the production of cytolysin. Differences in opinion exist regarding the role virulence genes may play in the colonisation of epithelial cells. Lactobacillus johnsonii DPN184, isolated from the cecum, produced hydrogen peroxide. Lactobacillus salivarius DPN181, isolated from the colon, produced hydrogen peroxide and high levels of lactic acid. Lactobacillus crispatus DPN167 was isolated from the crop, proventriculus and ventriculus, and produced hydrogen peroxide and bile salt hydrolase. Lactobacillus gallinarum DPN164 was isolated from the jejunum and ileum. A multi-species probiotic, consisting of L. johnsonii DPN184, L. salivarius DPN164, L. crispatus DPN167, L. gallinarum DPN164, E. faecalis DPN94 and B. amyloliquefaciens DPN123, was added to the feed of broilers and its effect on growth performance, size of the lymphoid organs, gizzard mass, mineral content of the tibia bones and red blood cell parameters determined. A separate group of broilers was administered a combination of sulphadiazine, colistin and trimethoprim through their feed and subjected to the same tests. A third group of birds received standard feed without additives and served as control. On day 19, the villi of broilers on antibiotics had larger surface areas, and higher lymphocyte and basophil counts compared to broilers from probiotic and control treatment groups. On day 29, the cecal microbiome of broilers from the control and probiotic treatment groups were similar but differed significantly from broilers that received antibiotics. Probiotic administration did not alter homeostasis of the normal GIT microbiome, suggesting that probiotics rather modulate the microbiome by preventing dysbiosis induced by pathogenic microorganisms. Birds on antibiotics had lower levels of Enterobacteriaceae and higher levels of unidentified Clostridiales, Brucellaceae, Synergistaceae, Erysipelotrichaceae and Coriobacteriaceae in their ceca. The multi-species probiotic repressed the growth of Listeria monocytogenes EDGE in vivo, most probably by lowering the cell’s metabolic activity, by competing with Listeria for receptor sites on the gut wall or mucosa, or by production of antimicrobial compounds such as short-chain fatty acids, hydrogen peroxide and lipopeptides. Salmonella Enteritidis 147 invaded Caco-2 cells and altered claudin-3 tight junctions between the cells, leading to monolayer disruption. Salmonella decreased tight junctions by invading eukaryotic cells which led to cell death. Interaction of S. Enteritidis with broiler epithelial cells led to the up-regulation of lysozyme C and G, cathelicidin 2 and 3, myeloid protein 1, trypsin inhibitor CITI-1, gallinacin-2 and ubiquitin-fold modifier 1, and the down-regulation of glutaredoxin-1, gallicin-7 and vigilin. Up-regulated proteins acted as chemotactic compounds, inhibitors of microbial enzymes, and played critical roles during stress. Down-regulated proteins activated natural killer cells, and regulated apoptosis and antimicrobial defence systems. The multi-species probiotic was not cytotoxic, but the metabolic end products were. The probiotic bacteria adhered to Caco-2 cells but did not invade them, and decreased claudin-3 tight junctions but did not disrupt the monolayer. Probiotics decreased claudin-3 tight junctions by producing short-chain fatty acids, hydrogen peroxide and antimicrobial lipopeptides. In broilers administered with the multi-species probiotic, transgelin 2/3, elongation factor-1 beta and anterior gradient 2 were up-regulated, but carnitine O-acetyltransferase, adenylate kinase 2, superoxide dismutase Cu-Zn and protein SET down-regulated. Upregulated proteins were involved in the proliferation, migration and healing of cells and regulation of the cytoskeleton, whereas down-regulated proteins were important in fatty acid transport, energy homeostasis, nucleotide metabolisms, free-radical elimination and signal transduction. Concluded from these studies, the multi-species probiotic was non-toxic and interacted with epithelial cells in a symbiotic manner. Feeding of Salmonella enterica serovar Enteritidis A9 to broilers had no effect on body mass, and no significant differences were observed with respect to immune organ weights, haematological parameters and serum interferon gamma levels. Colonisation of Salmonella in the cecum of broilers that received oxytetracycline was, however, lower on days 11 (one day post infection, dpi 1) and 14 (dpi 4), but then increased to levels corresponding to those of birds in the control and probiotic groups. At first, the antibiotics decreased the cell numbers of Salmonella in the cecum, but higher levels were recorded with continuous administration. The increase in cell numbers may be due to antibiotics disturbing the microbiome in the GIT, indirectly favouring the colonisation of Salmonella. On day 29 (dpi 19), the cell numbers of Salmonella in the cecum of broilers administered with the multi-species probiotic were similar to those of infected and uninfected birds. Broilers that received oxytetracycline displayed higher serum bactericidal activity against Salmonella on day 11 (dpi 1) compared to birds from the probiotic and control groups. In addition, on day 29 (dpi 19) birds on probiotics had higher serum bactericidal activity against Salmonella than birds in the control group. Broilers receiving the multi-species probiotic had higher levels of lysozyme in their serum on day 11 (dpi 1) compared to uninfected broilers. Broilers receiving the antibiotic and probiotic had higher T lymphocyte responses compared to broilers from the control treatment groups on day 17 (dpi 7). These results suggested that antibiotic and probiotic feed additives stimulated the immune response of broilers infected with Salmonella. The designed multi-species probiotic possessed numerous beneficial characteristics and its daily use as a feed additive was deemed safe, as probiotic use did not negatively affect the performance of healthy birds. The probiotic strains adhered to intestinal epithelial cells and crosstalk between these cells did not induce negative proteomic changes. The multi-species probiotic also increased broiler immune responses during Salmonella infection, which suggests that the strains may be used as an alternative feed additive to improve broiler health and performance.