Botrytis species affect over 300 host plants in all climate areas of the world, at both pre and post-harvest stages, leading to significant losses in agricultural produce. Therefore, the development of a rapid, sensitive and reliable method to assess the pathogen load of infected crops can help to prescribe an effective curing regime. Growers would then have the ability to predict and manage the full storage potential of their crops and thus provide an effective disease control and reduce post-harvest losses. A highly sensitive electrochemical immunosensor based on a screen-printed gold electrode (SPGE) with onboard carbon counter and silver / silver chloride (Ag/AgCl) pseudo-reference electrode was developed in this work for the detection and quantification of Botrytis species. The sensor utilised a direct sandwich enzyme-linked immunosorbent assay (ELISA) format with a monoclonal antibody against Botrytis immobilised on the gold working electrode. Two immobilisation strategies were investigated for the capture antibody, and these included adsorption and covalent immobilisation after self-assembled monolayer formation with 3-dithiodipropionic acid (DTDPA). A polyclonal antibody conjugated to the electroactive enzyme horseradish peroxidase (HRP) was then applied for signal generation. Electrochemical measurements were conducted using 3,3’, 5,5’-tetramethylbenzidine dihydrochloride / hydrogen peroxide (TMB/H2O2) as the enzyme substrate system at a potential of -200 mV. The developed biosensor was capable of detecting latent Botrytis infections 24 h post inoculation with a linear range from 150 to 0.05 μg fungal mycelium ml-1 and a limit of detection (LOD) as low as 16 ng ml-1 for covalent immobilisation and 58 ng ml-1 for adsorption, respectively. Benchmarked against the commercially available Botrytis ELISA kits, the optimised immuno-electrochemical biosensor showed strong correlation of the quantified samples (R2=0.998).

The aim of this study was to confirm that the polyunsaturated fatty acids n-3 (n-3 PUFA) have a positive effect on plasma lipids. These acids can reduce cholesterol by increasing gene expression Insig-1 while decreasing the expression of genes encoding Hmgcr and Ldlr. We tested in experimental rats, which were added to the feed mixture of 6 % safflower oil , 6 % fish oil or 6 % of the oil from the algae Schizochytrium. Relative gene expression was Insig-1 in the test group with addition of fish oil to 120% of controls (P<0.05) and in the group with addition of oils from algae Schizochytrium the relative expression of 170 % of control (P<0.05). These results confirm our hypothesis, only a part, as the relative expression of the gene and Hmgcr and Ldlr was in the test group with addition of fish oil 103% (P>0.05) and 101 % of control (P>0.05) and in the group with addition of oils from algae Schizochytrium the relative expression of 117% (P>0.05) and 156 % (P>0.05) compared to control. Thus, to reduce the relative expression of these genes did not. However, we have shown that n-3 PUFA contribute to a reduction in plasma cholesterol and in this case up to 20 % of control. The concentration of cholesterol in the group with addition of safflower oil was 1.35 mmol.l-1, the group with the addition of fish oil 0.98 mmol.l-1.

Botrytis species affect over 300 host plants in all climate areas of the world, at both pre and post-harvest stages, leading to significant losses in agricultural produce. Therefore, the development of a rapid, sensitive and reliable method to assess the pathogen load of infected crops can help to prescribe an effective curing regime. Growers would then have the ability to predict and manage the full storage potential of their crops and thus provide an effective disease control and reduce post-harvest losses. A highly sensitive electrochemical immunosensor based on a screen-printed gold electrode (SPGE) with onboard carbon counter and silver / silver chloride (Ag/AgCl) pseudo-reference electrode was developed in this work for the detection and quantification of Botrytis species. The sensor utilised a direct sandwich enzyme-linked immunosorbent assay (ELISA) format with a monoclonal antibody against Botrytis immobilised on the gold working electrode. Two immobilisation strategies were investigated for the capture antibody, and these included adsorption and covalent immobilisation after self-assembled monolayer formation with 3-dithiodipropionic acid (DTDPA). A polyclonal antibody conjugated to the electroactive enzyme horseradish peroxidase (HRP) was then applied for signal generation. Electrochemical measurements were conducted using 3,3’, 5,5’-tetramethylbenzidine dihydrochloride / hydrogen peroxide (TMB/H2O2) as the enzyme substrate system at a potential of -200 mV. The developed biosensor was capable of detecting latent Botrytis infections 24 h post inoculation with a linear range from 150 to 0.05 μg fungal mycelium ml-1 and a limit of detection (LOD) as low as 16 ng ml-1 for covalent immobilisation and 58 ng ml-1 for adsorption, respectively. Benchmarked against the commercially available Botrytis ELISA kits, the optimised immuno-electrochemical biosensor showed strong correlation of the quantified samples (R2=0.998) ... [cont.].

The transmission of human pathogens by faecally contaminated fruit and vegetables is well established, but the burden of disease caused by foodborne pathogens is unknown. Fresh produce can be contaminated through the use of polluted irrigation water or by the handling of the produce by infected individuals either pre- or post harvest. There is very little known regarding the extent of viral contamination of irrigation water and fresh produce in South Africa. Noroviruses (NoV) and hepatitis A virus (HAV) are recognized as leading causes of foodborne viral disease. These viruses are transmitted predominantly via the faecal–oral route, primarily person-to-person by direct contact with an infected person, or indirectly by ingestion of contaminated food and water. The detection of enteric viruses in food or water is problematical and complex as many foodborne viruses, including HAV and NoV, cannot be readily isolated in cell culture. The aim of this investigation was to develop and optimise simple and efficient methods for the concentration and detection of NoV GII and HAV in irrigation water and fresh produce. These methods would then be applied to field samples of irrigation water and fresh produce to try and establish a link between viral contamination detected in irrigation water and that on associated irrigated fresh produce. The efficiency of different commercial real-time reverse transcriptase-polymerase chain reaction amplification kits for the realtime detection of HAV, NoV GI and NoV GII was assessed, and standard curves for the quantitative detection of these viruses were constructed using the most appropriate kit. Using two types of fresh produce, three different elution buffers, each at two pHs, with two different elution times were compared to establish which buffer was the most efficient for the extraction of viruses from the fresh produce. The tris-glycine beef extract buffer (pH 9.5) with an elution time of 20 minutes most efficient for the extraction of the selected enteric viruses from fresh produce. From April 2008 to November 2009, 86 irrigation water and 72 fresh produce samples were collected from commercial and subsistence farms, street vendors and commercial outlets. All the irrigation water and fresh produce samples were analysed for HAV, NoV GI and NoV GII. Overall, 16.3 % (13/86) and 12.5 % (9/72) of irrigation water and fresh produce samples tested positive for one or more human pathogenic viruses, namely NoV GII and HAV, respectively. Nucleotide sequence and phylogenetic analysis of the HAV and NoV GII strains identified clinically relevant viruses in the irrigation water and on the fresh produce. A direct link between contaminated irrigation water and contamination of fresh produce could not be established, but irrigation water was identified as a possible source of contamination of the fresh produce. The results also suggested that food handlers contributed significantly to the viral contamination of the fresh produce. This study highlights the potential health risk posed by fresh produce to consumers in South Africa and highlights the need for further in depth studies to quantify the risk to consumers. This study represents new data on the occurrence of enteric viruses in food and water in South Africa and is crucial for the development of effective intervention and control strategies for food safety in South Africa. Copyright / Dissertation (MSc)--University of Pretoria, 2011. / Medical Virology / unrestricted

Monocytes are professional antigen presenting cells (APC). They serve as precursors of macrophages and dendritic cells (DC). We have used cytopathic (cp) and non-cytopathic (ncp) Bovine Viral Diarrhea Viruses (BVDV) to determine the genes and proteins expression levels in bovine monocytes. Four specific aims were accomplished in this study. The first aim was to assess the baseline expression of the proteins involved in professional antigen presentation in bovine monocytes. The results showed that the differential detergent fractionation (DDF) approach can provide interpretable and meaningful functional information in bovine monocytes. The second aim was to evaluate the role of in vitro cp and ncp BVDV infection in the expression of the selected bovine genes involved in professional antigen presentation. The results showed that both BVDV could escape innate immune responses by modulating toll-like receptor (TLR) gene expression, followed by pro-inflammatory, type I interferon (IFN), Th1/Th2 type cytokine genes expression, and decreasing the expression levels of CD80/CD86 in professional APC. The third objective was to determine how the two biotypes affect selective antigen uptake, receptor-mediated endocytosis and non-selective uptake, macropinocytosis in bovine monocytes. The results indicated that bovine monocytes use macropinocytosis for a bulklow uptake of soluble antigens. The final aim was to characterize protein profiles in peripheral blood monocytes infected with cp BVDV isolate in vitro. Comparative profiling of the membrane and cytosolic proteins related to professional antigen presentation were assessed. The results showed that 47 bovine proteins, involved in immune function of professional APC have been significantly altered after cp BVDV infection. Overall, we hypothesize that by modulating expression levels of multiple proteins and genes related to immune responses BVDV could significantly compromise immune defense mechanisms resulting in uncontrolled immune activation or suppression.

Proliferative gill disease (PGD) caused by the myxozoan parasite Henneguya ictaluri is one of the most devastating parasitic infections in channel catfish aquaculture. Currently, there is no effective treatment for H. ictaluri and the unpredictable outbreaks can result in 100% mortality. Management strategies have been developed to prevent losses in newly stocked fingerlings by evaluating the PGD status of a pond prior to stocking, which is difficult since resident fish may not show clinical signs even when actinospore levels are lethal to naive fish. Current diagnostic methods are limited to the identification of an active infection and methods of predicting potential outbreaks have several limitations. The PGD status of a pond to be stocked can be determined using sentinel fish exposures which are labor intensive and require a source of parasite free fish. These limitations necessitated the development of more rapid and efficient means of determining actinospore concentrations to determine the risk of losing fish prior to stocking. The development of a quantitative real-time polymerase chain reaction (QPCR) assay provided a more rapid, sensitive and quantitative method of diagnosing active infections and also provides a means to predict potential PD outbreaks and determine the PGD status of a pond prior to stocking. Another approach in the control of this parasite is the identification of a less susceptible culturable species or to identify traits that could be targeted in a selective breeding program. Challenge studies have shown that the closely related blue catfish (Ictalurus furcatus) does not exhibit as severe an inflammatory response to H. ictaluri and mortalities are significantly lower than in channel catfish. Comparisons of PGD severity and H. ictaluri infection in channel catfish, blue catfish and channel x blue catfish backcross hybrids by gross examination, histopathology and the newly developed H. ictaluri real-time PCR (QPCR) assay supported previous research suggesting the life cycle of the parasite can not be completed as efficiently through the blue catfish host. This dissertation describes the development and validation of a QPCR assay to detect H. ictaluri in both fish tissues and environmental samples and the application of this assay in both research and production settings.

No description available.

No description available.

Nutrient transporters in the small intestine are responsible for dietary nutrient assimilation and therefore the expression of these transporters can influence the overall nutrient status as well as the growth and development of the animal. This thesis examined correlated responses to selection in the developmental gene expression of the peptide transporter PepT1, the glutamate/aspartate transporter EAAT3, the sodium-dependent glucose transporter SGLT1, and the fructose transporter GLUT5 in the small intestine of chickens from lines divergently selected for high (HH) or low (LL) eight-week body weight and their reciprocal crosses, (HL and LH). Chicks were weighed and killed on embryonic day 20 (E20), day of hatch (DOH with no access to feed), and days 3 (D3), 7(D7), and 14 (D14) post hatch. Duodenum, jejunum, ileum and liver were collected. DNA extracted from liver was used to sex birds by PCR. RNA was extracted from the intestinal segments of four males and four females from each mating combination (MC) and time point except E20 HL males (n = 3) and D7 LL females (n = 2). Expression of nutrient transporters was assayed by real-time PCR using the relative quantification method. In comparing HH and LL males and females there was a line by segment interaction in PepT1 gene expression, with no segment difference in HH and greatest expression in the ileum of the LL (P < 0.05). There was also a MC by age by sex interaction for PepT1 gene expression (P < 0.0001) with peak gene expression occurring on DOH for LL females, on D7 for HH females, on D7 for LL males and D14 for HH males. Overall, females had greater EAAT3 expression (P < 0.03). Gene expression of EAAT3 was greatest in the ileum, intermediate in the jejunum, and least in the duodenum (P < 0.0007). There was an age by segment interaction for EAAT3 expression (P = 0.0002) and a MC by segment interaction (P < 0.02), with LL having greater expression than HH in the ileum. Females had greater SGLT1 expression than males (P < 0.0001). There was a sex by age interaction for the expression of SGLT1 (P < 0.0001). Females induced SGLT1 expression on DOH and maintained this level through D14, while males gradually increased expression through D7 and decreased expression by D14. These results indicate that expression of PepT1, EAAT3, SGLT1 are differentially expressed in male and female chickens regardless of selection for high or low juvenile body weight. These results also show a sexual dimorphism in the capacity to absorb peptides, anionic amino acids, and glucose from the intestine, which has implications for the poultry industry with regard to diet formulations for straight-run and sex-separate grow-out operations. In comparing male HH, HL, LH, and LL chicks, overall LL had the greatest level of expression (P <0.06), HH had the least level of expression (P < 0.006) and HL and LH had intermediate levels of expression (P < 0.06). Greatest PepT1 gene was expression in the ileum (P < 0.0003) and there was a MC by segment interaction with expression increasing from duodenum to ileum in LL, but there was no segment difference in any other MC (P < 0.08). Within each intestinal segment there was a MC difference (P < 0.02). There was an effect of sire for PepT1 expression, with progeny from low weight selected sires (LWS) having greater expression than progeny from high weight selected (HWS) sires (P = 0.0008). There was no difference between intestinal segments in progeny from HWS sires, however, greatest PepT1 gene expression was seen in the ileum of progeny from LWS sires (P < 0.0001). Overall, expression of EAAT3 was greatest in the ileum, intermediate in the jejunum and least in the ileum (P < 0.0001) and there was a segment by age interaction for EAAT3 expression (P < 0.0001). In all MCs except HH, EAAT3 gene expression increased from duodenum to ileum (P < 0.08). Within the ileum, the LL had greatest EAAT3 gene expression, LH and HL had intermediate gene expression, and HH had least expression (P < 0.08). Expression of SGLT1 gradually increased through D7 and decreased by D14 (P < 0.0001) and overall, was greatest in the distal small intestine (P < 0.0001). There was a MC by segment interaction, with SGLT1 gene expression being greatest in the distal small intestine in LL, LH, and HL, but greatest in the jejunum of HH (P < 0.04). Within the ileum, LL had greater SGLT1 gene expression than HH (P < 0.06). Overall, greatest GLUT5 expression was in the distal small intestine (P < 0.0001) and there was a MC by segment interaction, with expression being greatest in the distal small intestine in LL and HL (P < 0.02), greatest in the ileum of LH (P < 0.08), and greatest in the jejunum of HH (P < 0.09). Within the ileum there was a MC difference (P < 0.07). These results indicate that selection for high or low juvenile body weight may have influenced the gene expression pattern of these nutrient transporters in the small intestine, which may contribute to the overall differences in the growth and development of these lines of chickens. / Master of Science

Members of the alphavirus genus pose a serious or potential threat to public health in many areas of the world. Nearly all alphaviruses are maintained in nature by transmission cycles that involve alternating replication in a susceptible vertebrate and invertebrate host. The maintenance of this transmission cycle depends on the establishment of a life-long persistent infection in the invertebrate vector host. Although alphavirus replication has been extensively studied in vertebrate models, the strand-specific replication kinetics of alphaviruses during persistent infections of the invertebrate host have not been reported. We investigated the strand-specific replication of different alphavirus genotypes in invertebrate cells. By comparing different detection strategies and chemistries, we identified an optimal ssqPCR assay design for strand-specific quantification of viral RNAs in infected cells and tissues. We found that primer sets incorporating the use of a non-target tag sequence were able to avoid real-time PCR detection of amplicons that were falsely-primed during reverse-transcription. We also determined that DNA hydrolysis probes increased the sensitivity of ssqPCR assays when compared to a double-stranded DNA-specific dye, SYBR Green. Using this information, we determined the replication kinetics of two different genotypes of o'nyong nyong virus (ONNV) and chikungunya virus (CHIKV) in infected mosquito cells. We found that (-) strand viral RNAs persisted in invertebrate cells for up to 21 days after infection. We also found that significantly less (-) strand RNA was present in cells infected with opal variants of both ONNV and CHIKV than sense variants at several time points post infection, suggesting that the opal codon has a functional role in (-) strand RNA regulation. We also report the development of an ONNV replicon expression system. In total, the tools we developed for this report will facilitate future replication studies in the mosquito that may shed light on questions regarding the regulatory role of the opal codon and the persistence of (-) strand RNAs during long-term infections. The strand-specific replication kinetics of ONNV and CHIKV genotypes reported here will serve as a foundation for such investigations. / Master of Science in Life Sciences