Browsing by Subject "Aflatoxin"
Now showing 1 - 3 of 3
Results Per Page
Sort Options
Item B-G gene structure, genetic variation and expression in the turkey (Meleagris gallopavo) major histocompatibility complex(2013-02) Bauer, Miranda MarieThe major histocompatibility complex (MHC) is a genomic region encoding for genes with various immune functions in vertebrates. Although this region has been studied extensively in humans, mice and agriculturally important species such as cattle, horses and chickens, the organization and functional significance of the turkey MHC is relatively unknown. One set of loci located in the avian MHC, designated as B-G genes, have been identified and sequenced within the B-locus of the turkey, chicken and quail, with additional B-G loci identified in the extended MHC of the latter two species. These genes are linked to the class I and class II loci of the MHC and also show regions of extreme polymorphism (Miller, 1984). The total number, function and significance of the B-G genes are yet unknown in any bird species. In turkeys, three B-G genes were previously sequenced within the B-locus (Chaves et al., 2009a), with evidence suggesting additional functional B-G genes located past the 5' ends of the sequenced MHC region. Evidence in the chicken shows differential expression of B-G genes in various immune tissues, which suggests potential immune function. This research used 454 FLX Next Generation Sequencing technology (Roche) for sequencing a bacterial artificial chromosome (BAC) and identified two additional B-G genes located past the 5' end of the core B-locus of the turkey. These genes were annotated using in silico analysis and show organization similar to those in the chicken and quail. Using this information, sequence variation of the B-G genes was compared in different stocks of turkeys. Because each of the three groups, (commercial, heritage and wild), have been selected for different characteristics, variation within these loci was expected. This experiment found variation among loci within the three groups of turkeys. Lastly, B-G gene expression was investigated with quantitative real-time PCR using liver tissue of aflatoxin challenged birds. A low level of expression was observed for three of the four BG genes investigated, with BG5 expression invariant in all individuals across treatment groups. BG4 expression levels fluctuated within and between groups and a higher level of expression was measured in the lactobacillus + aflatoxin group. This work extended the turkey B-locus sequence past the homologous region in the chicken and marks the first examination of sequence variation and gene expression of multiple B-G genes in any species. Sequence variation and expression differences among loci support a hypothesis of distinct functions for these molecules.Item Hepatotoxic and Immunomodulatory Transcriptome Responses to Aflatoxin B1 in the Turkey (Meleagris gallopavo)(2015-05) Monson, MelissaHepatoxicity and immunotoxicity from dietary exposure to aflatoxin B1 (AFB1) adversely affect poultry health and production. Domestic turkeys (Meleagris gallopavo) are especially sensitive to AFB1 since they have a deficiency in glutathione-mediated detoxification of the reactive AFB1 intermediate. Changes in gene expression can be used to characterize the molecular mechanisms of toxicity; transcriptome analysis allows investigation of differential expression at the genome-wide level. In this research, Illumina RNA-sequencing (RNA-seq) was used to examine transcriptome responses to AFB1 exposure in the turkey. As the liver is the primary site of AFB1 activation and toxicity, the effects of dietary AFB1 on the domestic turkey liver transcriptome was first investigated by sequencing 4 pooled libraries representing 3 individuals for each of 4 treatment groups. As detailed in Chapter 2, predicted transcripts were de novo assembled and differential expression analysis identified significant effects on transcripts from genes involved in apoptosis, cell cycle regulation and lipid metabolism (like E3 ubiquitin-protein ligase Mdm2 and lipoprotein lipase). In Chapter 3, RNA-seq and de novo transcriptome assembly were performed on 3 individual spleen samples per treatment group (n = 12) collected from the same AFB1 challenge trial. Significant down-regulation of antimicrobial genes (like beta-defensin 1) and up-regulation of cytotoxic and antigen presentation genes (such as granzyme A) were observed after AFB1 treatment. Another aspect of these studies was to evaluate the ability of a Lactobacillus-based dietary probiotic to reduce AFB1-effects in the liver and spleen. Addition of probiotics during AFB1 exposure modulated expression in both tissues. Many AFB1-induced expression changes were not mitigated in liver, and although probiotics had some amelioratory effects in the spleen, they were also broadly suppressive of immune genes. Multiple genes impacted in the spleen transcriptome belonged to the Major Histocompatibility Complex (MHC), a region of the genome with genes essential to immune functions. The functions and expression patterns of many of the genes located in the turkey MHC have not been characterized. A single-gene investigation (Chapter 4) characterized expression patterns of 29 MHC genes in the domestic turkey and provided first evidence for expression of B-butyrophilin 2 in muscle tissue. Understanding these expression profiles will help determine MHC gene functions and provide background for expression changes from immunological challenges like AFB1. Unlike the domestic turkey, Eastern wild turkeys (M. g. silvestris) are more resistant to aflatoxicosis due at least in part to their ability to detoxify AFB1. In Chapter 5, an in ovo exposure model was utilized to directly compare the effects of AFB1 exposure in domestic and wild turkey embryos. Embryonic exposure has applicability to poultry production since AFB1 can be maternally transferred into eggs. RNA-seq datasets from embryonic liver tissue in domestic (n = 24) and wild (n =15) turkeys were mapped to a MAKER turkey gene set. Differential expression and pathway analysis identified conserved effects on cell cycle regulators (like E3 ubiquitin-protein ligase Mdm2) and variable effects in genes encoding detoxifying and anti-oxidant enzymes (like glutathione S-transferases) in domestic and wild turkeys. Overall, transcriptome analysis identified hepatic and splenic responses to AFB1, evaluated the use of probiotics, directly compared domestic and wild turkeys, and provided gene targets for future investigation of the molecular mechanisms of aflatoxicosis.Item Linear Polymer Affinity Agents for the Intrinsic SERS Detection of Food Safety Targets(2018-07) Szlag, VictoriaThis dissertation explores the use of polymer affinity agents for the surface-enhanced Raman spectroscopy (SERS) detection of food safety targets. First, current molecular motifs used as affinity agents in intrinsic surface-enhanced Raman spectroscopy (SERS) sensors are reviewed. By comparing antibody, aptamer, small molecule, and polymer affinity agents, the largely unresearched potential of polymer affinity agents (chemical customization, tunable length, ease of production, opportunity for multiplexing) is highlighted. The first proof of concept work of this dissertation targets the detection of the bioterror agent, ricin B-chain (RBC) in water and liquid food matrices. An N-acetyl-galactosamine glycopolymer capture layer was designed and applied to create a SERS sensor. The sensing scheme’s detection limit (20 ng/mL) is well below that of the predicted oral exposure limit. The RBC was detected in two types of juice, and a computed normal Raman spectrum of the glycomonomer supports polymer–RBC intermolecular interactions at the functional group level. Subsequent work focuses on the translation of this sensing scheme from the detection of proteins to the detection of small molecules relevant to food safety. Because interactions between a small molecule target and a polymer affinity agent are less specific than those that were leveraged in the RBC work, the development of a rapid affinity agent screening method was deemed necessary. A potent carcinogenic metabolite of a fungal pathogen that can infect food and feedstocks, aflatoxin B1 (AFB1), was used as a model target. Seven homopolymers of nitrogen-inclusive poly(N-(2-aminoethyl) methacrylamide) (pAEMA) and their oxygen analogs, poly(2-yydroxyethyl methacrylate) (pHEMA) were synthesized to be evaluated as AFB1 affinity agents based on hypothetical hydrogen bonding interactions and optimal polymer length. An isothermal titration calorimetry (ITC) method was development for rapid affinity agent screening and good agreement was observed between the ITC results and follow-on SERS sensing experiments. A final polymer series (poly(N-acryloyl glycinamide), pNAGA) was designed for the capture of AFB1 and was used to explore the influence of polymer molecular weight (2.0 – 5.2 kDa), attachment chemistry (thiol vs. trithiocarbonate), and order of addition (pre- vs. post- functionalization of the substrate) on the sensitivity of AFB1 detection. The best polymer chain length (pNAGA22), anchoring chemistry (thiol), and polymer/toxin assembly scheme (in-solution) allowed detection of 10 ppb AFB1 in water (below the FDA regulatory limit of 20 ppb), a hundred-fold improvement over SERS sensing without the pNAGA affinity agent. This dissertation concludes with the advantages, disadvantages, and future perspectives of polymers used as analytical affinity agents. Adjustment of surface attachment moieties, the use of crosslinkers with target affinity, and application to other signal transduction mechanisms are emphasized a potential areas for continuing work.