Aflatoxin B1 (AFB1), a secondary metabolite of Aspergillus flavus and Aspergillus parasiticus, is known for its potent carcinogenicity and immunosuppressive effects. It is also known that AFB1 toxicity appears in different degrees in different animal species and strains. The present study was performed to reveal the involvement of the hypothalamus-pituitary-adrenal gland (HPA) axis in the immunosuppressive effects of AFB1 on C57B/6 mice. Splenic lymphocy1es were assayed to investigate their phenotyping using flow cy1ometry, proliferative response against mitogen and allogenic lymphocy1es, cy1oly1ic cell activity, and IL-2 production. In addition, antibody-mediated immunocompetence was checked using sheep red blood cell (SRBC)-challenged animals by plaque-forming cell (PFC) assay and enzyme-linked immunosorbent assay (ELISA). Corticotropin releasing factor (CRF) in brain hypothalamus and cerebral cortex, plasma adrenocorticotropic hormone (ACTH), and corticosterone were determined by radioimmunoassay (RIA). Hypothalamic catecholamine and its metabolites were assayed by high-performance liquid chromatography (HPLC). The adrenalectomized animals and their respective control animals were used to evaluate corticosterone involvement in AFB1 immunosuppressive effects. A relatively higher dose was applied in the present study, compared to the previous studies that used different strains of mice. Immunosuppressive effects were observed in blastogenic response, IL-2 production, and primary antibody production of splenic cells. The amount of circulating anti-SRBC antibody was also affected. Decreases were observed in the helper-T cell and B cell percentage in phenotyping splenic lymphocyte. No significant changes were observed in natural killer cell activity, mixed lymphocyte response, brain biogenic amine concentrations, concentration of CRF in the hypothalamus, and those of ACTH and corticosterone in plasma. However, the expected effect of adrenalectomy to compensate for the immunosuppression of AFB1 was not observed. The results indicate that the HPA axis does not appear to have a major role in AFB1-induced immunotoxicity.

Plant breeders have focused on improving maize resistance to Aspergillus flavus infection and aflatoxin accumulation by breeding with genotypes having the desirable traits. Various maize inbred lines have been developed for the breeding of resistance. Identification of differentially expressed proteins among such maize inbred lines will facilitate the development of gene markers and expedite the breeding process. Computational biology and proteomics approaches on the investigation of differentially expressed proteins were explored in this research. The major research objectives included 1) application of computational methods in homology and comparative modeling to study 3D protein structures and identify single nucleotide polymorphisms (SNPs) involved in changes of protein structures and functions, which can in turn increase the efficiency of the development of DNA markers; 2) investigation of methods on total protein profiling including purification, separation, visualization, and computational analysis at the proteome level. Special research goals were set on the development of open source computational methods using Matlab image processing tools to quantify and compare protein expression levels visualized by 2D protein electrophoresis gel techniques.

Taking precautions to restrain aflatoxin M1 (AFM1) from milk is critical, particularly due to the health and economic impact AFM1 imposes. The predominant post-harvest means of reducing AFM1 in milk includes the addition of sequestering agents to feed to diminish the bioavailability of aflatoxin B1 (AFB1), the parent compound of AFM1 found in contaminated feed. Still, residual AFM1 has been found in the milk. Using sequestering agents added to raw milk, we found that activated carbon was the most effective binder to reduce AFM1 contamination. The combination of 0.75% granular activated carbon (GAC) and a flow rate of 0.4 mL/min to pump contaminated milk through a glass column were chosen as optimum conditions for the removal of AFM1. These conditions obtained a 98.4% reduction of 0.75 ng/mL AFM1 from raw milk. The treated milk was also analyzed to assess the effects of GAC on milk constituents. The results determined that GAC had no significant effect on major nutritive milk constituents: total protein, lactose, minerals, and fat. Additionally, we optimized an extraction method coupled to high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) that minimized matrix effects, lowered the levels of detection, and reduced analysis costs. The optimized extraction method was based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe). Results determined 5 mL milk (15°C) with 10 mL acetonitrile, 3200 g centrifugation, and 0.2 μm syringe filter were the optimum conditions for the extraction of 0.5 ng/mL AFM1 from raw milk. The method was validated according to AOAC guidelines. This study reports experimental results on AFM1 remediation from raw bovine milk. The use of GAC for the removal of AFM1 in raw milk has reduced the amount of AFM1 below the FDA action limit and European Union maximum regulatory level. This method could have a global health impact, particularly, for people in developing nations and for infants and children who are more susceptible to the adverse effects of AFM1.

Maize, Zea mays L., is the largest cereal grain crop grown in United States. Its yield and grain quality are adversely impacted by diseases every year. Aspergillus ear rot, caused by the fungus Aspergillus flavus, received little interest until its carcinogenic secondary metabolites, aflatoxins, were discovered. The objectives of this study were to introgress the quantitative trait loci (QTL) 2.04 from Mp313E and 5.03 from Mp715 into two commercial inbred lines, MonF and MonM; and evaluate their near isogenic lines (NILs) and testcrosses for preharvest aflatoxin accumulation and secondary agronomic traits. Marker assisted selection to create NILs and the testcross production was conducted by Bayer Company between 2015 and 2018. Field trials were conducted in summer 2019 as randomized complete block trials at three locations. The entry list of inbred trials included two donor parents (DP), two recurrent parents (RP), and their 58 NILs, and that of hybrid trials included 114 NIL testcrosses and 8 parental testcrosses. The top ear of each plant in every plot was inoculated with a 3.4 ml of A.flavus conidial suspension 13 days after mid-silk. All the inoculated ears were harvested at maturity, dried, machine shelled, ground, and aflatoxin concentration was determined by plot. Separate hybrid yield trials were conducted in four locations to measure the grain yield including an additional commercial check. Data on aflatoxin and other secondary traits was analyzed using SAS software. Overall, MonF NILs improved significantly more than MonM NILs in terms of their resistance to aflatoxin accumulation with the introgression of QTL 2.04 from Mp313E, but there were no differences with the introgression of QTL 5.03 from Mp715. Overall, Mp313E NILs improved more than Mp715 NILs when the recurrent parent was MonF, but the response was opposite when the recurrent parent was MonM. Compared to their respective recurrent parents, there were at least two NILs from each of the three out of four RP x DP crosses that significantly improved their resistance to aflatoxin accumulation with a minimal loss of their agronomic performance and testcross grain yields. These NILs could be considered as parents in future introgression projects.

<p>In the presented work, immunosensors for detection of Aflatoxin B1 based on different immobilization platforms were studied. Synthesis of an electroactive hydrogel was also carried out. Aflatoxins are a group of mycotoxins that have deleterious effects on humans and are produced during fungal infection of plants or plant products. Electrochemical immunosensor for the determination of Aflatoxin B1 (AFB1) was developed with anti-aflatoxin B1 antibody immobilized on Pt electrodes modified with polyaniline (PANi) and polystyrene sulphonic acid (PSSA). Impedimetric analysis shows that the electron transfer resistances of Pt/PANi-PSSA electrode, Pt/PANi-PSSA/AFB1-Ab immunosensor and Pt/PANi-PSSA/AFB1-Ab incubated in BSA were 0.458, 720 and 1066 k&Omega / , respectively. These results indicate that electrochemical impedance spectroscopy (EIS) is a suitable method for monitoring the change in electron-transfer resistance associated with the immobilization of the antibody. Modelling of EIS data gave equivalent circuits which showed that the electron transfer resistance increased from 0.458 k&Omega / for Pt/PANi-PSSA electrode to 1066 k&Omega / for Pt/PANi-PSSA/AFB1-Ab immunosensor, indicating that immobilization of the antibody and incubation in BSA introduced an electron transfer barrier. The AFB1 immunosensor had a detection limit of 0.1 mg/L and a sensitivity of 869.6 k &Omega / L/mg.</p>

<p>In the presented work, immunosensors for detection of Aflatoxin B1 based on different immobilization platforms were studied. Synthesis of an electroactive hydrogel was also carried out. Aflatoxins are a group of mycotoxins that have deleterious effects on humans and are produced during fungal infection of plants or plant products. Electrochemical immunosensor for the determination of Aflatoxin B1 (AFB1) was developed with anti-aflatoxin B1 antibody immobilized on Pt electrodes modified with polyaniline (PANi) and polystyrene sulphonic acid (PSSA). Impedimetric analysis shows that the electron transfer resistances of Pt/PANi-PSSA electrode, Pt/PANi-PSSA/AFB1-Ab immunosensor and Pt/PANi-PSSA/AFB1-Ab incubated in BSA were 0.458, 720 and 1066 k&Omega / , respectively. These results indicate that electrochemical impedance spectroscopy (EIS) is a suitable method for monitoring the change in electron-transfer resistance associated with the immobilization of the antibody. Modelling of EIS data gave equivalent circuits which showed that the electron transfer resistance increased from 0.458 k&Omega / for Pt/PANi-PSSA electrode to 1066 k&Omega / for Pt/PANi-PSSA/AFB1-Ab immunosensor, indicating that immobilization of the antibody and incubation in BSA introduced an electron transfer barrier. The AFB1 immunosensor had a detection limit of 0.1 mg/L and a sensitivity of 869.6 k &Omega / L/mg.</p>

Philosophiae Doctor - PhD / In the presented work, immunosensors for detection of Aflatoxin B1 based on different immobilization platforms were studied. Synthesis of an electroactive hydrogel was also carried out. Aflatoxins are a group of mycotoxins that have deleterious effects on humans and are produced during fungal infection of plants or plant products. Electrochemical immunosensor for the determination of Aflatoxin B1 (AFB1) was developed with anti-aflatoxin B1 antibody immobilized on Pt electrodes modified with polyaniline (PANi) and polystyrene sulphonic acid (PSSA). Impedimetric analysis shows that the electron transfer resistances of Pt/PANi-PSSA electrode, Pt/PANi-PSSA/AFB1-Ab immunosensor and Pt/PANi-PSSA/AFB1-Ab incubated in BSA were 0.458, 720 and 1066 kΩ, respectively. These results indicate that electrochemical impedance spectroscopy (EIS) is a suitable method for monitoring the change in electron-transfer resistance associated with the immobilization of the antibody. Modelling of EIS data gave equivalent circuits which showed that the electron transfer resistance increased from 0.458 kΩ for Pt/PANi-PSSA electrode to 1066 kΩ for Pt/PANi-PSSA/AFB1-Ab immunosensor, indicating that immobilization of the antibody and incubation in BSA introduced an electron transfer barrier. The AFB1 immunosensor had a detection limit of 0.1 mg/L and a sensitivity of 869.6 kΩL/mg. / South Africa

Philosophiae Doctor - PhD / In the presented work, immunosensors for detection of Aflatoxin B1 based on different immobilization platforms were studied. Synthesis of an electroactive hydrogel was also carried out. Aflatoxins are a group of mycotoxins that have deleterious effects on humans and are produced during fungal infection of plants or plant products. Electrochemical immunosensor for the determination of Aflatoxin B1 (AFB1) was developed with anti-aflatoxin B1 antibody immobilized on Pt electrodes modified with polyaniline (PANi) and polystyrene sulphonic acid (PSSA). Impedimetric analysis shows that the electron transfer resistances of Pt/PANi-PSSA electrode, Pt/PANi-PSSA/AFB1-Ab immunosensor and Pt/PANi- PSSA/AFB1-Ab incubated in BSA were 0.458, 720 and 1066 kΩ, respectively. These results indicate that electrochemical impedance spectroscopy (EIS) is a suitable method for monitoring the change in electron-transfer resistance associated with the immobilization of the antibody. Modelling of EIS data gave equivalent circuits which showed that the electron transfer resistance increased from 0.458 kΩ for Pt/PANi-PSSA electrode to 1066 kΩ for Pt/PANi- PSSA/AFB1-Ab immunosensor, indicating that immobilization of the antibody and incubation in BSA introduced an electron transfer barrier. The AFB1 immunosensor had a detection limit of 0.1 mg/L and a sensitivity of 869.6 k ΩL/mg. In the second platform an immunosensor based on gold nanoparticles (AuNP) and polythionine-modified glassy carbon electrode (GCE) for the determination of aflatoxin B1 (AFB1) was developed. Aflatoxin B1-BSA conjugate was immobilised on the modified GCE. Horseradish peroxidase (HRP) or Bovine serum albumin (BSA) were used to block sites against non-specific binding of the AFB1- conjugate with other compounds such as the salts used in preparing the buffer when the antibody interacts with the AFB1 conjugate and free AFB1. Competition reaction was allowed to take place between the free AFB1 and AFB1-conjugate for the binding sites of the anti-aflatoxin B1 antibody. Cyclic voltammetry (CV) was employed to characterize the electrochemical properties of the modified process. The peak separation of the immunosensor (ΔEp) was 62 mV indicating a quasi reversible process. Differential pulse voltammetry (DPV) was used to monitor the analytical signal. The response decreased with an increase in AFB1 concentration in the range of 0.6-2.4 ng/mL with a limit of detection of 0.07 and 0.16 ng/mL for HRP and BSA blocked immunosensors respectively. Significantly the low detection limit of 0.07 ng/mL is within the limits set by worl health organization (WHO) for AFB1 and its derivatives which is 2 ng/mL The proposed method eliminates the use of secondary antibody enzymatic labels. Synthesis and characterization of (p-(HEMA)-polyaniline hydrogels were investigated. The hydrogels were synthesized using: 2-Hydroxyeththyl methacrylate (HEMA), N-Tris (hydroxymethyl) methyl] acrylamide, 3- Sulfopropyl methacrylate potassium salt, Tetraethylene glycol diacrylate, Poly-(2- hydroxyethyl methacrylate), 2, 2-Dimethoxy-2-phenylacetophenone and aniline by UV irradiation. Two sets of the hydrogels were prepared using water / 1, 3, 3, 3-(tetramethyl butyl phenyl polyethylene glycol [Triton X-100] and water / ethylene glycol as the solvent. Scanning electron microscopy (SEM) revealed a more uniform pore size when Triton X 100 (TX-100 HG) was used as compared to ethylene glycol (EG-HG). Thermogravimetric analysis (TGA) showed that both hydrogels were stable up to 270 oC. Fourier transform-Infra red (FTIR) spectrum confirmed the incorporation of polyaniline (PANi) and HEMA in the composite. Electrochemical properties of the hydrogels evaluated using Cyclic Voltammetry and Electrochemical Impedance Spectroscopy (EIS) demonstrated the electroactivity and conductivity.

This study has examined how cooking affects the levels of the toxicantsaflatoxin, arsenic, lead, dioxins, cadmium, mercury, perfluorinatedcompounds and polybrominated diphenyl ethers in rice, potatoes and fish.Not every toxicant was examined for all three types of food, they were onlyexamined for those types of food where current levels of the toxicant in thattype of food are relevant from a risk assessment perspective. To determineif there is a danger of negative effects due to the exposure of thesetoxicants from food, articles from the Swedish National Food Agency andother scientific articles have been compiled. A minor experimentalsubstudy was also performed where rice bought in Uppsala was rinsed andboiled in different ways to see if that affected the residue levels of arsenic.The results of this study indicate that the levels of some toxic substancescan be lowered on a dry weight basis by cooking, and that this reduction isdependent on the cooking method used, the properties of the food andtoxin. The levels of cadmium and mercury were generally unchanged bycooking whereas the results for lead and perfluorinated compounds wereconflicting. Cooking can lover the levels of aflatoxin, arsenic, dioxins andpolybrominated diphenyl ethers on a dry weight basis. The effects ofcooking on toxicant levels should be considered when performing riskassessments, but further studies are needed to achieve a better basis for decision-making.

<p>Maize is a major calorie source for people living in Sub-Sahara Africa. In this region, <i>Aspergillus flavus</i> causes ear rot diseases in maize, contributing to food insecurity due to aflatoxin contamination. The biological control principle of competitive exclusion has been applied in both the United States and Africa to effectively reduce aflatoxin levels in maize at harvest by introducing atoxigenic strains that out-compete toxigenic strains. The goal of this study was to determine if the efficacy of preharvest biocontrol treatments carry over into the drying period, which is often delayed in Sub-Sahara Africa by the complexities of postharvest drying practices and lack of modern drying machinery. Maize was collected from fields in Texas and North Carolina that were treated with commercial biocontrol, and control fields that were untreated. To simulate moisture conditions similar to those experienced by farmers during drying in Sub-Sahara Africa, we adjusted the grain to 20% moisture content and incubated it at 28 ℃ for 6 days. Although the initial number of infected kernels in most samples were high, less than 24% of kernels were infected with <i>Aspergillus flavus</i> and aflatoxin levels were low (<4ppb). Both toxigenic and atoxigenic strains increased and spread through the grain over the incubation period, and aflatoxin levels increased, even in samples from biocontrol-treated fields. Our molecular analysis suggests that applied biocontrol strains from treated fields migrate to untreated fields. The results also indicate that the population of toxigenic <i>A. flavus</i> in the harvested grain will grow and produce aflatoxin during the drying period when moisture is high. Therefore, any potential postharvest reduction in aflatoxin accumulation will depend on how effective the biocontrol strain was at displacing the toxigenic populations prior to harvest.</p>