College of Agriculture & Natural Resources

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    QUANTIFICATION OF IONOPHORE ANTIMICROBIALS ASSOCIATED WITH POULTRY LITTER AND THEIR DYNAMICS IN THE SOILS OF THE MID-ATLANTIC USA
    (2014) Biswas, Saptashati; McGrath, Joshua M; Sapkota, Amir; Environmental Science and Technology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Anticoccidants, biochemically known as ionophores are added to poultry feed for growth promotion, prophylactic and therapeutic purposes to better sorb nutrients and against coccidiosis caused by parasite Eimeria sp. Ionophores belong to the class of emerging contaminants, as they are not regularly monitored in the environment and not specifically treated in the effluents. Potentially, this can cause ionophores to enter into the environment freely. There is little information regarding the dynamics of ionophores in the environment. This has been related to the lack of reliable, sensitive and robust methods that can measure their trace levels from complex environmental matrices like soil, natural water and animal manure. Studies show ionophore toxicity exhibited in flora and fauna, even reported in humans above the dose of 1 mg kg-1. Hence accumulation of ionophores in the environmental can be detrimental. Our multi-scale investigation of ionophores involved, a) method development for trace analysis of ionophores in poultry manure using liquid chromatography triple quadrupole mass spectrometry (HPLC-–MS/MS), b) batch equilibrium studies of ionophores using soils from mid-Atlantic region of the USA and c) influence of soil physico-chemical parameters on dynamics of ionophores in soil-water systems. Our HPLC-–MS/MS method was successful in quantifying ionophores ranging from (19.19 ±± 6.6) µg kg-1 to (97.86 ±± 19.19) µg kg-1 with concentrations of monensin being the highest. This method was further used to investigate partitioning of monensin in soil-ndash;water systems relevant to the occurrence of ionophores in the natural environment. Sorption and desorption isotherms were developed and influence of soil physico-chemical parameters on the sorption-desorption processes were analyzed. C-–type linear isotherms were generated with partition coefficients ranging from (6.41±± 1.34) to (343.83 ±± 5.68) LKg-1. Soil parameters such as cation exchange capacities, pH, organic matter, sand and silt content were found to correlate with sorption in different conditions. A major focus of this dissertation was to develop novel methodologies and design experiments to execute our research objectives.
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    Proteomic Profiling and Label-Free Quantification of Bovine Milk Proteins during Experimentally Induced Escherichia coli Mastitis
    (2009) Boehmer, Jamie Layne; Peters, Robert R; Bannerman, Douglas D; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Coliform mastitis has been a primary focus of dairy cattle disease research due to staggering affiliated losses, severe systemic complications arising from host inflammatory response to lipopolysaccharide, and the poor response of coliform pathogens to antimicrobials. Reliable biomarkers are needed to evaluate the efficacy of adjunctive therapies for the treatment of inflammation associated with coliform mastitis, and to aid in the approval of new veterinary drugs. The aims of the current analyses were to utilize proteomic methodologies to evaluate protein expression in whey from cows with experimentally induced coliform mastitis, and to employ label-free quantification strategies to estimate changes in relative abundance of proteins identified in milk over the course of clinical infection. Two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI- TOF) mass spectrometry (MS) resulted in the identification of complement factors, antimicrobial proteins, and acute phase proteins in mastitic milk. Analysis using liquid chromatography (LC) inline with electrospray ionization - quadrupole TOF tandem mass spectrometry (MS/MS) resulted primarily in the identification of abundant whey and casein proteins, and the transient detection of proteins related to host response. Nano-LC- nanospray-MS/MS using a linear ion trap, however, led to the robust discovery of over fifty inflammatory proteins in whey from mastitic milk, including the novel markers kininogen-2 and inter-alpha trypsin inhibitor heavy chain-4. Normalized spectral counts were compared to enzyme-linked immunosorbant assay (ELISA) for select proteins to assess the accuracy of the spectral count data. Similar expression patterns were detected using spectral counts and ELISA. Results indicate that proteomic methodologies can detect biomarkers of coliform mastitis in bovine milk during clinical infections, and that spectral counts are a viable means of evaluating relative changes in protein biomarkers of mastitis, including those for which no antibody currently exists.