College of Agriculture & Natural Resources
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The collections in this community comprise faculty research works, as well as graduate theses and dissertations.
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Item Kinetics of Tetrachloroethene-Respiring Dehalobacter and Dehalococcoides Strains and Their Effects on Competition for Growth Substrates(2010) Lai, Yenjung; Becker, Jennifer G; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The chlorinated solvents tetrachloroethene (PCE) and trichloroethene (TCE) are common groundwater contaminants. Reductive dechlorination of PCE and TCE at contaminated sites is commonly carried out by dehalorespiring bacteria that utilize these compounds as terminal electron acceptors, but often results in the accumulation of cis-1,2-dichloroethene (cDCE) and vinyl chloride (VC), rather than non-toxic ethene. This project focused on evaluating how interactions among dehalorespiring populations that may utilize the same electron acceptors, electron donors and/or carbon source may affect the extent of PCE dechlorination in situ. These interactions may be particularly important if both Dehalococcoides ethenogenes (Dhc. ethenogenes) and Dehalobacter restrictus (Dhb. restrictus) are present because these bacteria utilize the same electron donor (H2) and both respire PCE and TCE. However, unlike Dhc. ethenogenes, Dhb. restrictus cannot dechlorinate PCE beyond cDCE. Therefore, the outcome of the population interactions may determine the extent of detoxification achieved. Monod kinetic parameter estimates that describe chlorinated ethene and electron donor utilization by Dhc. ethenogenes and Dhb. restrictus at non-inhibitory substrate concentrations were obtained in batch assays. Substrate inhibition effects on both populations were also evaluated. Highly chlorinated ethenes negatively impacted dechlorination of the lesser chlorinated ethenes in both populations. In Dhc. ethenogenes, cometabolic transformation of VC was also inhibited by the presence of other chlorinated ethenes. PCE and TCE dechlorination by Dhb. restrictus was strongly inhibited by VC. The microbial interactions between Dhc. ethenogenes and Dhb. restrictus was investigated using reactors and mathematical models under engineered bioremediation and natural attenuation conditions. Under engineered bioremediation conditions, Dhc. ethenogenes became the dominant population, and the modeling predictions suggested that the inhibition of Dhb. restrictus by VC was a key factor in determining this outcome. Dechlorination rates by Dhb. restrictus appeared to be affected very little by low acetate concentrations under natural attenuation conditions, giving it an advantage over Dhc. ethenogenes, which requires relatively high acetate concentrations. This study highlighted that substrate interactions among dehalorespiring bacteria can influence their performance and contaminant fate under common bioremediation scenarios. A better understanding of the factors affecting the outcomes of these microbial interactions was achieved, which should aid in the design of successful bioremediation strategies.Item Assessment of Foodborne Pathogen Survival During Production and Pre-harvest Application of Compost and Compost Tea(2009) Ingram, David Thomas; Joseph, Sam W; Meng, Jianghong; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The use of compost in crop production systems as a soil amendment is recognized by both conventional and organic plant production practitioners as a means to increase yields and reduce the incidence of foliar diseases. Compost tea (CT), an aqueous extract of the biological components of compost, is also recognized as a means to broadcast the phytopathogen-reducing components of compost directly to the surfaces of plants where many foliar diseases become established. CT has been shown to control the proliferation of a variety of foliar diseases in many turf, crop and horticulture production systems when applied directly to the foliar surfaces of plants. This dissertation research was designed to address several pre-harvest food safety issues concerning compost and compost teas. Three objectives were pursued to establish whether the use of compost and compost teas as pre-harvest practices may introduce foodborne pathogens into the food supply and, therefore, contribute to the incidence of foodborne illness. The first objective involved a microbiological survey of commercially available compost in the U.S. to determine the prevalence of fecal coliforms, Escherichia coli, Salmonella and enterococci that might be reaching consumers through contaminated fruit and vegetables. The second objective was to investigate the ability of these foodborne pathogens to propagate during the production of CT. The third objective involved a field study examining the potential of CT to disseminate E. coli into organic and conventional strawberry production systems. The effects of CT on the fruit yield, phytopathogen suppression, as well as the potential for foodborne pathogen survival on the fruit surfaces were examined. This project provided important information and recommendations for the safe production and pre-harvest application of compost and compost teas. It was contended that, with proper attention to the manufacture and storage of compost and with simple modification of current trends in CT production systems, the current threshold of pre-harvest introduction of foodborne pathogens could be significantly reduced.Item Microbial Ecology and Horticultural Sustainability of Organically and Conventionally Managed Apples(2008) Ottesen, Andrea; Walsh, Christopher S; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Objectives: Organically and conventionally managed apple trees (Malus domestica Borkh) were evaluated for three growing seasons (2005-2007) to examine the impact of organic and conventional pesticide applications on the microbial ecology of phyllosphere and soil microflora. An important objective was to establish if organic or conventional selection pressures contribute to an increased presence of enteric pathogens in phyllosphere microflora. The horticultural and economic sustainability of the organic crop was also compared to the conventional crop with regard to fruit yield and input costs. Methods: Microbial populations from phyllosphere and soil environments of apple trees were evaluated using clone libraries of 16S rRNA gene fragments. Clones were sequenced and software was used to assess diversity indices, identify shared similarities and compute statistical differences between communities. These measurements were subsequently used to examine treatment effects on the microbial libraries. Phyllosphere Results: Eight bacterial phyla and 14 classes were found in this environment. A statistically significant difference between organically and conventionally managed phyllosphere bacterial microbial communities was observed at four of six sampling time points. Unique phylotypes were found associated with each management treatment but no increased human health risk could be associated with either treatment with regard to enteric pathogens. Soil Results: Seventeen bacterial phyla spanning twenty-two classes, and two archaeal phyla spanning eight classes, were seen in the 16S rRNA gene libraries of organic and conventional soil samples. The organic and conventional soil libraries were statistically different from each other although the sampling depth was not sufficient to make definitive inference about this environment. Horticultural Results: Fruit yields from organically managed apple trees were from one half to one third of the yields from conventionally managed trees. Based on input costs, organic fruit was about twice as expensive to produce. Asian pears (Prunus serotina) were also included in this horticultural analysis and showed greater field tolerance as an organic specialty niche crop than apples.Item FUNCTIONAL CHARACTERIZATION OF THE INTERACTION OF HEPATITIS E VIRUS ORF3 PRODUCT WITH THE CYTOSKELETON(2008) Kannan, Harilakshmi; Zhang, Yan-Jin; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Hepatitis E virus (HEV) causes several outbreaks of hepatitis in humans. Many aspects of HEV pathogenesis are not well understood. The HEV ORF3 product (henceforth known as vp13) is a multifunctional protein essential for infection of animals. To better understand the vp13 functions, this study was performed. We observed that vp13 protein was associated with the microtubules (MT) in transfected cells. Mutational studies revealed that both hydrophobic domains at the N-terminal region of vp13 are required for the vp13-MT interaction. Our studies also showed that HEV vp13 protein increased the stability of the MT, activated the apoptotic pathway, and, increased the levels of tumor suppressor gene p53 and its downstream effector p21Cip/WAF1 in the transfected cells. However, no noticeable effect on cell survival was observed. These results indicated that HEV vp13 protein may act as a viral regulatory protein.Item Regulation of factors contributing to virulence in Escherichia coli(2008-04-25) Mitra, Arindam; Mukhopadhyay, Suman; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Extraintestinal pathogenic strains of Escherichia coli cause a wide range of diseases including colibacillosis in chickens and urinary tract infections in humans. Persistent infections in E. coli and other gram-negative species are associated with population-dependent physiological processes such as cell-cell signaling and biofilm formation. Such social behaviors require careful coordination and modulation of gene expression in response to environmental cues. Adaptive response of bacteria in new environment is predominantly achieved through a signaling cascade called two-component regulatory systems. The function of the BarA/UvrY two-component regulatory system and its downstream factors in controlling virulence associated processes, specifically regulation of AI-2 based signaling and biofilm formation was investigated. In E. coli, a type of cell-cell signaling termed Quorum Sensing involves release, detection, and response to small molecule called autoinducer (AI-2), synthesis of which is dependent on luxS gene products via methyl cycle. The BarA-UvrY and Csr system displayed dual regulation on luxS expression at the level of transcription and post-transcription. The uptake of AI-2 by the Lsr transporter is also modulated by the signaling cascade suggested a balance between AI-2 synthesis and uptake in the cell. The role of transcriptional regulator uvrY in biofilm formation in Uropathogenic Escherichia coli was also studied. Mutation of uvrY reduced expression of fimA and papA, major fimbrial subunit of Type 1 and Pap pilus respectively. Acidic exopolysaccharide accumulation and the ability to swarm are also being impaired. Finally, uvrY mutants demonstrated poor colonization in kidneys and bladders in an ascending model of UTI. Overall, the effect of uvrY on biofilm formation seems to be multi-factorial and might play a critical role in adaptation and colonization of UPEC. The fine tuning of processes associated with cell-cell communication and biofilm formation at the level of transcription and post-transcription by the BarA/UvrY/CsrA signaling cascade indicated that this system might be crucial for quick adaptation, social behavior, colonization and virulence attributes in Escherichia coli.Item Regulation of Virulence by BarA-UvrY Two-Component system and LuxS in Extraintestinal Pathogenic Escherichia coli.(2007-12-05) Palaniyandi, Senthilkumar; Mukhopadhyay, Suman; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Pathogenic E. coli cause intestinal or extraintestinal infections in many host species. E. coli strains enter the intestinal tract through food and colonize the intestinal epithelium to cause infections. In animals and humans, E. coli causes gastroenteritis, neonatal meningitis and urinary tract infections. In birds, E. coli causes a complex syndrome called avian colibacillosis. The orthologs of BarA-UvrY two-component (TCS) system is known to regulate a number of phenotypic traits in gamma proteobacteria, although their role in Extraintestinal pathogenic Escherichia coli (ExPEC) virulence is yet to be determined. The barA gene is membrane bound sensor kinase protein and the uvrY gene encodes the cognate response regulator in E. coli. Work in this study has focused how the BarA-UvrY and LuxS system regulates in vivo virulence in uropathogenic E. coli (UPEC) and avian pathogenic E. coli (APEC) during infection. The main goal of this study is to look at how BarA-UvrY TCS and LuxS regulate virulence in APEC 7122 and UPEC CFT073. In this study, we studied the role of BarA-UvrY TCS system in regulation of virulence in the aforementioned ExPEC strains using animal model and tissue culture system and the role of LuxS in regulation of virulence determination in ExPEC. Our results indicate that BarA-UvrY regulates multiple virulence properties in APEC 7122 and UPEC CFT073 and that LuxS regulates partial virulence properties in APEC 7122 and UPEC CFT073.Item Campylobacter jejuni/coli - Host Intestinal Epithelial Cell Interaction(2006-08-15) zheng, jie; meng, jianghong; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Campylobacter jejuni/coli have been known to be major bacterial causes of human gastroenteritis worldwide for decades. Regarding its pathogenicity, little is known yet. A better understanding of the pathogenic mechanisms will provide important information, not only for generating molecular markers to differentiate pathogenic strains versus non-pathogenic ones; but also for developing rational strategies to prevent and control Campylobacter-caused disease. The objectives of this study were to characterize the pathogenic abilities of various C. jejuni/coli retail meat isolates, including their abilities to adhere to, invade into and transmigrate across human epithelial cells, to examine the role of NF-κB pathway in IL-8 secretion induced by Campylobacter, and to identify C. jejuni-specific adherence/invasion genes during host pathogen interaction. It was found that the adherence and invasiveness of total 43 Campylobacter retail meat isolates in human intestinal epithelial T84 cell model indicated that C. jejuni/coli present in retail meat were considerably diverse in their ability to adhere to and invade human epithelial cells. Meanwhile, eight putative virulence genes, determined by PCR, were shown to be widespread among the Campylobacter isolates. C. jejuni /coli-induced proinflammatory cytokine Interleukin (IL)-8 secretion in polarized human colonic epithelial cells T84 was examined, and the role of NF-κB pathway in Campylobacter-induced IL-8 secretion was determined. Data suggested that C. jejuni/coli induce basolateral-polarized secretion of IL-8 in human intestinal epithelial cells, and C. jejuni-induced IL-8 secretion is NF-κB-dependent. The effort to identify C. jejuni-specific adherence/invasion genes during host pathogen interaction by using restriction fragment differential display PCR (RFDD-PCR) has been made. As a result, it was not successful. However this study still provides useful information and experience on the application of this technique for prokaryotic gene expression analysis during host pathogen interaction, which remains an unexplored area. In summary, Campylobacter retail meat isolates exhibited wide diversity in cell culture model in the ability of adherence, invasion and transmigration. As the first line defense, intestinal epithelium activates NF-κB and secretes proinflammatory cytokine IL-8 in response to Campylobacter infection. Multiple virulence factors have roles in Campylobacter-intestinal epithelial cell interaction.Item Variations in 11B-Hydroxysteroid Dehydrogenase Type1 in Two Rat Models of Obesity(2006-05-04) Parsons, Erica Zager; Castonguay, Thomas W; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Obesity is an epidemic that has been estimated to cost the United States over $117 billion every year. Glucocorticoids have long been implicated in the maintenance of energy homeostasis due to their involvement with the hypothalamic-pituitary-adrenal axis. 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1) is an enzyme that interconverts the glucocorticoids cortisol and cortisone, and their rat counterparts corticosterone and 11-dehydrocorticosterone. We predicted an association between hepatic levels of this enzyme and either genetic obesity or dietary-induced obesity from any of two fat levels (high fat, low fat) or three fat sources (saturated, polyunsaturated, or monounsaturated). Our results indicated that hepatic 11β-HSD-1 is downregulated in genetic obesity, as has been previously found. High fat diets also caused reductions in hepatic enzyme message, although these were not statistically significant. There did not appear to be any effect of fat type.Item Detection of norovirus and indicator organisms on fresh produce(2005-12-06) Williams, Karen Elizabeth; Meng, Jianghong; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Food borne illness is a major problem around the world. Recently, more food borne outbreaks involve produce as the vehicle and viruses as the source of contamination. Norovirus is a common food borne viral pathogen. Genetic diversity among the viruses has made detection difficult. Due to the difficulties in detection, the norovirus is an ideal candidate for having an indicator organism. FRNA bacteriophages share several similarities with enteric viruses and would be an ideal candidate. In this study, we evaluated reverse transcriptase polymerase chain reaction (RT-PCR) detection of norovirus and evaluated using FRNA bacteriophages, E. coli, and Enterococcus as indicator organisms for the virus on produce. Of the five RT-PCR methods tested, only two worked with both controls. Of the 180 produce samples tested, 37.2% were positive for FRNA bacteriophage, 17.2% were positive for Enterococcus, and 0% were positive for E. coli. We conclude that RT-PCR is not an efficient method for screening norovirus on produce and including FRNA bacteriophages as indicator organisms for viruses may help decrease outbreaks.Item REVERSE GENETICS OF AVIAN METAPNEUMOVIRUS(2005-12-06) Dhanasekaran, Govindarajan; Samal, Siba K; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Avian metapneumovirus (AMPV) causes an acute respiratory disease in turkeys and is associated with "swollen head syndrome" in chickens, contributing to significant economic losses to the US turkey industry. With a long-term goal of developing a better vaccine for controlling AMPV in the US, a reverse genetics system to produce infectious AMPV entirely form cloned cDNA was established. To achieve this, the unpublished sequences of the G gene, the L gene, the leader and trailer region were first determined to complete the entire genome sequence of AMPV subgroup C strain Colorado (AMPV/CO). Our results showed that the full-length AMPV/CO genome was 14,150 nucleotides (nt) in length, denoting that AMPV/CO possessed the longest genome among known metapneumoviruses. Subsequently, a cDNA clone encoding the entire 14,150 nt genome was generated by assembling 5 cDNA fragments, representing the entire genome, between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme of a low-copy number transcription plasmid pBR 322. Transfection of this plasmid, along with the expression plasmids encoding the N, P, M2-1 and L proteins of AMPV/CO, into cells stably expressing T7 RNA polymerase resulted in the recovery of infectious AMPV/CO. The recovered virus was observed to contain the genetic markers that were artificially introduced during cloning. Characterization of the recombinant AMPV/CO showed that its growth characteristics in tissue culture were similar to those of the parental virus. These results demonstrate that infectious AMPV can be generated entirely from cloned DNA using reverse genetics techniques. The potential of AMPV/CO to serve as a viral-vector was examined using green fluorescent protein (GFP) as a reporter. The recovered rAMPV/CO-GFP virus stably expressed GFP for at least five serial passages and showed characteristics similar to that of the parental virus, except that there was a one-log reduction in the virus titer. These results demonstrated that the established reverse genetics system can be utilized effectively for various studies involving AMPV molecular biology, pathogenesis and vaccine development.