<p> The Spring Ladies&rsquo; Tresses orchid&rsquo;s (<i>Spiranthes vernalis </i>) range extends throughout eastern North America. Although the taxon&rsquo;s distribution is reportedly widespread the species is considered threatened or endangered in many areas throughout its range and is reportedly only found in small populations in old prairie remnants, mainly next to railways. One goal for any conservation program is to successfully increase population numbers, possibly by re-introduction of plants into existing populations. It is commonly known that orchids form unique relationships with mycorrhizal fungi and the association is often essential for orchid seed germination and enhances seedling survival and growth rate. The intent of this study is to isolate and identify mycorrhizal fungi associated with <i>Spiranthes vernalis</i> individuals from a population in Madison County, Illinois. The fungi were isolated by excising fungal pelotons from the roots of an adult orchid because of the growth rate and morphology of the fungal isolates,. Fungi assignable to the ubiquitous genera <i>Ceratorhiza</i> and <i>Epulorhiza</i> were isolated. After ITS sequencing of fungal DNA BioEdit Sequence Alignment Editor an NCBI GenBank BLAST (blastn) search was used to confirm the identities of the mycorrhizal fungi found to be in association with the Spiranthes vernalis were <i>Ceratorhiza (Ceratobasidium</i> sp. UAMH 9847). Seeds from three individuals were plated with four fungal types on specialized media to see if any fungi and seed combinations had greater germination rates than other combinations. The percent of germination was calculated based on seed counts and the stage of seed development. Identification of the superior fungi will allow more seeds to be germinated and seedlings grown for eventual transplantation into their native Illinois populations. One of the fungal isolations did in fact germinate seeds with higher success with one of the seed sets used. Overall, each of the <i>Ceratorhiza</i> spp. isolates germinate well with at least one seed set with higher success than the other two seed sets. In addition, after nine months (&plusmn;one week) the seed germination experiment was split into two groups for one month; cold stratified at 4&deg;C with no light, and room temperature &plusmn;21&deg;C with a photoperiod of 8 hours a day. This was done to investigate if additional cold stratification was beneficial to the germinated seedlings or if that was the right time to expose germinated seedlings to light. This cold stratification and light exposure treatment resulted in having no a distinct effect on the germination rates. </p><p> To further confirm that the <i>Ceratorhiza</i> spp. used in the germination experiment was truly responsible for success in germination, re-isolation of the fungi was attempted and successful, but yet unconfirmed by ITS sequencing.</p>

The studies presented here evaluate the survival of different types of pathogens in a variety of environments. The study Appendix A focuses on the presence of enteric bacteria in reusable shopping bags. We demonstrated that ninety-seven percent of individuals surveyed never washed their reusable shopping bags and that this lack of washing can lead to the buildup of potentially harmful bacteria such as Salmonella spp. The hand-washing of the bags was shown to reduce seeded organisms to below detectable limits. Appendix B examines the survival of Bacillus altrophaeus endospores during household laundering. It was demonstrated that detergent alone didn't significantly reduce the number of viable endospores or their spread to other garments and that bleach is necessary to significantly reduce the number of viable endospores and their spread. Risks for infection were significantly lower when bleach was used during laundering. Appendix C details the survival of Ascaris ova in biosolid-amended Brazito sandy loam and clay loam. Survival of Ascaris ova was significantly higher in clay soil and ova inactivation increased with increasing temperature. The risk for Ascaris infection from consuming raw lettuce grown on such soils was calculated and it was found that annual risks for infection decreased significantly with time after harvest.

Campylobacter spp. is one of the two major causes of foodborne illness throughout the world. Campylobacter accounts for the most common causes of diarrheal illness caused by bacterial pathogens worldwide and in the United States. It is estimated that Campylobacter diarrheal illness affects about 2.4 million persons every year with an estimated cost of treatment and loss of productivity exceeding $1 billion annually. Previous work in our laboratory on biofilms has demonstrated the presence pilus-like surface-associated structures disseminating from the cell wall of C. jejuni isolates not expressing flagella (flaAB mutants). To further investigate this finding, bioinformatics analysis, purification and identification of genes involved in the expression of surface-associated structures as well as mutational analysis of putative genes were performed. We identified two important poultry colonization factors in C. jejuni. These studies might provide insights in understanding the pathogenesis of C. jejuni. Moreover, it will provide a new target for potential vaccine development against C. jejuni infection in poultry production system. Thus directly impacting the number of C. jejuni infection in humans.

The CDC estimates that 48 million illnesses, 128,000 hospitalizations, and 3,000 deaths annually are attributable to foodborne illnesses, making their impact significant in terms of both human health and economic losses (3). Estimates vary, but it is frequently stated that Campylobacter species affect 2.4 million people annually (28). Among bacterial foodborne pathogens it is second in the US only to Salmonella, which in recent years has consistently been the most frequently reported, most likely to cause hospitalization, and deadliest foodborne bacterial illness in the US (3, 106).In order to reduce the burden of illness caused by these pathogens and improve the safety of our food supply, continued investigation of the epidemiology, transmission and interactions of these organisms with their environments is necessary. Additionally, prevention of colonization within natural reservoirs of these bacteria which contribute to contamination of foods is an important step in the reduction of the burden of foodborne illness. This work examines the relationship of Salmonella to oysters and the aquatic environment in which they are raised, the interactions of Salmonella in a small feedlot environment, and the reduction of colonization of broiler chickens by Campylobacter jejuni through vaccination with recombinant attenuated Salmonella vectors into which novel Campylobacter genes had been cloned. It was found that while Salmonella is still sporadically present on the West Coast of the US, an area where oysters were previously found to be positive for the organism, the strain which predominated in the last study of that area is reduced in prevalence. Additionally, it was found that that strain does not possess special fitness in oysters or the aquatic environments in which they are raised, though Salmonella survives in oysters and water samples longer than a representative coliform. Salmonella is also present in the small feedlot environment sampled, and animal stress appears to play a role in the shedding of the organism in that environment, leading to the potential contamination of beef carcasses during processing. Reduction of colonization by C. jejuni in broilers was achieved in the case of both vaccines, with a maximum reduction of four logs as compared to controls.

Studies were conducted in DUB/ICR mice with minute virus of mice (MVM), a single stranded DNA virus, and Colorado tick fever (CTF) virus, a double stranded RNA Virus, to determine if CTF virus had teratogenic potential and to characterize further MVM and its teratogenic effects. MVM is a known teratogen, whereas CTF virus had never been investigated previously for teratogenic potential. In characterization studies of MVM infectivity, the following were determined: (a) Rat embryo cells proved to be superior to six cell lines in assaying for MVM infectivity; (b) MVM infectivity could be inactivated with common laboratory disinfectants, ultraviolet radiation, temperatures 45 C to 100 C, and acid treatment: although in some cases a resistant population of MVM was detected; (0) MVM was relatively stable at 4 C and was very stable when stored in liquid nitrogen; (d) In the infected mouse, MVM was found to be firmly associated with the erythrocyte fraction of the blood; (e) MVM was a poor inducer of interferon, but was sensitive to the action of interferon. In teratogenic studies with MVM infection in mice, incidences of 5% stillbirths and 12 to 25% neonatal deaths were noted. In addition, between 77% to 91% of fetuses, and up to 51% of surviving neonates (to four weeks of age) had detectable MVM. Growth rates, weight gains, and developmental characteristics were normal for mice delivered of mothers inoculated with MVM. Teratogenic studies with Colorado tick fever (CTF) virus infection in mice revealed a high incidence of stillbirths (6 to 45%) and neonatal deaths (26 to 50%). CTF was demonstrated to cross the placenta and to replicate in the placenta and fetus and could be isolated from stillborns, newborns, and dead neonates. Several developmental aberrations were noted in neonates; however, there was no weight nor growth rate abnormality noted in the survivors. The teratogenic effects of CTF virus were inhibited significantly by neutralization with specific CTF antiserum, proving that the teratogenic effects seen were due to CTF virus.

Infection with Human Immunodeficiency Virus-1(HIV) results in a disease process characterized by three stages: an acute phase characterized by viremia, a clinically latent stage with little or no detection of virus in the blood, and the last stage, AIDS, which. is characterized. by marked immunodeficiency. During clinical latency, CD4+ T cells decline over a period lasting from a few to several years. Throughout this period, HIV is found trapped on the surface of follicular dendritic cells(FDC) in the germinal centers of secondary lymphoid tissues and this is the primary site of active viral replication. We hypothesize that FDC, and the unique microenvironment they help provide, play a critical role in HIV pathogenesis. The objective of these studies was to begin to characterize the role of FDC in HIV pathogenesis by determining if HIV immune complexes trapped on FDC are infectious. To begin to test this, HIV(IIIB) immune complexes were formed by incubating' virus with serum from. HIV infected individuals as a source of virus-specific antibody. Highly enriched tonsilar FDC (from non-infected individuals)were then incubated with these HIV-complexes to allow FDC trapping in Vitro. HIV binding to FDC was confirmed by electron microscopy(EM). Unbound HIV was removed by washing the cells and FDC bearing HIV immune complexes or control FDC were cultured for four days with superantigen activated, FACS sorted, autologous CD4+ tonsilar T lymphocytes to determine if the T cells could be infected by the FDC trapped virus. HIV infection was detected using PCR amplification of proviral gag sequences that would be present in DNA isolated from the cultures. To ensure that our in Vitro cultures were representative of in vivo events, we used a xenogeneic model where HIV immune complexes were formed and trapped on murine FDC in Vivo. Immune complexes were formed by injection of virus-specific antibody followed by HIV (IIIB). Murine FDC bearing HIV trapped in vivo were isolated and incubated with activated human CD4+ T cells as the only source of virus for infection. Infection was assessed as before. HIV infection of T cells was detected in cultures containing FDC bearing HIV immune complexes trapped in vitro whereas no infection was detected in controls. Furthermore, murine FDC bearing in vivo trapped HIV immune complexes also infected human CD4+ T cells. In some in Vivo experiments, HIV immune complexes were formed using a neutralizing antibody that could block infection. FDC bearing these neutralized HIV immune complexes also transmitted infection to T cells. This prompted the hypothesis that FDC may be able to negate the effects of neutralizing antibody. To test this, we formed HIV immune complexes with several doses (picogram to milligram) of neutralizing antibody and cultured these with T cells ± FDC. No infection was present in cultures of immune complexes and T cells without FDC, however, infection was clearly seen when FDC were added. Furthermore, neither macrophages, dendritic cells, nor FDC depleted populations of tonsilar cells could replace FDC in negating the effect of neutralizing antibody and this effect could be observed with different neutralizing antibodies and several strains of virus including a primary isolate. These data indicate that FDC associated HIV is infectious and that FDC can negate the effects of high levels of neutralizing antibody thus permitting infection to occur. This finding may help explain why HIV infected individuals with neutralizing antibody still have ongoing infection. In addition, this data may cause us to reshape our thinking about vaccination and treatment strategies. Finally, this work supports our hypothesis that FDC play an important role in HIV pathogenesis.

<p> A perpetual spring containing an orange biofilm composed of microbial sheaths was examined utilizing both geochemical and biological techniques. Big Bend Ranch State Park, the largest state park in Texas, covers over 120,000 hectares in the Chihuahuan Desert. While most surface features in this rugged, remote, and unpopulated setting are volcanic, underlying sedimentary features are found throughout the area. The presence of an orange biofilm containing a matrix of microbial sheaths was observed at two distinct springs within the park: Las Cuevas Amarillas and Ojo Mexicano. These springs contain low concentrations of hydrocarbons and abundant iron oxides in the water. This study presents the results of a biological analysis of the Las Cuevas Amarillas site in order to better understand the microbial consortia and metabolic strategies associated with these unique microbes. Basic chemical analysis of the water and soil of the site, and scanning electron microscopy with energy- dispersive x-ray spectroscopy analysis and x-ray diffraction analysis of the sheaths themselves, were performed. Total DNA was extracted from the biofilm matrix and universal primer set combinations were utilized to amplify both bacterial and archaeal 16s rRNA genes. These amplicons were subsequently cloned, sequenced, and phylogenetically analyzed. High-throughput sequencing (Roche 454 platform) was also performed in order to provide a deeper data set and a better representation of the diversity within this site. These analyses revealed numerous <i>Sphaerotilus</i> and <i>Leptothrix</i> spp., both genera known to be capable of sheath production. Iron- and manganese-oxidizing bacteria were determined to be abundant. Other beta- and gamma-proteobacteria, including <i>Thiobacillus</i> spp., <i>Curvibacter</i> spp., <i>Ideonella</i> spp., <i>Chromatiaceae</i> spp. and both Crenarchaeota and Euryarchaeota, were also detected.</p>

<p> Numerous pathogenic bacterial species have been found in many freshwater systems around the world. These pathogens affect the overall water quality of these systems and may cause diseases in both aquatic and terrestrial animals which may lead to loss of species diversity and abundance in their environments. This study sought to identify and document pathogenic bacterial species that may inhabit the streams that flow through Great Smoky Mountains National Park. Bacterial cells were collected by filtering water from four streams (Oconaluftee River, Kephart Prong, Little Pigeon River and Hickory King Branch Stream) through separate capsule filters. The cells were later backflushed from the filters and cultured on various selective and differential media. Ten isolates were selected based on phenotypic characteristics such as colony color and growth on specific media type, and sample origin. The nearly full 16S rDNA was sequenced for all ten isolates and analyzed to determine their identity. </p><p> Out of the ten isolates, four isolates were from the phylum Firmicutes while the other six were in the phylum <i>Proteobacteria</i>. Phylogenetic analysis of these isolates showed eight out of the ten isolates were related to known opportunistic pathogens. The other two were related to a ubiquitous <i>Bacillus</i> species that is considered to be a probiotic. Although none of the isolates had a 100% match to a known obligate or opportunistic pathogen, many isolates matched > 97% to opportunistically pathogenic species. Follow up molecular and metabolic tests need to be employed to determine the pathogenicity of each isolate.</p>

Foodborne outbreaks associated with leafy greens are attributed to many factors including cross-contamination between harvesting equipment and leafy greens. The objectives of the first study were a) to evaluate the efficacy of organic sanitizers and plant antimicrobials on these tools, and b) to determine if modified designs of coring knives are easier to decontaminate in comparison to the original design. Recently plant extracts and essential oils are gaining popularity due to their antimicrobial properties and being viewed as natural compounds. Studies have shown that plants compounds have antimicrobial activities both in vitro and in foods. However, studies regarding the effects of these antimicrobials on the organoleptic properties of foods are limited. The objectives of the second study were to a) perform sensory analysis to identify plant extracts or essential oils with highest preference liking by consumers in organic leafy greens; b) identify the effects of these compounds on sensory attributes of treated organic leafy greens; and c) determine changes in firmness and color properties of treated organic leafy greens. In order to reduce the strong aroma and flavor characteristics associated with essentials oils and plants extracts, these compounds can be incorporated into edible films. Edible films are thin layer of films made using fruit or vegetable pulp containing plant antimicrobials. The main objective of the last study was to determine preference liking by consumers and changes in physical properties of organic baby spinach treated with antimicrobial edible films. Three different designs of coring tools were evaluated. Coring tools were inoculated with S. Newport and treated with one of the following: deionized water, 50 ppm bleach, 3% hydrogen peroxide, 5% Chico wash™, 0.1% Oregano oil, 0.4% SaniDate 5.0®, 3% fulvic acid, or 0.1% oregano oil for 5 min. The surviving Salmonella populations on the tools were determined by swabbing four different locations on the tools and plating onto xylose lysine desoxycholate (XLD) agar. After inoculation with Salmonella overnight culture (8 log CFU/ml), an average of 6.35 log CFU/cm² attached onto the original coring tool, 6.31 log CFU/cm² on modified design 1, and 6.26 log CFU/cm² on modified design 2 coring tools. When comparing the efficacy of sanitizers, 3% H₂O₂ had the highest reductions of 5.98±0.56-6.22±0.29 log CFU/cm² in Salmonella population. Treatments with 0.39% SaniDate 5.0® and 0.1% oregano oil were comparable (to hydrogen peroxide) which yielded reductions of 5.89±0.80-6.19±0.22 log CFU/cm² and 5.51±0.58-5.90±0.46 log CFU/cm², respectively. When comparing the four locations on these tools, the greatest reduction was seen at location 2/3 on all three designs of coring tools. Organic iceberg lettuce and baby spinach were washed with various essential oils, plant extracts, and their combinations in tap water for 2 min. After wash treatment, each sample was stored at 4°C for 20-24 h before performing sensory evaluation and measuring changes in physical properties (color and texture) of leafy greens. A randomized block design with an affective test was used and 60 panelists were asked to evaluate each sample for preference liking based on a 9-point hedonic scale where 9 was extremely liked and 1 not liked at all. Preference liking was evaluated for the following parameters: aroma, color, freshness, mouthfeel, flavor, and overall acceptability. Additionally, panelists quantified each sample using a 5-point hedonic scale for the following attributes: pungency, browning, bitterness, off-odor, and sourness. Changes in firmness values and color of leafy greens were measured using Texture Analyzer and Chroma meter, respectively. Similar procedure was followed for sensory analysis of baby spinach treated with antimicrobial edible films wherein the edible films were added to bagged spinach. Edible films were made from hibiscus, apple, or carrot pulp which included 0.5%, 1.5%, or 3% of carvacrol or cinnamaldehyde. Forty panelists were asked to evaluate each sample based on preference liking and identify intensity of sensory attributes (pungency, browning, bitterness, off-odor, and sourness). Changes in color and firmness values were measured for organic baby spinach treated with edible films in plastic bags.Sensory analysis showed that washing organic iceberg lettuce and baby spinach with 0.1% cinnamon oil had the highest preference liking (7-moderately liked) and the least impact on sensory attributes (1-not affected at all) of these leafy greens. Similar results were observed for spinach leaves treated with cinnamaldehyde containing edible films showing higher preference liking values in comparison to those treated with carvacrol containing edible films. Our results also indicated that essential oils had higher impact on the firmness values and plant extracts had higher impact on the color properties of leafy greens.For textural analysis, washing iceberg lettuce with 0.1% oregano oil in combination with 10% olive extract yielded the highest firmness value (F=783.1±53.8). For spinach, samples washed with 0.1% lemongrass oil in combination with 1% apple extract yielded the highest firmness value (F=939.30±35.2). Additionally, no significant difference (p≤0.05) was found in firmness or color values of baby spinach treated with edible films containing plant antimicrobials. Results from the coring tool study will provide alternative organic sanitizer options for washing these tools which are more effective than currently used chemical sanitizers such as bleach. Findings from the sensory study will help in identifying appropriate antimicrobial treatments for washing organic leafy greens. Additionally, use of edible films with essential oils may prevent the adverse effects due to the direct application of essential oils on organic leafy greens.

<p> <i>Mycobacterium marinum</i> (Mm) shares at least 80% amino acid sequence identity with over 3,000 orthologous genes of Mycobacterium tuberculosis (Mtb) and is thus used as a surrogate pathogen for Mtb research. Our laboratory investigates mycobacteriosis using Japanese medaka (<i> Oryzias latipes</i>) as an aquatic animal model. Mm disease presentation in medaka is similar to Mtb disease presentation in humans, including growth in macrophages, granulomatous lesions, and lifelong chronic disease. We have previously shown that a major route of infection in fish is through an oral route and have thus developed methods to infect medaka with Mm utilizing mosquito larvae as vectors. Recently, our lab was able to show that Mm is able to cross the gut epithelia of medaka in a relatively short-time frame and travel to the underlying submucosa. Therefore, Mm must have the ability to attach to the gut mucosal layer and evade killing by GALT immune cells. Mm is apparently able to exploit macrophages of the mucosal immune system to transport the bacteria to target organs like the head kidney, liver, and spleen for a systemic infection. Utilizing an Mm strain engineered to carry a deletion in the RD-1 region, known to include a number of virulence genes, our lab has shown that mucosal immunity against Mm can be induced in medaka. We have shown that exposure to the mutant RD-1 strain offers some protection against a chronic wild-type oral challenge. Since we know that mutant RD-1 can elicit a mucosal immune response, I tested to see if sensitizing mucosal immunity would also induce systemic immunity by first priming fish with mutant RD-1 and then subsequently challenging them with wild type Mm via an IP route. This thesis demonstrates that mucosal immunity is limited to the gut and thus does not appear to provide broad systemic immunity. Additionally, I tested to see if systemic vaccination would protect against a systemic virulent wild-type challenge by vaccinating and challenging fish via an IP route of infection. Results showed that systemic vaccination does not induce systemic immunity and thus does not protect against an IP injected virulent challenge. Collectively, results from this thesis have shown mutant RD-1 to only be effective as a vaccine against mycobacteriosis if given orally since it was shown to only induce a mucosal immune response and only be protective against an oral virulent wild type challenge.</p>