Microbiological Sciences

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Research from the Department of Microbiological Sciences. The department website may be found at https://www.ndsu.edu/micro/

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Glucose Uptake by the Cellulolytic Rumen Anaerobe Bacteroides Succinogenes
(North Dakota State University, 1986) Franklund, Clifton Victor
Glucose uptake by the cellulclytic rumen anaerobe, Bacteroides succinogenes S85, was measured under conditions that maintained anaerobiosis and osmotic stability. This organism was found to possess a highly specific, active transport mechanism for glucose. Evidence for a phosphoenol-pyruvate:g1ucose phosphotransferase system was not detected. Compounds that inhibit electron transport systems (non-heme iron chelators, and sulfhydryl reagents) were effective inhibitors of glucose uptake. The strongest inhibitors were compounds (proton and metal ionophores) that interfere with maintenance of the proton motive force. Compounds which interfere with ATP synthesis also inhibited glucose uptake, but a role for ATP in energizing uptake could not be inferred from these results. Oxygen prevented glucose uptake (75% inhibition), reflecting possible active sulfhydryl centers (above) or autooxidation of electron transport components. The results suggest the fumarate reductase-coupled electron transport system of B. succinogenes can generate a proton motive force that is used to energize glucose uptake. Na+ and Li+. but not K+, stimulated glucose uptake and may partly account for the growth requirement of B. succinogenes for Na+. However, the data were insufficient to conclude that glucose uptake occurs by a Na+ symport mechanism. Spheroplasts of B. succinogenes transported glucose as well as whole cells, indicating glucose uptake is not dependent on a periplasmic glucose binding protein. A variety of sugars including the nonmetabolizable analog, [inversely proportional symbol]-methylglucoside. did not inhibit glucose uptake. Only cellobiose and 2-deoxyglucose were active and neither behaved as a competitive inhibitor. Metabolism of both sugars was probably responsible for the inhibition. Cellobiose-grcwn B. succinogenes showed a reduced ability to transport glucose compared to glucose-grown cells. This may indicate regulation of synthesis of the glucose carrier protein by cellobiose through a mechanism other than catabolite repression. Differences in the ability to transport glucose were detected between transition cells (transition from lag to log phase of growth) and log-phase cells. However, the differences were not due to different glucose transport mechanisms. Alterations in the structural integrity of the cell envelope, as reflected by osmotic- and cold-sensitivity features of transition and log cells, may have affected the glucose uptake abilities in these cell types.
lntraspecific Variation In Pathogenic Cryptosporidium parvum
(North Dakota State University, 2010) Herges, Grant Richard
Cryptosporidium causes cryptosporidiosis, an infectious diarrheal disease, which can become chronic and life-threatening in immunocompromised individuals. Cryptosporidium parvum and C. hominis are the primary causes of human cryptosporidiosis. Of these species, C. hominis only infects humans while C. parvum additionally infects ruminants, in particular neonatal calves. Therefore, understanding the transmission dynamics of C. parvum, particularly the specific contribution of zoonotic and anthroponotic transmission, is critical to the control of this pathogen. Cryptosporidium parvum genotypes have been identified which appear to be restricted to a single host, which suggests that this may be a heterogeneous species, with varying infection and transmission dynamics. The first objective to this thesis was to determine the population structure of pathogenic C. parvum in the upper Midwest United States. A total of 216 isolates from cases of human cryptosporidiosis in Minnesota and Wisconsin and 64 isolates from diarrheic calves in Minnesota, Wisconsin, and North Dakota were genotyped at 8 polymorphic loci. A total of 213 isolates, 167 from humans and 46 from calves, had complete multilocus types (MLT). There were 93 different Ml Ts and sixty of those Ml Ts were only represented by one isolate. Population analysis revealed a highly recombining, panmictic population that does not have any genetic, geographic, or host sub-structuring. The second objective to this thesis was to determine the variability in the in vitro infectivity of different C. parvum strains, IOWA II and Moredun. A quantitative RT-PCR approach was used to quantify expression of target genes during infection of HCT-8 cells. Fluorescence microscopy was used to quantify life cycle stages during infection. Our data showed that the IOWA II reached its sexual stages earlier and had a greater number of trophozoites/meronts at 24 h p.i. than Moredun. The host used for propagation of C. parvum also affected subsequent infectivity of HCT-8 cells.
Phenotypic and Genotypic Effects of FlhC Mediated Gene Regulation in Escherichia Coli O157:H7
(North Dakota State University, 2011) Sule, Preeti
Escherichia coli (E.coli) 0157:H7, a pathogen belonging to the enterohemorrhagic group of E.coli, has long been a concern to human health. The pathogen causes a myriad of symptoms in humans, ranging from diarrhea and malaise to renal failure. Human infection with the spread of the pathogen is mainly attributed to consumption of contaminated food material such as meat. Decontamination of meat via sprays have to date been the most commonly practiced method to reduce contamination, which now has little relevance in the face of developing resistance by the pathogen. In the following study we investigated FlhC mediated gene regulation in E. coli 0157:H7 on the surface of meat, in an attempt to recognize FlhC regulated targets, which may ultimately serve as targets for the development of novel decontaminating sprays. Microarray experiments were conducted to compare gene expression levels between a parental E. coli 0157:H7 strain and its isogenic flhC mutant, both grown on meat. Putative FlhC targets were then grouped based on their function. Realtime PCR experiment was done to confirm the regulation. Additionally, experiments were done to investigate the phenotypic effects of the regulation. To test the effect of FlhC on biofilm formation, an ATP based assay was first developed in E.coli K-12, which has been detailed in the following dissertation. This assay was used to quantify biofilm biomass in E. coli 0157. Microarray experiments revealed 287 genes as being down regulated by FlhC. These genes belonged to functions relating to cell division, metabolism, biofilm formation and pathogenicity. Real-time PCR confirmed the regulation of 87% of the tested genes. An additional 13 genes were tested with real-time PCR. These belonged to the same functional groups, but were either not spotted on the microarray chips or had missing data points and were hence not included in the analysis. All 13 of these genes appeared to be regulated by FlhC. The phenotypic experiments performed elucidated that the FlhC mutants divided to 20 times higher cell densities, formed five times more biofilm biomass and were twice as pathogenic in a chicken embryo lethality assay, when compared to the parental strain. The following dissertation also reports the development of a combination assay for the quantification of biofilm that takes advantage of the previously mentioned ATP assay and the PhenotypeMicroarray TM (PM) system. The assay was developed using the parental E. coli strain AJW678 and later applied to its isogenic flhD mutant to elaborate on the differences in nutritional requirements between the two strains during biofilm formation. Metabolic modeling and statistical testing was also applied to the data obtained. This assay will be used in the future to study biofilm formation by the parental strain E. coli 0157:H7 strain and its isogenic FlhC mutants on single carbon sources, hence identifying potential metabolites which differentially support biofilm formation in the parental and the mutant strain.
Gene Regulation in Biofilms
(North Dakota State University, 2011) Samanta, Priyankar
Sessile bacterial communities which form on the solid surface or solid-liquid interface are known as biofilms. Both single species and multispecies biofilms are characterized by an extracellular matrix of polymeric substances which gives them several hundred times more antibiotic resistances than a planktonic bacterial culture. Though bacteria are the most common causative agent of various diseases, because of the high antibiotic resistance, biofilms cause complications of various diseases like cystic fibrosis, prosthetic valve endocarditis, chronic pulmonary diseases, catheter-associated urinary tract infections and several other diseases. From past studies, quorum sensing has been established as a novel target mechanism against biofilms; in this study, the two-component signal transduction systems (2CSTSs) have been focused. Once better understood, 2CSTSs can serve as a novel drug target and prevention mechanism for biofilm associated diseases. According to prior high-throughput experiments and phenotype microarray experiments by our lab, several 2CSTSs like OmpR-EnvZ, RcsCDB along with the global regulator FlhD/FlhC were hypothesized to have an important effect on various developmental stages of biofilm formation. From that past study, we postulated that acetate metabolism may be an important aspect for biofilm formation. In this study, we tested and confirmed this hypothesis. We observed biofilms formed by several mutants in 2CSTS, as well as mutants in acetate metabolism, using Scanning Electron Microscopy (SEM). We found quantitative and qualitative differences in the biofilm of the acetate mutants when compared to their isogenic parental Escherichia coli strain. An additional mutation in rcsB with acetate mutant strains forms less clumpy biofilms whereas an additional mutation in dcuR results in the formation of less biofilms. So the structural and the quantitative differences of acetate mutant biofilms depend on additional mutations in rcsB and dcuR. Though a number of studies have been done on the temporal gene expression within biofilms, spatial gene expression of the mature biofilm is a big gap of knowledge. The future aim of this study is to study the temporal as well as the spatial gene expression of different 2CSTSs in the biofilm. In my MS thesis, I have constructed selected promoter fused GFP /RFP plasmids and some other fusion plasmids were purchased from the promoter collections from Open Biosystems, lastly E. coli AJW678 bacterial strains were transformed with these GFP /RFP fused plasmids. A 96 well microtiter plate assay was performed to study the temporal expression from the promoters by quantifying the fluorescence intensity in the planktonic culture. According to this experiment, the highest expression of flhD was after 20 hours whereas, the expression of ompR increases up to 7 days, which indicates that the flhD expresses earlier than ompR. The decreasing phase of flhD expression was paralleled by the sharpest increase in ompR expression as phosphorylated OmpR is an inhibitor of flhD expression.
Evaluation of a Climate-Sensitive Disease Control Strategy and Investigation of Multi-drug Resistance in Infectious Bacterial Diseases: A US-Africa Experience
(North Dakota State University, 2012) Mukiibi, Herbert
This paper presents two research projects that explore avenues of controlling infectious diseases both in Africa and the United States. In Uganda, a retrospective study of Otuboi Sub County patient data to evaluate the impact of Stamp Out Sleeping sickness (SOS) intervention was performed. Polymerase Chain Reaction to detect the conjugatively transferred virulence factors from MDR E. coli to Salmonella was performed in North Dakota. Human African Trypanosomiasis prevalence was significantly reduced at intervention year (2006) compared to the pre-intervention years; 2004 (P = 0.00024) and 2005 (P = 0.000001). Of the 22 screened virulence factor genes, eight genes were PCR detected in MDR E. coli 2077 isolate. Six of the detected genes were found to be received by Salmonella transconjugates. The protective effect of SOS intervention was sustained for only two years (2007 and 2008) post intervention. MDR E.coli 2077 isolate conjugatively transferred its virulence factors to Salmonella strains.
Drivers of Infectious Disease Outbreaks: How Climate, Environment and Disease Control Programs Influence Occurence of Infectious Disease Outbreaks
(North Dakota State University, 2012) Muleme, Michael
This research study described the factors driving infectious disease outbreaks using Foot-and-mouth disease (FMD) in Uganda and Lyme disease in North Dakota (ND), Minnesota (MN) and Wisconsin (WI) as case studies. Retrospective data on FMD vaccines and outbreaks in Uganda (2001 – 2010) and Lyme disease in ND, MN and WI (1990 – 2011) was used. The time (7.5 weeks) taken to respond to FMD outbreaks, limited serotyping/subtyping (9/121) of outbreaks and the low percentage of cattle vaccinated (2.1 – 21.2%) portray ineffective control programs. Similarly, increase in fall temperature (P = 0.0189) and annual precipitation (P = 0.0250) were associated with increased human Lyme cases. Shrub land coverage and human population also increased in WI, MN and ND while forest coverage increased in ND. These favor tick and deer proliferation leading to increased human exposure to Lyme Borreliosis. Therefore ineffective disease control programs, climate and environment factors influence infectious disease occurrence.
Emerging Infectious Diseases with Limited Treatment Options: The Case of Ebola Hemorrhagic Fever in Uganda and Shiga Toxin Producing Escheria Coli in the United States
(North Dakota State University, 2012) Gemmeda, Rahel Dubiwak
Emerging infectious diseases are diseases that newly emerge in a population or change the frequency or spatial distribution of their occurrence. Ebola Hemorrhagic Fever (EHF) and Shiga Toxin-producing Escherichia coli (STEC) infections are among diseases that emerged in the 1970s. The two diseases have limited treatment options with no vaccines. This paper is based on two case studies. The first case study utilized data from the 2007/2008 EHF outbreak in Uganda and investigated the epidemiological and clinical aspects of the outbreak. The second case study was based on a study done on STEC isolates collected from beef cattle at the North Dakota State University Research Extension Center in Dickinson. The study investigated the prevalence of the common pathogenic STEC serotypes. The driving factors for the emergence of EHF and STEC, their prevention and control strategies and their challenges were discussed based on the case studies.
Brucellosis Epidemiology, Virulence Factors, Control and Molecular Targets to Prevent Bacterial Infectious Diseases
(North Dakota State University, 2012) Mugabi, Robert
Brucellosis is a bacterial zoonosis that infects both professional phagocytic and nonphagocytic cells in the hosts. Brucella intracellular survival is important for its virulence. In a study to establish the seroprevalence and risk factors of brucellosis in livestock in Kazo and Buremba sub-counties of Kiruhura district, Uganda, fifty goat and 112 bovine serum samples were tested for Brucella antibodies. The prevalence of Brucella antibodies in goats and cattle was 26.0% and 38.4% respectively, while individual seroprevalence rates by livestock breeds were 10.7% (cross-breed goats), 45.5% (local goat breeds), 49.1% (cross-breed cattle), 31.0% (local cattle breeds), and 17.4% (exotic cattle breeds) (p = 0.001). Sharing of watering points, using surface water for livestock, presence of wildlife on pasture, lack of vaccination was significantly correlated with Brucella seropositivity in livestock. The molecular study on biofilm in Escherichia coli included in this paper revealed that pflA knock out mutations had a significant effect on biofilm amounts when biofilms formed on D-serine and acetate. The ldhA formed generally high bacterial biofilm amounts on all carbon sources as compared to the wild type.
Hepatitis Virus B and Hepatitis Virus C Co-Infection Among HIV Patients and Development of an Enzyme Linked Immunosorbent Assay (ELISA) for Diagnosis of Equine Protozoa Myeloencephalitis
(North Dakota State University, 2012) Kyomuhangi, Annet
Study 1: HIV patients with chronic HBV and/or HCV are more likely to die of liver disease and have a more rapid progression to Acquired Immunodeficiency Syndrome (AIDS) than patients solely infected with HIV. Blood samples were assayed for the presence of HIV 1/2 antibodies, Hepatitis B Surface Antigen (HBsAg) and Hepatitis C antibodies (HCVab). The prevalence of HBV and HCV among HIV patients was 13% and 10%, respectively. This calls for integration of HBV and HCV prevention, and treatment into HIV programs. Study 2: The current techniques used to diagnose EPM have low sensitivities and some are expensive. To improve serologic diagnosis of EPM, a trivalent antigen (rSAG2/4D1/3D2) was expressed, purified and later incorporated into indirect ELISAs. rSAG2 and rSAG4D1/3D2 reported two and one false negative (s), respectively while rSAG2/4D1/3D2 reported none at a cut off of 15% positivity. rSAG2/4D1/3D2 could be more accurate and reliable than rSAG2 and rSAG4D1/3D2.
Preliminary Investigation of Escherichia Coli K12 Biofilm Inhibition on an Antimicrobial Polysiloxane Coating using Whole Transcriptome Profiling
(North Dakota State University, 2012) Stafslien, Shane J.
Whole transcriptome profiling was examined in E. coli K12 when cultured on the surface of a pure polysiloxane coating (Sil) and a polysiloxane coating containing a tethered quaternary ammonium compound (QSil) shown to inhibit biofilm formation. An optimized protocol was developed for isolating high quality RNA from the surface of these coatings prepared in multi-well plates. DNA microarray data obtained from the Sil and QSil coatings revealed that 222 genes were differentially expressed between these two surfaces by a factor of at least 2-fold and with a 90% level of confidence. Several genes of the lsr operon, which encode the various components of the AI-2 based quorum sensing system, were repressed on the QSil coating surface. The QSil coatings ability to effectively interfere with the AI-2 based quorum sensing system was most likely the primary factor that contributed to the impairment of E. coli K12 biofilm formation on that surface.
International Infectious Disease Management and its Role in the 'One World, One Health, One Medicine' Concept
(North Dakota State University, 2012) Swanson, Emma Louise
The knowledge that almost 75% of all new human pathogens have animal origins, requires health professionals from all fields, (i.e. human medicine, veterinary medicine, and public health professionals), to work on solving the major problems associated with infectious disease threats by utilizing the ‘One World, One Health, One Medicine’ approach. It is clear that the lack of surveillance systems, proper training, and communication presents the biggest obstacles when dealing with pathogens. In accordance with the goals of the Master’s of International Infectious Disease Management and Biosecurity program at North Dakota State University, the objectives of this paper are to examine two small projects completed by the student to understand the contribution to the growth and enrichment of the student’s career development path. These projects, though different in form, clearly demonstrate the importance of surveillance, education, and multi-sectoral approach to infectious disease.
Lung Mucosal Response to Repeated Inhalational Insults with Immunomodulatory Agents in a Murine Model of Fungal Asthma: Airway Epithelium Takes the Center Stage
(North Dakota State University, 2013) Pandey, Sumali
Asthma is a debilitating disease of the lungs affecting 235 million people worldwide. Fungus-associated asthma leads to a particularly severe type of disease, and exposure to environmental fungi and their products is unavoidable due to the ubiquitous nature of fungal species. Besides being allergenic, fungi are opportunistic pathogens, and anti-fungal and/or allergic pathways may be modified through repeated inhalation of immunomodulatory agents, affecting the outcome of fungus-induced asthma. Our aim in this project was to investigate the extent to which repeated inhalation of immunomodulatory agents influence the lung mucosal responses in a naïve murine host or in one that had been sensitized to fungal proteins (allergic). The immunomodulatory substances chosen hold relevance to human inhalational exposure, and included live or irradiation-killed Aspergillus fumigatus (a fungi) spores, deoxyxnivalenol (a mycotoxin), and fluticasone propionate (an inhalationally administered corticosteroid, commonly prescribed for allergic asthma). In a naïve host, inhalation of live A. fumigatus spores showed pathological features of fungal asthma. However, in an allergen-sensitized lung, both dead and live A. fumigatus spores established fungal airway disease, albeit to different extents. Next, we tested the effect of deoxynivalenol in an allergic host and found that its repeated inhalation did not affect pulmonary disease pathology, but did lead to a dose- and time- dependent increase in mucosal and systemic total IgA. Finally, we tested the effect of fluticasone propionate, and found that it did not influence the development of fungal airway disease, but did induce dynamic changes in lung physiology and antibody titers. Besides mimicking human inhalational exposures, inhalation ensures direct interaction of the inhaled substances with airway epithelium, which plays an important role in defense against inhaled substances and in asthma pathophysiology. By analyzing various mechanisms involved in murine lung-mucosal response to the inhaled substances, a critical involvement of airway epithelium as an orchestrator of immune responses is highlighted, and this would inform mechanism-based future studies. In conclusion, this project is likely to aid in establishing evidence based standards for fungus-related exposures and in making informed therapeutic decisions for fungus-associated diseases.
Investigations in Asthma Heterogeneity: The Roles of Aspergillus Fumigatus-Derived Eicosanoid Synthases and Occupational Exposures to Grain Dusts on the Development of Fungal Allergic Asthma
(North Dakota State University, 2013) Sharma, Akshat
Allergic asthma is an inflammatory syndrome of the respiratory system which changes the airway wall architecture. Using an aeroallergen, murine model of A. fumigatus-mediated asthma, the two studies herein examine the development of asthma in the contexts of host-allergen interactions via A. fumigatus knock-outs of eicosanoid synthases and occupational exposures to corn and soybean dusts. The lack of difference between control and treatment groups seen in post-methacholine airway responses, goblet cell metaplasia, peribronchial inflammation, and fibrosis in the first study show that fungus-derived eicosanoid synthases are dispensable in the development of fungal allergic asthma. However, the same set of respiratory parameters in the grain dust study reveals an increase in BAL neutrophilia and serum IgE titer. The study also underscores a need for modifications of dust exposure times and of time-points of data analysis. These two studies represent unique perspectives on asthma pathogenesis and emphasize the heterogeneity of the syndrome.
Inhibition of Fusarium Growth and Trichothecene Accumulation in Grain by Antifungal Compounds from Lactic Acid Bacteria
(North Dakota State University, 2013) Zhao, Hui
Fusarium head blight (FHB) is a widely occurring plant disease, which is caused by fungi in the genus Fusarium. FHB leads to mycotoxin accumulation on grain, which causes food safety risk and economic loss. In addition to chemical treatments, biological strategies, like application of lactic acid bacteria (LAB), could be useful in preventing and/or eradicating mycotoxigenic Fusarium growth and mycotoxin production.After comparision of the anti-Fusarium activities by a microdilution assay against Fusarium graminearum 08/RG/BF/51, Lactobacillus rhamnosus VT1 was found to have the highest anti-Fusarium activity. Response surface methodology (RSM) was employed to optimize the incubation conditions for the production of cell-free Lactobacillus culture supernatant (CFLCS) from the strain. The best combination included 34¨¬C, 55 hours, and shaking at 170 rpm for production of CFLCS from L. rhamnosus VT1. Under these incubation conditions, a 10% cell-free culture of Lactobacillus rhamnosus VT1 inhibited 83.7% of the Fusarium growth on microplate. MIC value of the CFLCS with a 104 conidia /well inoculum concentration is 18%.To identify the mechanisms of anti-Fusarium activity, a stepwise regression, with ¥á to enter = 0.15 and ¥á to remove = 0.15, was performed to analyze the data of the RSM design. It was indicated that pH, total acidity, and 3-phenyllactic acid were the most important factors and could be used to explain 39.2% variation of the anti-Fusarium activity. In addition, proteinaceous compounds might be important due to the possible synergistic effect in the CFLCS. CFLCS applied directly to grain not only prevented Fusarium growth, but also changed mycotoxin accumulation. Fusarium growth was inhibited completely by a 50% concentration (V/V) of the CFLCS applied on rice media after 14 days incubation, and almost no mycotoxins were detected. Concentrations of 15%, 30% and 50% of CFLCS as steeping water inhibited Fusarium growth and mycotoxin accumulation on barley in the malting process. Almost no mycotoxins were detected in the samples treated by 50% CFLCS. However, the germination ability of the barley samples was inhibited. In general, the CFLCS showed potential effective anti-Fusarium activity. However, the strategies of application of the CFLCS on grain should be further investigated.
Studies in Pathogenesis of a Novel Isolate of Cronobacter Sakazakii using an In Vitro Blood Brain Barrier Model
(North Dakota State University, 2013) Welker, Elliott Weston
Genus Cronobacter is a member of the family Enterobacteriaceae consisting of several opportunistic species. The primary focus of this study was to utilize an in vitro co-culture model of the blood brain barrier to investigate a bovine fecal strain of C. sakazakii to investigate pathogenicity. The strain was found to have the same effect on the barrier’s integrity as the positive Escherichia coli control. Additionally, C. sakazakii strain BAA-894 was found to have the same effect as the negative E. coli control. This study also focused on the development of a site-specific mutagenesis procedure for C. sakazakii. A procedure using linear transformation was able to replace the putative virulence gene zpx (zinc-containing metalloprotease) in C. sakazakii. A future virulence study would involve using this mutagenesis procedure to induce a mutation in genes of C. sakazakii speculated to play a role in BBB translocation followed by challenge in the BBB model.
Rhomboid Proteases and Surface Adhesins During Cryptosporidium Development
(North Dakota State University, 2013) Tabe, Ebot Sahidu
Cryptosporidium parvum, a primary cause of cryptosporidiosis in humans and livestock worldwide, has a complex life cycle that includes an environmental oocyst stage, and stages of merogony, gametogony, and sporogony that are completed in a single host. Development within the host takes place in a protected intracellular but extracytoplasmic niche at the apical surface of epithelial cells. The life cycle can be described as having alternating extracellular invasive and intracellular replicative stages. With no effective chemotherapeutics, understanding the mechanism of host cell invasion by this pathogen is paramount. The first aim dissertation was to identify functions of sporozoite surface proteins and rhomboid proteases (CpROMs) during motility and invasion of host cells. We demonstrate that two CpROMs distinctively and collectively cleaved five thrombospondin-family proteins (TSPs) and a mucin-like glycoprotein in a heterologous assay. Further, there was differential co-expression and co-localization of the CpROMs and their substartes during in vitro life cycle development; CpROM4 and CpTSP10 proteins colocalized to the anterior, middle and posterior of sporozoites and in developing intracellular stages while CpROM5 and TRAP-C1 colocalized to intact and non-intact oocyst walls, the anterior of sporozoites, and intracellular stages as early as 2 h post infection. CpTSP7, also localized to the oocyst wall, the anterior and posterior of sporozoites and intracellular stages from 6 h post infection. Similar to CpTSP10, CpTSP9 was not present in the oocyst wall; however, it was expressed in sporozoites and intracellular stages from 6 h post infection. Short synthetic peptides derived from adhesive ectodomains in thrombospondins including a TRAP-C1 apple domain (TAAP), thrombospondin type I domains in CpTSP7 (7TS) and CpTSP9 (9TS), and a kringle domain in CpTSP10 (10K1) as well as their corresponding antibodies demonstrated competitive and neutralization inhibition effect of C. parvum infection of host cells. Polyclonal antibodies against TAAP caused sporozoites to agglutinate in a concentrationdependent manner, suggesting a contribution to reduced infectivity. In conclusion, the specificity and expression profiles of CpROM4 and CpROM5 indicate that they have distinct functions in shedding surface adhesins during excystation, motility, invasion, and intracellular development.
Happy Beef: The Development of ß-Phenylethylamine as a Novel Nutrient Treatment Reducing Bacterial Cell Count by Escherichia Coli O157H7 on Beef Meat
(North Dakota State University, 2013) Lynnes, Ty Cordell
Since its emergence in 1980's, Escherichia coli O157:H7 has often been associated with the consumption of contaminated meat. E. coli O157:H7 continues to persist as a food borne pathogen not only in beef but many other food products as well. One of the reasons for its persistence is its ability to overcome many of the current control effort including citric acid treatments. This research looks at the use of nutrients as a novel way to control E. coli O157:H7. In this research we used Phenotype MicroArray ™ technology from BioLog (Hayward, CA) technology to screen 95 carbon and 95 nitrogen nutrient sources for their ability to reduce respiration, biofilm amounts and cell number. The top eight performing nutrients were then screened a second time to look at the effects of concentration on their ability to reduce biomass, biofilm amounts and cell number in beef broth. The second screening allowed for the calculation of the concentrations needed to inhibit these factors by 50%. This screening reduced the number of chemicals from eight to two chemicals, acetoacetic acid and ß-phenylethylamine, both of which were characterized by low inhibitory concentrations (<10 mg/ml). In a final experiment, these two chemicals were used in various concentrations as treatment on beef, which was then inoculated with E. coli O157:H7. Only ß-phenylethylamine was able to reduce the bacterial cell counts of E. coli O157:H7 over the seven day incubation period. ß-phenylethylamine, a natural trace amine found in chocolate after fermentation, was able to reduce the recovered colony forming units by >74%. This shows that a nutrient can be used as a novel way to control phenotypic traits in E. coli O157:H7 in a preventative manner.
Gene Expression and Evolution in Escherichia Coli Biofilm
(North Dakota State University, 2014) Samanta, Priyankar
Biofilms can be defined as a complex aggregation of bacterial communities that involves many gene regulatory mechanisms, as well as evolutionary processes to increase biodiversity. Specific Aim 1 used a gene regulation approach to identity novel targets for the development of biofilm prevention and treatment techniques. The goal was to determine genes that get expressed early in biofilm development (prevention targets) and genes that get expressed late and in the outer layer of the biofilm (treatment targets). Biofilm formation is regulated by numerous regulators, including the two-component osmoregulator system EnvZ/OmpR, the colanic acid activator rcsCDB and the global regulator FlhD/FlhC. In this study, we determined the temporal and spatial expression of flhD, ompR and rcsB in E. coli k-12 AJW678 biofilm, as well as the gene expression of flhD in isogenic ompR and rcsB mutants. Results indicated that flhD was expressed early, and in the outer layer of the mature biofilm. We concluded that FlhD/FlhC would be the first target for novel prevention and treatment technique. One mechanism to increase biodiversity in biofilm is the insertion of transposon elements, which was investigated as Specific Aim 2. Insertion of IS elements into the flhD promoter resulted in increased motility in numerous E. coli K-12 strains has been shown in previous study. In this study, we recovered isolates from biofilm, where IS1 had inserted in the flhD promoter further downstream than in previously described strains. These isolates showed reduced motility. We also wanted to determine the effect of an IS element insertion on regulation of flhD expression by OmpR and RcsB in biofilm. Temporal and spatial gene expression of three different GFP-tagged flhD promoters was measured. The results indicated that IS5 insertion in the flhD promoter at the published hotspot did not have any effect on regulation of flhD expression by OmpR and RcsB in biofilm.
Cronobacter Sakazakii Characterization and Analysis of Cytotoxicity in Microvascular Endothelial Cells
(North Dakota State University, 2014) Hafner, Hilary Jayne
Contamination of powdered infant formulas by the bacteria Cronobacter sakazakii can pose serious risk to infants and neonates who consume the formula and subsequently develop C. sakazakii related illnesses such as sepsis and meningitis (1). The Gibbs’ lab assesses C. sakazakii isolates’ ability to cross the blood brain barrier and cause meningitis. This thesis research investigated C. sakazakkii cytotoxicity towards microvascular endothelial cells which comprise the first cell line encountered in the barrier. Understanding the mechanisms used to affect these cells will contribute to our understanding of early stages of invasion. Cytotoxicity assays performed for this research found that the cell line used could not sustain confluency when co-cultured with C. sakazakii isolates over periods beyond 24 hours of incubation. In addition, cell-free cytotoxicity assays found that live cells are not necessary to cause damage suggesting a toxin mediated effect.
Characterization of a Novel Cryptosporidium Genotype in Red-Winged Black Birds
(North Dakota State University, 2014) Jesudoss Chelladurai, Jeba Rose Jennifer
Cryptosporidium species cause cryptosporidiosis, characterized by acute gastroenteritis in humans and animals worldwide. Knowledge of the diversity of Cryptosporidium among mammals and birds is incomplete, especially in North American passerines. In this first molecular study of Cryptosporidium in a North American passerine, C. parvum and a novel genotype, called the red-winged black bird genotype were isolated. Genetic characteristics and phylogenetic analyses of the red-winged black bird genotype were described at the 18S rRNA, actin and HSP70 loci, and it was distinct from previously described species and genotypes. The novelty of this genotype was also supported by propagation studies in chickens, zebra finches and cockatiels that failed to produce patent infections. The study adds to our understanding of the co-evolution of the parasite with its hosts and potential sources of C. parvum transmission to susceptible human and animal hosts.

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