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Browsing by Author "Hoover, Edward A., advisor"

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    Biologic and biochemical features of prion pathogenesis
    (Colorado State University. Libraries, 2016) Hoover, Clare Elizabeth, author; Hoover, Edward A., advisor; Zabel, Mark D., advisor; Avery, Anne, committee member; Tjalkens, Ronald, committee member
    Prions are the causative agents of a group of fatal neurodegenerative diseases known as transmissible spongiform encephalopathies. Prions are unique in that disease is initiated when the normal prion protein (PrPC) undergoes a conformational change and propagates through a process of templated conversion to an infectious, misfolded, isoform (PrPRES, PrPCWD, or PrPSc) which can assemble into oligomers and amyloid fibrils. Disease is associated with prion accumulation in the central nervous system, causing the pathologic lesions of neurodegeneration, white matter spongiosis, and a reactive astrogliosis. Previous work has demonstrated the process of prion propagation and disease pathogenesis can be influenced by conversion cofactors, inhibitors, and biologic systems. Heat shock proteins have been shown to protect against the toxic disease effects of denatured and aggregated proteins in several models of neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, and spinocerebellar ataxia. In this dissertation, I investigated if heat shock protein 72 (HSP72) expression in neurons could protect against prion disease-associated pathology through a cell culture and mouse model of murine-adapted scrapie strain RML. In contrast to the role in other neurodegenerative diseases, HSP72 did not alter the prion disease course or amount of prion conversion in either disease model. Chronic wasting disease (CWD) is a naturally occurring, horizontally transmitted prion disease affecting wild and captive cervid populations that is rapidly expanding into new states and countries. Studies investigating the distribution of PrPCWD during early subclinical CWD infection have detected prions in the oropharyngeal lymphoid tissues as early as 1.5 months; however, the complete tissue distribution of PrPCWD immediately following prion exposure and the chronological progression of prion tissue accumulation remains unknown. Here, I show prions initially accumulate in the oropharyngeal lymphoid tissues following mucosal exposure and rapidly disseminate to all systemic lymphoid tissues prior to neuroinvasion. These findings will help better understand the early pathogenesis of CWD prior to clinical disease and potentially identify therapeutic targets. Prion disease diagnosis relies on demonstration of the misfolded isoform by immunodetection, amyloid seeding assays, or animal bioassays, all assays which may require separate sample preparations precluding examination by multiple tests. To address this limitation, I developed a new technique to detect PrPCWD amyloid seeding in fixed paraffin-embedded (FPE) tissues by real-time quaking induced conversion (RT-QuIC). FPE RT-QuIC proved to be more sensitive than IHC for prion detection and the use of RT-QuIC amyloid formation kinetics yielded a semi-quantitative estimate of the prion burden in samples without the cost and time of animal bioassays. The normal cellular prion protein resides in cell membrane lipid rafts, which has been shown to be a site of pathogenic conversion. Previous in vitro assays have highlighted the ability of lipids to promote prion formation but knowledge is limited regarding the capacity of lipids to inhibit prion formation. Here, I show endogenous polar brain lipids directly inhibit prion amyloid formation in RT-QuIC in a dose-dependent manner. This work is the first to identify an inhibitory role of lipids and suggests the prion conversion process is influenced by a balance of pro-conversion and inhibitory molecules.
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    Chronic wasting disease: a model for prion transmission via saliva and urine
    (Colorado State University. Libraries, 2010) Haley, Nicholas James, author; Hoover, Edward A., advisor; Zabel, Mark Douglas, committee member; Ross, Eric D., committee member; Mason, Gary L., committee member
    To view the abstract, please see the full text of the document.
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    Epidemiological investigation of antimicrobial resistance in beef production using metagenomic sequencing
    (Colorado State University. Libraries, 2019) Doster, Enrique, author; Hoover, Edward A., advisor; Morley, Paul S., advisor; Belk, Keith E., committee member; Abdo, Zaid, committee member; Gow, Sheryl P., committee member
    Globally, the emergence of antimicrobial resistance (AMR) resulting in treatment failure is recognized as a growing public health threat. Antimicrobial use practices used in beef production are thought to be a direct driver of increasing antimicrobial resistance in pathogens and the environment, in part due to the higher volumes of antimicrobial drug necessary to treat cattle weighing 10 times more than an average person. This has led policy makers and public health organizations to promote "judicious use" or outright ban of antimicrobial drugs in livestock production. Use of antimicrobials is unavoidable for the treatment of disease and we must therefore learn how we can best adjust our AMD use to reduce selection of AMR pathogens. However, outside of important indicator organisms and pathogens, little is known about how different antimicrobial drug use practices affect communities of microorganisms, or microbiomes, and the AMR gene determinants, or resistome, shared between pathogen and non-pathogens alike. With advances in high-throughput sequencing (HTS), we can perform culture-independent studies and gain a better understanding of how antimicrobial drug use practices in livestock production affect AMR epidemiology. This dissertation consists of five studies that employ HTS to characterize the microbiome and resistome of samples with differing AMD exposure along the beef production line. Projects begin with a look into the short-term effects on the microbiome and resistome of feedlot cattle following treatment with a macrolide drug, tulathromycin, in the manuscript "Investigating Effects of Tulathromycin Metaphylaxis on the Fecal Resistome and Microbiome of Commercial Feedlot Cattle Early in the Feeding Period". Fecal samples collected in this project also were processed with aerobic culture, polymerase chain reaction (PCR), and lateral flow immunoassay for identification of Salmonella enterica and the comparison of these results are presented in "A Cautionary Report for Pathogen Identification Using Shotgun Metagenomics; a Comparison to Aerobic Culture and Polymerase Chain Reaction for Salmonella enterica Identification". Samples collected as part of a longitudinal study in feedlot cattle were analyzed to characterize the associations between AMD use and AMR in two bacterial species. These archived samples are leveraged for a broader understanding of AMR dynamics by adding a community-level perspective to results from aerobic culture. Results in individual cattle are presented in "Antimicrobial Drug Use in Beef Feedlots; Effects on the Microbiome and Resistome Dynamics in Individual Cattle" and results at the pen-level in "Metagenomic Investigation of the Effects of Antimicrobial Drug Use Practices on the Microbiome and Resistome of Beef Feedlot Cattle". Finally, in "Metagenomic Characterization of the Microbiome and Resistome in Retail Ground Beef" we examined the end of the beef production line by comparing the microbiome and resistome of retail ground beef products from either conventional production systems or those labeled as "raised with antibiotics" (RWA). The five studies presented in this dissertation each contribute to the collective understanding of how AMD use in livestock production system can affect the ecology of AMR in microbial communities. These projects are useful first steps in learning to manage AMR in beef production systems; encompassing a targeted look at the use of one type of AMD, characterizing the resistome dynamics in individual cattle and pens over time in a feedlot, a comparison of the resistome in ground beef products, and many other aspects of AMR epidemiology. The final study, describing limits to incorporating HTS for pathogen identification, serves as a cautionary reminder that with new technologies come new challenges and that research must keep pace.
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    In vitro amplification and enhanced trans-species transmission of chronic wasting disease prions
    (Colorado State University. Libraries, 2009) Kurt, Timothy Daniel, author; Hoover, Edward A., advisor; Wilusz, Jeffrey, advisor
    Chronic wasting disease (CWD) is a prion disease of deer, elk and moose that is spreading rapidly in North America. Like all prion diseases, CWD is associated with conversion of a normal protein, PrPC, to a protease-resistant conformer, PrPRES (or PrPCWD). Little is known about the mechanisms of prion conversion or how it could lead to the rapid spread of CWD among cervids in nature. In this dissertation, I demonstrate the in vitro conversion of PrPC to PrPCWD via two protocols: non-denaturing amplification and serial protein misfolding cyclic amplification (sPMCA). Serial PMCA using brain substrate from transgenic mice that express cervid PrPC [Tg(CerPrP)1536 mice] produced PrPCWD amplification of >6.5 x 109-fold after six rounds. Efficient in vitro amplification of PrPCWD is a significant step toward potential ante-mortem detection of PrPCWD in CWD-infected animals. Whether CWD presents a threat to non-cervid species is not known. To predict non-cervid susceptibility to CWD, I used sPMCA to amplify PrP CWD in normal brain substrates from several non-cervid species. I show that brain homogenates from several CWD-susceptible species, such as ferrets and hamsters, support amplification of PrPCWD by sPMCA, whereas brain homogenates from CWD-resistant species, such as laboratory mice and transgenic mice expressing human PrPC, do not. Three common rodent species (including prairie voles and field mice) that share the environment with infected cervids supported PrPCWD amplification, whereas several other species (including prairie dogs, cats and coyotes) did not. Analysis of PrP sequences suggests that ability to support amplification of PrPCWD in trans-species sPMCA correlates with the presence of asparagine at position 170 of the substrate species PrP. I then inoculated CWD from deer into prairie voles (Microtus ochrogaster) and found they are somewhat susceptible to CWD. Inoculation of prairie voles with trans-species sPMCA products resulted in shorter and more consistent incubation periods. Furthermore, immunohistochemical analysis revealed an altered pattern of CWD prion deposition in infected voles compared to infected Tg(CerPrP)1536 mice, suggesting a different CWD strain. These results indicate that sPMCA can be used to increase PrPCWD detection sensitivity, predict susceptibility to CWD, accelerate adaptation in non-cervid species, and create new strains of CWD.
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    Tracking infectious prions in the body fluids of deer infected with chronic wasting disease
    (Colorado State University. Libraries, 2010) Mathiason, Candace Kay, author; Hoover, Edward A., advisor; Avery, Anne, committee member; Bamburg, James R., committee member; Zabel, Mark, committee member
    Chronic wasting disease (CWD) is a prion disease of cervid (elk, moose and deer) with unusually high transmission efficiency. While the nidus of disease was described in a captive herd of cervid in northern Colorado/southeastern Wyoming in the late 60's, it has now been detected in both captive and free-ranging populations in 17 states and 2 Canadian provinces of North America and one Asian country. CWD is unique in being the only transmissible spongiform encephalopathy (TSE) described in a free-ranging population of animals. The etiology of CWD, like all prion diseases, is the conversion of the normal host-encoded cellular prion protein (PrPC) to an aberrantly folded protease resistant isoform (PrPRES/PrPCWD). An intriguing aspect of prion diseases is their ability to be transmitted from one organism to the next. In this dissertation work, we ask-By what means are infectious prions transmitted from one host to the next? In particular to CWD-What do infected cervids share or leave behind that contain sufficient infectious particles to initiate disease in the next cervid? We addressed this question by bioassay of body secretions and excretions- ' secreta '- (saliva, blood, urine and feces) in the native white-tailed deer host and in transgenic mice expressing the normal cervid prion protein (Tg(CerPrP) mice). Cohorts of deer were exposed by oral (PO) ingestion of 'secreta', or intraperitoneal (IP)/intravenous (IV) transfusion of blood components. To replicate a more natural/realistic exposure to CWD in which a deer might travel into a contaminated area and feed for a short period of time, an additional cohort of deer was exposed to fomites (bedding, feed and water buckets) from the suites of CWD-infected deer-without direct contact with infected deer. These variously exposed deer were monitored for a minimum of 19 months post inoculation (mo pi) for CWD infection and disease by immunohistochemical (IHC) and western blot (WB) detection of PrPCWD in serial tonsil biopsies and in multiple tissues after necropsy. Parallel studies were conducted in Tg(CerPrP) mice with the addition of an intracranial (IC) inoculation group for each body fluid. We found that sufficient infectious prions were present in the saliva, whole blood, the B cell- and platelet-enriched fractions of blood, and in fomites from infected deer premises to transmit CWD. Conversely, PrPCWD was not detected in the brain or lymphoid tissues of deer or mice inoculated with urine and feces, cell-free plasma or CD14+ monocytes from CWD-infected donor deer. The results of this work: 1) suggest that the efficient transmission of CWD may be due in part to the sharing of saliva between cervids and its deposition upon surfaces frequented by cervids; 2) establish a hematogenous dissemination of infectious prions in CWD associated with the cellular fraction of blood- in particular B cells and platelets— in CWD-infected deer; 3) extend previous work localizing PrPCWD to the interface of follicular B cells and dendritic cells; 4) provide insights to PrPCWD trafficking and CWD pathogenesis; and 5) establish saliva and blood cells as viable substrates for PrPCWD antemortem detection.