- Bartz, Jason
- Creighton University
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- The goal of this proposal is to develop a spectrofluorometric assay using the amyloid binding dye thioflavine T (ThT) to distinguish different conformations of PrPSc in a strain-specific manner. This will provide a rapid method for identification of transmissible spongiform encephalopathy strains in livestock.
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- Identification of transmissible spongiform encephalopathy (TSE) strains has taken on increased importance with the emergence of bovine spongiform encephalopathy in the United Kingdom and its subsequent transmission to other species including humans. Chronic wasting disease is an emerging TSE of deer and elk in the United States that has an unknown host range and may pose a risk to sheep and cattle since they share pasturelands. Currently, no method exists to monitor for the prevalent or emerging TSE strains of livestock and wild animals. The objective of this proposal is to develop a rapid method for surveillance of TSE strains in livestock. An assay will be developed to identify TSE strains based on the ability of a fluorescent dye to interact with a TSE-specific protein in a strain-specific manner. First, reaction conditions will be optimized. Second, the number of distinct strains that can be identified in a single host species and in how many host species can the same TSE strain can be identified. Finally, isolates of naturally occurring TSEs from the U.S. will be examined in order to investigate TSE strain diversity in livestock. The ability to monitor endemic and emerging TSE strains in domestic animals can facilitate control measures to reduce the spread of TSEs and to assess the risk that emerging strains pose to animal and human health.
thioflavine T (ThT) based fluorometric assay will be developed to identify TSE strains based on the conformation of the disease-specific amyloid isoform of the prion protein, PrPSc. ThT fluorometry is a conformational assay used to detect amyloid proteins that is based on the binding of ThT to amyloid B-sheet structure. ThT is a benzothiazole dye that undergoes a spectral shift in excitation and emission maxima in the presence of amyloid protein but not in the presence of precursor amyloid, amorphous aggregates, and non-amyloid proteins with high B-sheet content. Since the molecular basis of TSE strain diversity is throught to be based on PrPSc conformation ThT fluorometry will be used to distinguish different conformations of PrPSc. First, ThT fluorometry will be optimized for identification of TME strains based on ThT binding to the PrPSc conformations (Aim 1). In Aim 2, ThT fluorometry will be used to determine i) the number of distinct strains (i.e. PrPSc conformations) that can be identified in a single host species, and ii) in how many distinct host species (i.e. different prion protein genotypes) can the same TME strain be identified. In aim 3 isolates of scrapie and CWD from U.S. outbreaks will be examined by ThT fluorometry in order to investigate TSE strain diversity in livestock and cervids.
PROGRESS: 2000/10 TO 2002/09
As a postdoctoral fellow, I was awarded a fellowship from the United States Department of Agriculture. This support has led to the demonstration that following peripheral nerve inoculation, prions were transported within distinct motor tracts and transynaptically spread according to known neuronal circuitry (Bartz et al., 2002). Additional studies on oral prion pathogenesis have identified an alternative, highly efficient, route of neuroinvasion that directly travels along cranial nerve XII to the brainstem (Bartz et al., 2003). These studies suggest that wounds on the tongue can increase the risk of prion transmission, especially in natural settings where low doses of prions would most likely occur. Further experiments are being performed to investigate the mechanism responsible for the high efficiency of prion infection by this route. In a related series of experiments, the prion agent was identified in the tongues of hamsters infected with 6 different prion strains. This data suggests that prion infectivity in tongue tissue may be a common event in prion diseases. In light of this data, it is concerning that tongue is still included for human consumption in Great Britain.
IMPACT: 2000/10 TO 2002/09
The identification of an alternative, highly efficient, route of prion neuroinvasion has direct implications on the assessment of risk factors associated with prion infection in animals and humans. In addition, a reexamination of allowing cattle tongue for human consumption in Great Britain is warranted based on the identification of prion infectivity in the tongue of prion-infected rodents.
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- Bacterial Pathogens
- Sanitation and Quality Standards
- Meat, Poultry, Game