Page Banner

United States Department of Agriculture

Agricultural Research Service

Research Project: DETECTION OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHY AGENTS IN LIVESTOCK, WILDLIFE, AGRICULTURAL PRODUCTS, AND THE ENVIRONMENT
2009 Annual Report


1a.Objectives (from AD-416)
We will develop highly sensitive diagnostic tests to detect transmissible spongiform encephalopathy (TSE) in livestock and wildlife animal species prior to the onset of clinical disease. We will also develop tests to confirm the presence or absence of TSE disease agents in ingredients of animal origin and decontaminated environments.


1b.Approach (from AD-416)
The threat of Bovine Spongiform Encephalopathy(BSE) continues to affect export economics for US meat. Meanwhile scrapie continues to influence sheep profits and herd biosecurity, and CWD is spreading throughout North America. Thus U.S. animal industry stakeholders have identified detection of the TSE infectious agent (prions) as a priority biosecurity research issue essential for prevention of TSE diseases.

We will build on our previous successes using mass spectrometry (MS) for high-sensitivity and specificity in detection of PrPsc as a marker for TSE infectivity in blood using a hamster scrapie model. We will also develop a novel PrP-null mouse strain and related myeloma cell culture system for production of monoclonal antibodies (MAb), which may be specific for PrPsc. We will then choose MS or MAb and validate our novel diagnostic for preclinical diagnosis of scrapie in sheep blood.

Whereas MS and MAb methods rely on dissolved samples, contamination of agricultural products and environmental surfaces is associated with solid samples. So we will produce a cell culture based assay for TSE infectivity that is surface-adsorbed. After using the relatively convenient hamster model for early development, we will validate this technology for detection of scrapie in sheep brain on meat-and-bone meal and stainless steel. Replacing 5325-32000-007-00D (3/19/2008).


3.Progress Report
In FY09 we continued to experiment with new combinations of detergents, temperature, and centrifugation conditions, to refine our method for enzyme-free isolation of prions from animal tissues. An enzyme-free method is required, because prions in blood are not resistant to the proteinase K enzyme cocktail that is typically used to isolate prions from brain. We have now demonstrated that our purified prion material remains infectious.

Getting our method for hamster brain to work on sheep brain, this year, required a surprisingly large amount of work. We found that sheep brains contain significantly more fibrous tissue. This feature apparently evolved to meet the needs of a species remarkable for head-butting behavior.

We are studying plasma and buffy coat as blood fractions to develop for prion diagnosis. Each of these fractions contains about half the infectivity present in blood. Buffy coat is more concentrated, while plasma may prove more robust for handling and storage.

We have completed our evaluation of cell culture for detection of prions in commodities and environmental samples. This work involved collaboration with the world’s leading prion cell biologists. We found this method gave very poor agreement between experiments due to instability of the cell lines.

We have continued to pursue a Contingency on covalent modification of PrP to generate disease-specific molecular differences that subsequently can be recognized by antibodies. This work attracted interest and collaboration with the Canadian company Amorfix.


4.Accomplishments
1. New targets for prion diagnosis and treatment. Molecular interactions with unknown partners inside the cell are involved in the conversion of PrP-c (the non-infective particle) in uninfected animals to PrP-Sc (the infective particle) in animals infected with Transmissible Spongiform Encephalopathy diseases (TSE) and need to be identified. ARS scientists in Albany, CA identified a short list of new proteins that bind to the PrP protein. These bind in a disease-specific manner. Identification of disease-specific interactions should help in understanding the biological processes involved in the disease.

2. An agent that delays "Mad Cow" disease in infected animals. Transmissible Spongiform Encephalopathy (TSE) disease can have a significant economical impact. There is currently no treatment for any of the closely related TSE. ARS scientists at Albany, CA found that a synthetic RADA hydrogel peptide caused a significant delay in the time required for prion-infected animals to come down with disease. This may lead to new preventive treatments or cures for TSE diseases such as scrapie.

3. Preventing “mad cow” disease while making biodiesel. Although it was formerly valuable as a highly nutritious supplement for animal feed and pet food, the agricultural commodity Meat-and-Bone Meal was responsible for initiating and perpetuating the bovine spongiform encephalopathy (BSE) epidemic of the 1990s, because even high-temperature rendering failed to disinfect prions. Using scrapie as a model ARS scientists in Albany, CA found a cheap room-temperature chemical reaction that destroys 99.9999% of the infective dose, while producing biodiesel as a co-product. This technology promises to help reduce the huge burden of disposal created by the Food and Drug Administration’s new "enhanced feed ban".


6.Technology Transfer

Number of New Patent Applications Filed2

Review Publications
Harman, J.L., Silva, C.J. 2009. Bovine Spongiform Encephalopathy. Journal of the American Veterinary Medical Association. (2009)234(1):59-72

Hnasko, R.M., Bruederle, C.E. 2009. Inoculation of Scrapie with the Self-Assembling RADA-Peptide Disrupts Prion Accumulation and Extends Hamster Survival. PLoS ONE. 4(2): http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004440

Silva, C.J. 2009.Prion Diseases:Sequelae and Long Term Consequences of Infectious Diseases. District of Columbia:ASM Press. 442p.

Last Modified: 8/31/2014
Footer Content Back to Top of Page