Location: Produce Safety and Microbiology Research
2021 Annual Report
Objectives
Transmissible spongiform encephalopathies (TSEs) are animal diseases caused by infectious prion proteins that result in a slow progressive neurodegeneration that is fatal. The observation that prion diseases can be transmitted between animals, including humans, by consumption of contaminated food necessitates strategies to mitigate their occurrence in the food supply. In support of existing public health and food safety measures the USDA conducts TSE surveillance of suspect animals to monitor the incidence of the disease in the livestock population. TSE diagnostic surveillance is dependent on the detection of infectious prions in post mortem brain samples by immunoassay. The limitations of current prion immunoassays necessitate the development of improved prion detection methods that can reliably monitor the: 1) effectiveness of the ruminant feed ban, 2) spontaneous occurrence of disease, and 3) zoonotic transmission of TSE from endemic reservoirs of disease.
The objective of this research is to develop immunodiagnostic methodologies that improve the effectiveness of TSE surveillance of livestock.
Objective 1: Define methodologies for tissue specific prion sample enrichment to increase immunoassay sensitivity.
Subobjective 1.1: Define methods to enrich prions from fresh or frozen tissues.
Subobjective 1.2: Define methods to enhance prion detection from aldehyde preserved tissues.
Subobjective 1.3: Define methods to enrich prions from decomposed tissues.
Objective 2: Generate and validate improved prion monoclonal antibodies to achieve increased selectivity and sensitivity.
Subobjective 2.1: Inoculate Prnp(0/0) Balbc/J mice with purified infectious prions and perform hybridoma fusions to generate conformation specific anti-prion monoclonal antibodies.
Subobjective 2.2: Characterize the biochemical properties and validate binding specificity of anti-prion monoclonal antibodies.
Objective 3: Develop and deploy applied immunoassays for prion detection.
Subobjective 3.1: Develop prion immunoassays and evaluate detection sensitivity in agriculturally relevant models.
The project will generate transferable technologies useful in the diagnosis of TSEs and the detection of low-level infectious prions in livestock tissues. These technologies will facilitate ante mortem TSE detection tests that will enhance our understanding of TSE disease prevalence in captive and wild animal populations. An effective ante mortem test for prion disease would also be valuable in the diagnosis of the closely related human Creutzfeldt-Jakob disease (CJD) and aid in the discovery of effective therapeutic interventions.
Approach
To overcome the obstacle of detecting low-level prions as a result of slow prion propagation following initial infection and allow sampling of non-neuronal tissues for evaluation we will define methodologies for tissue specific prion sample enrichment. These enrichment methods will include the biochemical isolation of prions with lipid rafts from fresh or frozen tissue, the use of chemically mediated antigen retrieval from aldehyde fixed tissue, proteolytic degradation of interfering proteins from decomposing tissues, high molecular weight dialysis to retain large aggregate prion amyloid, and chemical precipitation to concentrate prion enriched samples. The application of these methods will result in an increased yield of prions from target tissues and improve the reliability of prion detection measures.
The properties of prion antibodies dictate the sensitivity and selectivity of prion immunoassays used in the determination of disease status. To generate improved prion monoclonal antibodies (mAbs) we will use highly purified prion immunogens, genetically engineered prion-knockout mice, hybridoma technology, and stringent screening methods for the identification of high-affinity anti-prion mAbs. Identified mAbs will be evaluated for prion binding that includes: epitope mapping, affinity measurements, species and strain specificity, and immunoassay application. Rigorous selection criteria will be used to identify high-affinity conformation- dependent anti-prion mAbs for development of enhanced prion immunoassays.
Effective and reliable TSE surveillance depends on the sensitive detection of infectious prions by immunoassay. Applied prion tissue enrichment methodologies along with improved anti-prion monoclonal antibodies will be used to develop and optimize immunoassays for prion detection. Construction and deployment of various immunoassay platforms and antibody conjugated reporters (enzymatic, colorimetric, and fluorescent) will address end user needs for sensitive tissue specific prion detection that include: enzyme-linked immunosorbant assay (ELISA), field deployable lateral flow immunoassay (LFIA), Western blotting (WB), and immunohistochemistry.
Progress Report
This report is for a bridging project that began on March 1, 2018, and continues research from project 2030-32000-009-00D. Scientific resources were also put toward addressing Objective 5, "Development of immuno-, bacteriophage-, and mass spectrometry-based methods for rapid detection of foodborne pathogens," in the 2030-42000-050-0D project. Please see that project's report for additional information.
Research continued on distinguishing among prions by their conformation. Prion strains differ by conformation from one another and from the normal cellular prion protein. Researchers at Albany, California, collaborated with Spanish scientists to develop a working model of the prion’s structure and demonstrated that infectious recombinant prions and wild-type prions share common structural features. The researchers showed that the covalent modification of a prion did not substantially alter its structure. The researchers used small molecule reagents to trap conformational differences in five well-characterized hamster-adapted prion strains. Mass spectrometry and Western blot-based analysis revealed the trapped strain-dependent differences. The researchers used mass spectrometry to quantify the composition of prions. The prions were isolated from heterozygous sheep naturally infected with classical scrapie and strains of chronic wasting disease (CWD) from experimentally infected heterozygous white-tailed deer. The researchers analyzed samples of heterozygous sheep naturally infected with atypical scrapie. They showed that atypical prions replicate at similar rates, regardless of the polymorphism, suggesting that there is unlikely to be a protective genotype. The tissue contained similar amounts of cellular prion protein (PrPC) and equal amounts of both polymorphisms, indicating that atypical scrapie is not a consequence of PrPC overexpression. This work defines an approach to determining the structural differences in prion conformations. Using this method, zoonotic prions can be identified before they are detected in a patient as part of a Creutzfeldt-Jakob disease surveillance program.
Planning for a new Animal Health program project was initiated in fiscal year 2021. New targets necessitated scientific literature review, planning, and outreach activities. New collaborations have been established. Research has been initiated on the development of rapid diagnostic technologies based on aptamers and antibodies for the early detection of emerging pathogens. These pathogens that affect animal health include influenza virus A – H1N1 in swine, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), and Senecavirus A. Viral targets and their variants have been identified and confirmed after discussing them with stakeholders (Animal and Plant Health Inspection Service, veterinarians, and virologists). Similarly, target proteins of individual virus groups have been identified. For rapid immunodetection assays using portable platforms, research activity has focused on the generation of novel antibody biologics. For aptamer-based lateral flow assay, primer sequences that are essential for generating highly specific aptamers have been designed and used to generate preliminary data. Preliminary results have shown that the aptamers that could target Senecavirus A protein (VP1) are 100 – 125 bp in size. Future efforts will be directed to construct diagnostic tools for pen-side testing.
Accomplishments
1. Covalent modification of prions does not alter their structure. Prion diseases afflict farmed and wild animals. A prion’s pathogenicity is encrypted enciphered in its shape. Reacting prions with chemical reagents will reveal differences in their shapes. ARS researchers in Albany, California, used chemical reagents to determine a prion’s shape. The reagents chemically changed the prion, and the prion remained infectious. This means that the information derived from this analysis is specifically related to a prion’s shape. Researchers can now use chemical reagents to define the shape of a prion, which is essential to characterize what makes a prion infectious. This information can be used to develop new diagnostics, potential vaccines, and refine existing prion structural models.
2. Detecting prions over a time course. Prions cause serious animal diseases and transmit diseases by their shape, so they are difficult to detect. ARS researchers in Albany, California, in collaboration with Spanish scientists, developed a mass spectrometry-based method of detecting prions. They measured the amounts of prions in the brains of experimental animals during the disease. They showed that prions were detectable at the same time as they would be detectable by bioassay. These results indicate that this mass spectrometry-based approach can detect prions early in the disease. It is a valuable means of detecting prions in animals.
3. Biosensor platform facilitates rapid pen-side detection. Emerging disease-causing pathogens negatively impact animal health, global trade, and food security. Rapid pen-side testing facilitates the early detection of disease-causing agents and promotes effective mitigation strategies. ARS researchers at Albany, California, have engineered a disposable biosensor for use with immunoassay test strips to simplify pen-side testing. This technology provides a valuable new tool for veterinarians and inspectors to protect livestock and improve animal health. A patent application for this invention was filed in 2021 (Serial Number 17/154,539).
Review Publications
Silva, C.J., Onisko, B.C., Dynin, I.A., Erickson-Beltran, M.L., Requena, J.R. 2021. Time of detection of prions in the brain by nanoscale liquid chromatography coupled to tandem mass spectrometry is comparable to animal bioassay. Journal of Agricultural and Food Chemistry. 69(7):2279-2286. https://doi.org/10.1021/acs.jafc.0c06241.
Silva, C.J., Erickson-Beltran, M.L., Dynin, I.C. 2020. Quantifying the role of lysine in prion replication by Nano-LC mass spectrometry and bioassay. Frontiers in Bioengineering and Biotechnology. 8. Article 562953. https://doi.org/10.3389/fbioe.2020.562953.
Busche, M., Scarpin, R.M., Hnasko, R.M., Brunkard, J.O. 2021. TOR coordinates nucleotide availability with ribosome biogenesis in plants. The Plant Cell. 33(5):1615-1632. https://doi.org/10.1093/plcell/koab043.
Manhas, P.K., Quintela, I.A., Wu, V.C. 2021. Enhanced detection of major pathogens and toxins in poultry and livestock with zoonotic risks using nanomaterials-based diagnostics. Frontiers in Veterinary Science. 8. Article 673718. https://doi.org/10.3389/fvets.2021.673718.
