1. Aya Murakami

2. Background Transmissible Spongiform Encephalopathies and Prion Other for TSEs CWD Distribution in the US Symptoms and Diagnosis Biomarkers Research Goals Results Conclusion

3. Transmissible spongiform encephalopathy (TSE) is a group of progressive diseases that affect the nervous systems and brains of many mammals, including human. Caused by accumulation of a misfolded isoform of the protein called prion protein (PrP) Prion is an infectious agent which forms aggregates in nervous tissues.

4. Two protein conformations exist… PrP sen = native conformation of PrP present in all mammals and is sensitive to digestion with protease. It is a membrane associated protein but its function is unknown. PrP res = misfolded conformation and is resistant to digestion with protease

5. Mad Cow Disease (Bovine Spongiform Encephalophathy; BSE) Scrapie Chronic Wasting Disease (CWD) Creutzfeldt- Jakob Disease (CJD) Cows and other mammals Sheep and goats Deer and elk humans

7. Symptoms Weight loss Behavioral changes: Decreased interactions with other animals Listlessness Lowering of the head Blank facial expression Repetitive walking in set patterns A smell like meat starting to rot

8. Diagnosis Post-mortem: detection of PrP res and vacuolization in nervous tissues. Ante-mortem: RAMALT (recto-anal mucosal associated lymphoid tissue) to detect PrP res.

9. Biomarkers= indicators of diseases/ conditions Pregnancy Albumin in urine to measure the kidney functions ALT/AST level to evaluate the liver functions many other… Easy, fast, non-invasive testing: use of urine/ feces/ blood

10. And others… The Mission: To detect these biomarkers in feces using Western Blotting Why feces? -Easy to collect in the field. -Non- invasive For method development, we used feces samples from captive white tail deer that were inoculated with chronic wasting disease.

11. Western blot is a common technique to detect a protein of interest in a sample using antibodies specific to the protein. Separate proteins on SDS-PAGE ProteinTransfer proteins to nitrocellulose membrane Membrane is exposed to specific antibodies Protein bands detected by specific antibodies

12. Banshee 2Banshee 1Dundee 2Dundee 1 Kool aid 2Kool Aid 1 Red 2Red 1 Niblet 2Niblet 1 LokiIndy SwiftMarker 250 150 100 75 50 37 20 15 kD This is very DIRTY!! We would like to see nice clear bands… Our Goals: To make this better looking… By altering extraction buffer dilution of the primary and secondary antibodies Extraction time/ methods

13. Alteration of primary antibody: decided 1:300 dilution was the best Alteration of secondary antibody: decided 1:10,000 was the best This is a blot with 1:300 of 1°antbody and 1: 10,000 of 2°antibody There is not much improvement. Banshee 2Banshee 1Dundee 2Dundee 1 Kool aid 2Kool Aid 1 Red 2Red 1 Niblet 2Niblet 1 LokiIndy SwiftMarker 250 150 100 75 50 37 20 15 kD Banshee 2Banshee 1Dundee 2Dundee 1 Kool aid 2Kool Aid 1 Red 2Red 1 Niblet 2Niblet 1 LokiIndy SwiftMarker kD 250 150 100 75 50 37 20 15

14. Indy 1 hr We previously incubated the samples for 15 hrs to extract the mucus layers. In this experiment, 1, 3 and 5 hr extraction was performed. 250 150 100 75 50 37 20 15 10 Dundee5 hrDundee 3hrDundee 1hr Red 5 hr Red 3hr Red 1 hr Red (cont’l)Indy 5hrIndy 3hr Swift 5hrSwift 3hrSwift 1hrSwift (cont’l) Marker Banshee 2Banshee 1Dundee 2Dundee 1 Kool aid 2Kool Aid 1 Red 2Red 1 Niblet 2Niblet 1 LokiIndy SwiftMarker 250 150 100 75 50 37 20 15 kD 1 hr extraction seemed to work the best.

15. We previously extracted the feces using tumbler shaker (360°rotation). In this experiment, we used a rotary shaker instead. 904520904520904520904520 Marker kD 250 150 100 75 50 37 20 15 10 CWD +CWD - Kool Aid Red LokiIndy 1 hr extraction on the rotary shaker Kool Aid 2Kool Aid 1Niblet 2Niblet 1Red 2 Red 1Banshee 2Banshee 1 Dundee 2Dundee 1 Indy Swift LokiMarker kD 250 150 100 75 50 37 20 15 10 1 hr extraction on the tumbler shaker

16. Tried our methods on feces from deer of unknown CWD status. Blot was a little ugly. Decided to alter the buffer to have less salt and detergent. 1A 1B 2A 2B 3A 3B 4A 4B 5A 5B 6A 6B CWD+CWD –Marker 250 150 100 75 50 37 20 15 10 kD

17. Alter the salt and detergent concentration. NaCl: 130 mM → 10 mM Detergent: 0.5% → 0.05% Addition of phenyl methyl sulfonyl fluoride (protease inhibitor) 1A 1B 2A 2B 3A 3B 4A 4B 5A 5B 6A 6B CWD+CWD –Marker 250 150 100 75 50 37 20 15 10 kD NaCl: 130 mM Detergent: 0.5% 1A 1B 2A 2B 3A 3B 4A 4B 5A 5B 6A 6B CWD+CWD –Marker kD 250 150 100 75 50 37 20 15 10 NaCl: 10 mM Detergent: 0.05%

18. Improved the method for protein extraction from deer feces for biomarker detection. Furthermore, these methods seemed to work for deer feces with unknown CWD status. This method has potential to serve as a non-invasive screen for CWD in the field.

19. I would like to thank all the faculty members in Dr. Lewis’ Lab, especially Ted John for walking through the experiments with me and being a great mentor.