1. Workshop on Bovine Spongiform Encephalopathy (BSE) control measures in EU member states Franco Mutinelli, Anna Granato Istituto Zooprofilattico Sperimentale delle Venezie Legnaro (PD), Italy Kiev (Ukraina), 4-5 April 2011 TSE Diagnostics Development and diagnosis of atypical TSE

2. Mabbott et al. Nature Reviews Microbiology advance online publication; published online 06 February 2006 | doi:10.1038/nrmicro1346

3. Prion Diseases in animals Natural Sheep scrapie CattleBovine Spongiform Encephalopathy (BSE) DeerChronic Wasting Disease (CWD) Elk MinkTransmissible mink encephalopathy (TME) CatFeline spongiform encephalopathy (FSE) Experimental Rodents (mice, rats, hamsters, guinea pigs) Pigs Non-human primates all species with naturally occurring prion disease

4. Etiological hypotheses Transmissible Spongiform Encephalopathies Prion hypothesis: infectivity is caused by a structurally modified form of the PrP protein which promotes conversion of other PrP molecules into the same form, and these accumulate to interfere with the function of nerve cells ”Virino”: an infectious pathogen containing a core of nucleic acid associated with host derived cellular proteins “Filamentous virus”: nucleic acid + encoded protein

5. PrP c 254 aminoacids 2 sites of glicosilation Secondary structure characterized by α-elix Easily soluble Not resistant to proteases Codified by PRNP gene

6. PrP Sc PrP Sc o PrP res Aminoacid sequence and primary structure as for PrP C Secondary structure characterized by β-sheets PrP Sc in contact with PrP C causes its transformation into PrP Sc

7. Proteinasi K ISS 2000

8. Passive surveillance (UK) Real feed ban

9. Diagnosis of BSE On live animals: clinical 14-3-3 protein (SF) Serology (not applicable) in vivo tests (?) On dead animals: histopathology immunohistochemistry western blot electron microscopy inoculation of laboratory rodents RAPID TESTS

10. TSE Diagnosis Rapid Tests Decision 2001/8/CE of 29/12/2000 of the European Commission modifying Decision 2000/764/CE Active surveillance Regulation (EC) n. 315/2008 approved 12 tests for the diagnosis of BSE and 9 for Scrapie SPECIFIC MARKER PRP Sc

11. Reg. (EC) N. 315/2008-Reg. (EU) N. 956/2010 TSE in sheep and goat: 1.Enfer TSE kit versione 2.0 2.Enfer TSE kit versione 3.0 3.Bio-Rad TeSeE test 4.Bio-Ra Sheep /Goat test 5.Prionics-Check LIA Small Ruminants test 6.Beckam Coulter InPro CDI test 7.IDEXX HerdCheck BSE-Scrapie Antigen Test kit 8.POURQUIER’S-LIA Scrapie BSE in cattle: 1.Prionics Check Western test 2.Enfer test e Enfer test TSE Kit versione 2.0, authomated sample preparation 3.Enfer TSE versione 3 4.Bio-Rad TeSeE test 5.Prionics-Check LIA test 6.IDEXX HerdCheck BSE Antigen Test Kit, EIA & IDEXX HerdChek BSE-Scrapie Antigen Test Kit, EIA 7.Prionics Check PrioSTRIP 8.Roboscreen Beta Prion BSE EIA test Kit 9.Roche Applied Science PrionScreen

12. Histopathology Medulla oblongata: one half of the sample fixed into 10% buffered formalin and one half frozen Histopathology and IHC on formalin-fixed, paraffin embedded 5 m thick sections of pons at the cerebellar peduncles and obex For histopathology, sections were stained with hematoxilyn and eosin (HE)

13. Histopathological findings Mild to severe diffuse spongiform changes of the neuropil and neuronal vacuolation Vacuolar changes in the neuropil in the solitary tract nucleus and in the spinal tract nucleus of the trigeminal nerve Vacuolization of neuronal perikarya of vestibular nuclear complex; in reticular formation; in dorsal nucleus of the vagus nerve Astrocytosis Neuronal loss

14. IMMUNOHISTOCHEMISTRY (principle) Antigen in tissue section Primary antibody Secondary antibody Detection system Chromogen

15. Immunohistochemistry Immunohistochemical staining for the detection of PrP: avidin/biotin peroxidase method or EnVision TM (DAKO) and the 3-3' diaminobenzidine (DAB) as chromogen Unmasking treatment: 98% formic acid solution bath for 20 min and hydrated autoclaving at 122°C for 30 min Primary antibodies: polyclonal rabbit antiserum, P7/7 (Dr. Pocchiari), raised against purified PrP 27-30 of hamster brain infected with 263 K scrapie strain; monoclonal antibody, 34C9 (Dr. Oesch) monoclonal antibody, F99/97.6.1 cell line (M152 24, VMRD Inc., Pullman, WA), Astrocytosis: IHC using an anti Glial Fibrillary Acidic Protein (GFAP) polyclonal antibody (Dako, 1:250)

16. Immunohistochemical findings PrP immunolocalization in all examined cerebral areas, showing a common pattern, characterized by fine or coarse, widespread deposition within the neuropil A punctate pattern of PrP immunostaining was constantly observed in the dorsal nucleus of the vagus nerve Another common pattern of PrP immunoreactivity was the "beaded" linear tracts, noticed in all cases Small PrP plaque-like deposits in the neuropil were also present

17. Immunohistochemical findings PrP immunolocalization in all examined cerebral areas, showing a common pattern, characterized by fine or coarse, widespread deposition within the neuropil A punctate pattern of PrP immunostaining was constantly observed in the dorsal nucleus of the vagus nerve Another common pattern of PrP immunoreactivity was the "beaded" linear tracts, noticed in all cases Small PrP plaque-like deposits in the neuropil were also present