2006 Annual Report 4d.Progress report. This report serves to document research conducted under a specific cooperative agreement between ARS and the Italian Reference Centre for Animal TSE (CEA) at the Istituto Zooprofilattico Sperimentale, Turin, Italy. Additional details of research can be found in the report for the parent project 3625-32000-073-00D, Transmission, Differentiation, and Pathobiology of Transmissible Spongiform Encephalopathies (TSEs). The aim of the cooperative research project is (i) to compare the U.S. Bovine Spongiform Encephalopathy (BSE) isolates and the atypical BSE isolates identified in Italy and (ii) to determine whether diagnostic methods routinely used at the USDA are able to identify atypical BSE cases. Within FY06, formalin fixed and frozen brain materials from animals with typical and atypical Italian (BASE) BSE have been sent by CEA to the USDA-ARS-NADC laboratory in Ames, IA. The serial brain material sections of BSE and BASE (consecutively numbered) will be analyzed in Ames using the USDA immunohistochemistry (IHC) protocol. To evaluate its reproducibility in Italian laboratories and to standardize the method, the USDA IHC protocol is being established at the CEA neuropathology laboratory. As soon as the Ventana NexES IHC Staining System is available to the CEA, the same samples (different cut numbers) will be examined by the CEA using the CEA in-house and the USDA IHC protocol. In order to evaluate the USDA Western blot method, about 2 gram of typical Italian BSE and about 2 gram of atypical BASE brain tissue have been sent to the USDA-ARS-NADC laboratory. These samples and U.S. typical and atypical BSE samples have been analyzed in parallel using both the USDA and CEA Western blot methods and three different monoclonal antibodies (6H4, P4, SAF 84). These studies were performed during the visit by a CEA collaborator to the USDA-ARS-NADC. The latter studies revealed that both the Italian and USDA extraction and Western blot methods allowed the identification of the typical and atypical BSE samples tested.