RT-QuIC detection of pathological prion protein in subclinical goats following experimental oral transmission of L-type BSE

Objective The spread of bovine spongiform encephalopathy (BSE) agent to small ruminants is still a major issue in the surveillance of transmissible spongiform encephalopathies (TSEs). L-type bovine spongiform encephalopathy (L-BSE) is an atypical form of BSE with an unknown zoonotic potential that is transmissible to cattle and small ruminants. Our current knowledge of bovine atypical prion strains in sheep and goat relies only on experimental transmission studies by intracranial inoculation. To assess oral susceptibility of goats to L-BSE, we orally inoculated five goats with cattle L-BSE brain homogenates and investigated pathogenic prion protein (PrPsc) distribution by an ultrasensitive in vitro conversion assay known as Real-Time Quaking Induced Conversion (RT-QuIC). Results Despite a prolonged observation period of 80 months, all these animals and the uninfected controls did not develop clinical signs referable to TSEs and tested negative by standard diagnostics. Otherwise, RT-QuIC analysis showed seeding activity in five out of five examined brain samples. PrPsc accumulation was also detected in spinal cord and lymphoreticular system. These results indicate that caprine species are susceptible to L-BSE by oral transmission and that ultrasensitive prion tests deserve consideration to improve the potential of current surveillance systems against otherwise undetectable forms of animal prion infections.

The Microscopic Detection of Animal Proteins in Animal Feed Regarding Bovine Spongiform Encephalopathy

Due to the actuality of spongiform encephalopathies and their proven spreading by means of animal feed containing meat and bone meal, the description and measurement of osteocytic lacunae contributes to more easily distinguish bone fragments in meat and bone meal. Transmissible spongiform encephalopathies (TSEs) have attracted a lot of attention, especially after 1986, when the first case of BSE (bovine spongiform encephalopathy) was detected. Since the outbreak of spongiform encephalopathy (BSE), the use of animal protein including bone meal as an ingredient in animal feed has been controlled by several regulations including Regulation (EC) 999/2001, Regulation (EC) 1774/2002, and Regulation (EC) 1234/2003. The classical microscopic method is the only official method for detecting animal protein in animal feed in the European Union (Commission Regulation (EC) 152/2009). By applying the microscopic method to the animal feed samples, we performed detection in order to determine the presence of animal proteins that originate from mammals and fish. The microscopic analysis of all 421 samples, of which 115 were raw materials for the production of animal feed, 230 were concentrates for ruminant nutrition and 76 were concentrates for non-ruminant nutrition (32 concentrates for laying hens and 44 concentrates for pigs), did not provide positive results, that is, no remains of animal tissues of mammalian origin were found in any specimen. Whereas in 10 out of 32 (31.25%) concentrates intended for non-ruminant nutrition (laying hens), pieces of fish tissue were found. In these samples, we usually detected the presence of fish bones, gills and scales.

The ultrastructure of infectious L-type bovine spongiform encephalopathy prions constrains molecular models

Bovine spongiform encephalopathy (BSE) is a prion disease of cattle that is caused by the misfolding of the cellular prion protein (PrPC) into an infectious conformation (PrPSc). PrPC is a predominantly α-helical membrane protein that misfolds into a β-sheet rich, infectious state, which has a high propensity to self-assemble into amyloid fibrils. Three strains of BSE prions can cause prion disease in cattle, including classical BSE (C-type) and two atypical strains, named L-type and H-type BSE. To date, there is no detailed information available about the structure of any of the infectious BSE prion strains. In this study, we purified L-type BSE prions from transgenic mouse brains and investigated their biochemical and ultrastructural characteristics using electron microscopy, image processing, and immunogold labeling techniques. By using phosphotungstate anions (PTA) to precipitate PrPSc combined with sucrose gradient centrifugation, a high yield of proteinase K-resistant BSE amyloid fibrils was obtained. A morphological examination using electron microscopy, two-dimensional class averages, and three-dimensional reconstructions revealed two structural classes of L-type BSE amyloid fibrils; fibrils that consisted of two protofilaments with a central gap and an average width of 22.5 nm and one-protofilament fibrils that were 10.6 nm wide. The one-protofilament fibrils were found to be more abundant compared to the thicker two-protofilament fibrils. Both fibrillar assemblies were successfully decorated with monoclonal antibodies against N- and C-terminal epitopes of PrP using immunogold-labeling techniques, confirming the presence of polypeptides that span residues 100–110 to 227–237. The fact that the one-protofilament fibrils contain both N- and C-terminal PrP epitopes constrains molecular models for the structure of the infectious conformer in favour of a compact four-rung β-solenoid fold.