Does the Presence of Scrapie Affect the Ability of Current Statutory Discriminatory Tests To Detect the Presence of Bovine Spongiform Encephalopathy?

Abstract

e Current European Commission (EC) surveillance regulations require discriminatory testing of all transmissible spongiform encephalopathy (TSE)-positive small ruminant (SR) samples in order to classify them as bovine spongiform encephalopathy (BSE) or non-BSE. This requires a range of tests, including characterization by bioassay in mouse models. Since 2005, naturally occurring BSE has been identified in two goats. It has also been demonstrated that more than one distinct TSE strain can coinfect a single animal in natural field situations. This study assesses the ability of the statutory methods as listed in the regulation to identify BSE in a blinded series of brain samples, in which ovine BSE and distinct isolates of scrapie are mixed at various ratios ranging from 99% to 1%. Additionally, these current statutory tests were compared with a new in vitro discriminatory method, which uses serial protein misfolding cyclic amplification (sPMCA). Western blotting consistently detected 50% BSE within a mixture, but at higher dilutions it had variable success. The enzyme-linked immunosorbent assay (ELISA) method consistently detected BSE only when it was present as 99% of the mixture, with variable success at higher dilutions. Bioassay and sPMCA reported BSE in all samples where it was present, down to 1%. sPMCA also consistently detected the presence of BSE in mixtures at 0.1%. While bioassay is the only validated method that allows comprehensive phenotypic characterization of an unknown TSE isolate, the sPMCA assay appears to offer a fast and cost-effective alternative for the screening of unknown isolates when the purpose of the investigation was solely to determine the presence or absence of BSE.

Four types of scrapie in goats differentiated from each other and bovine spongiform encephalopathy by biochemical methods

“…CH1641 scrapie exhibits similarities with BSE since in both types distinct N - terminal PrP epitopes are protease sensitive [31]. In addition, mixtures of TSE forms could be present in a single animal, which hamper recognition of low BSE levels [32].…”

BSE can propagate in sheep co-infected or pre-infected with scrapie

“…During the study, a sPMCA method became available that can detect very low amounts of BSE PrP Sc in a background of an excess of scrapie 41 , 42 . The assay does not detect scrapie PrP Sc .…”

“…We used natural scrapie, SSBP/1 experimental scrapie and cattle BSE; the latter can be distinguished from the scrapie strains by incubation period, IHC and western blotting 39 , 40 . Furthermore, a specific in vitro replication assay (sPMCA) that can detect BSE PrP Sc even in the presence of a large excess of scrapie PrP Sc was also applied 41 , 42 . Sheep of different PRNP genotypes underwent co-challenge with BSE and scrapie (SSBP/1) or pre-infection with scrapie (either SSBP/1 or natural scrapie 43 ) followed by BSE, and incubation periods were indicative of either scrapie or BSE disease.…”

Methods for Differentiating Prion Types in Food-Producing Animals

“…These observations have been developed into a sensitive assay using a combination of the VRQ/VRQ and AHQ/AHQ brain homogenate substrates [ 113 ]. These two substrates encourage the amplification of BSE over scrapie and when used in sequential rounds of PMCA have demonstrated amplification specificity to BSE, even when BSE is present with a vast excess of different scrapie isolates [ 113 , 114 ]. The assay was shown to be far superior to ELISA and western blot tests for detecting ovine BSE in the presence of a large excess of scrapie agent [ 114 ].…”

“…These two substrates encourage the amplification of BSE over scrapie and when used in sequential rounds of PMCA have demonstrated amplification specificity to BSE, even when BSE is present with a vast excess of different scrapie isolates [ 113 , 114 ]. The assay was shown to be far superior to ELISA and western blot tests for detecting ovine BSE in the presence of a large excess of scrapie agent [ 114 ]. Such an assay could be applied to monitor the status of animals for mixed infection where the levels of BSE may be very low, and whose detection may be obscured by the presence of scrapie.…”