Infection of cell lines with experimental and natural ovine scrapie agents.

Mouse bioassay remains the gold standard for determining proof of infectivity, strain type, and infectious titer estimation in prion disease research. The development of an approach using ex vivo cell-based assays remains an attractive alternative, both in order to reduce the use of mice and to hasten results. The main limitation of a cell-based approach is the scarcity of cell lines permissive to infection with natural transmissible spongiform encephalopathy strains. This study combines two advances in this area, namely, the standard scrapie cell assay (SSCA) and the Rov9 and MovS6 cell lines, which both express the ovine PrP VRQ allele, to assess to what extent natural and experimental ovine scrapie can be detected ex vivo. Despite the Rov9 and MovS6 cell lines being of different biological origin, they were both permissive and resistant to infection with the same isolates of natural sheep scrapie as detected by SSCA. Rov9 subclones that are 20 times more sensitive than Rov9 to SSBP/1-like scrapie infection were isolated, but all the subclones maintained their resistance to isolates that failed to transmit to the parental line. The most sensitive subclone of the Rov9 cell line was used to estimate the infectious titer of a scrapie brain pool (RBP1) and proved to be more sensitive than the mouse bioassay using wild-type mice. Increasing the sensitivity of the Rov9 cell line to SSBP/1 infection did not correlate with broadening susceptibility, as the specificity of permissiveness and resistance to other scrapie isolates was maintained.

A retrospective immunohistochemical study reveals atypical scrapie has existed in the United Kingdom since at least 1987.

Atypical scrapie is a relatively recent discovery, and it was unknown whether it was a new phenomenon or whether it had existed undetected in the United Kingdom national flock. Before 1998, the routine statutory diagnosis of transmissible spongiform encephalopathy (TSE) in sheep relied on the presence of TSE vacuolation in the brainstem. This method would not have been effective for the detection of atypical scrapie. Currently, immunohistochemistry (IHC) and Western blot are commonly used for the differential diagnosis of classical and atypical scrapie. The IHC pattern of PrPd deposition in atypical scrapie is very different from that in classical scrapie using the same antibody. It is thus possible that because of a lack of suitable diagnostic techniques and awareness of this form of the disease, historic cases of atypical scrapie remain undiagnosed. Immunohistochemistry was performed on selected formalin-fixed, paraffin-embedded (FFPE) blocks of ovine brain from the Veterinary Laboratories Agency archives that were submitted for various reasons, including suspect neurological disorders, between 1980 and 1989. It was found that PrPd deposits in a single case were consistent with atypical scrapie. A method was developed to obtain a PrP genotype from FFPE tissues and was applied to material from this single case, which was shown to be AHQ/AHQ. This animal was a scrapie suspect from 1987, but diagnosis was not confirmed by the available techniques at that time.

Real-time PCR detection and identification of prohibited mammalian and avian material in animal feeds.

A method for the detection and identification of "prohibited" mammalian or avian material in animal feed was developed and assessed through the analysis of DNA. A generic real-time PCR assay was designed to detect the presence of mammalian and avian mitochondrial DNA 16S rRNA genes in animal feed samples. Samples positive with this screening method were further investigated using identification assays to detect the 16S rRNA gene from bovine, ovine, porcine, and avian species and to determine whether the DNA originated from species whose material is prohibited from inclusion in farmed animal feed. An internal positive control was coamplified in the 16S real-time PCR assays to monitor PCR amplification efficiency and avoid potential false-negative results. Using vegetable-based feed standards spiked with meat and bone meal generated with a commercial rendering process, 0.1% meat and bone meal could be detected using the general and species-specific 16S assays. The species-specific assays had 100% specificity for the homologous target species. The 16S real-time PCR assays were evaluated alongside existing tests based on protein evaluation or microscopic examination for a wide range of commercial animal feed samples. In total, 111 (0.76%) of 14,678 samples examined contained prohibited material based on the results from at least one of these tests. However, most positive results did not represent noncompliance because they were associated with samples of pet food, which can legitimately contain material prohibited for use in food for farmed animals. The species-specific 16S assays confirmed the presence of prohibited material in 75% of the 111 samples, whereas the existing protein and microscope tests confirmed the presence of this material in 25 and 54% of the samples, respectively.

Ovine PRNP untranslated region and promoter haplotype diversity.

The diversity and possible contribution of non-coding regions of the prion protein (PrP) gene (PRNP) to transmissible spongiform encephalopathy susceptibility and PrP regulation are not fully known. This study defined ten ovine PRNP promoters and five untranslated region (UTR) haplotypes found in atypical and classical scrapie cases and healthy control sheep. A greater diversity of promoter and UTR haplotypes was observed in conjunction with the ARQ PrP allele (seven promoter and four UTR haplotypes), while it was observed that the other alleles were linked with a limited number of haplotypes, such as ARR, found to be linked to only two promoter and one UTR haplotypes. In silico analysis identified potential transcription factor binding sites that differed in the promoter haplotype variants. Furthermore, a 5' UTR internal ribosome entry site motif was identified in exon 2 and highlights a possible role for this exon in regulating PrP expression at the translational level.

Clinical findings in two cases of atypical scrapie in sheep: a case report.

BACKGROUND: Atypical scrapie is a recently recognised form of transmissible spongiform encephalopathy of sheep that differs from classical scrapie in its neuropathological and biochemical features. Most cases are detected in apparently healthy sheep and information on the clinical presentation is limited. CASE PRESENTATION: This report describes the clinical findings in two sheep notified as scrapie suspects and confirmed as atypical scrapie cases by immunohistochemistry and Western immunoblotting. Although both sheep displayed signs suggestive of a cerebellar dysfunction there was considerable variation in the individual clinical signs, which were similar to classical scrapie. CONCLUSION: Any sheep presenting with neurological gait deficits should be assessed more closely for other behavioural, neurological and physical signs associated with scrapie and their presence should lead to the suspicion of scrapie.