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.

Caprine prion gene polymorphisms are associated with decreased incidence of classical scrapie in goat herds in the United Kingdom.

The application of genetic breeding programmes to eradicate transmissible spongiform encephalopathies in goats is an important aim for reasons of animal welfare as well as human food safety and food security. Based on the positive impact of Prnp genetics on sheep scrapie in Europe in the past decade, we have established caprine Prnp gene variation in more than 1100 goats from the United Kingdom and studied the association of Prnp alleles with disease phenotypes in 150 scrapie-positive goats. This investigation confirms the association of the Met142 encoding Prnp allele with increased resistance to preclinical and clinical scrapie. It reveals a novel association of the Ser127 encoding allele with a reduced probability to develop clinical signs of scrapie in goats that are already positive for the accumulation of disease-specific prion protein in brain or periphery. A United Kingdom survey of Prnp genotypes in eight common breeds revealed eleven alleles in over thirty genotypes. The Met142 encoding allele had a high overall mean allele frequency of 22.6%, whereas the Ser127 encoding allele frequency was considerably lower with 6.4%. In contrast, a well known resistance associated allele encoding Lys222 was found to be rare (0.9%) in this survey. The analysis of Prnp genotypes in Mexican Criollas goats revealed nine alleles, including a novel Phe to Leu substitution in codon 201, confirming that high genetic variability of Prnp can be found in scrapie-free populations. Our study implies that it should be feasible to lower scrapie prevalence in goat herds in the United Kingdom by genetic selection.

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.

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.

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.

PrP gene polymorphisms in Cyprus goats and their association with resistance or susceptibility to natural scrapie.

In contrast to scrapie in sheep, the genetic basis of susceptibility to scrapie in goats is not well understood. To study the association of prion protein (PrP) alleles with susceptibility to scrapie in goats in Cyprus, the coding sequence of the caprine PrP gene was determined in 717 goats, including 218 scrapie positive animals. Several novel polymorphisms were detected, such as a novel octarepeat variant and a stop codon mutation. Amino acids at codons 146 and 154 were associated with susceptibility to goat scrapie. Animals heterozygous for serine (S) and aspartate (D) at codon 146 were significantly under-represented in scrapie positive animals and no positive animals were found that were homozygous for these amino acids at codon 146. These results might provide the basis for genetic control of scrapie in Cypriot goats.

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.

High prevalence of scrapie in a dairy goat herd: tissue distribution of disease-associated PrP and effect of PRNP genotype and age.

Following a severe outbreak of clinical scrapie in 2006-2007, a large dairy goat herd was culled and 200 animals were selected for post-mortem examinations in order to ascertain the prevalence of infection, the effect of age, breed and PRNP genotype on the susceptibility to scrapie, the tissue distribution of diseaseassociated PrP (PrP(d)), and the comparative efficiency of different diagnostic methods. As determined by immunohistochemical (IHC) examinations with Bar224 PrP antibody, the prevalence of preclinical infection was very high (72/200; 36.0%), with most infected animals being positive for PrP(d) in lymphoreticular system (LRS) tissues (68/72; 94.4%) compared to those that were positive in brain samples (38/72; 52.8%). The retropharyngeal lymph node and the palatine tonsil showed the highest frequency of PrP(d) accumulation (87.3% and 84.5%, respectively), while the recto-anal mucosa-associated lymphoid tissue (RAMALT) was positive in only 30 (41.7%) of the infected goats. However, the efficiency of rectal and palatine tonsil biopsies taken shortly before necropsy was similar. The probability of brain and RAMALT being positive directly correlated with the spread of PrP(d) within the LRS. The prevalence of infection was influenced by PRNP genetics at codon 142 and by the age of the goats: methionine carriers older than 60 months showed a much lower prevalence of infection (12/78; 15.4%) than those younger than 60 months (20/42; 47.6%); these last showed prevalence values similar to isoleucine homozygotes of any age (40/80; 50.0%). Two of seven goats with definite signs of scrapie were negative for PrP(d) in brain but positive in LRS tissues, and one goat showed biochemical and IHC features of PrP(d) different from all other infected goats. The results of this study have implications for surveillance and control policies for scrapie in goats.