Experimental transmission of ovine atypical scrapie to cattle.

Classical bovine spongiform encephalopathy (BSE) in cattle was caused by the recycling and feeding of meat and bone meal contaminated with a transmissible spongiform encephalopathy (TSE) agent but its origin remains unknown. This study aimed to determine whether atypical scrapie could cause disease in cattle and to compare it with other known TSEs in cattle. Two groups of calves (five and two) were intracerebrally inoculated with atypical scrapie brain homogenate from two sheep with atypical scrapie. Controls were five calves intracerebrally inoculated with saline solution and one non-inoculated animal. Cattle were clinically monitored until clinical end-stage or at least 96 months post-inoculation (mpi). After euthanasia, tissues were collected for TSE diagnosis and potential transgenic mouse bioassay. One animal was culled with BSE-like clinical signs at 48 mpi. The other cattle either developed intercurrent diseases leading to cull or remained clinical unremarkable at study endpoint, including control cattle. None of the animals tested positive for TSEs by Western immunoblot and immunohistochemistry. Bioassay of brain samples from the clinical suspect in Ov-Tg338 and Bov-Tg110 mice was also negative. By contrast, protein misfolding cyclic amplification detected prions in the examined brains from atypical scrapie-challenged cattle, which had a classical BSE-like phenotype. This study demonstrates for the first time that a TSE agent with BSE-like properties can be amplified in cattle inoculated with atypical scrapie brain homogenate.

Preliminary optimisation of a simplified sample preparation method to permit direct detection of SARS-CoV-2 within saliva samples using reverse-transcription loop-mediated isothermal amplification (RT-LAMP).

We describe the optimisation of a simplified sample preparation method which permits rapid and direct detection of SARS-CoV-2 RNA within saliva, using reverse-transcription loop-mediated isothermal amplification (RT-LAMP). Treatment of saliva samples prior to RT-LAMP by dilution 1:1 in Mucolyse™, followed by dilution in 10 % (w/v) Chelex© 100 Resin and a 98 °C heat step for 2 min enabled detection of SARS-CoV-2 RNA in positive saliva samples. Using RT-LAMP, SARS-CoV-2 RNA was detected in as little as 05:43 min, with no amplification detected in 3097 real-time reverse transcription PCR (rRT-PCR) negative saliva samples from staff tested within a service evaluation study, or for other respiratory pathogens tested (n = 22). Saliva samples can be collected non-invasively, without the need for skilled staff and can be obtained from both healthcare and home settings. Critically, this approach overcomes the requirement for, and validation of, different swabs and the global bottleneck in obtaining access to extraction robots and reagents to enable molecular testing by rRT-PCR. Such testing opens the possibility of public health approaches for effective intervention during the COVID-19 pandemic through regular SARS-CoV-2 testing at a population scale, combined with isolation and contact tracing.

Codon 141 polymorphisms of the ovine prion protein gene affect the phenotype of classical scrapie transmitted from goats to sheep.

BACKGROUND: A study to investigate transmission of classical scrapie via goat milk was carried out in sheep: firstly, lambs were challenged orally with goat scrapie brain homogenate to confirm transmission of scrapie from goats to sheep. In the second study phase, milk from scrapie-infected goats was fed to lambs. Lambs were selected according to their prion protein gene (PRNP) genotype, which was either VRQ/VRQ or ARQ/ARQ, with or without additional polymorphisms at codon 141 (FF141, LF141 or LL141) of the ovine PRNP. This report describes the clinical, pathological and molecular phenotype of goat scrapie in those sheep that progressed to clinical end-stage. RESULTS: Ten sheep (six VRQ/VRQ and four ARQ/ARQ, of which three FF141 and one LL141) challenged with one of two scrapie brain homogenates, and six pairs of sheep (ARQ, of which five LL141 and seven LF141) fed milk from six different goats, developed clinical disease, which was characterised by a pruritic (all VRQ/VRQ and LL141 sheep) or a non-pruritic form (all LF141 and FF141 sheep). Immunohistochemical (IHC) examination revealed that the pattern of intra- and extracellular accumulation of disease-associated prion protein in the brain was also dependent on PRNP polymorphisms at codon 141, which was similar in VRQ and LL141 sheep but different from LF141 and FF141 sheep. The influence of codon 141 was also seen in discriminatory Western blot (WB), with LF141 and FF141 sheep showing a bovine spongiform encephalopathy-like profile (diminished reactivity with P4 antibody) on brain tissue. However, discriminatory WB in lymphoid tissues, and IHC pattern and profile both in lymphoid and brain tissue was consistent with classical scrapie in all sheep. CONCLUSIONS: This study provided further evidence that the clinical presentation and the pathological and molecular phenotypes of scrapie in sheep are influenced by PRNP polymorphisms, particularly at codon 141. Differences in the truncation of disease-associated prion protein between LL141 sheep and those carrying the F141 allele may be responsible for these observations.

Abnormalities in Brainstem Auditory Evoked Potentials in Sheep with Transmissible Spongiform Encephalopathies and Lack of a Clear Pathological Relationship.

Scrapie is transmissible spongiform encephalopathy (TSE), which causes neurological signs in sheep, but confirmatory diagnosis is usually made postmortem on examination of the brain for TSE-associated markers like vacuolar changes and disease-associated prion protein (PrP(Sc)). The objective of this study was to evaluate whether testing of brainstem auditory evoked potentials (BAEPs) at two different sound levels could aid in the clinical diagnosis of TSEs in sheep naturally or experimentally infected with different TSE strains [classical and atypical scrapie and bovine spongiform encephalopathy (BSE)] and whether any BAEP abnormalities were associated with TSE-associated markers in the auditory pathways. BAEPs were recorded from 141 clinically healthy sheep of different breeds and ages that tested negative for TSEs on postmortem tests to establish a reference range and to allow comparison with 30 sheep clinically affected or exposed to classical scrapie (CS) without disease confirmation (test group 1) and 182 clinically affected sheep with disease confirmation (test group 2). Abnormal BAEPs were found in 7 sheep (23%) of group 1 and 42 sheep (23%) of group 2. The proportion of sheep with abnormalities did not appear to be influenced by TSE strain or PrP(Sc) gene polymorphisms. When the magnitude of TSE-associated markers in the auditory pathways was compared between a subset of 12 sheep with and 12 sheep without BAEP abnormalities in group 2, no significant differences in the total PrP(Sc) or vacuolation scores in the auditory pathways could be found. However, the data suggested that there was a difference in the PrP(Sc) scores depending on the TSE strain because PrP(Sc) scores were significantly higher in sheep with BAEP abnormalities infected with classical and L-type BSE, but not with CS. The results indicated that BAEPs may be abnormal in sheep infected with TSEs but the test is not specific for TSEs and that neither vacuolation nor PrP(Sc) accumulation appears to be responsible for the clinical abnormalities.

Further characterisation of transmissible spongiform encephalopathy phenotypes after inoculation of cattle with two temporally separated sources of sheep scrapie from Great Britain.

BACKGROUND: The infectious agent responsible for the bovine spongiform encephalopathy (BSE) epidemic in Great Britain is a transmissible spongiform encephalopathy (TSE) strain with uniform properties but the origin of this strain remains unknown. Based on the hypothesis that classical BSE may have been caused by a TSE strain present in sheep, cattle were inoculated intracerebrally with two different pools of brains from scrapie-affected sheep sourced prior to and during the BSE epidemic to investigate resulting disease phenotypes and characterise their causal agents by transmission to rodents. RESULTS: As reported in 2006, intracerebral inoculation of cattle with pre-1975 and post-1990 scrapie brain pools produced two distinct disease phenotypes, which were unlike classical BSE. Subsequent to that report none of the remaining cattle, culled at 10 years post inoculation, developed a TSE. Retrospective Western immunoblot examination of the brains from TSE cases inoculated with the pre-1975 scrapie pool revealed a molecular profile similar to L-type BSE. The inoculation of transgenic mice expressing the bovine, ovine, porcine, murine or human prion protein gene and bank voles with brains from scrapie-affected cattle did not detect classical or atypical BSE strains but identified two previously characterised scrapie strains of sheep. CONCLUSIONS: Characterisation of the causal agents of disease resulting from exposure of cattle to naturally occurring scrapie agents sourced in Great Britain did not reveal evidence of classical or atypical BSE, but did identify two distinct previously recognised strains of scrapie. Although scrapie was still recognizable upon cattle passage there were irreconcilable discrepancies between the results of biological strain typing approaches and molecular profiling methods, suggesting that the latter may not be appropriate for the identification and differentiation of atypical, particularly L-type, BSE agents from cattle experimentally infected with a potential mixture of classical scrapie strains from sheep sources.

The pathological and molecular but not clinical phenotypes are maintained after second passage of experimental atypical bovine spongiform encephalopathy in cattle.

BACKGROUND: Atypical bovine spongiform encephalopathies (BSEs), classified as H-type and L-type BSE based on the Western immunoblot profiles, are naturally occurring diseases in cattle, which are phenotypically different to classical BSE. Transmission studies in cattle using the intracerebral route resulted in disease where the phenotypes were maintained irrespective of BSE type but clinically affected cattle with a shorter survival time displayed a nervous form whereas cattle with a longer survival time displayed a dull form. A second transmission study is reported here where four cattle were intracerebrally inoculated with brain tissue from experimentally infected cattle presenting with either the nervous or dull form of H- or L-type BSE to determine whether the phenotype is maintained. RESULTS: The four inoculated cattle were culled at 16.5-19.5 months post inoculation after presenting with difficulty getting up, a positive scratch response (all) and dullness (three cattle), which was not observed in two non-inoculated control cattle, each housed with either group of inoculated cattle. Only the inoculated cattle had detectable prion protein in the brain based on immunohistochemical examination, and the Western immunoblot profile was consistent with the H-type or L-type BSE of the respective donor cattle. CONCLUSIONS: Second passage of H-type and L-type BSE in cattle produced a TSE where the majority of cattle displayed the dull form regardless of clinical disease form of the donor cattle. The pathological and molecular phenotypes of H- and L-type BSE were maintained.

Bovine spongiform encephalopathy: the effect of oral exposure dose on attack rate and incubation period in cattle - an update.

BACKGROUND: To provide information on dose-response and aid in modelling the exposure dynamics of the BSE epidemic in the United Kingdom groups of cattle were exposed orally to a range of different doses of brainstem homogenate of known infectious titre from clinical cases of classical bovine spongiform encephalopathy (BSE). Interim data from this study was published in 2007. This communication documents additional BSE cases, which occurred subsequently, examines possible influence of the bovine prion protein gene on disease incidence and revises estimates of effective oral exposure. FINDINGS: Following interim published results, two further cattle, one dosed with 100 mg and culled at 127 months post exposure and the other dosed with 10 mg and culled at 110 months post exposure, developed BSE. Both had a similar pathological phenotype to previous cases. Based on attack rate and incubation period distribution according to dose, the dose estimate at which 50% of confirmed cases would be clinically affected was revised to 0.15 g of the brain homogenate used in the experiment, with a 95% confidence interval of 0.03-0.79 g. Neither the full open reading frame nor the promoter region of the prion protein gene of dosed cattle appeared to influence susceptibility to BSE, but this may be due to the sample size. CONCLUSIONS: Oral exposure of cattle to a large range of doses of a BSE brainstem homogenate produced disease in all dose groups. The pathological presentation resembled natural disease. The attack rate and incubation period were dependent on the dose.

Experimental H-type and L-type bovine spongiform encephalopathy in cattle: observation of two clinical syndromes and diagnostic challenges.

BACKGROUND: The majority of atypical bovine spongiform encephalopathy (BSE) cases so far identified worldwide have been detected by active surveillance. Consequently the volume and quality of material available for detailed characterisation is very limiting. Here we report on a small transmission study of both atypical forms, H- and L-type BSE, in cattle to provide tissue for test evaluation and research, and to generate clinical, molecular and pathological data in a standardised way to enable more robust comparison of the two variants with particular reference to those aspects most relevant to case ascertainment and confirmatory diagnosis within existing regulated surveillance programmes. RESULTS: Two groups of four cattle, intracerebrally inoculated with L-type or H-type BSE, all presented with a nervous disease form with some similarities to classical BSE, which progressed to a more dull form in one animal from each group. Difficulty rising was a consistent feature of both disease forms and not seen in two BSE-free, non-inoculated cattle that served as controls. The pathology and molecular characteristics were distinct from classical BSE, and broadly consistent with published data, but with some variation in the pathological characteristics. Both atypical BSE types were readily detectable as BSE by current confirmatory methods using the medulla brain region at the obex, but making a clear diagnostic distinction between the forms was not consistently straightforward in this brain region. Cerebellum proved a more reliable sample for discrimination when using immunohistochemistry. CONCLUSIONS: The prominent feature of difficulty rising in atypical BSE cases may explain the detection of naturally occurring cases in emergency slaughter cattle and fallen stock. Current confirmatory diagnostic methods are effective for the detection of such atypical cases, but consistently and correctly identifying the variant forms may require modifications to the sampling regimes and methods that are currently in use.

Use of murine bioassay to resolve ovine transmissible spongiform encephalopathy cases showing a bovine spongiform encephalopathy molecular profile.

Two cases of unusual transmissible spongiform encephalopathy (TSE) were diagnosed on the same farm in ARQ/ARQ PrP sheep showing attributes of both bovine spongiform encephalopathy (BSE) and scrapie. These cases, UK-1 and UK-2, were investigated further by transmissions to wild-type and ovine transgenic mice. Lesion profiles (LP) on primary isolation and subpassage, incubation period (IP) of disease, PrP(Sc) immunohistochemical (IHC) deposition pattern and Western blot profiles were used to characterize the prions causing disease in these sheep. Results showed that both cases were compatible with scrapie. The presence of BSE was contraindicated by the following: LP on primary isolation in RIII and/or MR (modified RIII) mice; IP and LP after serial passage in wild-type mice; PrP(Sc) deposition pattern in wild-type mice; and IP and Western blot data in transgenic mice. Furthermore, immunohistochemistry (IHC) revealed that each case generated two distinct PrP(Sc) deposition patterns in both wild-type and transgenic mice, suggesting that two scrapie strains coexisted in the ovine hosts. Critically, these data confirmed the original differential IHC categorization that these UK-1 and UK-2 cases were not compatible with BSE.

Monitoring of clinical signs in goats with transmissible spongiform encephalopathies.

BACKGROUND: As there is limited information about the clinical signs of BSE and scrapie in goats, studies were conducted to describe the clinical progression of scrapie and BSE in goats and to evaluate a short clinical protocol for its use in detecting scrapie-affected goats in two herds with previously confirmed scrapie cases. Clinical assessments were carried out in five goats intracerebrally infected with the BSE agent as well as five reported scrapie suspects and 346 goats subject to cull from the two herds, 24 of which were retained for further monitoring. The brain and selected lymphoid tissue were examined by postmortem tests for disease confirmation. RESULTS: The sensitivity and specificity of the short clinical protocol in detecting a scrapie case in the scrapie-affected herds was 3.9% and 99.6%, respectively, based on the presence of tremor, positive scratch test, extensive hair loss, ataxia and absent menace response. All BSE- and scrapie-affected goats displayed abnormalities in sensation (over-reactivity to external stimuli, startle responses, pruritus, absent menace response) and movement (ataxia, tremor, postural deficits) at an advanced clinical stage but the first detectable sign associated with scrapie or BSE could vary between animals. Signs of pruritus were not always present despite similar prion protein genotypes. Clinical signs of scrapie were also displayed by two scrapie cases that presented with detectable disease-associated prion protein only in lymphoid tissues. CONCLUSIONS: BSE and scrapie may present as pruritic and non-pruritic forms in goats. Signs assessed for the clinical diagnosis of scrapie or BSE in goats should include postural and gait abnormalities, pruritus and visual impairment. However, many scrapie cases will be missed if detection is solely based on the display of clinical signs. PrPd accumulation in the brain appeared to be related to the severity of clinical disease but not to the display of individual neurological signs.

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.