Extended application of the rapid post-mortem test kit for bovine spongiform encephalopathy to chronic wasting disease.

Chronic wasting disease (CWD) is a prion disease affecting cervid species primarily in the United States of America and Canada; however, it is now emerging in Scandinavian countries. Although CWD cases have not been reported in Japan, in case of a CWD outbreak occuring, it is critical to prepare for testing a large number of specimens. The present study showed that a rapid post-mortem test kit, which is used for bovine spongiform encephalopathy surveillance in Japan, is valid for the detection of CWD prion.

Discrimination between L-type and C-type bovine spongiform encephalopathy by the strain-specific reactions of real-time quaking-induced conversion.

Real-time quaking-induced conversion (RT-QUIC) assays using Escherichia coli-derived purified recombinant prion protein (rPrP) enable us to amplify a trace amount of the abnormal form of PrP (PrPSc) from specimens. This technique can be useful for the early diagnosis of both human and animal prion diseases and the assessment of prion contamination. In the present study, we demonstrated that there are strain-specific differences in the RT-QUIC reactions between an atypical form of bovine spongiform encephalopathy (BSE), l-BSE, and classical BSE (C-BSE). Whereas mouse rPrP (rMoPrP) was efficiently converted to amyloid fibrils in the presence of PrPSc seed derived from either l-BSE or C-BSE, hamster rPrP (rHaPrP) was converted only in l-BSE, not C-BSE. These characteristics were preserved in the second round reaction, but gradually weakened in the subsequent rounds and were completely lost by the fifth round, most likely due to the selective growth advantage of nonspecific rPrP amyloid fibrils in the RT-QUIC. Our findings further enhance the discrimination of prion strains using RT-QUIC, and further our understanding of the molecular basis of prion strains.

Comparison between plasma and cerebrospinal fluid biomarkers for the early diagnosis and association with survival in prion disease.

OBJECTIVE: To compare the diagnostic accuracy and the prognostic value of blood and cerebrospinal fluid (CSF) tests across prion disease subtypes. METHODS: We used a single-molecule immunoassay to measure tau and neurofilament light chain (NfL) protein levels in the plasma and assessed CSF total(t)-tau, NfL and protein 14-3-3 levels in patients with prion disease (n=336), non-prion rapidly progressive dementias (n=106) and non-neurodegenerative controls (n=37). We then evaluated each plasma and CSF marker for diagnosis and their association with survival, taking into account the disease subtype, which is a strong independent prognostic factor in prion disease. RESULTS: Plasma tau and NfL concentrations were higher in patients with prion disease than in non-neurodegenerative controls and non-prion rapidly progressive dementias. Plasma tau showed higher diagnostic value than plasma NfL, but a lower accuracy than the CSF proteins t-tau and 14-3-3. In the whole prion cohort, both plasma (tau and NfL) and CSF (t-tau, 14-3-3 and NfL) markers were significantly associated with survival and showed similar prognostic values. However, the intrasubtype analysis revealed that only CSF t-tau in sporadic Creutzfeldt-Jakob disease (sCJD) MM(V)1, plasma tau and CSF t-tau in sCJD VV2, and plasma NfL in slowly progressive prion diseases were significantly associated with survival after accounting for covariates. CONCLUSIONS: Plasma markers have lower diagnostic accuracy than CSF biomarkers. Plasma tau and NfL and CSF t-tau are significantly associated with survival in prion disease in a subtype-specific manner and can be used to improve clinical trial stratification and clinical care.

Diffuse Leptomeningeal Oligodendrogliomatosis in a Cow.

A 4.5-year-old cow showing neurological signs consistent with predictors of bovine spongiform encephalopathy (BSE) was investigated as a potential BSE-suspect case and proved to be negative. Macroscopic analysis revealed a tan neoplastic mass growing along the leptomeninges of the caudal brain and extending into the third (III) ventricle without significantly involving the neuroparenchyma. Pathological features (uniform round hyperchromatic neoplastic cells embedded in abundant myxoid matrix, microcysts, microvascular proliferation) and diffuse Olig2 expression were most consistent with diffuse high-grade leptomeningeal oligodendrogliomatosis. In line with former reports of extensive leptomeningeal involvement in bovine oligodendroglioma, this report suggests that bovine oligodendroglial tumors have a strong propensity to grow within the leptomeningeal space. In addition, it indicates that Olig2 is a useful marker to confirm glial lineage in formalin-fixed, paraffin-embedded bovine tissue.

Role of prion protein glycosylation in replication of human prions by protein misfolding cyclic amplification.

Prion diseases are transmissible neurological disorders associated with the presence of abnormal, disease-related prion protein (PrPD). The detection of PrPD in the brain is the only definitive diagnostic evidence of prion disease and its identification in body fluids and peripheral tissues are valuable for pre-mortem diagnosis. Protein misfolding cyclic amplification (PMCA) is a technique able to detect minute amount of PrPD and is based on the conversion of normal or cellular PrP (PrPC) to newly formed PrPD, sustained by a self-templating mechanism. Several animal prions have been efficiently amplified by PMCA, but limited results have been obtained with human prions with the exception of variant-Creutzfeldt-Jakob-disease (vCJD). Since the total or partial absence of glycans on PrPC has been shown to affect PMCA efficiency in animal prion studies, we attempted to enhance the amplification of four major sporadic-CJD (sCJD) subtypes (MM1, MM2, VV1, and VV2) and vCJD by single round PMCA using partially or totally unglycosylated PrPC as substrates. The amplification efficiency of all tested sCJD subtypes underwent a strong increase, inversely correlated to the degree of PrPC glycosylation and directly related to the matching of the PrP polymorphic 129 M/V genotype between seed and substrate. This effect was particularly significant in sCJDMM2 and sCJDVV2 allowing the detection of PK-resistant PrPD (resPrPD) in sCJDMM2 and sCJDVV2 brains at dilutions of 6 × 107 and 3 × 106. vCJD, at variance with the tested sCJD subtypes, showed the best amplification with partially deglycosylated PrPC substrate reaching a resPrPD detectability at up to 3 × 1016 brain dilution. The differential effect of substrate PrPC glycosylations suggests subtype-dependent PrPC-PrPD interactions, strongly affected by the PrPC glycans. The enhanced PMCA prion amplification efficiency achieved with unglycosylated PrPC substrates may allow for the developing of a sensitive, non-invasive, diagnostic test for the different CJD subtypes based on body fluids or easily-accessible-peripheral tissues.

Oral Transmission of L-Type Bovine Spongiform Encephalopathy Agent among Cattle.

To determine oral transmissibility of the L-type bovine spongiform encephalopathy (BSE) prion, we orally inoculated 16 calves with brain homogenates of the agent. Only 1 animal, given a high dose, showed signs and died at 88 months. These results suggest low risk for oral transmission of the L-BSE agent among cattle.

L-Arginine ethylester enhances in vitro amplification of PrP(Sc) in macaques with atypical L-type bovine spongiform encephalopathy and enables presymptomatic detection of PrP(Sc) in the bodily fluids.

Protease-resistant, misfolded isoforms (PrP(Sc)) of a normal cellular prion protein (PrP(C)) in the bodily fluids, including blood, urine, and saliva, are expected to be useful diagnostic markers of prion diseases, and nonhuman primate models are suited for performing valid diagnostic tests for human Creutzfeldt-Jakob disease (CJD). We developed an effective amplification method for PrP(Sc) derived from macaques infected with the atypical L-type bovine spongiform encephalopathy (L-BSE) prion by using mouse brain homogenate as a substrate in the presence of polyanions and L-arginine ethylester. This method was highly sensitive and detected PrP(Sc) in infected brain homogenate diluted up to 10(10) by sequential amplification. This method in combination with PrP(Sc) precipitation by sodium phosphotungstic acid is capable of amplifying very small amounts of PrP(Sc) contained in the cerebrospinal fluid (CSF), saliva, urine, and plasma of macaques that have been intracerebrally inoculated with the L-BSE prion. Furthermore, PrP(Sc) was detectable in the saliva or urine samples as well as CSF samples obtained at the preclinical phases of the disease. Thus, our novel method may be useful for furthering the understanding of bodily fluid leakage of PrP(Sc) in nonhuman primate models.

Detection of Atypical H-Type Bovine Spongiform Encephalopathy and Discrimination of Bovine Prion Strains by Real-Time Quaking-Induced Conversion.

Prion diseases of cattle include the classical bovine spongiform encephalopathy (C-BSE) and the atypical H-type BSE (H-BSE) and L-type BSE (L-BSE) strains. Although the C- and L-BSE strains can be detected and discriminated by ultrasensitive real-time quaking-induced conversion (RT-QuIC) assays, no such test has yet been described for the detection of H-BSE or the discrimination of each of the major bovine prion strains. Here, we demonstrate an RT-QuIC assay for H-BSE that can detect as little as 10(-9) dilutions of brain tissue and neat cerebrospinal fluid samples from clinically affected cattle. Moreover, comparisons of the reactivities with different recombinant prion protein substrates and/or immunoblot band profiles of proteinase K-treated RT-QuIC reaction products indicated that H-, L-, and C-BSE have distinctive prion seeding activities and can be discriminated by RT-QuIC on this basis.

Preclinical detection of variant CJD and BSE prions in blood.

The emergence of variant Creutzfeldt Jakob Disease (vCJD) is considered a likely consequence of human dietary exposure to Bovine Spongiform Encephalopathy (BSE) agent. More recently, secondary vCJD cases were identified in patients transfused with blood products prepared from apparently healthy donors who later went on to develop the disease. As there is no validated assay for detection of vCJD/BSE infected individuals the prevalence of the disease in the population remains uncertain. In that context, the risk of vCJD blood borne transmission is considered as a serious concern by health authorities. In this study, appropriate conditions and substrates for highly efficient and specific in vitro amplification of vCJD/BSE agent using Protein Misfolding Cyclic Amplification (PMCA) were first identified. This showed that whatever the origin (species) of the vCJD/BSE agent, the ovine Q171 PrP substrates provided the best amplification performances. These results indicate that the homology of PrP amino-acid sequence between the seed and the substrate is not the crucial determinant of the vCJD agent propagation in vitro. The ability of this method to detect endogenous vCJD/BSE agent in the blood was then defined. In both sheep and primate models of the disease, the assay enabled the identification of infected individuals in the early preclinical stage of the incubation period. Finally, sample panels that included buffy coat from vCJD affected patients and healthy controls were tested blind. The assay identified three out of the four tested vCJD affected patients and no false positive was observed in 141 healthy controls. The negative results observed in one of the tested vCJD cases concurs with results reported by others using a different vCJD agent blood detection assay and raises the question of the potential absence of prionemia in certain patients.

L-BSE experimentally transmitted to sheep presents as a unique disease phenotype.

Apart from prion protein genotype, the factors determining the host range and susceptiblity for specific transmissible spongiform encephalopathy agents remain unclear. It is known that bovine atypical L-BSE can transmit to a range of species including primates and humanised transgenic mice. It is important, therefore, that there is as broad an understanding as possible of how such isolates might present in food animal species and how robust they are on inter- and intra-species transmission to inform surveillance sytems and risk assessments. This paper demonstrates that L-BSE can be intracerebrally transmitted to sheep of several genotypes, with the exception of ARR/ARR animals. Positive animals mostly present with a cataplectic form of disease characterized by collapsing episodes and reduced muscle tone. PrP accumulation is confined to the nervous system, with the exception of one animal with lymphoreticular involvement. In Western blot there was maintenance of the low molecular mass and glycoform profile associated with L-BSE, irrespective of ovine host genotype, but there was a substantially higher N-terminal antibody signal relative to the core-specific antibody, which is similar to the ratio associated with classical scrapie. The disease phenotype was maintained on experimental subpassage, but with a shortened survival time indicative of an original species barrier and subsequent adaptation. Passive surveillance approaches would be unlikely to identify such cases as TSE suspects, but current statutory active screening methods would be capable of detecting such cases and classifying them as unusual and requiring further investigation if they were to occur in the field.

Atypical variants of bovine spongiform encephalopathy: rare diseases with consequences for BSE surveillance and control.

INTRODUCTION: Occurring for the first time in 1986 in the United Kingdom, bovine spongiform encephalopathy (BSE), the so-called "mad-cow disease", has had unprecedented consequences in veterinary public health. The implementation of drastic measures, including the ban of meat-and-bone-meal from livestock feed and the removal of specified risk materials from the food chain has eventually resulted in a significant decline of the epidemic. The disease was long thought to be caused by a single agent, but since the introduction of immunochemical diagnostic techniques, evidence of a phenotypic variation of BSE has emerged. Reviewing the literature available on the subject, this paper briefly summarizes the current knowledge about these atypical forms of BSE and discusses the consequences of their occurrence for disease control measures.

INTRODUCTION: L'encéphalopathie spongiforme bovine (ESB) dite aussi "maladie de la vache folle", apparue pour la première fois en 1996 au Royaume-Uni, a eu des conséquences sans équivalent pour le service public vétérinaire. La mise en application de mesures de lutte drastique, telle l'interdiction d'affourager les animaux de rente avec des farines animales et le retrait de la chaine des aliments de matériels à risque a conduit à un recul significatif de l'épidémie. Durant longtemps on a considéré que la maladie n'était causée que par un seul type de l'agent infectieux. Avec l'introduction de techniques de diagnostic immunochimiques, on a toutefois des indices de variantes phénotypiques de l'ESB. Le présent article résume la littérature disponible et fait le point des connaissances quant à ces variantes atypiques de l'ESB; on y discute également les conséquences possibles de leur apparition quant à la lutte contre la maladie.

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

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.

Detection and discrimination of classical and atypical L-type bovine spongiform encephalopathy by real-time quaking-induced conversion.

Statutory surveillance of bovine spongiform encephalopathy (BSE) indicates that cattle are susceptible to both classical BSE (C-BSE) and atypical forms of BSE. Atypical forms of BSE appear to be sporadic and thus may never be eradicated. A major challenge for prion surveillance is the lack of sufficiently practical and sensitive tests for routine BSE detection and strain discrimination. The real-time quaking-induced conversion (RT-QuIC) test, which is based on prion-seeded fibrillization of recombinant prion protein (rPrPSen), is known to be highly specific and sensitive for the detection of multiple human and animal prion diseases but not BSE. Here, we tested brain tissue from cattle affected by C-BSE and atypical L-type bovine spongiform encephalopathy (L-type BSE or L-BSE) with the RT-QuIC assay and found that both BSE forms can be detected and distinguished using particular rPrPSen substrates. Specifically, L-BSE was detected using multiple rPrPSen substrates, while C-BSE was much more selective. This substrate-based approach suggests a diagnostic strategy for specific, sensitive, and rapid detection and discrimination of at least some BSE forms.

The Canadian Management of Bovine Spongiform Encephalopathy in Historical and Scientific Perspective, 1990-2014.

On February 11, 2015, the Canadian Food Inspection Agency announced that a cow born and raised in Alberta had tested positive for bovine spongiform encephalopathy (BSE), commonly known as mad cow disease. BSE is a prion disease of cattle that, when transmitted to humans, produces a fatal neurodegenerative disease known as variant Creutzfeldt-Jakob disease. We believe that this latest case of BSE in Canadian cattle suggests the timeliness of a review of the management of BSE in Canada from a historically and scientifically informed perspective. In this article, we ask: how did the Canadian management of BSE between 1990 and 2014 engage with the contemporary understanding of BSE's human health implications? We propose that Canadian policies largely ignored the implicit medical nature of BSE, treating it as a purely agricultural and veterinary issue. In this way, policies to protect Canadians were often delayed and incomplete, in a manner disturbingly reminiscent of Britain's failed management of BSE. Despite assurances to the contrary, it is premature to conclude that BSE (and with it the risk of variant Creutzfeldt-Jakob disease) is a thing of Canada's past: BSE remains very much an issue in Canada's present.

Defining an emerging disease.

Defining an emerging disease is not straightforward, as there are several different types of disease emergence. For example, there can be a 'real' emergence of a brand new disease, such as the emergence of bovine spongiform encephalopathy in the 1980s, or a geographic emergence in an area not previously affected, such as the emergence of bluetongue in northern Europe in 2006. In addition, disease can emerge in species formerly not considered affected, e.g. the emergence of bovine tuberculosis in wildlife species since 2000 in France. There can also be an unexpected increase of disease incidence in a known area and a known species, or there may simply be an increase in our knowledge or awareness of a particular disease. What all these emerging diseases have in common is that human activity frequently has a role to play in their emergence. For example, bovine spongiform encephalopathy very probably emerged as a result of changes in the manufacturing of meat-and-bone meal, bluetongue was able to spread to cooler climes as a result of uncontrolled trade in animals, and a relaxation of screening and surveillance for bovine tuberculosis enabled the disease to re-emerge in areas that had been able to drastically reduce the number of cases. Globalisation and population growth will continue to affect the epidemiology of diseases in years to come and ecosystems will continue to evolve. Furthermore, new technologies such as metagenomics and high-throughput sequencing are identifying new microorganisms all the time. Change is the one constant, and diseases will continue to emerge, and we must consider the causes and different types of emergence as we deal with these diseases in the future.

Highly sensitive detection of small ruminant bovine spongiform encephalopathy within transmissible spongiform encephalopathy mixes by serial protein misfolding cyclic amplification.

It is assumed that sheep and goats consumed the same bovine spongiform encephalopathy (BSE)-contaminated meat and bone meal that was fed to cattle and precipitated the BSE epidemic in the United Kingdom that peaked more than 20 years ago. Despite intensive surveillance for cases of BSE within the small ruminant populations of the United Kingdom and European Union, no instances of BSE have been detected in sheep, and in only two instances has BSE been discovered in goats. If BSE is present within the small ruminant populations, it may be at subclinical levels, may manifest as scrapie, or may be masked by coinfection with scrapie. To determine whether BSE is potentially circulating at low levels within the European small ruminant populations, highly sensitive assays that can specifically detect BSE, even within the presence of scrapie prion protein, are required. Here, we present a novel assay based on the specific amplification of BSE PrP(Sc) using the serial protein misfolding cyclic amplification assay (sPMCA), which specifically amplified small amounts of ovine and caprine BSE agent which had been mixed into a range of scrapie-positive brain homogenates. We detected the BSE prion protein within a large excess of classical, atypical, and CH1641 scrapie isolates. In a blind trial, this sPMCA-based assay specifically amplified BSE PrP(Sc) within brain mixes with 100% specificity and 97% sensitivity when BSE agent was diluted into scrapie-infected brain homogenates at 1% (vol/vol).

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.

Infectious Diseases of the Brain and Spine: Parasitic and Other Atypical Transmissible Diseases.

Atypical infections of the brain and spine caused by parasites occur in immunocompetent and immunosuppressed hosts, related to exposure and more prevalently in endemic regions. In the United States, the most common parasitic infections that lead to central nervous system manifestations include cysticercosis, echinococcosis, and toxoplasmosis, with toxoplasmosis being the most common opportunistic infection affecting patients with advanced HIV/AIDS. Another rare but devastating transmittable disease is prion disease, which causes rapidly progressive spongiform encephalopathies. Familiarity and understanding of various infectious agents are a crucial aspect of diagnostic neuroradiology, and recognition of unique features can aid timely diagnosis and treatment.

Foodborne transmission of bovine spongiform encephalopathy to nonhuman primates.

Risk for human exposure to bovine spongiform encephalopathy (BSE)-inducing agent was estimated in a nonhuman primate model. To determine attack rates, incubation times, and molecular signatures, we orally exposed 18 macaques to 1 high dose of brain material from cattle with BSE. Several macaques were euthanized at regular intervals starting at 1 year postinoculation, and others were observed until clinical signs developed. Among those who received ≥5 g BSE-inducing agent, attack rates were 100% and prions could be detected in peripheral tissues from 1 year postinoculation onward. The overall median incubation time was 4.6 years (3.7-5.3). However, for 3 macaques orally exposed on multiple occasions, incubation periods were at least 7-10 years. Before clinical signs were noted, we detected a non-type 2B signature, indicating the existence of atypical prion protein during the incubation period. This finding could affect diagnosis of variant Creutzfeldt-Jakob disease in humans and might be relevant for retrospective studies of positive tonsillectomy or appendectomy specimens because time of infection is unknown.

Evaluation of a test for its suitability in the diagnosis of variant Creutzfeldt-Jakob disease.

BACKGROUND AND OBJECTIVES: Evaluation of variant Creutzfeldt-Jakob disease (vCJD) diagnostic/donor screening tests is made complicated by the very limited supply of blood samples from clinically confirmed cases of vCJD. To determine appropriate access for test developers to rare Creutzfeldt-Jakob disease (CJD) blood samples, the oversight committee of the NIBSC CJD Resource Centre has developed a process and protocols detailing minimum requirements for both test sensitivity and specificity. This protocol is broadly similar to that outlined in the common technical specification (European Directive 98/79/EC). MATERIALS AND METHODS: Tests are subjected to a stepwise evaluation (step 1). vCJD tissue homogenates spiked into pooled human plasma (step 2). Blood samples from animals known to be incubating (Transmissible spongiform encephalopathy) TSE disease (scrapie/Bovine Spongiform encephalopathy (BSE)-infected sheep, BSE-infected primates) and appropriate controls (step 3). Fresh or frozen plasma from normal UK blood donors and (step 4). Plasma samples from individuals with confirmed clinical stage variant CJD (transfusion transmission) or sporadic CJD (no evidence of blood transmission). RESULTS: The assay evaluated performed with good sensitivity with vCJD-spiked tissue homogenates, poor sensitivity for ovine TSE-infected blood samples and failed with plasma from BSE-infected non-human primates and with true vCJD clinical samples. CONCLUSIONS: The test evaluated here is currently unsuitable for use in blood donor screening or diagnosis using blood.