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.

Development of a sensitive real-time quaking-induced conversion (RT-QuIC) assay for application in prion-infected blood.

Efforts to prevent human-to-human transmission of variant Creutzfeldt-Jakob disease (vCJD) by contaminated blood would be aided by the development of a sensitive diagnostic test that could be routinely used to screen blood donations. As blood samples from vCJD patients are extremely rare, here we describe the optimisation of real-time quaking-induced conversion (RT-QuIC) for detection of PrPSc (misfolded prion protein, a marker of prion infection) in blood samples from an established large animal model of vCJD, sheep experimentally infected with bovine spongiform encephalopathy (BSE). Comparative endpoint titration experiments with RT-QuIC, miniaturized bead protein misfolding cyclic amplification (mb-PMCA) and intracerebral inoculation of a transgenic mouse line expressing sheep PrP (tgOvARQ), demonstrated highly sensitive detection of PrPSc by RT-QuIC in a reference sheep brain homogenate. Upon addition of a capture step with iron oxide beads, the RT-QuIC assay was able to detect PrPSc in whole blood samples from BSE-infected sheep up to two years before disease onset. Both RT-QuIC and mb-PMCA also demonstrated sensitive detection of PrPSc in a reference vCJD-infected human brain homogenate, suggesting that either assay may be suitable for application to human blood samples. Our results support the further development and evaluation of RT-QuIC as a diagnostic or screening test for vCJD.

Detection of Abnormal Prion Protein by Immunohistochemistry.

Abnormal prion proteins (PrPSc) are the disease-associated isoform of cellular prion protein and diagnostic markers of transmissible spongiform encephalopathies (TSEs). These neurodegenerative diseases affect humans and several animal species and include scrapie, zoonotic bovine spongiform encephalopathy (BSE), chronic wasting disease of cervids (CWD), and the newly identified camel prion disease (CPD). Diagnosis of TSEs relies on immunodetection of PrPSc by application of both immunohistochemistry (IHC) and western immunoblot methods (WB) on encephalon tissues, namely, the brainstem (obex level). IHC is a widely used method that uses primary antibodies (monoclonal or polyclonal) against antigens of interest in cells of a tissue section. The antibody-antigen binding can be visualized by a color reaction that remains localized in the area of the tissue or cell where the antibody was targeted. As such, in prion diseases, as in other fields of research, the immunohistochemistry techniques are not solely used for diagnostic purposes but also in pathogenesis studies. Such studies involve detecting the PrPSc patterns and types from those previously described to identify the new prion strains. As BSE can infect humans, it is recommended that biosafety laboratory level-3 (BSL-3) facilities and/or practices are used to handle cattle, small ruminants, and cervid samples included in the TSE surveillance. Additionally, containment and prion-dedicated equipment are recommended, whenever possible, to limit contamination. The PrPSc IHC procedure consists of a formic acid epitope-demasking step also acting as a prion inactivation measure, as formalin-fixed and paraffin-embedded tissues used in this technique remain infectious. When interpreting the results, care must be taken to distinguish non-specific immunolabeling from target labeling. For this purpose, it is important to recognize artifacts of immunolabeling obtained in known TSE-negative control animals to differentiate those from specific PrPSc immunolabeling types, which can vary between TSE strains, host species, and prnp genotype, further described herein.

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.

In-depth examination of PrPSc in Holstein cattle carrying the E211K somatic mutation of the bovine prion protein gene (PRNP).

Prion diseases are transmissible spongiform encephalopathies caused by deleterious prion protein (PrPSc ) derived from normal prion protein (PrPC ), which is encoded by the prion protein gene (PRNP). We performed an in-depth examination to detect PrPSc by using enzyme immunoassay (EIA), real-time quaking-induced conversion reactions (RT-QuIC) and protein misfolding cyclic amplification (PMCA) in nine brain tissues derived from three Holstein cattle carrying the E211K somatic mutation of the bovine PRNP gene. The EIA, RT-QuIC and PMCA analyses were not able to detect the PrPSc band in any tested samples. To the best of our knowledge, this report is the first to describe an in-depth examination of PrPSc in cattle carrying the E211K somatic mutation of the bovine PRNP gene.

RT-QuIC detection of pathological prion protein in subclinical goats following experimental oral transmission of L-type BSE.

OBJECTIVE: The spread of bovine spongiform encephalopathy (BSE) agent to small ruminants is still a major issue in the surveillance of transmissible spongiform encephalopathies (TSEs). L-type bovine spongiform encephalopathy (L-BSE) is an atypical form of BSE with an unknown zoonotic potential that is transmissible to cattle and small ruminants. Our current knowledge of bovine atypical prion strains in sheep and goat relies only on experimental transmission studies by intracranial inoculation. To assess oral susceptibility of goats to L-BSE, we orally inoculated five goats with cattle L-BSE brain homogenates and investigated pathogenic prion protein (PrPsc) distribution by an ultrasensitive in vitro conversion assay known as Real-Time Quaking Induced Conversion (RT-QuIC). RESULTS: Despite a prolonged observation period of 80 months, all these animals and the uninfected controls did not develop clinical signs referable to TSEs and tested negative by standard diagnostics. Otherwise, RT-QuIC analysis showed seeding activity in five out of five examined brain samples. PrPsc accumulation was also detected in spinal cord and lymphoreticular system. These results indicate that caprine species are susceptible to L-BSE by oral transmission and that ultrasensitive prion tests deserve consideration to improve the potential of current surveillance systems against otherwise undetectable forms of animal prion infections.

Analysis of German BSE Surveillance Data: Estimation of the Prevalence of Confirmed Cases versus the Number of Infected, but Non-Detected, Cattle to Assess Confidence in Freedom from Infection.

Quantitative risk assessments for Bovine spongiform encephalopathy (BSE) necessitate estimates for key parameters such as the prevalence of infection, the probability of absence of infection in defined birth cohorts, and the numbers of BSE-infected, but non-detected cattle entering the food chain. We estimated three key parameters with adjustment for misclassification using the German BSE surveillance data using a Gompertz model for latent (i.e., unobserved) age-dependent detection probabilities and a Poisson response model for the number of BSE cases for birth cohorts 1999 to 2015. The models were combined in a Bayesian framework. We estimated the median true BSE prevalence between 3.74 and 0.216 cases per 100,000 animals for the birth cohorts 1990 to 2001 and observed a peak for the 1996 birth cohort with a point estimate of 16.41 cases per 100,000 cattle. For birth cohorts ranging from 2002 to 2013, the estimated median prevalence was below one case per 100,000 heads. The calculated confidence in freedom from disease (design prevalence 1 in 100,000) was above 99.5% for the birth cohorts 2002 to 2006. In conclusion, BSE surveillance in the healthy slaughtered cattle chain was extremely sensitive at the time, when BSE repeatedly occurred in Germany (2000-2009), because the entry of BSE-infected cattle into the food chain could virtually be prevented by the extensive surveillance program during these years and until 2015 (estimated non-detected cases/100.000 [95% credible interval] in 2000, 2009, and 2015 are 0.64 [0.5,0.8], 0.05 [0.01,0.14], and 0.19 [0.05,0.61], respectively).

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

To understand the possible role of mixed-prion infections in disease presentation, the current study reports the co-infection of sheep with bovine spongiform encephalopathy (BSE) and scrapie. The bovine BSE agent was inoculated subcutaneously into sheep with ARQ/ARQ or VRQ/ARQ PRNP genotypes either at the same time as subcutaneous challenge with scrapie, or three months later. In addition, VRQ/VRQ sheep naturally infected with scrapie after being born into a scrapie-affected flock were challenged subcutaneously with BSE at eight or twenty one months-of-age. Sheep were analysed by incubation period/attack rate, and western blot of brain tissue determined the presence of BSE or scrapie-like PrPSc. Serial protein misfolding cyclic amplification (sPMCA) that can detect very low levels of BSE in the presence of an excess of scrapie agent was also applied to brain and lymphoreticular tissue. For VRQ/ARQ sheep challenged with mixed infections, scrapie-like incubation periods were produced, and no BSE agent was detected. However, whilst ARQ/ARQ sheep developed disease with BSE-like incubation periods, some animals had a dominant scrapie western blot phenotype in brain, but BSE was detected in these sheep by sPMCA. In addition, VRQ/VRQ animals challenged with BSE after natural exposure to scrapie had scrapie-like incubation periods and dominant scrapie PrPSc in brain, but one sheep had BSE detectable by sPMCA in the brain. Overall, the study demonstrates for the first time that for scrapie/BSE mixed infections, VRQ/ARQ sheep with experimental scrapie did not propagate BSE but VRQ/VRQ sheep with natural scrapie could propagate low levels of BSE, and whilst BSE readily propagated in ARQ/ARQ sheep it was not always the dominant PrPSc strain in brain tissue. Indeed, for several animals, a dominant scrapie biochemical phenotype in brain did not preclude the presence of BSE prion.

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.

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.

PMCA Applications for Prion Detection in Peripheral Tissues of Patients with Variant Creutzfeldt-Jakob Disease.

Prion diseases are neurodegenerative and invariably fatal conditions that affect humans and animals. In particular, Creutzfeldt-Jakob disease (CJD) and bovine spongiform encephalopathy (BSE) are paradigmatic forms of human and animal prion diseases, respectively. Human exposure to BSE through contaminated food caused the appearance of the new variant form of CJD (vCJD). These diseases are caused by an abnormal prion protein named PrPSc (or prion), which accumulates in the brain and leads to the onset of the disease. Their definite diagnosis can be formulated only at post-mortem after biochemical and neuropathological identification of PrPSc. Thanks to the advent of an innovative technique named protein misfolding cyclic amplification (PMCA), traces of PrPSc, undetectable with the standard diagnostic techniques, were found in peripheral tissues of patients with vCJD, even at preclinical stages. The technology is currently being used in specialized laboratories and can be exploited for helping physicians in formulating an early and definite diagnosis of vCJD using peripheral tissues. However, this assay is currently unable to detect prions associated with the sporadic CJD (sCJD) forms, which are more frequent than vCJD. This review will focus on the most recent advances and applications of PMCA in the field of vCJD and other human prion disease diagnosis.

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.

Deciphering the BSE-type specific cell and tissue tropisms of atypical (H and L) and classical BSE.

After the discovery of two atypical bovine spongiform encephalopathy (BSE) forms in France and Italy designated H- and L-BSE, the question arose whether these new forms differed from classical BSE (C-BSE) in their pathogenesis. Samples collected from cattle in the clinical stage of BSE during an intracranial challenge study with L- and H-BSE were analysed using biochemical and histological methods as well as in a transgenic mouse bioassay. Our results generally confirmed what had been described for C-BSE to be true also for both atypical BSE forms, namely the restriction of the pathological prion protein (PrPSc) and BSE infectivity to the nervous system. However, analysis of samples collected under identical conditions from both atypical H- and L-BSE forms allowed us a more precise assessment of the grade of involvement of different tissues during the clinical end stage of disease as compared to C-BSE. One important feature is the involvement of the peripheral nervous and musculoskeletal tissues in both L-BSE and H-BSE affected cattle. We were, however, able to show that in H-BSE cases, the PrPSc depositions in the central and peripheral nervous system are dominated by a glial pattern, whereas a neuronal deposition pattern dominates in L-BSE cases, indicating differences in the cellular and topical tropism of both atypical BSE forms. As a consequence of this cell tropism, H-BSE seems to spread more rapidly from the CNS into the periphery via the glial cell system such as Schwann cells, as opposed to L-BSE which is mostly propagated via neuronal cells.

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.

Early and Pre-Clinical Detection of Prion Seeding Activity in Cerebrospinal Fluid of Goats using Real-Time Quaking-Induced Conversion Assay.

Since 2005, two cases of natural bovine spongiform encephalopathies (BSE) have been reported in goats. Furthermore, experimental transmissions of classical (C-BSE) and atypical (L-BSE) forms of BSE in goats were also reported. To minimize further spreading of prion diseases in small ruminants the development of a highly sensitive and specific test for ante-mortem detection of infected animals would be of great value. Recent studies reported high diagnostic value of a second generation of cerebrospinal fluid (CSF) Real-Time Quaking-Induced Conversion (RT-QuIC) assay across a wide spectrum of human prions. Here, we applied this improved QuIC (IQ-CSF) for highly efficient detection of TSEs prion protein in goat cerebrospinal fluid. IQ-CSF sensitivity and specificity were evaluated on CSF samples collected at disease endpoint from goats naturally and experimentally infected with scrapie or bovine isolates of C-BSE and L-BSE, respectively. Next, CSF samples collected from L-BSE infected goats during pre-symptomatic stage were also analysed. PrPL-BSE associated seeding activity was detected at early time points after experimental inoculation, with an average time of 439 days before clinical symptoms appeared. Taken together these data are indicative of the great potential of this in vitro prion amplification assay as ante-mortem TSE test for live and asymptomatic small ruminants.

Atypical and classic bovine spongiform encephalopathy.

This chapter describes the prion diseases of cattle, or bovine transmissible spongiform encephalopathies (BoTSEs). "Classic" bovine spongiform encephalopathy (C-BSE), the major prion protein disorder of Bovidae, was first described in 1986. We also describe the spatiotemporal correlation of C-BSE to a novel form of human prion disease, variant Creutzfeldt-Jakob disease (vCJD), which led to the classification of BSE as a zoonotic disease (and the "cause" of vCJD) in 1996. From isolated cases first identified retrospectively in May 1985, a major bovine spongiform encephalopathy (BSE) epidemic peaked within the British Isles in 1991, and has so far led to over 195,000 confirmed cases in cattle, and several thousand more cases within Europe and a few elsewhere. By 2017, the disease had been essentially eradicated below the level of surveillance detection in Europe, although sporadic cases are still predicted to show up for several years to come. By 2004, other forms of BSE, H-type and L-type, had been recognized and, in the absence of information to the contrary, are also included in the classification of BSE as a zoonotic prion disease of bovine origin. This chapter aims to cover the clinical, epidemiologic, neuropathologic, biochemical, biomarker, and pathogenetic aspects of the bovine prion disease and gives a brief, but important, description of the surveillance of BSE and other animal TSEs in Europe.