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Neurodegenerative diseases (NDs) in mammals, such as Alzheimer's disease (AD), Parkinson's disease (PD), and transmissible spongiform encephalopathies (TSEs), are characterized by the accumulation of misfolded proteins in the central nervous system (CNS). Despite the presence of these pathogenic proteins, the immune response in affected individuals remains notably muted. Traditional immunological strategies, particularly those reliant on monoclonal antibodies (mAbs), face challenges related to tissue penetration, blood-brain barrier (BBB) crossing, and maintaining protein stability. This has led to a burgeoning interest in alternative immunotherapeutic avenues. Notably, single-domain antibodies (or nanobodies) and aptamers have emerged as promising candidates, as their reduced size facilitates high affinity antigen binding and they exhibit superior biophysical stability compared to mAbs. Aptamers, synthetic molecules generated from DNA or RNA ligands, present both rapid production times and cost-effective solutions. Both nanobodies and aptamers exhibit inherent qualities suitable for ND research and therapeutic development. Cross-seeding events must be considered in both traditional and small-molecule-based immunodiagnostic and therapeutic approaches, as well as subsequent neurotoxic impacts and complications beyond protein aggregates. This review delineates the challenges traditional immunological methods pose in ND research and underscores the potential of nanobodies and aptamers in advancing next-generation ND diagnostics and therapeutics.

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OBJECTIVE: The goal of the research presented here is to determine if methods previously developed for the aqueous extraction of PrPSc from formalin-fixed paraffin-embedded tissue (FFPET) are applicable to the detection PrPSc by real-time quaking induced conversion (RT-QuIC). Previous work has utilized aqueous extraction of FFPET for detection of transmissible spongiform encephalopathies (TSEs) utilizing western blot and ELISA. This research extends the range of suitable methods for detection of TSEs in FFPET to RT-QuIC, which is arguably the most sensitive method to detect TSEs. RESULTS: We found complete agreement between the TSE status and the results from RT-QuIC seeded with the aqueous extract of FFPET samples. The method affords the diagnostic assessment TSE status by RT-QuIC of FFPET without the use of organic solvents that would otherwise create a mixed chemical-biological waste for disposal.

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Atypical scrapie is a transmissible spongiform encephalopathy that is rarely diagnosed in living animals. In March 2022, a 7-y-old Herdwick ewe was referred to the Scottish Centre for Production Animal Health and Food Safety because of circling behavior and ill thrift. The ewe had a low body condition score, was obtunded, with a wide-based stance of the pelvic limbs, and was circling to the left. Hematologic, biochemical, and CSF analyses were unremarkable, but postmortem magnetic resonance imaging (MRI) findings were consistent with diffuse, bilateral, and symmetrical atrophy of the forebrain and ventriculomegaly. The clinical signs, the involvement of an individual older ewe, and the MRI results led to the clinical diagnosis of scrapie. Immunohistochemistry on the fixed brain, performed by the U.K. Animal and Plant Health Agency, revealed deposits of PrPSc, which is a specific disease marker of transmissible spongiform encephalopathies, mainly in the cerebellum and at lower concentrations in the cerebrum and obex, consistent with the diagnosis of atypical scrapie. MRI findings in a sheep with atypical scrapie have not been described previously, to our knowledge. Scrapie should be included in the list of clinical differential diagnoses when veterinarians are presented with sheep with progressive neurologic signs of several weeks' duration.

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Scrapie is a transmissible spongiform encephalopathy affecting sheep and goats. The prion protein-encoding gene (PRNP) plays a crucial role in determining susceptibility and resistance to scrapie. At the European level, surveillance of scrapie is essential to prevent the spread of the disease to livestock. According to the Regulation EU 2020/772 polymorphisms K222, D/S146 could function as resistance alleles in the genetic management of disease prevention. In Italy, a breeding plan for scrapie eradication has not been implemented for goats. However, surveillance plans based on the PRNP genotype have been developed as a preventive measure for scrapie. This research aimed to describe the polymorphisms at 7 positions within the PRNP gene in 956 goats of the Alpine, Saanen and mixed populations farmed in the Lombardy Region in Italy. PRNP polymorphisms were detected using single nucleotide polymorphism markers included in the Neogen GGP Goat 70 k chip. The K222 allele occurred in all populations, with frequencies ranging from 2.1 to 12.7%. No animals carried the S/D146 resistance allele. However, it has been demonstrated that polymorphisms in the other positions analysed could influence resistance or susceptibility to scrapie outbreaks in different ways. Ten potentially distinct haplotypes were found, and the most prevalent of the three populations was H2, which differed from the wild type (H1) in terms of mutation (S vs P) at codon 240. This study provided additional information on the genetic variability of the PRNP gene in these populations in the Lombardy region of Italy, contributing to the development of genetic control measures for disease prevention.

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After the detection of bovine spongiform encephalopathy (BSE), and a zoonotic transmissible spongiform encephalopathy (TSE) caused by the pathological prion protein (PrPSc) in two goats, the investigation of goat prions became of greater interest. Therefore, a broad collection of European goat TSE isolates, including atypical scrapie, CH1641 and goat BSE as reference prion strains were biochemically characterised and subsequently inoculated into seven rodent models for further analysis (already published results of this comprehensive study are reviewed here for comparative reasons). We report here the histopathological and immunohistochemical data of this goat TSE panel, obtained after the first passage in Tgshp IX (tg-shARQ) mice, which overexpress the ovine prion protein. In addition to the clear-cut discrimination of all reference prion strains from the classical scrapie (CS) isolates, we were further able to determine three categories of CS strains. The investigation further indicates the occurrence of sub-strains that slightly resemble distant TSE strains, such as BSE or CH1641, reinforcing the theory that CS is not a single strain but a mixture of sub-strains, existing at varying extents in one isolate. This study further proved that Tgshp IX is a potent and reliable tool for the in-depth characterisation of prion strains.

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Key Clinical Message: Creutzfeldt-Jakob disease is a neurodegenerative disorder caused by brain accumulation of a misfolded form of the cellular prion protein, whose diagnosis is challenging, particularly in early stages, due to the variability and nonspecificity of the clinical and radiological features. 18F-fluorodeoxyglucose positron-emitted tomography has the potential to be considered a crucial investigation in these patients, revealing metabolic abnormalities earlier than the conventional neuroimaging analysis. Abstract: A 59-year-old man, the military officer, was referred to our Units for the onset of neurological symptoms rapidly evolving within a month, characterized by akinetic mutism, constructional apraxia, and disorders of spatial orientation. Brain 18F-fluorodeoxyglucose (18F-FDG) positron-emitted tomography (PET)/CT depicted an asymmetric hypometabolism in the left fronto-temporo-parietal cortex, as well as in the left thalamus and the right cerebellar hemisphere, while the glucose metabolism appears to be preserved in the somatosensory cortex and the basal ganglia. Laboratory routine analyses, cerebrospinal fluid routine, infective tests, electroencephalography (EEG), and brain magnetic resonance (MR) were all unremarkable. A positive RT-QuIC result on cerebro-spinal fluid (CSF) was subsequently shown, without any pathogenic gene mutations and, therefore, the result was consistent with a diagnosis of sporadic Creutzfeld-Jacob disease. The clinical evolution was quickly unfavorable, and the patient died about 4 months after hospital admission. FDG PET/computed tomography (CT) has the potential to be considered a crucial investigation in these patients, documenting metabolic changes long time before other diagnostic investigations such as CSF, EEG, brain CT, and brain MR, thus suggesting a greater sensitivity of glucose metabolic evaluation in the early stage of the disease in question.

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The first case of CWD in a Norwegian red deer was detected by a routine ELISA test and confirmed by western blotting and immunohistochemistry in the brain stem of the animal. Two different western blotting tests were conducted independently in two different laboratories, showing that the red deer glycoprofile was different from the Norwegian CWD reindeer and CWD moose and from North American CWD. The isolate showed nevertheless features similar to the classical BSE (BSE-C) strain. Furthermore, BSE-C could not be excluded based on the PrPSc immunohistochemistry staining in the brainstem and the absence of detectable PrPSc in the lymphoid tissues. Because of the known ability of BSE-C to cross species barriers as well as its zoonotic potential, the CWD red deer isolate was submitted to the EURL Strain Typing Expert Group (STEG) as a BSE-C suspect for further investigation. In addition, different strain typing in vivo and in vitro strategies aiming at identifying the BSE-C strain in the red deer isolate were performed independently in three research groups and BSE-C was not found in it. These results suggest that the Norwegian CWD red deer case was infected with a previously unknown CWD type and further investigation is needed to determine the characteristics of this potential new CWD strain.

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Integrating host movement and pathogen data is a central issue in wildlife disease ecology that will allow for a better understanding of disease transmission. We examined how adult female mule deer (Odocoileus hemionus) responded behaviorally to infection with chronic wasting disease (CWD). We compared movement and habitat use of CWD-infected deer (n = 18) to those that succumbed to starvation (and were CWD-negative by ELISA and IHC; n = 8) and others in which CWD was not detected (n = 111, including animals that survived the duration of the study) using GPS collar data from two distinct populations collared in central Wyoming, USA during 2018-2022. CWD and predation were the leading causes of mortality during our study (32/91 deaths attributed to CWD and 27/91 deaths attributed to predation). Deer infected with CWD moved slower and used lower elevation areas closer to rivers in the months preceding death compared with uninfected deer that did not succumb to starvation. Although CWD-infected deer and those that died of starvation moved at similar speeds during the final months of life, CWD-infected deer used areas closer to streams with less herbaceous biomass than starved deer. These behavioral differences may allow for the development of predictive models of disease status from movement data, which will be useful to supplement field and laboratory diagnostics or when mortalities cannot be quickly retrieved to assess cause-specific mortality. Furthermore, identifying individuals who are sick before predation events could help to assess the extent to which disease mortality is compensatory with predation. Finally, infected animals began to slow down around 4 months prior to death from CWD. Our approach for detecting the timing of infection-induced shifts in movement behavior may be useful in application to other disease systems to better understand the response of wildlife to infectious disease.

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Acute ischemic cerebrovascular accident (CVA) is a time-sensitive emergent diagnosis, requiring rapid diagnosis and consideration of thrombolytic administration. However, a myriad of cerebrovascular mimics creates a diagnostic challenge. A rare CVA mimic is Creutzfeldt-Jakob disease (CJD), a rapidly progressive fatal dementia due to protein misfolding. Magnetic resonance imaging (MRI) and neurology consultation for electroencephalogram (EEG) and specialized cerebrospinal fluid (CSF) studies are diagnostic while the patient is alive. All forms are fatal within months, and diagnosis can be confirmed on postmortem brain testing. While incredibly uncommon, emergency clinicians should consider this diagnosis in the proper patient to advocate for specialized CSF testing and potential palliative care consultation.

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Heroin-induced leukoencephalopathy (HLE) is a rare toxic encephalopathy associated primarily with heroin inhalation, commonly referred to as "chasing the dragon." This study presents a clinical case of a 27-year-old polydrug user diagnosed with HLE during hospitalization for rapidly progressive flaccid tetraplegia and aphasia. The clinical manifestations encompassed cerebellar and bulbar dysfunction, coupled with motor impairment and altered consciousness. Based on the clinical data and MRI results, HLE was identified as the most likely cause. This article aims to provide insights into the clinical and radiological aspects of HLE, emphasizing the diagnostic significance of radiological findings. The gold standard examination for diagnosis is MRI, crucial due to the difficulties in obtaining histological confirmation for this rare condition.

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As chronic wasting disease (CWD) continues to spread across North America, the relationship between CWD and host genetics has become of interest. In Rocky Mountain elk (Cervus elaphus nelsoni), one or two copies of a leucine allele at codon 132 of the prion protein gene (132L*) has been shown to prolong the incubation period of CWD. Our study examined the relationship between CWD epidemiology and codon 132 evolution in elk from Wyoming, USA, from 2011 to 2018. Using PCR and Sanger sequencing, we genotyped 997 elk and assessed the relationship between genotype and CWD prevalence estimated from surveillance data. Using logistic regression, we showed that each 1% increase in CWD prevalence is associated with a 9.6% increase in the odds that an elk would have at least one copy of leucine at codon 132. In some regions, however, 132L* variants were found in the absence of CWD, indicating that evolutionary and epidemiologic patterns can be heterogeneous across space and time. We also provide evidence that naturally occurring CWD is not rare in 132L* elk, which merits the study of shedding kinetics in 132L* elk and the influence of genotype on CWD strain diversity. The management implications of cervid adaptations to CWD are difficult to predict. Studies that investigate the degree to which evolutionary outcomes are shaped by host spatial structure can provide useful epidemiologic insight, which can in turn aid management by informing scale and extent of mitigation actions.

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In 2006, a case of atypical H-type BSE (H-BSE) was found to be associated with a germline mutation in the PRNP gene that resulted in a lysine substitution for glutamic acid at codon 211 (E211K). The E211K amino acid substitution in cattle is analogous to E200K in humans, which is associated with the development of genetic Creutzfeldt-Jakob disease (CJD). In the present study, we aimed to determine the effect of the EK211 prion protein genotype on incubation time in cattle inoculated with the agent of H-BSE; to characterize the molecular profile of H-BSE in KK211 and EK211 genotype cattle; and to assess the influence of serial passage on BSE strain. Eight cattle, representing three PRNP genotype groups (EE211, EK211, and KK211), were intracranially inoculated with the agent of H-BSE originating from either a case in a cow with the EE211 prion protein genotype or a case in a cow with E211K amino acid substitution. All inoculated animals developed clinical disease; post-mortem samples were collected, and prion disease was confirmed through enzyme immunoassay, anti-PrPSc immunohistochemistry, and western blot. Western blot molecular analysis revealed distinct patterns in a steer with KK211 H-BSE compared to EK211 and EE211 cattle. Incubation periods were significantly shorter in cattle with the EK211 and KK211 genotypes compared to the EE211 genotype. Inoculum type did not significantly influence the incubation period. This study demonstrates a shorter incubation period for H-BSE in cattle with the K211 genotype in both the homozygous and heterozygous forms.

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The emergence of bovine spongiform encephalopathy (BSE) prions from atypical scrapie has been recently observed upon experimental transmission to rodent and swine models. This study aimed to assess whether the inoculation of atypical scrapie could induce BSE-like disease in cattle. Four calves were intracerebrally challenged with atypical scrapie. Animals were euthanized without clinical signs of prion disease and tested negative for PrPSc accumulation by immunohistochemistry and western blotting. However, an emergence of BSE-like prion seeding activity was detected during in vitro propagation of brain samples from the inoculated animals. These findings suggest that atypical scrapie may represent a potential source of BSE infection in cattle.

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Prion diseases are fatal neurodegenerative disorders in which the main pathogenic event is the conversion of the cellular prion protein (PrPC) into an abnormal and misfolded isoform known as PrPSc. Most prion diseases and their susceptibility and pathogenesis are mainly modulated by the PRNP gene that codes for PrP. Mutations and polymorphisms in the PRNP gene can alter PrPC amino acid sequence, leading to a change in transmission efficiency depending on the place where it occurs. Horses are animals that are considered to be highly resistant to prions. Several studies have attempted to identify polymorphisms in the PRNP gene that explain the reason for this high resistance. In this study, we have analysed 207 horses from 20 different breeds, discovering 3 novel PRNP polymorphisms. By using computer programmes such as PolyPhen-2, PROVEAN, PANTHER, Meta-SNP and PredictSNP, we have predicted the possible impact that these new polymorphisms would have on the horse prion protein. In addition, we measured the propensity for amyloid aggregation using AMYCO and analysed the lack of hydrogen bridges that these changes would entail together with their electrostatic potentials using Swiss-PdbViewer software, showing that an increased amyloid propensity could be due to changes at the level of electrostatic potentials.

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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.

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Prion diseases are a group of neurodegenerative, transmissible, and fatal disorders that affect several animal species. They are characterized by the conformational conversion of the cellular prion protein (PrPC) into the pathological prion protein (PrPSc). In 2016, chronic wasting disease (CWD) gained great importance at European level due to the first disease detection in a wild reindeer (Rangifer tarandus) in Norway. The subsequent intensive CWD surveillance launched in cervids resulted in the detection of CWD in moose (Alces alces), with 11 cases in Norway, 3 in Finland and 4 in Sweden. These moose cases differ considerably from CWD cases in North American and reindeer in Norway, as PrPSc was detectable in the brain but not in lymphoid tissues. These facts suggest the occurrence of a new type of CWD. Here, we show some immunohistochemical features that are clearly different from CWD cases in North American and Norwegian reindeer. Further, the different types of PrPSc deposits found among moose demonstrate strong variations between the cases, supporting the postulation that these cases could carry multiple strains of CWD.

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Sporadic Creutzfeldt-Jakob Disease (sCJD) is a rare neurodegenerative prion disease that presents with symptoms of rapid neuropsychiatric decline including dementia, behavioural abnormalities, and loss of higher cortical function. Patients commonly present with rapidly progressive neuromotor symptoms such as ataxia and myoclonus. Very few cases of CJD have been reported in which the patient initially presents with stroke symptoms such as hemiparesis as their primary presenting symptom. We present a case of a 56-year-old male who initially presented to the stroke unit with waxing and waning left-sided weakness and a non-corresponding ipsilateral left-sided acute parietal infarct on diffusion-weighted MRI. Over four weeks, his condition progressively worsened with declining cognitive function, motor dysfunction, sphincter dysfunction, and eventual death.

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Three retrospective lymphoreticular tissue studies (Appendix I, II, and III) aimed to estimate the UK prevalence of variant Creutzfeldt-Jakob disease (vCJD), following exposure of the population to the bovine spongiform encephalopathy (BSE) agent, in the late 1980s and 1990s. These studies evaluated the presence of abnormal prion protein aggregates, in archived formalin-fixed paraffin-embedded (FFPE) appendectomy samples, by immunohistochemical detection. Although there was concordance in the estimated prevalence of vCJD from these studies, the identification of positive specimens from pre- and post-BSE-exposure periods in Appendix III study has raised questions regarding the nature and origin of the detected abnormal prion protein. We applied a robust and novel approach in the extraction of disease-associated prion protein (PrPSc) present in frozen and FFPE samples of brain and appendix from a patient with pathologically confirmed vCJD. The extracted material was used to seed the highly sensitive protein misfolding cyclic amplification assay (hsPMCA) to investigate the in vitro and in vivo propagation properties of the extracted abnormal prion protein. We demonstrate that PrPSc can be successfully extracted from FFPE appendix tissue and propagated in vitro. Bioassay in wild-type and gene-targeted mouse models confirmed that the extracted and amplified product is infectious and retains strain properties consistent with vCJD. This provides a highly sensitive and reliable platform for subsequent analysis of the archived FFPE appendix tissue derived from the Appendix II and III surveys, to further evaluate the nature of the abnormal PrP detected in the positive samples.

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Creutzfeldt-Jakob disease surveillance in Australia: update to 31 December 2022: Nationwide surveillance of Creutzfeldt-Jakob disease (CJD) and other human prion diseases is performed by the Australian National Creutzfeldt-Jakob Disease Registry (ANCJDR). National surveillance encompasses the period since 1 January 1970, with prospective surveillance occurring from 1 October 1993. Over this prospective surveillance period, considerable developments have occurred in pre-mortem diagnostics; in the delineation of new disease subtypes; and in a heightened awareness of prion diseases in healthcare settings. Surveillance practices of the ANCJDR have evolved and adapted accordingly. This report summarises the activities of the ANCJDR during 2022. Since the ANCJDR began offering diagnostic cerebrospinal fluid (CSF) 14-3-3 protein testing in Australia in September 1997, the annual number of referrals has steadily increased. In 2022, a total of 599 domestic CSF specimens were referred for diagnostic testing and 79 persons with suspected human prion disease were formally added to the national register. As of 31 December 2022, just under half of the 79 suspect case notifications (36/79) remain classified as 'incomplete'; 15 cases were classified as 'definite' and 23 as 'probable' prion disease; five cases were excluded through neuropathological examination. For 2022, fifty-five percent of all suspected human-prion-disease-related deaths in Australia underwent neuropathological examination. No cases of variant or iatrogenic CJD were identified. The SARS-CoV-2 pandemic did not affect prion disease surveillance outcomes in Australia during 2022.

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Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disease that belongs to a group of diseases known as transmissible spongiform encephalopathies (TSEs). It is believed that the infectious agent responsible for prion diseases is abnormally folded prion protein (PrPSc), which derives from a normal cellular protein (PrPC), which is a cell surface glycoprotein predominantly expressed in neurons. There are three different types of BSE, the classical BSE (C-type) strain and two atypical strains (H-type and L-type). BSE is primarily a disease of cattle; however, sheep and goats also can be infected with BSE strains and develop a disease clinically and pathogenically indistinguishable from scrapie. Therefore, TSE cases in cattle and small ruminants require discriminatory testing to determine whether the TSE is BSE or scrapie and to discriminate classical BSE from the atypical H- or L-type strains. Many methods have been developed for the detection of BSE and have been reported in numerous studies. Detection of BSE is mainly based on the identification of characteristic lesions or detection of the PrPSc in the brain, often by use of their partial proteinase K resistance properties. The objective of this paper was to summarize the currently available methods, highlight their diagnostic performance, and emphasize the advantages and drawbacks of the application of individual tests.

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