Transmissible Spongiform Encephalopathies Cases Research Articles

Conventional and State-of-the-Art Detection Methods of Bovine Spongiform Encephalopathy (BSE).

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.

Transmissible Spongiform Encephalopathies: An Underrecognized Infection Control Issue

Background: Transmissible spongiform encephalopathies comprise a class of rapidly progressive and inevitably fatal degenerative brain disorders. The pathogenesis of these diseases is thought to be due to a change in the structure of the normal prion protein to an abnormal structure, leading to propagation of the abnormal protein. This abnormal protein is highly transmissible; thus, appropriate infection control measures should be put in place if the diagnosis is suspected. However, the diagnosis is often not considered at all, and many hospitals do not have protocols in place. Our hospital missed a case of familial fatal insomnia in a 45-year-old male. He was diagnosed with fatal familial insomnia by autopsy. The autopsy was performed without appropriate infection control measures, leading to costly contamination of medical instruments and exposure of multiple staff. This occurrence led our institution to re-evaluate hospital protocols and guidelines regarding workup and management of transmissible spongiform encephalopathies (TSEs). Methods: We reviewed cases of TSEs or Creutzfeldt-Jakob Disease (CJD)-like illness presenting to our hospital over a 30-month period. Patients were considered for inclusion based on clinical suspicion. CDC diagnostic criteria were used. Infection control measures were employed, including an alert in the EMR. MRI was then performed. If clinical or diagnostic suspicion was high, the patient underwent lumbar puncture. CSF results were reviewed based on criteria Creutzfeldt-Jakob Disease Foundation criteria. Infection control measures were maintained throughout hospitalization. Results: In total, 34 patients met the inclusion criteria: 8 patients had confirmed CJD and 25 were negative. Medical records were not available for 1 patient, who was excluded. Lumbar puncture was performed on all suspected cases. Of those confirmed cases, the 7 patients who underwent lumber puncture had a positive result for 14-3-3 protein. Also, 5 patients underwent RT-QuIC testing and were found to have a positive result. No further cases of contamination occurred using our protocol. Additionally, 1 patient with suspected CJD underwent a brain biopsy with appropriate precautions after an inconclusive lumbar puncture. Although biospy was negative, the case exemplifies how the initiation of a protocol can optimize the workflow and prevent potentially dangerous exposure. Conclusion: Diagnosis of TSEs remains difficult and is often missed. In our case, lack of suspicion for TSE led to a waste of resources and unnecessary exposure of staff member. It is of utmost importance to consider TSEs in rapidly progressive dementia and to employ appropriate sterile guidelines to prevent contamination of equipment and potential subsequent transmission. Healthcare providers should consider a similar protocol in cases suspicious for TSEs.Funding: NoneDisclosures: None

Role of Folding Intermediates in Initiating Aggregation of the Prion Protein

The spontaneous conformational conversion of the constitutively expressed, non‐infectious, cellular prion protein, PrPC, from its natively‐folded functional state to infectious, mis‐folded, amyloidogenic, PrPSc forms is the primary cause of most cases of transmissible spongiform encephalopathies (TSEs). TSEs are typically characterized by severe motor‐neuronal and cognitive impairments and progressive dementia accompanied by spongiform degeneration of the mammalian brain. Although the mechanism of this spontaneous conformational conversion process is not known, various studies in vitro have implicated that the conformational flexibility of the native (N) state of the prion protein is a crucial factor in the initiation of aggregation.High conformational flexibility of N states of proteins facilitates fluctuations to aberrant high energy intermediate conformations with altered topology and exposure of hydrophobic surfaces capable of initiating aggregation. However, owing to their transient nature, detection and structural characterization of such aggregation‐prone conformations has been difficult. Nevertheless, such conformations have been shown to initiate aggregation in a few pathogenic and non‐pathogenic proteins. This study aims at understanding the molecular basis of initiation of prion misfolding and aggregation by identifying and structurally characterizing the monomeric precursor conformation which initiates aggregation of the prion protein.In this study, mutagenesis in the hydrophobic core of the globular domain of the mouse prion protein is observed to stabilize a monomeric, on‐pathway, unfolding intermediate conformation, which populates significantly at equilibrium. The rate of misfolding of the protein to cytotoxic β‐structured oligomers exhibiting partial proteinase‐K resistance, which is a hall‐mark feature of PrPSc aggregates, is found to correlate well with the extent to which the intermediate populates. Additionally, the oligomerization process is limited by dimerization of this intermediate suggesting that it is the direct monomeric precursor initiating misfolding and oligomerization. Structural and energetic characterization of the intermediate by native‐state hydrogen exchange monitored by mass spectrometry and NMR indicates that the intermediate is a partially unfolded form of the native state of the protein populated under misfolding‐prone solvent conditions, where the α1‐β2 subdomain is separated from the hydrophobic α2‐α3 subdomain thus exposing the aggregation‐prone α2‐α3 helices to solvent. Kinetic folding studies in such misfolding‐prone solvent conditions using rapid mixing (in μs‐timescales) techniques indicate that the prion protein folds through multiple pathways with the aggregation‐prone intermediate being obligatory to the folding of the protein.Thus, a partially unfolded conformation of the prion protein acts as a common monomeric intermediate between the folding and aggregation of the prion protein. Structural characterization of this intermediate carried out in this study may significantly contribute to the design of drugs to prevent the misfolding and aggregation of the prion protein.Support or Funding InformationFunded by Tata Institute of Fundamental Research and by the Department of Science and Technology, Government of India

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

BackgroundThe 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.ResultsAs 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.ConclusionsCharacterisation 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1260-3) contains supplementary material, which is available to authorized users.

Ability of wild type mouse bioassay to detect bovine spongiform encephalopathy (BSE) in the presence of excess scrapie.

IntroductionScrapie and bovine spongiform encephalopathy (BSE) are transmissible spongiform encephalopathies (TSEs) which naturally affect small and large ruminants respectively. However, small ruminants, which are susceptible to BSE under experimental conditions, have been exposed to the same or similar contaminated food additives as cattle. To date two natural cases of BSE in small ruminants have been reported. As a result surveillance projects, combined with appropriate control measures, have been established throughout the European Union (EU) to minimize the overall incidence of small ruminant TSEs. Although BSE can be differentiated from classical scrapie (subsequently referred to as scrapie) if appropriate discriminatory tests are applied, the value of these tests in BSE/scrapie co-infection scenarios has not been evaluated fully. Mouse bioassay is regarded as the gold standard regarding differentiation of distinct TSE strains and has been used as to resolve TSE cases were laboratory tests produced equivocal results. However, the ability of this method to discriminate TSE strains when they co-exist has not been examined systematically. To address this issue we prepared in vitro mixtures of ovine BSE and scrapie and used them to challenge RIII, C57BL/6 and VM mice.ResultsDisease phenotype analysis in all three mouse lines indicated that most phenotypic parameters (attack rates, incubation periods, lesion profiles and Western blots) were compatible with scrapie phenotypes as were immunohistochemistry (IHC) data from RIII and C57BL/6 mice. However, in VM mice that were challenged with BSE/scrapie mixtures a single BSE-associated IHC feature was identified, indicating the existence of BSE in animals where the scrapie phenotype was dominant.ConclusionsWe conclude that wild type mouse bioassay is of limited value in detecting BSE in the presence of scrapie particularly if the latter is in relative excess.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-015-0194-2) contains supplementary material, which is available to authorized users.

Laboratory activities involving transmissible spongiform encephalopathy causing agents

Since the appearance in 1986 of epidemic of bovine spongiform encephalopathy (BSE), a new form of neurological disease in cattle which also affected human beings, many diagnostic and research activities have been performed to develop detection and therapeutic tools. A lot of progress was made in better identifying, understanding and controlling the spread of the disease by appropriate monitoring and control programs in European countries. This paper reviews the recent knowledge on pathogenesis, transmission and persistence outside the host of prion, the causative agent of transmissible spongiform encephalopathies (TSE) in mammals with a particular focus on risk (re)assessment and management of biosafety measures to be implemented in diagnostic and research laboratories in Belgium. Also, in response to the need of an increasing number of European diagnostic laboratories stopping TSE diagnosis due to a decreasing number of TSE cases reported in the last years, decontamination procedures and a protocol for decommissioning TSE diagnostic laboratories is proposed.

Species-barrier phenomenon in prion transmissibility from a viewpoint of protein science

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal infectious neurodegenerative disorders. Their causative agents are prions, which are composed of disease-associated forms of prion protein (PrP(Sc)). Naturally occurring cases of TSEs are found in several mammalian species including humans, sheep, goats, minks, cattle and deer. Prions are also experimentally transmissible to other mammals such as mice, hamsters and monkeys, but interspecies transmission is often inefficient due to the 'species-barrier'. Studies have suggested that the barrier is not only simply determined by differences in amino acid sequences of cellular PrP (PrP(C)) among animal species, but also by prion strains which are closely associated with conformational properties of PrP(Sc) aggregates. Although the conformational properties of PrP(Sc) remain largely unknown, recent investigation of local structures of PrP(C) and, in particular, structural modelling of PrP(Sc) aggregates have provided molecular insight into this field. In this review, we discuss the species-barrier phenomenon in terms of the protein science.

Selection of Distinct Strain Phenotypes in Mice Infected by Ovine Natural Scrapie Isolates Similar to CH1641 Experimental Scrapie

A few cases of transmissible spongiform encephalopathies in sheep have been described in France in which the protease-resistant prion protein (PrP(res)) exhibited some features in Western blot of experimental bovine spongiform encephalopathy in sheep. Their molecular characteristics were indistinguishable from those produced in the CH1641 experimental scrapie isolate. Four of these CH1641-like isolates were inoculated intracerebrally into wild-type C57Bl/6 mice. In striking contrast to previous results in ovine transgenic mice, CH1641 transmission in wild-type mice was efficient. Several components of the strain signature, that is, PrP(res) profile, brain distribution, and morphology of the deposits of the disease-associated prion protein, had some similarities with "classical" scrapie and clearly differed from both bovine spongiform encephalopathy in sheep and CH1641 transmission in ovine transgenic mice. These results on CH1641-like isolates in wild-type mice may be consistent with the presence in these isolates of mixed conformers with different abilities to propagate and mediate specific disease phenotypes in different species.

Heart rate variability analysis in sheep affected by transmissible spongiform encephalopathies

BackgroundThe function of the autonomic nervous system can be assessed by determining heart rate variability (HRV), which is impaired in some brainstem diseases in humans. Transmissible spongiform encephalopathies (TSEs) in sheep are diseases characterised by accumulation of disease-associated prion protein in the brainstem, including nuclei of the parasympathetic nervous system. This study was undertaken to assess whether analysis of HRV can be used as an aid in the diagnosis of TSEs in clinically affected, naturally or experimentally infected sheep.FindingsWhen HRV indices were compared between 41 clinical TSE cases (18 sheep infected with scrapie and 23 sheep infected with bovine spongiform encephalopathy), 11 control sheep and six sheep reported as scrapie suspects or dosed with BSE brain homogenate, which were not confirmed as TSE cases by postmortem tests, no significant differences were found between the groups. Median heart rate was significantly different but only when sheep were grouped by gender: it was higher in female TSE cases than in control sheep and higher in female than castrated male ovine classical BSE cases.ConclusionsHRV analysis was not useful as a diagnostic aid for TSEs of sheep.

Creutzfeldt-Jakob Disease Surveillance in Argentina, 1997–2008

Background: Epidemiological data on Creutzfeldt-Jakob disease (CJD) from Latin America are limited. We present a comprehensive epidemiological survey on CJD patients in Argentina based on systematic surveillance between 1997 and 2008. Methods: A CJD Surveillance Referral Center (SRC) was established in Argentina in 1997; previously a Neuropathology Referral Center was used from 1983 to 1996. All suspected cases referred to the SRC were classified using established criteria on the basis of information derived from the following: clinical data form, EEG, MRI (both for central review), cerebrospinal fluid (CSF) for protein 14-3-3 Western blot (WB), autopsy or biopsy material for neuropathology, prion protein (PrP) immunohistochemistry and PrP WB, as well as blood for DNA studies (when brain tissue was not available). Results: Of the 517 patients referred to the SRC between 1997 and 2008, 211 (40.8%) had CJD or other transmissible spongiform encephalopathies (TSEs) (definite or probable). Possible cases totaled 14.5%, while cases with no WHO criteria accounted for 16.4%. Non-CJD cases excluded by biopsy/autopsy or during follow-up corresponded to 28.2% of the 517 referrals. Main differential diagnoses included neurodegenerative diseases such as Alzheimer’s disease, frontotemporal dementia, vascular, metabolic or viral encephalopathy, and Hashimoto’s disease. Five percent of referred patients ultimately recovered. Eighty-three percent of TSE cases were sporadic CJD; 17% were genetic, mainly E200K (15.6%); the remaining 1.4% included an octarepeat insertion and two Gerstmann-Sträussler-Scheinker cases (P102L). Seventy-four of 100 definite cases had frozen tissue available for molecular subtyping (PrP^Sc/codon 129). CSF protein 14-3-3 WB sensitivity was 72.3% and specificity was 92.1%. Clinical diagnostic criteria for probable CJD when compared to definite diagnosis by neuropathology showed 71.3% sensitivity, 86.2% specificity, 94.4% positive predictive value and 48% negative predictive value. Country incidence increased over time and reached 0.85 cases per million in 2008, with the highest rate detected in the city of Buenos Aires (1.8). Districts with 6% of the total population have never reported suspected cases. Conclusion: In spite of an increase in incidence observed over time, the difference between Buenos Aires city, where the incidence is comparable to that of smaller European countries with higher population density, and the incidence observed in the rest of the country suggests underreporting in nonmetropolitan areas, probably due to a lack of access to specialized medical facilities. CSF WB sensitivity results for protein 14-3-3 were probably linked to the fact that testing was not routinely repeated during the course of the disease, when earlier test results had been negative. The spectrum of molecular CJD subtypes observed did not differ from other countries in Europe. No iatrogenic or variant CJD cases were identified. The sensitivity and negative predictive value of clinical diagnostic criteria for probable CJD (which includes EEG and/or CSF protein 14-3-3 levels) may have been resulted from confirmed cases not meeting probable criteria before autopsy, due to a lack of ancillary tests such as EEG and/or CSF 14-3-3 WB, or because negative tests were not repeated during follow-up.

Comprehensive Neuropathologic Analysis of Genetic Prion Disease Associated With the E196K Mutation inPRNPReveals Phenotypic Heterogeneity

The genetic forms of human transmissible spongiform encephalopathies (TSEs) are linked to mutations in the gene encoding the prion protein (PRNP) and account for 10% to 15% of human TSE cases. Some are distinct with respect to clinical signs, disease onset/duration, and diagnostic findings, whereas others closely resemble sporadic Creutzfeldt-Jakob disease (sCJD). We report a comprehensive analysis of 4 patients carrying the rare E196K (GAG→AAG) mutation who presented with clinical features of CJD. To date, information on this PRNP mutation is limited to clinical and genetic data. Consequently, the E196K mutation could not be unequivocally assigned to human prion disease. We report histopathologic and biochemical findings in addition to clinical observations, thus providing a more comprehensive analysis of this presumably genetic prion disease. Our data indicate that (i) the E196K mutation is causally linked to human prion disease, (ii) there is a complex phenotypic spectrum of this mutation that includes nonspecific symptoms at onset and features typical of sCJD during disease progression, and (iii) the corresponding histologic picture comprises both cases with atypical neuropathology and cases that closely resemble subtypes of sCJD corresponding to the classification of Parchi et al, with subtle modifications in hippocampal regions CA1-4.

Surveillance and simulation of bovine spongiform encephalopathy and scrapie in small ruminants in Switzerland

BackgroundAfter bovine spongiform encephalopathy (BSE) emerged in European cattle livestock in 1986 a fundamental question was whether the agent established also in the small ruminants' population. In Switzerland transmissible spongiform encephalopathies (TSEs) in small ruminants have been monitored since 1990. While in the most recent TSE cases a BSE infection could be excluded, for historical cases techniques to discriminate scrapie from BSE had not been available at the time of diagnosis and thus their status remained unclear. We herein applied state-of-the-art techniques to retrospectively classify these animals and to re-analyze the affected flocks for secondary cases. These results were the basis for models, simulating the course of TSEs over a period of 70 years. The aim was to come to a statistically based overall assessment of the TSE situation in the domestic small ruminant population in Switzerland.ResultsIn sum 16 TSE cases were identified in small ruminants in Switzerland since 1981, of which eight were atypical and six were classical scrapie. In two animals retrospective analysis did not allow any further classification due to the lack of appropriate tissue samples. We found no evidence for an infection with the BSE agent in the cases under investigation. In none of the affected flocks, secondary cases were identified. A Bayesian prevalence calculation resulted in most likely estimates of one case of BSE, five cases of classical scrapie and 21 cases of atypical scrapie per 100'000 small ruminants. According to our models none of the TSEs is considered to cause a broader epidemic in Switzerland. In a closed population, they are rather expected to fade out in the next decades or, in case of a sporadic origin, may remain at a very low level.ConclusionsIn summary, these data indicate that despite a significant epidemic of BSE in cattle, there is no evidence that BSE established in the small ruminant population in Switzerland. Classical and atypical scrapie both occur at a very low level and are not expected to escalate into an epidemic. In this situation the extent of TSE surveillance in small ruminants requires reevaluation based on cost-benefit analysis.

Creutzfeldt-Jakob Disease in Austria: An Autopsy-Controlled Study

Background: Definite diagnosis of prion diseases or transmissible spongiform encephalopathies (TSEs) requires neuropathology, usually at autopsy. Epidemiology of human TSEs has relied on definite as well as ‘probable’ cases in which neuropathological confirmation is lacking, usually because of low autopsy rates in most countries. Methods: In Austria, an active surveillance program for human prion diseases was established in 1996. Since then, more than 900 referrals were analyzed. Postmortem investigation of brain tissue is mandatory in every suspect case of TSE. Thus, epidemiological data on TSEs from Austria may serve as autopsy-controlled reference for countries with lower autopsy rates. Results: The total number of TSE cases in Austria since 1969 is 206. The average yearly mortality for the active surveillance period from 1996 to 30 June 2006 is 1.39 per million, with the highest rates for Vienna (2.37) compared with other provinces. Eighty-five percent of definite TSEs were classified as sporadic Creutzfeldt-Jakob disease (sCJD). We observed a significant linear increase in the mean age at death of 0.6 years per calendar year. Clinical diagnostic surveillance criteria had a sensitivity and specificity of 82.7 and 80.0% for probable CJD, respectively, and a positive predictive value of 80.5% for probable and 38.9% for ‘possible’ CJD. Alternative neuropathological diagnoses in suspect cases included Alzheimer’s disease with or without Lewy body pathology, vascular encephalopathy, metabolic encephalopathies and viral or limbic encephalitis. Conclusion: The steady increase in mortality rates, especially in old age groups, most likely reflects improved case ascertainment due to active surveillance causing higher awareness of the medical community. In comparison with other European countries, it is reassuring to note that the overall death rate of TSEs does not differ from the Austrian autopsy-controlled data, thus confirming the value of clinical surveillance criteria.

Safety procedures of coagulation factors

Two main types of safety procedures must be applied to biological products, including plasma derivatives: (i) preventive procedures and (ii) elimination procedures.Prevention includes epidemiological control of donor populations; checks on each donor’s health condition; analysis of each donation for the main pathogens using serological methods; additional analysis of all plasma for human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis A virus (HAV) and the B19 virus, using nucleic acid amplification techniques (NAT). A 60 days or longer inventory hold of all plasma donations is applied, to allow additional time to discard previous donations from potential seroconverting or otherwise rejectable donors.Elimination procedures minimize the low residual risk of transmitting pathogens, including unknown or previously undetected ones. Since the introduction 20 years ago of solvent‐detergent treatment, very effective against enveloped viruses (HIV, HBV, HCV, West Nile virus, SARS, avian influenza virus etc), there have been no known cases of transmission of this type of pathogens by products manufactured according to this procedure. Other inactivation procedures such as pasteurization, dry‐heat or nanofiltration may prove equally effective. In addition, dry‐heat treatment and nanofiltration are capable of effectively eliminating non‐enveloped viruses (HAV, B19 virus). Recent studies show that the B19 virus is much more sensitive to heat (in lyophilized state or by pasteurization) and acid pH than previously thought.Although there is no evidence for the transmission of classic transmissible spongiform encephalopathies (TSEs) through blood or blood‐products transfusion, four possible cases have been reported in the United Kingdom involving transmission by non‐leukoreduced blood components of the agent that causes variant Creutzfeldt‐Jakob Disease (vCJD), a disease linked to the outbreak of bovine spongiform encephalopathy (BSE) which took place in that country. However, there are no cases of human TSE (classic or variant) transmission by plasma‐derived products. Analytical methods capable of detecting the vCJD agent in patients’ brains (where high titres are found) and other tissues (such as the spleen, appendix and lymph nodes, where much lower concentrations are found) are unable to detect the agent in blood or plasma from patients with vCJD, even in the clinical phase of the disease. Experiments by Grifols and other groups show that the capacity of the production processes to eliminate vCJD agent models is many orders of magnitude greater than the maximum expected load of the agent. In this regard, the efficacy of precipitation, affinity chromatography, depth filtration and nanofiltration are particularly notable.

Revisiting the risks involved in using homograft ossicles in otological surgery

Despite the fact that cartilage, cortical bone and corneal homograft tissue is still widely used in South Africa and that there has never been a reported case of human immunodeficiency virus or Creutzfeldt-Jakob disease infection transmission via this route, otolaryngologists are still reluctant to use homograft ossicles. In third world countries, such as South Africa, where ossicular prostheses are not always readily available due to financial constraints, the question arises as to whether we should be reconsidering the use of homograft ossicles. This review examines the risk of developing Creutzfeldt-Jakob disease or acquiring human immunodeficiency virus following the use of homograft ossicles during ossicular reconstruction, and discusses sterilization techniques that have proven effective in eradicating the human immunodeficiency virus and prions. For decades, homograft ossicles have been used worldwide in otological surgery, especially in patients lacking suitable autograft ossicles. There has never been a reported case of transmission of the human immunodeficiency virus, and no cases of transmissible spongiform encephalopathy have been reported in the literature after otological surgery involving only the use of homograft ossicles. There have only been two documented otological cases of Creutzfeldt-Jakob disease; these involved the use of cadaveric dura mater and pericardium for tympanic membrane grafting. The human immunodeficiency virus is easily inactivated by simple sterilisation techniques, and there is a statistically insignificant risk of transmitting this virus if proper sterilisation protocols are followed.

Transmissible spongiform encephalopathy agent clearance by the immunoaffinity and anion‐exchange chromatography steps of the ReFacto® manufacturing process

ReFacto (moroctocog alfa), a recombinant factor VIII approved for the treatment of haemophilia A, is produced by a mammalian cell-culture process that includes therapeutic-grade human serum albumin (HSA) in the cell-culture medium. While to date there have been no cases of transmissible spongiform encephalopathy (TSE) resulting from the clinical use of HSA, Wyeth conducted a study to demonstrate that the ReFacto manufacturing process has significant capacity to remove a TSE agent if it were present as a contaminant in the HSA. The immunoaffinity (8A4 Sepharose) and anion-exchange (Q Sepharose) chromatography steps were evaluated for the clearance of the hamster TSE agent, strain 263K. This Good Laboratory Practice study was performed using appropriately qualified, laboratory-scale chromatography systems. Filtered brain homogenate from TSE-infected hamsters was added to loads of both chromatographic columns, and the concentration of TSE agent in the loads and product pools were determined using a validated western blot quantitation method. Replicate chromatography runs were consistent, as demonstrated by the < or =0.7 log(10) difference observed in TSE agent reduction between each pair of runs. The immunoaffinity and anion-exchanges steps demonstrated 3.8 log reduction and >5.2 log reduction respectively. These data provide a high degree of assurance that in the unlikely event of a TSE contamination of the HSA used in the ReFacto cell-culture process, the purification steps have the potential to remove the infectious agent to extremely low levels, thereby significantly reducing the risk to patients receiving ReFacto.

Diversity in Neuroanatomical Distribution of Abnormal Prion Protein in Atypical Scrapie

Scrapie is a transmissible spongiform encephalopathy (TSE) in sheep and goats. In recent years, atypical scrapie cases were identified that differed from classical scrapie in the molecular characteristics of the disease-associated pathological prion protein (PrPsc). In this study, we analyze the molecular and neuropathological phenotype of nine Swiss TSE cases in sheep and goats. One sheep was identified as classical scrapie, whereas six sheep, as well as two goats, were classified as atypical scrapie. The latter revealed a uniform electrophoretic mobility pattern of the proteinase K–resistant core fragment of PrPsc distinct from classical scrapie regardless of the genotype, the species, and the neuroanatomical structure. Remarkably different types of neuroanatomical PrPsc distribution were observed in atypical scrapie cases by both western immunoblotting and immunohistochemistry. Our findings indicate that the biodiversity in atypical scrapie is larger than expected and thus impacts on current sampling and testing strategies in small ruminant TSE surveillance.

Quantification of Peyer's patches in Cheviot sheep for future scrapie pathogenesis studies

Peyer's patches (PPs) are the most probable sites of intestinal uptake of the transmissible spongiform encephalopathy (TSE) agent. The amount of PP tissue varies considerably between different age groups of individuals, and whether this variation is related to susceptibility to TSE infection raises an intriguing possibility. The purpose of this study was to determine the surface area of PP tissue and the number of associated lymphoid follicles in different age groups of Neuropathogenesis Unit (NPU) Cheviot sheep. Terminal ilea were obtained from 33 sheep of different ages. Samples of ileal tissue were collected for immunocytochemistry and immunolabelled for prion protein (PrP). Specimens were then fixed in acetic acid, stained with methylene blue and transilluminated. Image analysis software was used to calculate the area of intestinal and PP tissue. The number of associated lymphoid follicles was determined using a dissecting microscope. Results showed a marked fall in surface area of PP tissue and lymphoid follicle density around puberty (about 8–9 months of age in NPU Cheviot sheep) and both measures remained low throughout adulthood. Using the Spearman's rank correlation coefficient, r s, these two measures were found to be closely correlated ( r s = 0.899, n = 33, P < 0.0001). There was also a significant (negative) correlation between age and the two respective measures (surface area of PP tissue versus age, r s = −0.879 ( n = 33, P < 0.0001); lymphoid follicle density versus age r s = −0.943 ( n = 33, P < 0.0001). Immunolabelling for PrP was observed primarily in the light zone of lymphoid follicles. Results obtained from this study are useful for future oral pathogenesis studies of the NPU Cheviot flock. They may also offer a possible biological explanation for the apparent age-susceptibility relationship observed in natural cases of TSEs and might help to explain the young age-distribution of cases.

Atypical transmissible spongiform encephalopathies (TSEs) in ruminants

Transmissible spongiform encephalopathies (TSEs) are associated with the accumulation in infected tissues of a disease-associated form of a host-encoded protein, the prion protein (PrP). Contrary to the normal form of the protein, this form of PrP is partially resistant to protease digestion (PrP(res)). Detailed characterisation of PrP(res) has been intensively investigated in recent years to try and decipher the diversity of TSEs in human and animals. This considerably and unexpectedly enlarged our knowledge about such diseases in ruminants. Previously, such a diversity was essentially shown by the demonstration that scrapie from sheep and goats could have different biological behaviours following transmission of the disease in mice, unlike bovine spongiform encephalopathy from cattle (BSE) which showed a distinct and unique behaviour. The properties of the BSE agent were also demonstrated to be very stable, following transmission to a variety of different species. Molecular studies of PrP(res), followed by transmission studies to mice, gave the first evidence for the accidental transmission of the BSE agent to humans where it induced a variant form of the fatal Creutzfeldt-Jakob disease (CJD) and also to different animal species including a goat in France. This last case was found among a few unusual cases of TSEs in small ruminants that showed some molecular similarities with BSE and which are currently under investigation by transmission studies in mice. The application of the molecular methods to characterise PrP(res) has most recently led to the unexpected discovery of deviant BSE forms in a few affected cattle in Europe and in the United States, which raises the question of a possible different origin at least of some cases of BSE in cattle. Finally, considerable numbers of a new TSE form in small ruminants, referred to as "atypical scrapie" or "Nor98", have meanwhile been identified in most European countries by TSE rapid testing using an assay which recognizes also comparatively less PK resistant PrP(res).

Amplified Immunohistochemical Detection of PrPsc in Animal Transmissible Spongiform Encephalopathies Using Streptomycin

Due to its sensitivity, immunohistochemistry (IHC) of abnormal prion protein (PrPsc) is used to study experimental and natural cases of transmissible spongiform encephalopathies (TSEs) such as Creutzfeldt-Jakob disease in humans or scrapie and bovine spongiform encephalopathy (BSE) in animals. The limits of detection are particularly critical when PrPsc IHC is used for diagnostic purposes. In this article, we describe for the first time the use of streptomycin sulfate in IHC, providing a novel original and easy way to amplify specifically PrPsc immunohistochemical detection in natural cases of BSE and scrapie, as well as in experimental TSEs in mice models using two different PrP antibodies.