Stability of BSE infectivity towards heat treatment even after proteolytic removal of prion protein.

The unconventional infectious agents of transmissible spongiform encephalopathies (TSEs) are prions. Their infectivity co-appears with PrPSc, aberrant depositions of the host's cellular prion protein (PrPC). Successive heat treatment in the presence of detergent and proteolysis by a keratinase from Bacillus licheniformis PWD-1 was shown before to destroy PrPSc from bovine TSE (BSE) and sheep scrapie diseased brain, however data regarding expected reduction of infectivity were still lacking. Therefore, transgenic Tgbov XV mice which are highly BSE susceptible were used to quantify infectivity before and after the bovine brain treatment procedure. Also four immunochemical analyses were applied to compare the levels of PrPSc. After heating at 115 °C with or without subsequent proteolysis, the original BSE infectivity of 106.2-6.4 ID50 g-1 was reduced to a remaining infectivity of 104.6-5.7 ID50 g-1 while strain characteristics were unaltered, even after precipitation with methanol. Surprisingly, PrPSc depletion was 5-800 times higher than the loss of infectivity. Similar treatment was applied on other prion strains, which were CWD1 in bank voles, 263 K scrapie in hamsters and sheep PG127 scrapie in tg338 ovinized mice. In these strains however, infectivity was already destroyed by heat only. These findings show the unusual heat resistance of BSE and support a role for an additional factor in prion formation as suggested elsewhere when producing prions from PrPC. Leftover material in the remaining PrPSc depleted BSE preparation offers a unique substrate for searching additional elements for prion infectivity and improving our concept about the nature of prions.

The zoonotic potential of animal prion diseases.

Bovine spongiform encephalopathy (BSE) is the only animal prion disease that has been demonstrated to be zoonotic, causing variant Creutzfeldt-Jakob disease (vCJD) in humans. The link between BSE and vCJD was established by careful surveillance, epidemiologic investigations, and experimental studies using in vivo and in vitro models of cross-species transmission. Similar approaches have been used to assess the zoonotic potential of other animal prion diseases, including atypical forms identified through active surveillance. There is no epidemiologic evidence that classical or atypical scrapie, atypical forms of BSE, or chronic wasting disease (CWD) is associated with human prion disease, but the limitations of the epidemiologic data should be taken into account when interpreting these results. Transmission experiments in nonhuman primates and human PrP transgenic mice suggest that classic scrapie, L-type atypical BSE (L-BSE), and CWD may have zoonotic potential, which for L-BSE appears to be equal to or greater than that of classic BSE. The results of in vitro conversion assays to analyze the human transmission barrier correlate well with the in vivo data. However, it is still difficult to predict the likelihood that an animal prion disease will transmit to humans under conditions of field exposure from the results of in vivo or in vitro experiments. This emphasizes the importance of continuing systematic surveillance for both human and animal prion diseases in identifying zoonotic transmission of diseases other than classic BSE.

Detection of PrPBSE and prion infectivity in the ileal Peyer's patch of young calves as early as 2 months after oral challenge with classical bovine spongiform encephalopathy.

In classical bovine spongiform encephalopathy (C-BSE), an orally acquired prion disease of cattle, the ileal Peyer's patch (IPP) represents the main entry port for the BSE agent. In earlier C-BSE pathogenesis studies, cattle at 4-6 months of age were orally challenged, while there are strong indications that the risk of infection is highest in young animals. In the present study, unweaned calves aged 4-6 weeks were orally challenged to determine the earliest time point at which newly formed PrPBSE and BSE infectivity are detectable in the IPP. For this purpose, calves were culled 1 week as well as 2, 4, 6 and 8 months post-infection (mpi) and IPPs were examined for BSE infectivity using a bovine PrP transgenic mouse bioassay, and for PrPBSE by immunohistochemistry (IHC) and protein misfolding cyclic amplification (PMCA) assays. For the first time, BSE prions were detected in the IPP as early as 2 mpi by transgenic mouse bioassay and PMCA and 4 mpi by IHC in the follicular dendritic cells (FDCs) of the IPP follicles. These data indicate that BSE prions propagate in the IPP of unweaned calves within 2 months of oral uptake of the agent.

Phenotypical Variability in Bovine Spongiform Encephalopathy: Epidemiology, Pathogenesis, and Diagnosis of Classical and Atypical Forms.

After thirty years, bovine spongiform encephalopathy (BSE) still represents the biggest crisis in the field of food safety. Initially detected in the United Kingdom in 1986, BSE spread to many other countries all over the world, involving approximately 200,000 cattle. The origin of BSE is uncertain, but epidemiological studies suggest that the source was cattle feed prepared from prion-infected animal tissues. The implementation of the drastic measures, including the ban of meat and bone meal from livestock feed and the removal of specified risk material from the food chain, has eventually resulted in a significant decline of the epidemic. For many years, it was believed that the disease was caused by transmission of a single prion strain. However, since 2004 two types of BSE, with distinct phenotypical characteristics, have been detected in Italy and France. These atypical types are characterized by distinct Western Blot profiles of abnormal protease-resistant prion protein, named high-type (H-BSE) and low-type (L-BSE). At present, there is no comprehensive information about the origin of the atypical BSEs (sporadic vs. acquired), and data about the pathogenesis of both atypical forms are very limited as compared to the classical type (C-BSE). This chapter will provide a well-organized overview of what is known about classical and atypical BSE. It will review information on the main epidemiological features, pathogenesis, and the criteria for routine diagnosis based on rapid tests, histological, immunohistochemical, and Western blot analysis. Furthermore, a brief overview about the most recently in vitro techniques will be also provided.

Is there a decline in bovine spongiform encephalopathy cases born after reinforced feed bans? A modelling study in EU member states.

Occasional cases of classical bovine spongiform encephalopathy (BSE) still continue to occur within the European Union (EU) for animals born after reinforced feed bans (BARBs), which should in theory have eliminated all risk of infection. The study aimed to determine (i) whether a common rate of decline of BSE infection was evident across EU member states, i.e. to determine whether control measures have been equally effective in all member states, (ii) whether there was any evidence of spontaneous occurrence of BSE in the data and (iii) the expected date for the last BSE case in UK. It was found that there was no significant difference in the rate of decline of BSE prevalence between member states, with a common rate of decline of 33·9% per annum (95% CI 30·9-37%) in successive annual birth cohorts. Trend analysis indicated an ultimate decline to 0 prevalence, suggesting that spontaneous occurrence does not explain the majority of cases. Projecting forward the trends from the back-calculation model indicated that there was approximately a 50% probability of further cases in the UK, and should the current rate of decline continue, there remains the possibility of further occasional cases up until 2026.

Assessing the sensitivity of European surveillance for detecting BSE in cattle according to international standards.

European surveillance for Bovine Spongiform Encephalopathy (BSE), initiated in 2001, has shown a steady exponential decline in the number of infected cattle, demonstrating that control measures have been effective. In 2016 23 European countries out of 28 demonstrated negligible risk status for the disease. The international standard setting body, the World Organization for Animal Health (OIE), prescribes that for countries where there is a non-negligible BSE risk, surveillance should allow the detection of one case per 100,000 in the adult cattle population with 95% confidence (Type A surveillance). The Cattle TSE Monitoring Model (C-TSEMM) was developed to estimate the sensitivity of surveillance systems to detect BSE in cattle. The model includes a cohort-based back calculation model to estimate the number and age of infected animals, the subset of those that are detectable by the diagnostic test, and the stream by which infected animals exit the standing population, that is, healthy slaughter, emergency slaughter, fallen stock and clinical suspects. Data collected by the European Commission (EC) and European Food Safety Authority (EFSA), containing the details of over 91 million cattle tested within the European Member States (EU25) surveillance scheme from 2002 to 2011, was used to populate the model. When considering the EU25 surveillance scheme as a single epidemiological unit, the model estimated that the surveillance scheme in place exceeded the OIE required threshold for Type A surveillance, and that a revised monitoring system excluding all healthy slaughter animals would also exceed this threshold. Results indicated a variation in individual country sensitivity of surveillance with seven countries (Germany, Spain, France, Ireland, Italy, Poland and the UK) exceeding the required threshold individually. Key assumptions included the fitting of an exponential distribution for all countries to describe the decline in prevalence over time and, for those members with no detected cases of BSE, an estimated area prevalence was used as a proxy. These results were part of the evidence base that lifted the testing requirement for healthy slaughter animals in European countries in 2013. The model has been maintained and updated by EFSA each year with the addition of new member states, and updated surveillance data.

Assessment of the PrPc Amino-Terminal Domain in Prion Species Barriers.

Chronic wasting disease (CWD) in cervids and bovine spongiform encephalopathy (BSE) in cattle are prion diseases that are caused by the same protein-misfolding mechanism, but they appear to pose different risks to humans. We are interested in understanding the differences between the species barriers of CWD and BSE. We used real-time, quaking-induced conversion (RT-QuIC) to model the central molecular event in prion disease, the templated misfolding of the normal prion protein, PrPc, to a pathogenic, amyloid isoform, scrapie prion protein, PrPSc We examined the role of the PrPc amino-terminal domain (N-terminal domain [NTD], amino acids [aa] 23 to 90) in cross-species conversion by comparing the conversion efficiency of various prion seeds in either full-length (aa 23 to 231) or truncated (aa 90 to 231) PrPc We demonstrate that the presence of white-tailed deer and bovine NTDs hindered seeded conversion of PrPc, but human and bank vole NTDs did the opposite. Additionally, full-length human and bank vole PrPcs were more likely to be converted to amyloid by CWD prions than were their truncated forms. A chimera with replacement of the human NTD by the bovine NTD resembled human PrPc The requirement for an NTD, but not for the specific human sequence, suggests that the NTD interacts with other regions of the human PrPc to increase promiscuity. These data contribute to the evidence that, in addition to primary sequence, prion species barriers are controlled by interactions of the substrate NTD with the rest of the substrate PrPc molecule. IMPORTANCE: We demonstrate that the amino-terminal domain of the normal prion protein, PrPc, hinders seeded conversion of bovine and white-tailed deer PrPcs to the prion forms, but it facilitates conversion of the human and bank vole PrPcs to the prion forms. Additionally, we demonstrate that the amino-terminal domain of human and bank vole PrPcs requires interaction with the rest of the molecule to facilitate conversion by CWD prions. These data suggest that interactions of the amino-terminal domain with the rest of the PrPc molecule play an important role in the susceptibility of humans to CWD prions.

Absence of Evidence for a Causal Link between Bovine Spongiform Encephalopathy Strain Variant L-BSE and Known Forms of Sporadic Creutzfeldt-Jakob Disease in Human PrP Transgenic Mice.

Prions are proteinaceous pathogens responsible for subacute spongiform encephalopathies in animals and humans. The prions responsible for bovine spongiform encephalopathy (BSE) are zoonotic agents, causing variant Creutzfeldt-Jakob disease (CJD) in humans. The transfer of prions between species is limited by a species barrier, which is thought to reflect structural incompatibilities between the host cellular prion protein (PrPC) and the infecting pathological PrP assemblies (PrPSc) constituting the prion. A BSE strain variant, designated L-BSE and responsible for atypical, supposedly spontaneous forms of prion diseases in aged cattle, demonstrates zoonotic potential, as evidenced by its capacity to propagate more easily than classical BSE in transgenic mice expressing human PrPC and in nonhuman primates. In humanized mice, L-BSE propagates without any apparent species barrier and shares similar biochemical PrPSc signatures with the CJD subtype designated MM2-cortical, thus opening the possibility that certain CJD cases classified as sporadic may actually originate from L-type BSE cross-transmission. To address this issue, we compared the biological properties of L-BSE and those of a panel of CJD subtypes representative of the human prion strain diversity using standard strain-typing criteria in human PrP transgenic mice. We found no evidence that L-BSE causes a known form of sporadic CJD. IMPORTANCE: Since the quasi-extinction of classical BSE, atypical BSE forms are the sole BSE variants circulating in cattle worldwide. They are observed in rare cases of old cattle, making them difficult to detect. Extrapolation of our results suggests that L-BSE may propagate in humans as an unrecognized form of CJD, and we urge both the continued utilization of precautionary measures to eliminate these agents from the human food chain and active surveillance for CJD phenotypes in the general population.

A stochastic model of the bovine spongiform encephalopathy epidemic in Canada.

Bovine spongiform encephalopathy (BSE) appeared in the United Kingdom in the mid 1980s, and has been attributed to the use of meat and bone meal (MBM) in cattle feed contaminated with a scrapie-like agent. Import of infectious materials from a country where BSE has occurred is believed to be the major factor underlying the spread of the BSE epidemic to other countries. This study presents a new stochastic model developed to estimate risk of BSE from importation of cattle infected with the BSE agent. The model describes the propagation of the BSE agent through the Canadian cattle herd through rendering and feeding processes, following importation of cattle with infectious prions. This model was used estimate the annual number of newly infected animals each year over the period 1980-2019. Model predictions suggested that the number of BSE infections in Canada might have been approximately 40-fold greater than the actual number of clinically diagnosed cases. Under complete compliance with the 2007 ban on feeding MBM, this model further predicts that BSE is disappearing from the Canadian cattle system. A series of sensitivity analyses was also conducted to test the robustness of model predictions to alternative assumptions about factors affecting the evolution of the Canadian BSE epidemic.

A Bayesian back-calculation method to estimate the risk of bovine spongiform encephalopathy (BSE) in Canada during the period 1996-2011.

Seventeen typical cases of bovine spongiform encephalopathy (BSE) were detected in Canada the period of 2003-2011. The clinical incidence of BSE was censored by early slaughter, death, or exportation of infected cattle due to the long incubation period of BSE disease. The aim of this study was to estimate the infection incidence of BSE in birth cohorts during 1996-2004 and project infection frequency through to 2007. An estimate of the number of asymptomatic infected cattle slaughtered for human consumption is also provided. The number of incident, asymptomatic cases was assumed to follow a Poisson process. A Bayesian back-calculation approach was used to project the risk of contracting BSE in those birth cohorts. Model parameters and inputs were taken from scientific literature and governmental data sources. The projected number of infected cattle in birth cohorts spanning the period 1996-2007 was 492, with median 95% credible interval 258-830. If the requirement to remove specified risk material (SRM) from cattle prior to entering the food chain was not in place, the predicted number of slaughtered infected in the human food chain from 1996-2010 was 298, with a 95% credible interval 156-500. The magnitude of the BSE epidemic in Canada for 1996-2007 birth cohorts was estimated to be approximately 28-fold higher than the number of clinical cases detected through to October 2011. Although some of those cattle were slaughtered for human consumption, the requirement of SRM removal may have prevented most of the infectious material from entering the food chain.

Valorization of rendering industry wastes and co-products for industrial chemicals, materials and energy: review.

Over the past decades, strong global demand for industrial chemicals, raw materials and energy has been driven by rapid industrialization and population growth across the world. In this context, long-term environmental sustainability demands the development of sustainable strategies of resource utilization. The agricultural sector is a major source of underutilized or low-value streams that accompany the production of food and other biomass commodities. Animal agriculture in particular constitutes a substantial portion of the overall agricultural sector, with wastes being generated along the supply chain of slaughtering, handling, catering and rendering. The recent emergence of bovine spongiform encephalopathy (BSE) resulted in the elimination of most of the traditional uses of rendered animal meals such as blood meal, meat and bone meal (MBM) as animal feed with significant economic losses for the entire sector. The focus of this review is on the valorization progress achieved on converting protein feedstock into bio-based plastics, flocculants, surfactants and adhesives. The utilization of other rendering streams such as fat and ash rich biomass for the production of renewable fuels, solvents, drop-in chemicals, minerals and fertilizers is also critically reviewed.

A Naturally Occurring Bovine Tauopathy Is Geographically Widespread in the UK.

Many human neurodegenerative diseases are associated with hyperphosphorylation and widespread intra-neuronal and glial associated aggregation of the microtubule associated protein tau. In contrast, animal tauopathies are not reported with only senescent animals showing inconspicuous tau labelling of fine processes albeit significant tau aggregation may occur in some experimental animal disease. Since 1986, an idiopathic neurological condition of adult cattle has been recognised in the UK as a sub-set of cattle slaughtered as suspect bovine spongiform encephalopathy cases. This disorder is characterised by brainstem neuronal chromatolysis and degeneration with variable hippocampal sclerosis and spongiform change. Selected cases of idiopathic brainstem neuronal chromatolysis (IBNC) were identified from archive material and characterised using antibodies specific to several tau hyperphosphorylation sites or different isoforms of the tau microtubule binding region. Labelling was also carried out for alpha synuclein, ubiquitin, TDP43, Aß 1-42, Aß 1-40. Widespread tau labelling was identified in all IBNC brains examined and with each of seven tau antibodies recognising different hyperphosphorylated sites. Labelling with each antibody was associated with dendrites, neuronal perikarya and glia. Thus IBNC is a sporadic, progressive neurological disease predominantly affecting aged cattle that occurs throughout the UK and is associated with hyperphosphorylation of tau, a rare example of a naturally-occurring tauopathy in a non-primate species. Secondary accumulation of alpha synuclein and ubiquitin was also present. The neuropathology does not precisely correspond with any human tauopathy. The cause of IBNC remains undetermined but environmental factors and exposure to agrochemicals needs to be considered in future aetiological investigations.

The 2008 US beef scare episode in South Korea: analysis of an unusual public reaction.

We investigated major factors underlying an unusual 2008 public 'candlelight protest' in South Korea about US beef imports related to concerns about bovine spongiform encephalopathy. Using a survey we explored determinants of consumer responses to negative publicity in mass media. Respondents (80.7 per cent) reduced consumption of imported beef during the scare; of those 62.5 per cent decreased consumption of US beef only. We explain the determinants in order of their importance and define the relevant terms from a theory of consumer behavior in economics. Our findings suggest that several effects worked jointly in their influence on most respondents who reduced beef consumption.

Estimating the BSE infection and detectable prevalence in cattle born after 2000 in Japan.

We estimated the infection prevalence of BSE in Japanese cattle born in the period 2000-2012, using maximum likelihood methods and BSE surveillance data of these birth cohorts. From this, we predicted the number of infected cattle and test positives in years 2004-2020. Assuming that the infection prevalence decayed exponentially over time from 2000, the infection prevalence of the 2000 birth cohort was estimated to be 0.00058 which declined exponentially by 0.115 times per year in the following years. The number of infected cattle was calculated to have peaked in 2005 and would be zero by 2020. The number of test positives was calculated to have peaked in 2005 and would be zero by 2012. The number of BSE cases actually detected was within the 95% confidence interval of the predicted numbers. The detectable prevalence (predicted number of test positives/number of cattle tested) was predicted to be highest in 2005. In this year it was predicted that one animal out of 160,000 tested would test positive. The detectable prevalence would decline exponentially to zero in the subsequent years.

Case-control study on the risks of BSE infections in Northern Germany.

This study was to identify risk factors for bovine spongiform encephalopathy (BSE) by means of individual case-control data. 43 BSE cases in a defined region in Lower Saxony and Schleswig-Holstein were compared with 84 control animals. Purchase of new breeding stock and cross contamination between feed on the farm did not seem to have influence on the BSE incidence in these regions. The results indicate independent risk patterns. Pattern 1: Cows with high milk yield seemed to be at risk on big farms with adjacent pig production and when they were not fed milk replacer. Pattern 2: Milk replacer (esp. from certain producers) is a risk factor for Non-Red Holstein cattle, low yielding cows and farms without pig production. Pattern 3: Red Holstein cattle not being fed milk replacer have a higher BSE risk than other breeds when they have a low milk yield and live on small farms with pig production. This study, like findings in Bavaria, Lower Saxony and Schleswig-Holstein, strengthens the hypothesis that BSE in Germany was caused by a feed mediated ubiquitous exposure to PrP(sc) during a confined time period. Producers, in need of buying animal derived feed components during that time slot, were more likely to spread the PrP(sc) than others. Their increased risk is not necessarily due to an inadequate purchasing policy, but can also be coincidental. The breed Red Holstein is not the risk factor itself but represents the risk from concentrated feed for animals during a susceptible age period (calves). Therefore, the authors suggest a continuous exclusion of animal-derived fat components from milk replacers.

The interpretation of disease phenotypes to identify TSE strains in mice: characterisation of BSE using PrPSc distribution patterns in the brain.

In individual animals affected by transmissible spongiform encephalopathies, different disease phenotypes can be identified which are attributed to different strains of the agent. In the absence of reliable technology to fully characterise the agent, classification of disease phenotype has been used as a strain typing tool which can be applied in any host. This approach uses standardised data on biological parameters, established for a single host, to allow comparison of different prion sources. Traditionally prion strain characterisation in wild type mice is based on incubation periods and lesion profiles after the stabilisation of the agent into the new host which requires serial passages. Such analysis can take many years, due to prolonged incubation periods. The current study demonstrates that the PrPSc patterns produced by one serial passage in wild type mice of bovine or ovine BSE were consistent, stable and showed minimal and predictable differences from mouse-stabilised reference strains. This biological property makes PrPSc deposition pattern mapping a powerful tool in the identification and definition of TSE strains on primary isolation, making the process of characterisation faster and cheaper than a serial passage protocol. It can be applied to individual mice and therefore it is better suited to identify strain diversity within single inocula in case of co-infections or identify strains in cases where insufficient mice succumb to disease for robust lesion profiles to be constructed. The detailed description presented in this study provides a reference document for identifying BSE in wild type mice.

Significant differences in incubation times in sheep infected with bovine spongiform encephalopathy result from variation at codon 141 in the PRNP gene.

The susceptibility of sheep to prion infection is linked to variation in the PRNP gene, which encodes the prion protein. Common polymorphisms occur at codons 136, 154 and 171. Sheep which are homozygous for the A(136)R(154)Q(171) allele are the most susceptible to bovine spongiform encephalopathy (BSE). The effect of other polymorphisms on BSE susceptibility is unknown. We orally infected ARQ/ARQ Cheviot sheep with equal amounts of BSE brain homogenate and a range of incubation periods was observed. When we segregated sheep according to the amino acid (L or F) encoded at codon 141 of the PRNP gene, the shortest incubation period was observed in LL(141) sheep, whilst incubation periods in FF(141) and LF(141) sheep were significantly longer. No statistically significant differences existed in the expression of total prion protein or the disease-associated isoform in BSE-infected sheep within each genotype subgroup. This suggested that the amino acid encoded at codon 141 probably affects incubation times through direct effects on protein misfolding rates.

Classification method for disease risk mapping based on discrete hidden Markov random fields.

Risk mapping in epidemiology enables areas with a low or high risk of disease contamination to be localized and provides a measure of risk differences between these regions. Risk mapping models for pooled data currently used by epidemiologists focus on the estimated risk for each geographical unit. They are based on a Poisson log-linear mixed model with a latent intrinsic continuous hidden Markov random field (HMRF) generally corresponding to a Gaussian autoregressive spatial smoothing. Risk classification, which is necessary to draw clearly delimited risk zones (in which protection measures may be applied), generally must be performed separately. We propose a method for direct classified risk mapping based on a Poisson log-linear mixed model with a latent discrete HMRF. The discrete hidden field (HF) corresponds to the assignment of each spatial unit to a risk class. The risk values attached to the classes are parameters and are estimated. When mapping risk using HMRFs, the conditional distribution of the observed field is modeled with a Poisson rather than a Gaussian distribution as in image segmentation. Moreover, abrupt changes in risk levels are rare in disease maps. The spatial hidden model should favor smoothed out risks, but conventional discrete Markov random fields (e.g. the Potts model) do not impose this. We therefore propose new potential functions for the HF that take into account class ordering. We use a Monte Carlo version of the expectation-maximization algorithm to estimate parameters and determine risk classes. We illustrate the method's behavior on simulated and real data sets. Our method appears particularly well adapted to localize high-risk regions and estimate the corresponding risk levels.