Low frequency of protective variants at regulatory region of PRNP gene indicating the genetically high risk of BSE in Ethiopian Bos indicus and Bos taurus africanus.

Susceptibility to classical bovine spongiform encephalopathy (BSE) has been linked to 23 bp indel in promoter and 12 bp indel in the first intron of cattle prion protein gene. This study aimed to investigate 23/12 bp indel polymorphisms in the polymorphisms in cattle prion protein (PRNP) gene to reveal the risk of BSE in Ethiopian cattle. Also, frequency of each polymorphism was compared to the other Bos taurus and Bos indicus breeds. According to results, the insertion variant was detected at a low frequency in all of the study populations at both loci. The 23 bp insertion allele in Fogera breed was relatively lower than Borona and Arsi and the same allele at the same locus in Afar breed was higher than the rest of the breeds (0.16). Due to high linkage disequilibrium (LD) of the deletion allele in Bos taurus, the frequencies of deletion allele at 23 bp (0.84) and 12 bp (0.86) loci in Afar breed were relatively closer than the rest of the breeds. In addition, DD/DD was found as the highly frequent diplotype in all of the breeds. The low frequency of insertion alleles at 23 and 12 bp indel sites demonstrate that Ethiopian cattle have a genetically high risk for BSE.

Glyceroltriheptanoate (GTH) occurrence in animal by-products: a monitoring study to minimise safety-related risk of misuse.

Early in this century, the crisis connected to the spread of bovine spongiform encephalopathy caused a great concern related to the use of animal by-products (ABPs). According to the Commission Regulation (EU) No 1069/2009, these materials are classified in three categories according to their related risk. In 2011 Commission Regulation (EU) No 142/2011 established that meat and bone meal (MBM) and fat deriving from ABPs not intended for human consumption (category 1 and 2) are required to be permanently marked with glyceroltriheptanoate (GTH), at a minimum concentration of 250 mg kg-1 of fat, while category 3 processed animal proteins (PAPs) must not contain this compound. PAPs are bio resources, which could be used in a renewable and regenerative way in a circular economy model for a conscious usage of raw materials. The aim of this study was to provide information on GTH occurrence in MBM and, if any, in PAPs. Samples were collected from 2017 to 2021 and analysed by GC-MS. Detected non-compliant samples were exclusively of MBM category 1 and 2, probably due to the addition of an inadequate amount of GTH during the manufacturing processes. These results highlighted the importance of National Monitoring Programs as a useful tool to minimise safety related risk due to the misuse of GTH. Thus, investigating the critical points in feed supply-chain and sharing the information on its occurrence may help to improve animal and human wellness and safety.

Epidemiological verification of the mechanism of occurrence of atypical L-type bovine spongiform encephalopathy.

Since 2004, a novel bovine spongiform encephalopathy (BSE), distinct from the conventional 'classical BSE' (C-BSE), has been reported as an atypical BSE. Atypical BSE is detected mostly in aged cattle, and it is suggested that atypical BSE may occur spontaneously. Relaxation of the relevant countermeasures such as feed ban, which prevents the use of bovine meat-and-bone meal as feed, has been discussed in recent years owing to the decrease in C-BSE cases. If atypical BSE occurs spontaneously without exposure to an agent called abnormal prion protein (PrPSc ), complete removal of these measures will be difficult. In this study, we verified the possibility that L-BSE, which is a subtype of atypical BSE, occurs spontaneously. We first hypothesized that L-BSE occurs only through the process of infection via oral exposure. If the hypothesis was true, the infection of L-BSE would be mostly limited to calves under 1 year of age due to their high susceptibility, and the feed ban would effectively reduce the number of infected calves by birth cohort. Thus, we created a mathematical model to estimate the number of infected calves by birth cohort and compared the effectiveness of the feed ban on C-BSE and L-BSE. The number of tested animals and detected cases in nine European countries were used for this analysis. Our results showed that the estimated number of infected calves in the birth cohort indicated that feed ban was less effective on L-BSE. This result supports the alternative hypothesis that at least a part of the L-BSE can occur without infection via oral exposure. Our results suggest that the complete abolition of countermeasures, such as feed ban, should be discussed carefully. As for the occurrence mechanism, although there remains uncertainty to reach conclusions, it is reasonable to assume that L-BSE can occur spontaneously at present.

Absence of proteinase K-resistant PrP in Korean Holstein cattle carrying potential bovine spongiform encephalopathy-related E211K somatic mutation.

Bovine spongiform encephalopathy (BSE) is a kind of prion disease caused by proteinase K-resistant prion protein (PrPSc ) in cattle. Although BSE has been reported worldwide, BSE-infected cases have never been reported in Korea. In a previous study, we identified BSE-related somatic mutation E211K in 3 Korean Holstein cattle. In Korea, the BSE surveillance system has been established. However, several genetic factors have not been controlled simultaneously thus far. In the present study, we performed enhanced surveillance of prion disease-related factors in Korean cattle, including Holstein cattle and Hanwoo (Korean native cattle), which is widely raised for meat. We investigated the germline mutation E211K at codon 211 of the PRNP gene and analysed genotype, allele and haplotype frequencies of the 23- and 12-bp insertion/deletion polymorphisms of the PRNP gene using direct DNA sequencing. In addition, we investigated linkage disequilibrium (LD) and compared haplotype distributions of polymorphisms among cattle breeds. Furthermore, we carried out BSE diagnosis in the medulla oblongata (MO) of Korean cattle including 3 Korean Holstein cattle carrying somatic mutation E211K using Western blotting analysis. We did not find the E211K mutation in the PRNP gene in any of the Korean cattle and found significantly different genotype, allele and haplotype distributions of the 23- and 12-bp insertion/deletion polymorphisms of the PRNP gene in male Holstein compared with male Hanwoo, female Hanwoo and total Hanwoo. In addition, only male Holstein showed weak LD between 23- and 12-bp insertion/deletion polymorphisms. Furthermore, the PrPSc bands were not detected in all Korean cattle tested. To the best of our knowledge, the enhanced surveillance system of BSE was conducted for the first time in Korean cattle.

Usage of meat and bone meal in animal, poultry and fish feeds: A survey and risk analysis for the occurrence of bovine spongiform encephalopathy in Bangladesh.

BACKGROUND: Bovine spongiform encephalopathy (BSE) is an emerging zoonotic disease of cattle associated with pathological prion protein (PrPsc ) transmitted via meat and bone meal (MBM). Although Bangladesh did not experience a BSE outbreak but the country could not export animal products to developed countries as has not yet been declared BSE free country by OIE due to lack of scientific risk evaluation for BSE. The objectives were identification of hazard, release and exposure pathways of pathological prion protein through MBM and analysis of risk for the occurrence of BSE in Bangladesh. METHODS: The scientific data were reviewed, hazards were scheduled and surveys were conducted on livestock production system, import of MBM and its use to identify the hazards present in Bangladesh context. The analysis was done by the 'OIE Risk Analysis Framework 2006 and European Union (EU) Scientific Steering Committee (SSC) 2003'. From the historical reviews, import of MBM and its use was identified, as external hazards. RESULTS: The analysis revealed that these hazards had negligible or moderate risk for the introduction of infectious PrPsc as Bangladeshi cattle are vegetarian cattle. No milk replacer was used and use of slaughtered waste in the animal feed industry is absent. Unconsumable bones are processed to produce bone chips, fertilizers and bone meal for poultry feeds. Scrapie was never prevalent in Bangladesh. Therefore, risk from the internal challenge was negligible in Bangladesh for the occurrence of classical BSE. These prevented the propagation of BSE infectivity and eliminated BSE infectivity from the system very fast, if that was present. CONCLUSIONS: It was concluded that introduction of PrPsc into cattle population of Bangladesh through MBM was very negligible. Therefore, Bangladesh can be considered as BSE negligible risk country.

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

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

Prion strains associated with iatrogenic CJD in French and UK human growth hormone recipients.

Treatment with human pituitary-derived growth hormone (hGH) was responsible for a significant proportion of iatrogenic Creutzfeldt-Jakob disease (iCJD) cases. France and the UK experienced the largest case numbers of hGH-iCJD, with 122 and 81 cases respectively. Differences in the frequency of the three PRNP codon 129 polymorphisms (MM, MV and VV) and the estimated incubation periods associated with each of these genotypes in the French and the UK hGH-iCJD cohorts led to the suggestion that the prion strains responsible for these two hGH-iCJD cohorts were different. In this study, we characterized the prion strains responsible for hGH-iCJD cases originating from UK (n = 11) and France (n = 11) using human PrP expressing mouse models. The cases included PRNP MM, MV and VV genotypes from both countries. UK and French sporadic CJD (sCJD) cases were included as controls. The prion strains identified following inoculation with hGH-iCJD homogenates corresponded to the two most frequently observed sCJD prion strains (M1CJD and V2CJD). However, in clear contradiction to the initial hypothesis, the prion strains that were identified in the UK and the French hGH-iCJD cases were not radically different. In the vast majority of the cases originating from both countries, the V2CJD strain or a mixture of M1CJD + V2CJD strains were identified. These data strongly support the contention that the differences in the epidemiological and genetic profiles observed in the UK and France hGH-iCJD cohorts cannot be attributed only to the transmission of different prion strains.

Prevalence in Britain of abnormal prion protein in human appendices before and after exposure to the cattle BSE epizootic.

Widespread dietary exposure of the population of Britain to bovine spongiform encephalopathy (BSE) prions in the 1980s and 1990s led to the emergence of variant Creutzfeldt-Jakob Disease (vCJD) in humans. Two previous appendectomy sample surveys (Appendix-1 and -2) estimated the prevalence of abnormal prion protein (PrP) in the British population exposed to BSE to be 237 per million and 493 per million, respectively. The Appendix-3 survey was recommended to measure the prevalence of abnormal PrP in population groups thought to have been unexposed to BSE. Immunohistochemistry for abnormal PrP was performed on 29,516 samples from appendices removed between 1962 and 1979 from persons born between 1891 through 1965, and from those born after 1996 that had been operated on from 2000 through 2014. Seven appendices were positive for abnormal PrP, of which two were from the pre-BSE-exposure era and five from the post BSE-exposure period. None of the seven positive samples were from appendices removed before 1977, or in patients born after 2000 and none came from individuals diagnosed with vCJD. There was no statistical difference in the prevalence of abnormal PrP across birth and exposure cohorts. Two interpretations are possible. Either there is a low background prevalence of abnormal PrP in human lymphoid tissues that may not progress to vCJD. Alternatively, all positive specimens are attributable to BSE exposure, a finding that would necessitate human exposure having begun in the late 1970s and continuing through the late 1990s.

Assessment of risk of variant creutzfeldt-Jakob disease (vCJD) from use of bovine heparin.

PURPOSE: In the late1990s, reacting to the outbreak of bovine spongiform encephalopathy (BSE) in the United Kingdom that caused a new variant of Creutzfeldt-Jakob disease (vCJD) in humans, manufacturers withdrew bovine heparin from the market in the United States. There have been growing concerns about the adequate supply and safety of porcine heparin. Since the BSE epidemic has been declining markedly, the US Food and Drug Administration reevaluates the vCJD risk via use of bovine heparin. METHODS: We developed a computational model to estimate the vCJD risk to patients receiving bovine heparin injections. The model incorporated information including BSE prevalence, infectivity levels in the intestines, manufacturing batch size, yield of heparin, reduction in infectivity by manufacturing process, and the dose-response relationship. RESULTS: The model estimates a median risk of vCJD infection from a single intravenous dose (10 000 USP units) of heparin made from US-sourced bovine intestines to be 6.9 × 10-9 (2.5-97.fifth percentile: 1.5 × 10-9 -4.3 × 10-8 ), a risk of 1 in 145 million, and 4.6 × 10-8 (2.5-97.fifth percentile: 1.1 × 10-8 -2.6 × 10-7 ), a risk of 1 in 22 million for Canada-sourced products. The model estimates a median risk of 1.4 × 10-7 (2.5-97.fifth percentile: 2.9 × 10-8 -9.3 × 10-7 ) and 9.6 × 10-7 (2.5-97.fifth percentile: 2.1 × 10-7 -5.6 × 10-6 ) for a typical treatment for venous thromboembolism (infusion of 2-4 doses daily per week) using US-sourced and Canada-sourced bovine heparin, respectively. CONCLUSIONS: The model estimates the vCJD risk from use of heparin when appropriately manufactured from US or Canadian cattle is likely small. The model and conclusions should not be applied to other medicinal products manufactured using bovine-derived materials.

Diagnostic Accuracy of Prion Disease Biomarkers in Iatrogenic Creutzfeldt-Jakob Disease.

Human prion diseases are classified into sporadic, genetic, and acquired forms. Within this last group, iatrogenic Creutzfeldt-Jakob disease (iCJD) is caused by human-to-human transmission through surgical and medical procedures. After reaching an incidence peak in the 1990s, it is believed that the iCJD historical period is probably coming to an end, thanks to lessons learnt from past infection sources that promoted new prion prevention and decontamination protocols. At this point, we sought to characterise the biomarker profile of iCJD and compare it to that of sporadic CJD (sCJD) for determining the value of available diagnostic tools in promptly recognising iCJD cases. To that end, we collected 23 iCJD samples from seven national CJD surveillance centres and analysed the electroencephalogram and neuroimaging data together with a panel of seven CSF biomarkers: 14-3-3, total tau, phosphorylated/total tau ratio, alpha-synuclein, neurofilament light, YKL-40, and real-time quaking induced conversion of prion protein. Using the cut-off values established for sCJD, we found the sensitivities of these biomarkers for iCJD to be similar to those described for sCJD. Given the limited relevant information on this issue to date, the present study validates the use of current sCJD biomarkers for the diagnosis of future iCJD cases.

Animal prion diseases: the risks to human health.

Transmissible spongiform encephalopathies (TSEs) or prion diseases of animals notably include scrapie in small ruminants, chronic wasting disease (CWD) in cervids and classical bovine spongiform encephalopathy (C-BSE). As the transmission barrier phenomenon naturally limits the propagation of prions from one species to another, and the lack of epidemiological evidence for an association with human prion diseases, the zoonotic potential of these diseases was for a long time considered negligible. However, in 1996, C-BSE was recognized as the cause of a new human prion disease, variant Creutzfeldt-Jakob disease (vCJD), which triggered an unprecedented public health crisis in Europe. Large-scale epidemio-surveillance programs for scrapie and C-BSE that were implemented in the EU after the BSE crisis revealed that the distribution and prevalence of prion diseases in the ruminant population had previously been underestimated. They also led to the recognition of new forms of TSEs (named atypical) in cattle and small ruminants and to the recent identification of CWD in Europe. At this stage, the characterization of the strain diversity and zoonotic abilities associated with animal prion diseases remains largely incomplete. However, transmission experiments in nonhuman primates and transgenic mice expressing human PrP clearly indicate that classical scrapie, and certain forms of atypical BSE (L-BSE) or CWD may have the potential to infect humans. The remaining uncertainties about the origins and relationships between animal prion diseases emphasize the importance of the measures implemented to limit human exposure to these potentially zoonotic agents, and of continued surveillance for both animal and human prion diseases.

Prion-related peripheral neuropathy in sporadic Creutzfeldt-Jakob disease.

OBJECTIVE: To assess whether the involvement of the peripheral nervous system (PNS) belongs to the phenotypic spectrum of sporadic Creutzfeldt-Jakob disease (sCJD). METHODS: We examined medical records of 117 sCJDVV2 (ataxic type), 65 sCJDMV2K (kuru-plaque type) and 121 sCJDMM(V)1 (myoclonic type) subjects for clinical symptoms, objective signs and neurophysiological data. We reviewed two diagnostic nerve biopsies and looked for abnormal prion protein (PrPSc) by western blotting and real-time quaking-induced conversion (RT-QuIC) in postmortem PNS samples from 14 subjects. RESULTS: Seventy-five (41.2%) VV2-MV2K patients, but only 11 (9.1%) MM(V)1, had symptoms or signs suggestive of PNS involvement occurring at onset in 18 cases (17 VV2-MV2K, 9.3%; and 1 MM(V)1, 0.8%) and isolated in 6. Nerve biopsy showed a mixed predominantly axonal and demyelinating neuropathy in two sCJDMV2K. Electromyography showed signs of neuropathy in half of the examined VV2-MV2K patients. Prion RT-QuIC was positive in all CJD PNS samples, whereas western blotting detected PrPSc in the sciatic nerve in one VV2 and one MV2K. CONCLUSIONS: Peripheral neuropathy, likely related to PrPSc deposition, belongs to the phenotypic spectrum of sCJDMV2K and VV2 and may mark the clinical onset. The significantly lower prevalence of PNS involvement in typical sCJDMM(V)1 suggests that the PNS tropism of sCJD prions is strain dependent.

Atypical and classic bovine spongiform encephalopathy.

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

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.

Prion Disease in Dromedary Camels, Algeria.

Prions cause fatal and transmissible neurodegenerative diseases, including Creutzfeldt-Jakob disease in humans, scrapie in small ruminants, and bovine spongiform encephalopathy (BSE). After the BSE epidemic, and the associated human infections, began in 1996 in the United Kingdom, general concerns have been raised about animal prions. We detected a prion disease in dromedary camels (Camelus dromedarius) in Algeria. Symptoms suggesting prion disease occurred in 3.1% of dromedaries brought for slaughter to the Ouargla abattoir in 2015-2016. We confirmed diagnosis by detecting pathognomonic neurodegeneration and disease-specific prion protein (PrPSc) in brain tissues from 3 symptomatic animals. Prion detection in lymphoid tissues is suggestive of the infectious nature of the disease. PrPSc biochemical characterization showed differences with BSE and scrapie. Our identification of this prion disease in a geographically widespread livestock species requires urgent enforcement of surveillance and assessment of the potential risks to human and animal health.

Using an epidemiological framework and bovine spongiform encephalopathy investigation questionnaire to investigate suspect bovine spongiform encephalopathy cases: an example from a bovine spongiform encephalopathy case in Ireland in 2015.

In several EU member states, bovine spongiform encephalopathy (BSE) cases have been identified in cattle born after the reinforced ban (BARB cases), for reasons that are not entirely clear. Epidemiological investigation of these cases has proved challenging. The European Food Safety Authority recently recommended the collection of a predefined set of epidemiological data from BSE suspects and confirmed BSE cases to aid future investigations. In this study, we present an epidemiological framework and BSE investigation questionnaire to aid the investigation of suspect BSE cases, and illustrate its application during the investigation of a BSE case in Ireland in 2015. It is recommended that the framework and questionnaire are used concurrently: the framework provides structure and focus, whereas the questionnaire (with 135 questions) aids data collection. The framework focuses on confirmation and discrimination, estimating the date and location of exposure, and determining the method/source of exposure. The BSE case in Ireland in 2015 was a BARB case born in 2010. It was identified with classical BSE at an authorised knackery as part of Ireland's targeted active surveillance programme for BSE. No definitive source of infection with the BSE agent could be attributed in this case.

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.