PrPTSE distribution in a primate model of variant, sporadic, and iatrogenic Creutzfeldt-Jakob disease

Human prion diseases, such as Creutzfeldt-Jakob disease (CJD), are neurodegenerative and fatal. Sporadic CJD (sCJD) can be transmitted between humans through medical procedures involving highly infected organs, such as the central nervous system. However, in variant CJD (vCJD), which is due to human contamination with the bovine spongiform encephalopathy (BSE) agent, lymphoreticular tissue also harbors the transmissible spongiform encephalopathy-associated prion protein (PrP(TSE)), which poses a particularly acute risk for iatrogenic transmission. Two blood transfusion-related cases are already documented. In addition, the recent observation of PrP(TSE) in spleen and muscle in sCJD raised the possibility that peripheral PrP(TSE) is not limited to vCJD cases. We aimed to clarify the peripheral pathogenesis of human TSEs by using a nonhuman primate model which mimics human diseases. A highly sensitive enzyme-linked immunosorbent assay was adapted to the detection of extraneural PrP(TSE). We show that affected organs can be divided into two groups. The first is peripheral organs accumulating large amounts of PrP(TSE), which represent a high risk of iatrogenic transmission. This category comprises only lymphoreticular organs in the vCJD/BSE model. The second is organs with small amounts of PrP(TSE) associated with nervous structures. These are the muscles, adrenal glands, and enteric nervous system in the sporadic, iatrogenic, and variant CJD models. In contrast to the first set of organs, this low level of tissue contamination is not strain restricted and seems to be linked to secondary centrifugal spread of the agent through nerves. It might represent a risk for iatrogenic transmission, formerly underestimated despite previous reports of low rates of transmission from peripheral organs of humans to nonhuman primates (5, 10). This study provides an additional experimental basis for the classification of human organs into different risk categories and a rational re-evaluation of current risk management measures.     

BSE prions propagate as either variant CJD-like or sporadic CJD-like prion strains in transgenic mice expressing human prion protein

Variant Creutzfeldt-Jakob disease (vCJD) has been recognized to date only in individuals homozygous for methionine at PRNP codon 129. Here we show that transgenic mice expressing human PrP methionine 129, inoculated with either bovine spongiform encephalopathy (BSE) or variant CJD prions, may develop the neuropathological and molecular phenotype of vCJD, consistent with these diseases being caused by the same prion strain. Surprisingly, however, BSE transmission to these transgenic mice, in addition to producing a vCJD-like phenotype, can also result in a distinct molecular phenotype that is indistinguishable from that of sporadic CJD with PrP(Sc) type 2. These data suggest that more than one BSE-derived prion strain might infect humans; it is therefore possible that some patients with a phenotype consistent with sporadic CJD may have a disease arising from BSE exposure.     

The expanding universe of prion diseases

Prions cause fatal and transmissible neurodegenerative disease. These etiological infectious agents are formed in greater part from a misfolded cell-surface protein called PrP(C). Several mammalian species are affected by the diseases, and in the case of "mad cow disease" (BSE) the agent has a tropism for humans, with negative consequences for agribusiness and public health. Unfortunately, the known universe of prion diseases is expanding. At least four novel prion diseases--including human diseases variant Creutzfeldt-Jakob disease (vCJD) and sporadic fatal insomnia (sFI), bovine amyloidotic spongiform encephalopathy (BASE), and Nor98 of sheep--have been identified in the last ten years, and chronic wasting disease (CWD) of North American deer (Odocoileus Specis) and Rocky Mountain elk (Cervus elaphus nelsoni) is undergoing a dramatic spread across North America. While amplification (BSE) and dissemination (CWD, commercial sourcing of cervids from the wild and movement of farmed elk) can be attributed to human activity, the origins of emergent prion diseases cannot always be laid at the door of humankind. Instead, the continued appearance of new outbreaks in the form of "sporadic" disease may be an inevitable outcome in a situation where the replicating pathogen is host-encoded.     

Generation of a Persistently Infected MDBK Cell Line with Natural Bovine Spongiform Encephalopathy (BSE)

Bovine spongiform encephalopathy (BSE) is a zoonotic transmissible spongiform encephalopathy (TSE) thought to be caused by the same prion strain as variant Creutzfeldt-Jakob disease (vCJD). Unlike scrapie and chronic wasting disease there is no cell culture model allowing the replication of proteinase K resistant BSE (PrP^BSE) and the further in vitro study of this disease. We have generated a cell line based on the Madin-Darby Bovine Kidney (MDBK) cell line over-expressing the bovine prion protein. After exposure to naturally BSE-infected bovine brain homogenate this cell line has shown to replicate and accumulate PrP^BSE and maintain infection up to passage 83 after initial challenge. Collectively, we demonstrate, for the first time, that the BSE agent can infect cell lines over-expressing the bovine prion protein similar to other prion diseases. These BSE infected cells will provide a useful tool to facilitate the study of potential therapeutic agents and the diagnosis of BSE.     

Sheep and goat BSE propagate more efficiently than cattle BSE in human PrP transgenic mice

A new variant of Creutzfeldt Jacob Disease (vCJD) was identified in humans and linked to the consumption of Bovine Spongiform Encephalopathy (BSE)-infected meat products. Recycling of ruminant tissue in meat and bone meal (MBM) has been proposed as origin of the BSE epidemic. During this epidemic, sheep and goats have been exposed to BSE-contaminated MBM. It is well known that sheep can be experimentally infected with BSE and two field BSE-like cases have been reported in goats. In this work we evaluated the human susceptibility to small ruminants-passaged BSE prions by inoculating two different transgenic mouse lines expressing the methionine (Met) allele of human PrP at codon 129 (tg650 and tg340) with several sheep and goat BSE isolates and compared their transmission characteristics with those of cattle BSE. While the molecular and neuropathological transmission features were undistinguishable and similar to those obtained after transmission of vCJD in both transgenic mouse lines, sheep and goat BSE isolates showed higher transmission efficiency on serial passaging compared to cattle BSE. We found that this higher transmission efficiency was strongly influenced by the ovine PrP sequence, rather than by other host species-specific factors. Although extrapolation of results from prion transmission studies by using transgenic mice has to be done very carefully, especially when human susceptibility to prions is analyzed, our results clearly indicate that Met129 homozygous individuals might be susceptible to a sheep or goat BSE agent at a higher degree than to cattle BSE, and that these agents might transmit with molecular and neuropathological properties indistinguishable from those of vCJD. Our results suggest that the possibility of a small ruminant BSE prion as vCJD causal agent could not be ruled out, and that the risk for humans of a potential goat and/or sheep BSE agent should not be underestimated.     

Preclinical deposition of pathological prion protein in muscle of experimentally infected primates

Prion diseases are transmissible fatal neurodegenerative disorders affecting humans and animals. A central step in disease progression is the accumulation of a misfolded form (PrP(Sc)) of the host encoded prion protein (PrP(C)) in neuronal and non-neuronal tissues. The involvement of peripheral tissues in preclinical states increases the risk of accidental transmission. On the other hand, detection of PrP(Sc) in non-neuronal easy-accessible compartments such as muscle may offer a novel diagnostic tool. Primate models have proven invaluable to investigate prion diseases. We have studied the deposition of PrP(Sc) in muscle and central nervous system of rhesus monkeys challenged with sporadic Creutzfeldt-Jakob disease (sCJD), variant CJD (vCJD) and bovine spongiform encephalopathy (BSE) in preclinical and clinical stage using biochemical and morphological methods. Here, we show the preclinical presence of PrP(Sc) in muscle and central nervous system of rhesus monkeys experimentally infected with vCJD.     

Evaluation of rapid post-mortem test kits for bovine spongiform encephalopathy (BSE) screening in Japan: Their analytical sensitivity to atypical BSE prions

A classical type of bovine spongiform encephalopathy (C-BSE), recognized in 1987, had a large impact on public health due to its zoonotic link to variant Creutzfeldt-Jakob disease by the human consumption of dietary products contaminated with the C-BSE prion. Thus, a number of countries implemented BSE surveillance using rapid post-mortem test kits that were approved for detection of the C-BSE prion in the cattle brain. However, as atypical BSE (L- and H-BSE) cases emerged in subsequent years, the efficacy of the kits for the detection of atypical BSE prions became a matter of concern. In response to this, laboratories in the European Union and Canada evaluated the kits used in their countries. Here, we carried out an evaluation study of NippiBL®, a kit currently used for BSE screening in Japan. By applying the kit to cattle brains of field cases of C-BSE and L-BSE, and an experimental case of H-BSE, we showed its comparable sensitivities to C, L-, and H-BSE prions, and satisfactory performance required by the European Food Safety Authority. In addition to NippiBL®, two kits (TeSeE® and FRELISA®) formerly used in Japan were effective for detection of the L-BSE prion, although the two kits were unable to be tested for the H-BSE prion due to the discontinuation of domestic sales during this study. These results indicate that BSE screening in Japan is as effective as those in other countries, and it is unlikely that cases of atypical BSE have been overlooked.     

Impact of vCJD on blood supply.

Variant Creutzfeldt-Jakob disease (vCJD) is an at present inevitably lethal neurodegenerative disease which can only be diagnosed definitely post mortem. The majority of the approximately 200 victims to date have resided in the UK where most contaminated beef materials entered the food chain. Three cases in the UK demonstrated that vCJD can be transmitted by blood transfusion. Since BSE and vCJD have spread to several countries outside the UK, it appears advisable that specific risk assessments be carried out in different countries and geographic areas. This review explains the approach adopted by Germany in assessing the risk and considering precautionary measures. A fundamental premise is that the feeding chain of cattle and the food chain have been successfully and permanently cleared from contaminated material. This raises the question of whether transmissions via blood transfusions could have the potential to perpetuate vCJD in mankind. A model calculation based on actual population data showed, however, that this would not be the case. Moreover, an exclusion of transfusion recipients from blood donation would add very little to the safety of blood transfusions, but would have a considerable impact on blood supply. Therefore, an exclusion of transfusion recipients was not recommended in Germany.     

Genome-wide scan identifies loci associated with classical BSE occurrence

Classical bovine spongiform encephalopathy (BSE) is an acquired prion disease that is invariably fatal in cattle and has been implicated as a significant human health risk. Sequence variations in the coding region of the prion gene (PRNP) have been associated with acquired transmissible spongiform encephalopathy (TSE) susceptibility in mammals; however, this is not the case in cattle. It has been hypothesized that genes, in addition to the prion gene, contribute to genetic susceptibility of acquired TSEs. Accordingly, genetic studies of classical BSE in cattle identified loci other than PRNP that are associated with disease incidence. The objective of this study was to utilize a genome-wide association study to test for genetic loci associated with classical BSE. The samples include 143 BSE affected (case) and 173 unaffected half sib (control) animals collected in the mid 1990s in Southern England. The data analysis identifies loci on two different chromosomes associated with BSE disease occurrence. Most notable is a single nucleotide polymorphism on chromosome 1 at 29.15 Mb that is associated with BSE disease (p = 3.09E-05). Additionally, a locus on chromosome 14, within a cluster of SNPs showed a trend toward significance (p = 5.24E-05). It is worth noting that in a human vCJD study markers on human chromosome 8, a region with shared synteny to the region identified on cattle chromosome 14, were associated with disease. Further, our candidate genes appear to have plausible biological relevance with the known etiology of TSE disease. One of the candidate genes is hypothetical gene LOC521010, similar to FK506 binding protein 2 located on chromosome 1 at 29.32 Mb. This gene encodes a protein that is a member of the immunophilin protein family and is involved in basic cellular processes including protein folding. The chromosomal regions identified in this study and candidate genes within these regions merit further investigation.     

Increased susceptibility of human-PrP transgenic mice to bovine spongiform encephalopathy infection following passage in sheep

The risk of the transmission of ruminant transmissible spongiform encephalopathy (TSE) to humans was thought to be low due to the lack of association between sheep scrapie and the incidence of human TSE. However, a single TSE agent strain has been shown to cause both bovine spongiform encephalopathy (BSE) and human vCJD, indicating that some ruminant TSEs are transmissible to humans. While the transmission of cattle BSE to humans in transgenic mouse models has been inefficient, indicating the presence of a significant transmission barrier between cattle and humans, BSE has been transmitted to a number of other species. Here, we aimed to further investigate the human transmission barrier following the passage of BSE in a sheep. Following inoculation with cattle BSE, gene-targeted transgenic mice expressing human PrP showed no clinical or pathological signs of TSE disease. However, following inoculation with an isolate of BSE that had been passaged through a sheep, TSE-associated vacuolation and proteinase K-resistant PrP deposition were observed in mice homozygous for the codon 129-methionine PRNP gene. This observation may be due to higher titers of the BSE agent in sheep or an increased susceptibility of humans to BSE prions following passage through a sheep. However, these data confirm that, contrary to previous predictions, it is possible that a sheep prion is transmissible to humans and that BSE from other species is a public health risk.     

Preclinical transmission of prions by blood transfusion is influenced by donor genotype and route of infection

Variant Creutzfeldt-Jakob disease (vCJD) is a human prion disease resulting from zoonotic transmission of bovine spongiform encephalopathy (BSE). Documented cases of vCJD transmission by blood transfusion necessitate on-going risk reduction measures to protect blood supplies, such as leucodepletion (removal of white blood cells, WBCs). This study set out to determine the risks of prion transmission by transfusion of labile blood components (red blood cells, platelets, plasma) commonly used in human medicine, and the effectiveness of leucodepletion in preventing infection, using BSE-infected sheep as a model. All components were capable of transmitting prion disease when donors were in the preclinical phase of infection, with the highest rates of infection in recipients of whole blood and buffy coat, and the lowest in recipients of plasma. Leucodepletion of components (<10⁶ WBCs/unit) resulted in significantly lower transmission rates, but did not completely prevent transmission by any component. Donor PRNP genotype at codon 141, which is associated with variation in incubation period, also had a significant effect on transfusion transmission rates. A sensitive protein misfolding cyclic amplification (PMCA) assay, applied to longitudinal series of blood samples, identified infected sheep from 4 months post infection. However, in donor sheep (orally infected), the onset of detection of PrP^Sc in blood was much more variable, and generally later, compared to recipients (intravenous infection). This shows that the route and method of infection may profoundly affect the period during which an individual is infectious, and the test sensitivity required for reliable preclinical diagnosis, both of which have important implications for disease control. Our results emphasize that blood transfusion can be a highly efficient route of transmission for prion diseases. Given current uncertainties over the prevalence of asymptomatic vCJD carriers, this argues for the maintenance and improvement of current measures to reduce the risk of transmission by blood products.     

BSE in France: epidemiological analysis and predictions

Attention throughout Europe continues to focus on bovine spongiform encephalopathy (BSE) with increasing evidence linking it to the new variant of Creutzfeldt-Jakob disease (vCJD) in humans. The age- and cohort-specific incidence of BSE in French cattle was modelled as a function of the survival distribution, the cohort-specific incidence of BSE infection, the underreporting rate of BSE cases, and the age-specific probability, conditional on survival, that an infected animal would experience clinical onset. The results reveal that thousands of French cattle were infected with BSE over the course of the epidemic. However, case incidence is predicted to decline in future years.     

Safety of milk and milk derivatives in relation to BSE: the lactoferrin example

Bovine Spongiform Encephalopathy (BSE) belongs to Transmissible Spongiform Encephalopathies (TSEs) or Prion diseases. BSE is a feed borne infection of cattle. Epidemiological and laboratory data suggest that the BSE infectious agent is responsible for the variant form of Creutzfeldt-Jakob Disease (vCJD) and that the oral route is the most plausible way of infection. Therefore there is concern that the BSE agent can be transmitted to humans by biological materials (i.e. meat products, blood, milk) from susceptible BSE animal species (mostly cows but possibly, sheep and goats). Lactoferrin (LF) can be produced by purification from large volumes of cow's milk or whey. Therefore, a potential BSE risk for milk and milk products needs to be evaluated by risk assessment. The Committee for proprietary Medicinal Products--CPMP of the European Commission and the WHO have categorized risk tissues from TSE susceptible ruminant species in different classes in relation to the BSE risk for medicinal products. Milk, colostrum, and tissues of the mammary gland have been classified in the category of no detectable infectivity. A secondary contamination of milk can be virtually excluded (i.e. milk is taken from living animals). In the light of current scientific knowledge and irrespective of the geographical origin, milk and milk derivatives (e.g. lactoferrin, lactose) are unlikely to present any risk of TSE contamination provided that milk is sourced from healthy animals in the same conditions as milk collected from human consumption. So the risk of milk and milk derivatives in relation to BSE is negligible.     

Prions: where do we stand 20 years after the appearance of bovine spongiform encephalopathy?

Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy (TSE) identified twenty years ago in the British cattle herds. Creutzfeldt-Jakob disease (CJD) is a TSE that occurs in humans. In 1996, scientists found a possible link between BSE and a new variant of CJD (vCJD). The fact that the non conventional infectious agent of TSE, named prions, could cross the species barrier from cattle to human through meat consumption, raised a tremendous concern for public safety in Europe. This led to the development in the following two decades of substantial and expensive measures to contain BSE and prevent its transmission to humans. In parallel, scientific programs have been funded to progress through the comprehension of the physiopathology of these fatal disorders. In Europe, the BSE epidemics is now ending and the number of cases is decreasing thanks to the strict control of animal foodstuff that was the main source of prion contamination. Only a small number of vCJD have been detected, however, additional concerns have been raised recently for public safety as secondary transmission of CJD through medical procedure and blood transfusion is possible. In addition, the possibility that the BSE was transmitted to other animals including small ruminants is also worrisome. Research efforts are now focussing on decontamination and ante mortem diagnosis of TSE to prevent animal and human transmission. However, needs for fundamental research are still important as many questions remain to be addressed to understand the mechanism of prion transmission, as well as its pathogenesis.     

The identification of candidate genes and SNP markers for classical bovine spongiform encephalopathy susceptibility

Classical bovine spongiform encephalopathy is a transmissible prion disease that is fatal to cattle and is a human health risk due to its association with a strain of Creutzfeldt-Jakob disease (vCJD). Mutations to the coding region of the prion gene (PRNP) have been associated with susceptibility to transmissible spongiform encephalopathies in mammals including bovines and humans. Additional loci such as the retinoic acid receptor beta (RARB) and stathmin like 2 (STMN2) have also been associated with disease risk. The objective of this study was to refine previously identified regions associated with BSE susceptibility and to identify positional candidate genes and genetic variation that may be involved with the progression of classical BSE. The samples included 739 samples of either BSE infected animals (522 animals) or non-infected controls (207 animals). These were tested using a custom SNP array designed to narrow previously identified regions of importance in bovine genome. Thirty one single nucleotide polymorphisms were identified at p < 0.05 and a minor allele frequency greater than 5%. The chromosomal regions identified and the positional and functional candidate genes and regulatory elements identified within these regions warrant further research.     

The identification of candidate genes and SNP markers for classical bovine spongiform encephalopathy susceptibility

Classical bovine spongiform encephalopathy is a transmissible prion disease that is fatal to cattle and is a human health risk due to its association with a strain of Creutzfeldt-Jakob disease (vCJD). Mutations to the coding region of the prion gene (PRNP) have been associated with susceptibility to transmissible spongiform encephalopathies in mammals including bovines and humans. Additional loci such as the retinoic acid receptor beta (RARB) and stathmin like 2 (STMN2) have also been associated with disease risk. The objective of this study was to refine previously identified regions associated with BSE susceptibility and to identify positional candidate genes and genetic variation that may be involved with the progression of classical BSE. The samples included 739 samples of either BSE infected animals (522 animals) or non-infected controls (207 animals). These were tested using a custom SNP array designed to narrow previously identified regions of importance in bovine genome. Thirty one single nucleotide polymorphisms were identified at p < 0.05 and a minor allele frequency greater than 5%. The chromosomal regions identified and the positional and functional candidate genes and regulatory elements identified within these regions warrant further research.     

The structural stability of wild-type horse prion protein

Prion diseases (e.g. Creutzfeldt-Jakob disease (CJD), variant CJD (vCJD), Gerstmann-Straussler-Scheinker syndrome (GSS), Fatal Familial Insomnia (FFI) and Kuru in humans, scrapie in sheep, bovine spongiform encephalopathy (BSE or 'mad-cow' disease) and chronic wasting disease (CWD) in cattles) are invariably fatal and highly infectious neurodegenerative diseases affecting humans and animals. However, by now there have not been some effective therapeutic approaches or medications to treat all these prion diseases. Rabbits, dogs, and horses are the only mammalian species reported to be resistant to infection from prion diseases isolated from other species. Recently, the β2-α2 loop has been reported to contribute to their protein structural stabilities. The author has found that rabbit prion protein has a strong salt bridge ASP177-ARG163 (like a taut bow string) keeping this loop linked. This paper confirms that this salt bridge also contributes to the structural stability of horse prion protein. Thus, the region of β2-α2 loop might be a potential drug target region. Besides this very important salt bridge, other four important salt bridges GLU196-ARG156-HIS187, ARG156-ASP202 and GLU211-HIS177 are also found to greatly contribute to the structural stability of horse prion protein. Rich databases of salt bridges, hydrogen bonds and hydrophobic contacts for horse prion protein can be found in this paper.