Conversion of the BASE prion strain into the BSE strain: the origin of BSE?

Atypical neuropathological and molecular phenotypes of bovine spongiform encephalopathy (BSE) have recently been identified in different countries. One of these phenotypes, named bovine "amyloidotic" spongiform encephalopathy (BASE), differs from classical BSE for the occurrence of a distinct type of the disease-associated prion protein (PrP), termed PrP(Sc), and the presence of PrP amyloid plaques. Here, we show that the agents responsible for BSE and BASE possess different biological properties upon transmission to transgenic mice expressing bovine PrP and inbred lines of nontransgenic mice. Strikingly, serial passages of the BASE strain to nontransgenic mice induced a neuropathological and molecular disease phenotype indistinguishable from that of BSE-infected mice. The existence of more than one agent associated with prion disease in cattle and the ability of the BASE strain to convert into the BSE strain may have important implications with respect to the origin of BSE and spongiform encephalopathies in other species, including humans.     

Distinct Transmissibility Features of TSE Sources Derived from Ruminant Prion Diseases by the Oral Route in a Transgenic Mouse Model (TgOvPrP4) Overexpressing the Ovine Prion Protein

Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases associated with a misfolded form of host-encoded prion protein (PrP). Some of them, such as classical bovine spongiform encephalopathy in cattle (BSE), transmissible mink encephalopathy (TME), kuru and variant Creutzfeldt–Jakob disease in humans, are acquired by the oral route exposure to infected tissues. We investigated the possible transmission by the oral route of a panel of strains derived from ruminant prion diseases in a transgenic mouse model (TgOvPrP4) overexpressing the ovine prion protein (A₁₃₆R₁₅₄Q₁₇₁) under the control of the neuron-specific enolase promoter. Sources derived from Nor98, CH1641 or 87V scrapie sources, as well as sources derived from L-type BSE or cattle-passaged TME, failed to transmit by the oral route, whereas those derived from classical BSE and classical scrapie were successfully transmitted. Apart from a possible effect of passage history of the TSE agent in the inocula, this implied the occurrence of subtle molecular changes in the protease-resistant prion protein (PrPres) following oral transmission that can raises concerns about our ability to correctly identify sheep that might be orally infected by the BSE agent in the field. Our results provide proof of principle that transgenic mouse models can be used to examine the transmissibility of TSE agents by the oral route, providing novel insights regarding the pathogenesis of prion diseases.     

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.     

Characterization of Syrian hamster adapted prions derived from L-type and C-type bovine spongiform encephalopathies

Atypical forms of bovine spongiform encephalopathy (BSE) may be caused by different prions from classical BSE (C-BSE). In this study, we examined the susceptibility of mice overexpressing mouse and hamster chimeric prion protein (PrP) to L-type atypical BSE (L-BSE). None of the transgenic mice showed susceptibility to L-BSE, except mice overexpressing hamster PrP. We also examined the transmission properties of L-BSE in hamsters. The incubation period of hamsters intracerebrally inoculated with L-BSE was 576.8 days, and that of the subsequent passage was decreased to 208 days. Although the lesion and glycoform profiles and relative proteinase K resistant core fragment of the abnormal isoform of PrP (PrPcore) of L-BSE were similar to that of C-BSE, the deposition of the abnormal isoform of PrP (PrPSc) and the molecular weight of PrPcore of L-BSE was different from than that of C-BSE. In hamster models, some prion strain characteristics of L-BSE were indistinguishable from those of C-BSE.     

Characterization of Syrian hamster adapted prions derived from L-type and C-type bovine spongiform encephalopathies

Atypical forms of bovine spongiform encephalopathy (BSE) may be caused by different prions from classical BSE (C-BSE). In this study, we examined the susceptibility of mice overexpressing mouse and hamster chimeric prion protein (PrP) to L-type atypical BSE (L-BSE). None of the transgenic mice showed susceptibility to L-BSE, except mice overexpressing hamster PrP. We also examined the transmission properties of L-BSE in hamsters. The incubation period of hamsters intracerebrally inoculated with L-BSE was 576.8 days, and that of the subsequent passage was decreased to 208 days. Although the lesion and glycoform profiles and relative proteinase K resistant core fragment of the abnormal isoform of PrP (PrPcore) of L-BSE were similar to that of C-BSE, the deposition of the abnormal isoform of PrP (PrP^Sc) and the molecular weight of PrPcore of L-BSE was different from than that of C-BSE. In hamster models, some prion strain characteristics of L-BSE were indistinguishable from those of C-BSE.Key words: prion, atypical, L-BSE, PrPcore, hamster, transmission     

Identification of a heritable polymorphism in bovine PRNP associated with genetic transmissible spongiform encephalopathy: evidence of heritable BSE

Background

Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy (TSE) of cattle. Classical BSE is associated with ingestion of BSE-contaminated feedstuffs. H- and L-type BSE, collectively known as atypical BSE, differ from classical BSE by displaying a different disease phenotype and they have not been linked to the consumption of contaminated feed. Interestingly, the 2006 US H-type atypical BSE animal had a polymorphism at codon 211 of the bovine prion gene resulting in a glutamic acid to lysine substitution (E211K). This substitution is analogous a human polymorphism associated with the most prevalent form of heritable TSE in humans, and it is considered to have caused BSE in the 2006 US atypical BSE animal. In order to determine if this amino acid change is a heritable trait in cattle, we sequenced the prion alleles of the only known offspring of this animal, a 2-year-old heifer.

Principal Findings

Sequence analysis revealed that both the 2006 US atypical BSE animal and its 2-year-old heifer were heterozygous at bovine prion gene nucleotides 631 through 633 for GAA (glutamic acid) and AAA (lysine). Both animals carry the E211K polymorphism, indicating that the allele is heritable and may persist within the cattle population.

Conclusions

This is the first evidence that the E211K polymorphism is a germline polymorphism, not a somatic mutation, suggesting BSE may be transmitted genetically in cattle. In the event that E211K proves to result in a genetic form of BSE, this would be the first indication that all 3 etiologic forms of TSEs (spontaneous, hereditary, and infectious) are present in a non-human species. Atypical BSE arising as both genetic and spontaneous disease, in the context of reports that at least some forms of atypical BSE can convert to classical BSE in mice, suggests a cattle origin for classical BSE.     

A C-terminal protease-resistant prion fragment distinguishes ovine "CH1641-like" scrapie from bovine classical and L-Type BSE in ovine transgenic mice

The protease-resistant prion protein (PrP(res)) of a few natural scrapie isolates identified in sheep, reminiscent of the experimental isolate CH1641 derived from a British natural scrapie case, showed partial molecular similarities to ovine bovine spongiform encephalopathy (BSE). Recent discovery of an atypical form of BSE in cattle, L-type BSE or BASE, suggests that also this form of BSE might have been transmitted to sheep. We studied by Western blot the molecular features of PrP(res) in four "CH1641-like" natural scrapie isolates after transmission in an ovine transgenic model (TgOvPrP4), to see if "CH1641-like" isolates might be linked to L-type BSE. We found less diglycosylated PrP(res) than in classical BSE, but similar glycoform proportions and apparent molecular masses of the usual PrP(res) form (PrP(res) #1) to L-type BSE. However, the "CH1641-like" isolates differed from both L-type and classical BSE by an abundant, C-terminally cleaved PrP(res) product (PrP(res) #2) specifically recognised by a C-terminal antibody (SAF84). Differential immunoprecipitation of PrP(res) #1 and PrP(res) #2 resulted in enrichment in PrP(res) #2, and demonstrated the presence of mono- and diglycosylated PrP(res) products. PrP(res) #2 could not be obtained from several experimental scrapie sources (SSBP1, 79A, Chandler, C506M3) in TgOvPrP4 mice, but was identified in the 87V scrapie strain and, in lower and variable proportions, in 5 of 5 natural scrapie isolates with different molecular features to CH1641. PrP(res) #2 identification provides an additional method for the molecular discrimination of prion strains, and demonstrates differences between "CH1641-like" ovine scrapie and bovine L-type BSE transmitted in an ovine transgenic mouse model.     

Intraspecies transmission of BASE induces clinical dullness and amyotrophic changes

The disease phenotype of bovine spongiform encephalopathy (BSE) and the molecular/ biological properties of its prion strain, including the host range and the characteristics of BSE-related disorders, have been extensively studied since its discovery in 1986. In recent years, systematic testing of the brains of cattle coming to slaughter resulted in the identification of at least two atypical forms of BSE. These emerging disorders are characterized by novel conformers of the bovine pathological prion protein (PrP(TSE)), named high-type (BSE-H) and low-type (BSE-L). We recently reported two Italian atypical cases with a PrP(TSE) type identical to BSE-L, pathologically characterized by PrP amyloid plaques and known as bovine amyloidotic spongiform encephalopathy (BASE). Several lines of evidence suggest that BASE is highly virulent and easily transmissible to a wide host range. Experimental transmission to transgenic mice overexpressing bovine PrP (Tgbov XV) suggested that BASE is caused by a prion strain distinct from the BSE isolate. In the present study, we experimentally infected Friesian and Alpine brown cattle with Italian BSE and BASE isolates via the intracerebral route. BASE-infected cattle developed amyotrophic changes accompanied by mental dullness. The molecular and neuropathological profiles, including PrP deposition pattern, closely matched those observed in the original cases. This study provides clear evidence of BASE as a distinct prion isolate and discloses a novel disease phenotype in cattle.     

PET-blot analysis contributes to BSE strain recognition in C57Bl/6 mice.

Identification of the strain of agent responsible for bovine spongiform encephalopathy (BSE) can be made histologically through the analysis of both distribution and intensity of brain vacuolar lesions after BSE transmission to mouse. Another useful way to distinguish the BSE agent from other prion strains is the study of the distribution of the abnormal prion protein (PrP(res)). For that purpose, paraffin-embedded tissue blot (PET-blot) method was applied on brains from C57Bl/6 mice infected with cattle BSE, experimental sheep BSE, or feline spongiform encephalopathy (FSE) from a cheetah. PrP(res) distribution was comparable, whichever of the three BSE agent sources was considered and was distinct from the PrP(res) distribution in C57Bl/6 mice inoculated with a French scrapie isolate or with a mouse-adapted scrapie strain (C506M3). These data confirm a common origin of infectious agent responsible for the British and French cattle BSE. They also indicate that PET-blot method appears as a precise complementary tool in prion strain studies because it offers easy and quick assessment of the PrP(res) mapping. Advantages and limits of the PET-blot method are discussed and compared with other established and validated methods of strain typing.     

Exploring the zoonotic potential of animal prion diseases

Following the discovery of a causal link between bovine spongiform encephalopathy (BSE) in cattle and variant Creutzfeldt–Jakob disease (vCJD) in humans, several experimental approaches have been used to try to assess the potential risk of transmission of other animal transmissible spongiform encephalopathies (TSEs) to humans. Experimental challenge of non-human primates, humanised transgenic mice and cell-free conversion systems have all been used as models to explore the susceptibility of humans to animal TSEs. In this review we compare and contrast in vivo and in vitro evidence of the zoonotic risk to humans from sheep, cattle and deer prions, focusing primarily on chronic wasting disease and our own recent studies using protein misfolding cyclic amplification.     

Molecular analysis of the protease-resistant prion protein in scrapie and bovine spongiform encephalopathy transmitted to ovine transgenic and wild-type mice

The existence of different strains of infectious agents involved in scrapie, a transmissible spongiform encephalopathy (TSE) of sheep and goats, remains poorly explained. These strains can, however, be differentiated by characteristics of the disease in mice and also by the molecular features of the protease-resistant prion protein (PrP(res)) that accumulates into the infected tissues. For further analysis, we first transmitted the disease from brain samples of TSE-infected sheep to ovine transgenic [Tg(OvPrP4)] and to wild-type (C57BL/6) mice. We show that, as in sheep, molecular differences of PrP(res) detected by Western blotting can differentiate, in both ovine transgenic and wild-type mice, infection by the bovine spongiform encephalopathy (BSE) agent from most scrapie sources. Similarities of an experimental scrapie isolate (CH1641) with BSE were also likewise found following transmission in ovine transgenic mice. Secondly, we transmitted the disease to ovine transgenic mice by inoculation of brain samples of wild-type mice infected with different experimental scrapie strains (C506M3, 87V, 79A, and Chandler) or with BSE. Features of these strains in ovine transgenic mice were reminiscent of those previously described for wild-type mice, by both ratios and by molecular masses of the different PrP(res) glycoforms. Moreover, these studies revealed the diversity of scrapie strains and their differences with BSE according to labeling by a monoclonal antibody (P4). These data, in an experimental model expressing the prion protein of the host of natural scrapie, further suggest a genuine diversity of TSE infectious agents and emphasize its linkage to the molecular features of the abnormal prion protein.     

Comparative analysis of Japanese and foreign L-type BSE prions

L-type bovine spongiform encephalopathy (BSE) is an atypical form of BSE. To characterize the Japanese L-type BSE prion, we conducted a comparative study of the Japanese and foreign L-type BSE isolates. The L-type BSE isolates of Japan, Germany, France and Canada were intracerebrally inoculated into bovinized prion protein-overexpressing transgenic mice (TgBoPrP). All the examined L-type BSE isolates were transmitted to TgBoPrP mice, and no clear differences were observed in their biological and biochemical properties. Here, we present evidence that the Japanese and Canadian L-type BSE prions are identical to those from the European cases.     

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.     

Possible Case of Maternal Transmission of Feline Spongiform Encephalopathy in a Captive Cheetah

Feline spongiform encephalopathy (FSE) is considered to be related to bovine spongiform encephalopathy (BSE) and has been reported in domestic cats as well as in captive wild cats including cheetahs, first in the United Kingdom (UK) and then in other European countries. In France, several cases were described in cheetahs either imported from UK or born in France. Here we report details of two other FSE cases in captive cheetah including a 2^nd case of FSE in a cheetah born in France, most likely due to maternal transmission. Complete prion protein immunohistochemical study on both brains and peripheral organs showed the close likeness between the two cases. In addition, transmission studies to the TgOvPrP4 mouse line were also performed, for comparison with the transmission of cattle BSE. The TgOvPrP4 mouse brains infected with cattle BSE and cheetah FSE revealed similar vacuolar lesion profiles, PrP^d brain mapping with occurrence of typical florid plaques. Collectively, these data indicate that they harbor the same strain of agent as the cattle BSE agent. This new observation may have some impact on our knowledge of vertical transmission of BSE agent-linked TSEs such as in housecat FSE, or vCJD.     

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

Background  

After 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.     

PRNP gene variation in Pakistani cattle and buffaloes

Bovine spongiform encephalopathy (BSE) is a neurodegenerative prion protein misfolding disorder of cattle. BSE is of two types, classical BSE and atypical BSE which in turn is of two types, H-type BSE and L-type BSE. Both H-type BSE and L-type BSE are primarily sporadic prion disorders. However, one case of H-type BSE has recently been associated with E211K polymorphism in the prion protein gene (PRNP). Two polymorphisms in the bovine PRNP are also associated with susceptibility to classical BSE: a 23 bp insertion/deletion (indel) in the PRNP promoter region and a 12 bp indel in the first intron. No information regarding BSE susceptibility in Pakistani cattle is available. The present study aimed at achieving this information. A total of 236 cattle from 7 breeds and 281 buffaloes from 5 breeds were screened for E211K polymorphism and 23 bp and 12 bp indels employing triplex PCR. The E211K polymorphism was not detected in any of the animals studied. The 23 bp insertion allele was underrepresented in studied cattle breeds while the 12 bp insertion allele was overrepresented. Both 23 bp and 12 bp insertion alleles were overrepresented in studied buffalo breeds. Almost 90% of alleles were insertion alleles across all studied buffalo breeds. The average frequency of 23 bp and 12 bp insertion alleles across all studied cattle breeds was found to be 0.1822 and 0.9407, respectively. There were significant differences between Pakistani and worldwide cattle in terms of allele, genotype and haplotype frequencies of 23 bp and 12 bp indels. The higher observed frequency of 12 bp insertion allele suggests that Pakistani cattle are relatively more resistant to classical BSE than European cattle. However, the key risk factor for classical BSE is the dietary exposure of cattle to contaminated feedstuffs.     

A Case of Bovine Spongiform Encephalopathy in Denmark

Bovine spongiform encephalopathy (BSE), a transmissible spongiform encephalopathy in cattle, was first described in England by Wells et al. (1987). The infection occurs mainly due to digestion of feedstuffs containing ruminant derived protein in form of meat and bone meal contaminated with a scrapie-like agent (Wilesmith et al. 1991). Later, cases of BSE were diagnosed in the Republic of Ireland, Oman, France, and Switzerland (Marinovic & Senn 1991). This report describes the first case of BSE in Denmark.     

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.     

Different prion disease phenotypes result from inoculation of cattle with two temporally separated sources of sheep scrapie from Great Britain

Background

Given the theoretical proposal that bovine spongiform encephalopathy (BSE) could have originated from sheep scrapie, this study investigated the pathogenicity for cattle, by intracerebral (i.c.) inoculation, of two pools of scrapie agents sourced in Great Britain before and during the BSE epidemic. Two groups of ten cattle were each inoculated with pools of brain material from sheep scrapie cases collected prior to 1975 and after 1990. Control groups comprised five cattle inoculated with sheep brain free from scrapie, five cattle inoculated with saline, and for comparison with BSE, naturally infected cattle and cattle i.c. inoculated with BSE brainstem homogenate from a parallel study. Phenotypic characterisation of the disease forms transmitted to cattle was conducted by morphological, immunohistochemical, biochemical and biological methods.

Results

Disease occurred in 16 cattle, nine inoculated with the pre-1975 inoculum and seven inoculated with the post-1990 inoculum, with four cattle still alive at 83 months post challenge (as at June 2006). The different inocula produced predominantly two different disease phenotypes as determined by histopathological, immunohistochemical and Western immunoblotting methods and biological characterisation on transmission to mice, neither of which was identical to BSE. Whilst the disease presentation was uniform in all scrapie-affected cattle of the pre-1975 group, the post-1990 inoculum produced a more variable disease, with two animals sharing immunohistochemical and molecular profile characteristics with animals in the pre-1975 group.

Conclusion

The study has demonstrated that cattle inoculated with different pooled scrapie sources can develop different prion disease phenotypes, which were not consistent with the phenotype of BSE of cattle and whose isolates did not have the strain typing characteristics of the BSE agent on transmission to mice.