Vincent Béringue

BSE agent signatures in a goat

SIR, – One of the concerns about BSE is the potential presence of the agent in small ruminants, sheep and goats, as well as cattle. With the objective of documenting this, seven French laboratories have analysed 438 brain samples from confirmed cases of TSE in sheep and goats. These comprised

Pathogenesis of the oral route of infection of mice with scrapie and bovine spongiform encephalopathy agents.

Transmissible spongiform encephalopathies can be transmitted via the oral route. The understanding of this mode of contamination has become a major issue since it is responsible for the appearance of bovine spongiform encephalopathy (BSE) and is probably implicated in new variant Creutzfeldt-Jakob disease. In this study, we addressed the questions of the propagation pathway and the strain specificity of the pathogenesis of oral contamination of mice with the C506M3 scrapie strain and the 6PB1 BSE strain. PrPres was used as a marker of infectivity and was searched for sequentially in 22 organs during the whole incubation period and clinical stage. PrPres was first detectable in the Peyers patches and mesenteric lymph nodes at 45 days post-inoculation. It became detectable 1 to 3 months later in the other tissues of the lymphoreticular system (LRS) such as the spleen and the lymph nodes not related to the digestive tract. These data indicate that after an oral route of entry, the infectious agent is propagated from the Peyers patches to the mesenteric lymph nodes by the lymphatic route, then enters the bloodstream and is distributed to the secondary replication site, the LRS. The major difference between the two agents is that PrPres could be detected in the digestive tract (from the stomach to the colon) with the scrapie agent only. This observation may have implications for the horizontal transmission of scrapie in endemically affected sheep flocks.

Role of spleen macrophages in the clearance of scrapie agent early in pathogenesis.

The involvement of spleen macrophages in the early stages of scrapie pathogenesis was studied by applying the ‘macrophage‐suicide technique’ to scrapie‐infected mice. This method comprises critically the intravenous administration to mice of dichloromethylene disphosphonate encapsulated into liposomes. Depletion of spleen macrophages before scrapie infection induced an increased amount of scrapie inoculum in the spleen, consequently leading to accelerated scrapie agent replication in the early phase of pathogenesis, as followed by PrPres accumulation, a specific hallmark of scrapie. The same effect was observed when spleen macrophages were depleted just before the beginning of scrapie agent replication. These findings suggest that macrophages may partly control scrapie infection in peripheral tissues by sequestration of the scrapie inoculum and may thus impair early scrapie agent replication in the spleen. In addition to macrophages, most follicular dendritic cells and B lymphocytes, which are thought to support scrapie agent replication, were also transiently depleted by dichloromethylene disphosphonate administration. This suggests that a compensatory mechanism is sufficient to ensure the persistence of infection in these early stages of pathogenesis. Copyright

Prion agent diversity and species barrier

Mammalian prions are the infectious agents responsible for transmissible spongiform encephalopathies (TSE), a group of fatal, neurodegenerative diseases, affecting both domestic animals and humans. The most widely accepted view to date is that these agents lack a nucleic acid genome and consist primarily of PrP(Sc), a misfolded, aggregated form of the host-encoded cellular prion protein (PrP(C)) that propagates by autocatalytic conversion and accumulates mainly in the brain. The BSE epizooty, allied with the emergence of its human counterpart, variant CJD, has focused much attention on two characteristics that prions share with conventional infectious agents. First, the existence of multiple prion strains that impose, after inoculation in the same host, specific and stable phenotypic traits such as incubation period, molecular pattern of PrP(Sc) and neuropathology. Prion strains are thought to be enciphered within distinct PrP(Sc) conformers. Second, a transmission barrier exists that restricts the propagation of prions between different species. Here we discuss the possible situations resulting from the confrontation between species barrier and prion strain diversity, the molecular mechanisms involved and the potential of interspecies transmission of animal prions, including recently discovered forms of TSE in ruminants.

Isolation from cattle of a prion strain distinct from that causing bovine spongiform encephalopathy.

To date, bovine spongiform encephalopathy (BSE) and its human counterpart, variant Creutzfeldt-Jakob disease, have been associated with a single prion strain. This strain is characterised by a unique and remarkably stable biochemical profile of abnormal protease-resistant prion protein (PrPres) isolated from brains of affected animals or humans. However, alternate PrPres signatures in cattle have recently been discovered through large-scale screening. To test whether these also represent separate prion strains, we inoculated French cattle isolates characterised by a PrPres of higher apparent molecular mass—called H-type—into transgenic mice expressing bovine or ovine PrP. All mice developed neurological symptoms and succumbed to these isolates, showing that these represent a novel strain of infectious prions. Importantly, this agent exhibited strain-specific features clearly distinct from that of BSE agent inoculated to the same mice, which were retained on further passage. Moreover, it also differed from all sheep scrapie isolates passaged so far in ovine PrP-expressing mice. Our findings therefore raise the possibility that either various prion strains may exist in cattle, or that the BSE agent has undergone divergent evolution in some animals.

The physical relationship between infectivity and prion protein aggregates is strain-dependent.

Prions are unconventional infectious agents thought to be primarily composed of PrPSc, a multimeric misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrPC). They cause fatal neurodegenerative diseases in both animals and humans. The disease phenotype is not uniform within species, and stable, self-propagating variations in PrPSc conformation could encode this ‘strain’ diversity. However, much remains to be learned about the physical relationship between the infectious agent and PrPSc aggregation state, and how this varies according to the strain. We applied a sedimentation velocity technique to a panel of natural, biologically cloned strains obtained by propagation of classical and atypical sheep scrapie and BSE infectious sources in transgenic mice expressing ovine PrP. Detergent-solubilized, infected brain homogenates were used as starting material. Solubilization conditions were optimized to separate PrPSc aggregates from PrPC. The distribution of PrPSc and infectivity in the gradient was determined by immunoblotting and mouse bioassay, respectively. As a general feature, a major proteinase K-resistant PrPSc peak was observed in the middle part of the gradient. This population approximately corresponds to multimers of 12–30 PrP molecules, if constituted of PrP only. For two strains, infectivity peaked in a markedly different region of the gradient. This most infectious component sedimented very slowly, suggesting small size oligomers and/or low density PrPSc aggregates. Extending this study to hamster prions passaged in hamster PrP transgenic mice revealed that the highly infectious, slowly sedimenting particles could be a feature of strains able to induce a rapidly lethal disease. Our findings suggest that prion infectious particles are subjected to marked strain-dependent variations, which in turn could influence the strain biological phenotype, in particular the replication dynamics.

A Bovine Prion Acquires an Epidemic Bovine Spongiform Encephalopathy Strain-Like Phenotype on Interspecies Transmission

Implementation in Europe of large-scale testing to detect bovine spongiform encephalopathy (BSE)-infected cattle and prevent the transmission of this prion disease to humans has recently led to the discovery of novel types of bovine prions. We characterized atypical isolates called BSE L-type by analyzing their molecular and neuropathological properties during transmission to several mouse lines transgenic for the prion protein (PrP). Unexpectedly, such isolates acquired strain features closely similar to those of BSE-type agents when propagated in mice expressing ovine PrP, although they retained phenotypic traits distinct from BSE in other lines, including bovine PrP mice. These findings further underline the relationship between the crossing of species barrier and prion strain diversification, and, although the origin of the epidemic BSE agent has only been speculative until now, they provide new insight into the nature of the events that could have led to the appearance of this agent.

Facilitated Cross-Species Transmission of Prions in Extraneural Tissue

Prion Problem Prion disease, like “mad cow disease,” has shown a frightening ability to cross the species transmission barrier, but, mercifully, with low efficiency. However, the role of different tissues in prion cross-species transmission is unclear. Béringue et al. (p. 472; see the cover; see the Perspective by Collinge) compared the ability of brain and lymphoid tissues from “ovinized” (sheeplike) and “humanized” transgenic mouse models to replicate prion transmission across a robust transmission barrier. Lymphoid tissue of these mice was consistently more permissive than brain tissue to prions such as those causing chronic wasting disease and bovine spongiform encephalopathy. Because previous measures of the transmission barrier have focused on the brain, this heightened susceptibility of lymphoid tissues could strongly impact estimates of the number of silent carriers of prion disease. Lymphoid tissue is more permissive than the brain to foreign prions. Prions are infectious pathogens essentially composed of PrPSc, an abnormally folded form of the host-encoded prion protein PrPC. Constrained steric interactions between PrPSc and PrPC are thought to provide prions with species specificity and to control cross-species transmission into other host populations, including humans. We compared the ability of brain and lymphoid tissues from ovine and human PrP transgenic mice to replicate foreign, inefficiently transmitted prions. Lymphoid tissue was consistently more permissive than the brain to prions such as those causing chronic wasting disease and bovine spongiform encephalopathy. Furthermore, when the transmission barrier was overcome through strain shifting in the brain, a distinct agent propagated in the spleen, which retained the ability to infect the original host. Thus, prion cross-species transmission efficacy can exhibit a marked tissue dependence.

Transmission of atypical bovine prions to mice transgenic for human prion protein.

To assess risk for cattle-to-human transmission of prions that cause uncommon forms of bovine spongiform encephalopathy (BSE), we inoculated mice expressing human PrP Met129 with field isolates. Unlike classical BSE agent, L-type prions appeared to propagate in these mice with no obvious transmission barrier. H-type prions failed to infect the mice.

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.