RESEARCH ARTICLE
A single amino acid residue in bank vole prion protein drives permissiveness to Nor98/atypical scrapie and the emergence of multiple strain variants
Laura Pirisinu,Michele Angelo Di Bari,Claudia D’Agostino,Ilaria Vanni,Geraldina Riccardi,Stefano Marcon,Gabriele Vaccari,Barbara Chiappini,Sylvie L. Benestad,Umberto Agrimi,Romolo Nonno
Version 2
Published: June 22, 2022
Abstract
Prions are infectious agents that replicate through the autocatalytic misfolding of the cellular prion protein (PrPC) into infectious aggregates (PrPSc) causing fatal neurodegenerative diseases in humans and animals. Prions exist as strains, which are encoded by conformational variants of PrPSc. The transmissibility of prions depends on the PrPC sequence of the recipient host and on the incoming prion strain, so that some animal prion strains are more contagious than others or are transmissible to new species, including humans. Nor98/atypical scrapie (AS) is a prion disease of sheep and goats reported in several countries worldwide. At variance with classical scrapie (CS), AS is considered poorly contagious and is supposed to be spontaneous in origin. The zoonotic potential of AS, its strain variability and the relationships with the more contagious CS strains remain largely unknown. We characterized AS isolates from sheep and goats by transmission in ovinised transgenic mice (tg338) and in two genetic lines of bank voles, carrying either methionine (BvM) or isoleucine (BvI) at PrP residue 109. All AS isolates induced the same pathological phenotype in tg338 mice, thus proving that they encoded the same strain, irrespective of their geographical origin or source species. In bank voles, we found that the M109I polymorphism dictates the susceptibility to AS. BvI were susceptible and faithfully reproduced the AS strain, while the transmission in BvM was highly inefficient and was characterized by a conformational change towards a CS-like prion strain. Sub-passaging experiments revealed that the main strain component of AS is accompanied by minor CS-like strain components, which can be positively selected during replication in both AS-resistant or AS-susceptible animals. These findings add new clues for a better comprehension of strain selection dynamics in prion infections and have wider implications for understanding the origin of contagious prion strains, such as CS.
Author summary
Prions are transmissible agents responsible for fatal neurodegenerative diseases in humans and animals. Prions exist as strains, exhibiting distinct disease phenotypes and transmission properties. Some prion diseases occur sporadically with a supposedly spontaneous origin, while others are contagious and give rise to epidemics, mainly in animals. We investigated the strain properties of Nor98/atypical scrapie (AS), a sporadic prion disease of small ruminants. We found that AS was faithfully reproduced not only in a homologous context, i.e. ovinised transgenic mice, but also in an unrelated animal species, the bank vole. A natural polymorphism of the bank vole prion protein, coding for methionine (BvM) or for isoleucine (BvI) at codon 109, dictated the susceptibility of voles to AS, with BvI being highly susceptible to AS and BvM rather resistant. Most importantly, the M109I polymorphism mediated the emergence of AS-derived mutant prion strains resembling classical scrapie (CS), a contagious prion disease. Finally, by sub-passages in bank voles, we found that the main strain component of AS is accompanied by minor CS-like strain components, which can be positively selected during replication in both AS-resistant or AS-susceptible vole lines. These findings allow a better understanding of strain selection dynamics and suggest a link between sporadic and contagious prion diseases.
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Discussion
Although it is unclear if AS has a spontaneous or acquired origin, AS isolates have been successfully transmitted in transgenic mice expressing sheep/goat-derived PrP sequences [51–54, 69, 73], in sheep [47–50] and, with a much lower efficiency, in transgenic mice expressing bovine [57] or porcine PrP [56]. Wild type inbred mouse lines were repeatedly shown to be resistant to experimental challenge with AS isolates [34, 51, 53, 72]. The picture emerging from these studies shows that AS faithfully propagated on sheep/goat PrP sequences, while in non-small ruminant PrP-bearing hosts a high transmission barrier was accompanied by inability to efficiently reproduce AS prions and by strain mutation, with a shift from PrPres of 8 kDa to PrPres 27–30 [56, 57]. In one instance, AS strain mutation has been observed even upon intra-species intracerebral challenge, where a single sheep showed a strain shift to the classical scrapie strain CH1641 [74].
In this study, we tested the transmissibility of AS in two genetic lines of bank voles, a rodent species known to be susceptible to a wide range of prion strains [8, 55, 59–68].
Unexpectedly, our findings were reminiscent of previous transmission studies with AS, as we observed the same dichotomy between a susceptible recipient model, BvI, able to faithfully reproduce AS prions, and the more resistant BvM, in which rare positive transmissions were characterized by strain mutation and the accumulation of PrPres 27–30 instead of 8 kDa PrPres. Thus, these data add to previous evidence showing that, in animal models unable to reproduce AS prions, long lasting and inefficient primary transmissions of AS isolates mediate the emergence of mutant prion strains, raising the question of their origin. Do they derive from the conversion of PrPSc to alternative conformations caused by PrP amino acid mismatches between donor and recipient host species, as posited by the “deformed templating” model [75], or are minor strain variants pre-existing in AS isolates and positively selected in animal species unable to reproduce bona fide AS prions, as postulated by the “conformational selection” model [18]? The recent experiments by Huor and colleagues [57] represent a cornerstone in support of the latter hypothesis, as they used an ultrasensitive in vitro replication method to directly demonstrate the presence of classical BSE prions as a minor component in AS isolates, which can be positively selected in transgenic mice expressing bovine PrP. Accordingly, Huor and colleagues also found that AS is accompanied by minute amounts of classical BSE prions even during replication in tg338 mice, a sheep-based transgenic mouse line. We consider that our results in bank voles can be better interpreted within the framework of the conformational selection model, thus supporting the hypothesis that minor strain variants are present in AS isolates.
The transmission of AS in BvI was highly efficient and without obvious transmission barrier, as secondary transmission did not lead to shortening of the survival time. Most importantly, BvI faithfully recapitulated the pathological features reported in small ruminants AS, including N- and C- terminal cleaved 8 kDa PrPres [34, 35, 72, 76, 77], PrPSc with very low conformational stability [27, 35], brain distribution of spongiform degeneration and PrPSc deposition mainly in neocortex, hippocampus, basal ganglia and cerebellum [6], involvement of white matter tracts and absence of intraneuronal PrPSc deposits [6, 71]. Accordingly, bioassay of BvI-adapted AS in tg338 reporter mice confirmed the full preservation of the AS strain in BvI. Thus, despite the rather diverging PrP sequence compared to sheep, BvI PrP was fully able to acquire the PrPSc conformation that characterize AS, behaving as expected for sheep/goat-derived PrPs and allowing disease kinetics similar or even faster than in transgenic mice over-expressing small ruminant PrPs [51–54, 69, 73]. These results prove that the small ruminant PrP sequence is not an absolute requirement for propagation of bona fide AS and that amino acid mismatches with sheep PrPs did not cause deformed templating of AS in BvI.
Unexpectedly, the simple met/ile substitution at PrP codon 109 in BvM made voles unable to reproduce bona fide AS, so that BvM behaved very similarly to other non-small ruminant PrP-bearing animal models. Despite the resistance of BvM, a single AS isolate induced disease in two voles at first passage, with a clear phenotype shift. The shifted phenotype was maintained at the second and third passages, leading to a BvM-adapted strain with CS-like features, such as PrPres 27–30, main subcortical involvement and intraneuronal PrPSc deposition in grey matter. In keeping with what observed in sheep [74], the AS-derived strain that emerged in BvM showed incubation time and neuropathological features reminiscent of BvM-adapted CH1641 [78]. In contrast, it is not surprising that classical BSE did not emerge in BvM, given the very low amounts of classical BSE prions detected in AS isolates [57] and the low susceptibility of BvM to classical BSE [59, 68]. In summary, the results in BvM are difficult to explain with deformed templating mechanisms. Indeed, the same or similar, CH1641 CS strain emerged in sheep and BvM, regardless of their diverging PrP sequences. Furthermore, as AS breed true in BvI, deformed templating in BvM could have only depended on the M109 amino acid residue, which is nonetheless the same of the corresponding position in sheep PrP (M112).
To find direct evidence that strain components characterized by PrPres 27–30 could be propagated in a context favoring AS replication, we then looked for the presence of PrPres 27–30 in all AS-affected BvI and found a single vole brain showing both 27–30 and 8 kDa PrPres. This finding is reminiscent of rare AS cases reported in sheep showing co-existence of CS and AS, either evinced by bioassay [79], or detected by WB [80], similarly to our finding in BvI. Furthermore, it demonstrates that PrPres 27–30 components can propagate in BvI along with bona fide AS and can become the main component due to positive selection during experimental transmissions. Sub-passaging of the mixed case led to a BvI-adapted strain characterized by survival times shorter than bona fide AS and by PrPres 27–30, thus supporting a positive selection mechanism. The presence of minimal amounts of PrPres 8 kDa only in two BvI from this experiment can be explained by the three times shorter survival time of the mutant strain compared to bona fide AS, which didn’t allow PrPres 8 kDa to replicate at detectable levels in all recipient BvI.
How a single amino acid variation in bank vole PrP sequence could determine the susceptibility to AS prions was further investigated through the inoculation of BvI-adapted AS into BvM. The phenotype obtained was different from the AS strain observed in BvI, showing that BvM was not able to reproduce the original AS strain properties regardless of whether the inoculum derived from natural hosts or BvI. The transmissions of BvI-adapted bona fide AS (i.e. without detectable phenotype shift) and of the mixed case to BvM led to the emergence of similar, although not identical, mutant PrPres 27–30 strains. Interestingly, the transmission of BvI-adapted AS was much less efficient than that originating from the mixed case, with incomplete attack rate and much longer survival times (284 vs 74 dpi), which is in agreement with the presence of PrPres 27–30 at detectable levels in the mixed case, but not in bona fide AS. Overall, these findings suggest that the inefficient transmission of BvI-adapted bona fide AS to BvM depends on the inability of BvM to replicate AS, while the accompanying strain mutation reflects the presence of minor prion variants, along with bona fide AS, in AS-affected BvI, as also observed in sheep [74] and tg338 mice [57].
Bank voles are the first wild type rodents showing susceptibility to AS. The peculiar susceptibility of bank voles to a wide range of prion sources has been mainly ascribed to features of vole PrP rather than to the vole genetic background [59, 63, 68, 81–83]. Indeed, the ability of bank voles to faithfully propagate AS was clearly dependent on a single PrP amino acid residue, i.e. I109. This finding represents a clear parallel with previous experiments showing that I109 in Bv PrP was key for the faithful propagation of human prion diseases characterized by PrPSc with atypical features similar to AS [35], such as GSS and VPSPr [66, 67, 84]. This suggests a specific role of this amino acid residue, located in the central region of PrP, in the ability to replicate atypical PrPSc conformations characterised by N- and C-terminal cleaved PrPres types. In AS natural hosts, the key role of single amino acid residues in this PrP region is highlighted by the strong association of the disease with specific sheep and goat PrP polymorphisms, such as F141 and H154 [41, 42]. Interestingly, small ruminants encode for methionine at codon 112, the corresponding of codon 109 of bank vole PrP, but this does not interfere with the susceptibility of natural hosts to AS. This sharp divergence might suggest that I109 plays a role exclusively in the vole PrP context, or that small ruminant PrP polymorphisms that are strongly associated with the development of AS in sheep, i.e. F141 and H154, allow to overtake a hypothetical inhibitory role of M109. Recent experiments favor the latter hypothesis, as transgenic mice expressing sheep PrP with isoleucine instead of methionine at codon 112 (corresponding to bank vole codon 109) are more susceptible to AS than those expressing wild type sheep PrP and develop spontaneous AS within the mice lifespan (Enric Vidal, personal communication). Overall, these considerations support the view that the central region of PrP plays a key role for the development, the transmissibility and the strain features of prions [85–89].
In conclusion, we show that BvI are uniquely susceptible to AS and are the first available wild type rodent model for experimental studies with this prion strain. The resistance of BvM to AS and the emergence of CS-like strains in this bank vole genetic line can be explained in the context of the conformational selection model, supporting evidences that AS isolates are composed of an ensemble of prion strain components, i.e. PrPSc conformations, with a main component characterized by 8 kDa PrPres and minor components by PrPres 27–30. Although it is a minor component, PrPres 27–30 can still play an important role as it might be endowed with the contagiousness that is typical of CS and can be positively selected under specific circumstances, as we have shown here in BvI. Recent data showing that classical BSE prions are present in AS isolates led to suggest that AS cases might be a possible source of classical BSE prions [57]. Thus, the present findings add new clues for a better comprehension of strain selection dynamics and transmission barrier and might have wider implications for public health, namely for understanding the origin of the more contagious prion strains, such as CS.
Atypical Nor98 Scrapie, Atypical BSE, CWD, Can Emerge As Different TSE PrP In Cross Species Transmission, A Volatile Situation For Human and Animal Health
Research Project: Pathobiology, Genetics, and Detection of Transmissible Spongiform Encephalopathies Location: Virus and Prion Research
Title: Passage of the CWD agent through meadow voles results in increased attack rates and decreased incubation periods in raccoons
Author item MOORE, SARA JO - Orise Fellow item CARLSON, CHRISTINA - Us Geological Survey (USGS) item SCHNEIDER, JAY - Us Geological Survey (USGS) item JOHNSON, CHRISTOPHER - Us Geological Survey (USGS) item Greenlee, Justin Submitted to: Emerging Infectious Diseases Publication Type: Peer Reviewed Journal Publication Acceptance Date: 12/13/2021 Publication Date: N/A Citation: N/A Interpretive
Summary: Transmissible spongiform encephalopathies (TSEs) are a group of fatal diseases caused by the accumulation of misfolded prion protein in the brain. Several livestock species including cattle, sheep, deer, and elk are afflicted by prion diseases. In sheep the disease is called scrapie. In deer and elk, the disease is called chronic wasting disease (CWD). Due to the human consumption of cervid meat products and intermingling of various livestock species with wild cervid populations, there is significant interest in characterizing the possible host range of CWD. This study reports the successful transmission of the CWD agent to raccoons, a ubiquitous omnivore present throughout North America. In addition, passage of the CWD agent from deer through meadow voles, a scavenger present in much of the range where CWD occurs, results in changes in the biological behavior of the CWD agent when that material is used to inoculate raccoons. This research is of interest to regulatory officials or anyone interested in controlling CWD in wildlife or captive cervid herds.
Technical Abstract: Chronic wasting disease (CWD) is a naturally-occurring neurodegenerative disease of cervids. Raccoons (Procyon lotor) and meadow voles (Microtus pennsylvanicus) have previously been shown to be susceptible to CWD and their scavenging habits could expose them to environmental CWD infectivity. To investigate the potential for transmission of the agent of CWD from white-tailed deer to voles and subsequently to raccoons, we intracranially inoculated raccoons with brain homogenate from a CWD-affected white-tailed deer (CWDWtd), or derivatives of this isolate after it had been passaged through voles one or five times. We found that passage of the CWDWtd isolate through voles led to a change in the biological behavior of the CWD agent, including increased attack rates and decreased incubation periods in raccoons. A better understanding of the dynamics of cross-species transmission of CWD prions will help us to better manage and control the spread of CWD in free-ranging and farmed cervid populations.
''We found that passage of the CWDWtd isolate through voles led to a change in the biological behavior of the CWD agent, including increased attack rates and decreased incubation periods in raccoons.''
Disturbing...terry
Volume 26, Number 6—June 2020
Research
Radical Change in Zoonotic Abilities of Atypical BSE Prion Strains as Evidenced by Crossing of Sheep Species Barrier in Transgenic Mice
Alba Marín-Moreno1, Alvina Huor1, Juan Carlos Espinosa, Jean Yves Douet, Patricia Aguilar-Calvo2, Naima Aron, Juan Píquer, Sévérine Lugan, Patricia Lorenzo, Cecile Tillier, Hervé Cassard, Olivier Andreoletti, and Juan María TorresComments to Author Author affiliations: Centro de Investigación en Sanidad Animal, Madrid, Spain (A. Marín-Moreno, J.C. Espinosa, P. Aguilar-Calvo, J. Píquer, P. Lorenzo, J.M. Torres); Interactions Hôte Agent Pathogène–École Nationale Vétérinaire de Toulouse, Toulouse, France (A. Huor, J.Y. Douet, N. Aron, S. Lugan, C. Tiller, H. Cassard, O. Andreoletti)
Abstract
Classical bovine spongiform encephalopathy (BSE) is the only zoonotic prion disease described to date. Although the zoonotic potential of atypical BSE prions have been partially studied, an extensive analysis is still needed. We conducted a systematic study by inoculating atypical BSE isolates from different countries in Europe into transgenic mice overexpressing human prion protein (PrP): TgMet129, TgMet/Val129, and TgVal129. L-type BSE showed a higher zoonotic potential in TgMet129 mice than classical BSE, whereas Val129-PrP variant was a strong molecular protector against L-type BSE prions, even in heterozygosis. H-type BSE could not be transmitted to any of the mice. We also adapted 1 H- and 1 L-type BSE isolate to sheep-PrP transgenic mice and inoculated them into human-PrP transgenic mice. Atypical BSE prions showed a modification in their zoonotic ability after adaptation to sheep-PrP producing agents able to infect TgMet129 and TgVal129, bearing features that make them indistinguishable of sporadic Creutzfeldt-Jakob disease prions.
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Our results and those provided by other studies indicate that L-BSE adapted to a VRQ sheep sequence resemble C-BSE in its molecular features (14). Moreover, L-BSE adapted to ARQ sheep sequence and H-BSE adapted to VRQ sheep sequence generate prions similar to classical scrapie, at least in terms of PrPres glycoprofile. Therefore, in the supposed case of atypical BSE transmission to sheep, early differentiation of both atypical BSE agents from other sheep prions like classical scrapie would be difficult. Nevertheless, the combination of the low incidence of atypical BSE (because of its supposed sporadic nature) and the continued prohibition of meat and bone meal recycling ameliorates the risk for transmission to sheep.
The transmission of atypical BSEs into sheep resulted in the emergence of prions similar to types 1 and 2 sCJD in terms of mean survival times, attack rates, PrPres profile, and PrPres deposition pattern in the brain of human-PrP transgenic mice. The similarities between the sheep-adapted atypical BSE prions propagated into our human-PrP transgenic mouse lines and sCJD prions could suggest a link between them. The well-established dogma that sCJD is a spontaneous disorder unrelated to animal prion disease has been questioned in a previous study given the resemblance of scrapie prions transmitted into human transgenic mouse models to sCJD strains (26); however, the data from that study do not unequivocally establish a causative link between exposure to sheep scrapie and the subsequent appearance of sCJD in humans, and the same could apply to our findings. An alternative explanation that cannot be ruled out is that, although being different strains, only a limited number of phenotypes could be generated for the human-PrP, indicating phenotypic convergence. Updates to old epidemiologic research is needed to reconsider all these results involving a possible infectious origin of sCJD. In any case, continuing the characterization of this newly emerged prion strain would be useful to finally discarding or refuting a link with sCJD prions.
Extrapolation of results from prion transmission studies based on transgenic mice should be done with caution, especially when human susceptibility to prions is analyzed. However, our results clearly indicate that atypical BSE adaptation to an ovine-PrP sequence could modify the prion agent to potentially infect humans, showing strain features indistinguishable from those of classic sCJD prions, even though they might or might not be different agents. The supposed sporadic nature of atypical BSE makes its transmission to sheep and later to humans unlikely. However, the expanding range of TSE agents displaying the capacity to transmit in human-PrP–expressing hosts warrants the continuation of the ban on meat and bone meal recycling and underscores the ongoing need for active surveillance.
Published: 28 November 2018
Interspecies transmission to bovinized transgenic mice uncovers new features of a CH1641-like scrapie isolate
Kohtaro Miyazawa, Kentaro Masujin, Yuichi Matsuura, Yoshifumi Iwamaru, Takashi Yokoyama & Hiroyuki Okada Veterinary Research volume 49, Article number: 116 (2018) Cite this article
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Abstract In animal prion diseases, including bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease in cervids, and scrapie in sheep and goats, a disease-associated isoform of prion protein (PrPd) accumulates in the brains of affected animals. Although the CH1641 scrapie isolate was experimentally established in the UK, a few natural CH1641-like scrapie cases have been reported in France and the UK. The molecular mass of the unglycosylated protease-resistant core of PrPd (PrPres) is known to be similar between CH1641-like scrapie and experimental BSE in sheep. We previously established an experimental CH1641-like scrapie isolate (Sh294) from a natural classical scrapie case. Here, we demonstrated that the Sh294 isolate was independent of both classical and atypical BSEs by cross-species transmission to bovine PrP overexpressing (TgBoPrP) mice and wild-type mice. Interestingly, we found that the Sh294 isolate altered its host range by the transmission to TgBoPrP mice, and we succeeded in the first stable reproduction of CH1641-like scrapie specific PrPres banding patterns with the ~12-kDa small C-terminal fragment in wild-type mice. This study provides new insight into the relationship between CH1641-like scrapie isolates and BSEs. In addition, interspecies transmission models such as we have demonstrated here could be a great help to investigate the origin and host range of animal prions.
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Thus, all our data demonstrate that the Sh294 isolate is independent of all three BSE strains, suggesting that CH1641-like scrapie isolates could not be candidates for the origin of BSEs. Indeed, several studies have suggested that spontaneously occurring atypical BSEs in cattle may have been the origin of C-BSE [28,29,30,31,32].
***> In the US, scrapie is reported primarily in sheep homozygous for 136A/171Q (AAQQ) AND the disease phenotype is similar to that seen with experimental strain CH1641. ...see below in another study...TSS
***> Atypical BSE prions showed a modification in their zoonotic ability after adaptation to sheep-PrP producing agents able to infect TgMet129 and TgVal129, bearing features that make them indistinguishable of sporadic Creutzfeldt-Jakob disease prions.
Circulation of Nor98 Atypical Scrapie in Portuguese Sheep Confirmed by Transmission of Isolates into Transgenic Ovine ARQ-PrP Mice
Mafalda Casanova 1,2, Carla Machado 3 , Paula Tavares 4 , João Silva 3 , Christine Fast 5 , Anne Balkema-Buschmann 5 , Martin H. Groschup 5 and Leonor Orge 3,6,*
1 Histopathology Facility, Instituto Gulbenkian de Ciência (IGC), 2780-156 Oeiras, Portugal; mccasanova@igc.gulbenkian.pt 2 Veterinary Medicine Department, University of Évora, 7004-516 Évora, Portugal 3 Pathology Laboratory, UEISPSA, National Institute for Agricultural and Veterinary Research (INIAV), I.P., 2780-157 Oeiras, Portugal; carlitaneves@gmail.com (C.M.); joao.silva@iniav.pt (J.S.) 4 Pathology Laboratory, UEISPSA, National Institute for Agricultural and Veterinary Research (INIAV), I.P., 4485-655 Vairão-Vila do Conde, Portugal; paula.tavares@iniav.pt 5 Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut, Insel Riems, 17493 Greifswald, Germany; Christine.Fast@fli.de (C.F.); Anne.Balkema-Buschmann@fli.de (A.B.-B.); martin.groschup@fli.de (M.H.G.) 6 Animal and Veterinary Research Centre (CECAV), Associate Laboratory for Animal and Veterinary Science—AL4AnimalS, University of Trás-os-Montes and Alto Douro (UTAD), 5000-801 Vila Real, Portugal * Correspondence: leonor.orge@iniav.pt
Abstract: Portugal was among the first European countries to report cases of Atypical Scrapie (ASc), the dominant form of Transmissible Spongiform Encephalopathy (TSE) in Portuguese small ruminants. Although the diagnostic phenotypes observed in Portuguese ASc cases seem identical to those described for Nor98, unequivocal identification requires TSE strain-typing using murine bioassays. In this regard, we initiated characterization of ASc isolates from sheep either homozygous for the ARQ genotype or the classical scrapie-resistant ARR genotype. Isolates from such genotypes were transmitted to TgshpXI mice expressing ovine PrPARQ. Mean incubation periods were 414 ± 58 and 483 ± 107 days in mice inoculated with AL141RQ/AF141RQ and AL141RR/AL141RR sheep isolates, respectively. Both isolates produced lesion profiles similar to French ASc Nor98 ‘discordant cases’, where vacuolation was observed in the hippocampus (G6), cerebral cortex at the thalamus (G8) level, cerebellar white matter (W1) and cerebral peduncles (W3). Immunohistochemical PrPSc deposition was observed in the hippocampus, cerebellar cortex, cerebellar white matter and cerebral peduncles in the form of aggregates and fine granules. These findings were consistent with previously reported cases of ASc Nor98 transmitted to transgenic TgshpXI mice, confirming that the ASc strain present in Portuguese sheep corresponds to ASc Nor98.
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Recent work revealed the possibility of the development of a classical-BSE (BSE-C) prion after inoculation of bovine PrP transgenic mice with ASc isolates [3]. That study found that BSE-C may be present in natural ASc isolates as a minor variant, and transmission of such isolates to transgenic bovine mice resulted in emergence of BSE-C as a dominant variant. The same phenomenon was not observed after inoculation of CSc isolates. Hence, there is concern regarding the possibility of ASc having a role in the emergence of BSE-C in cattle, and a possible role in the origin of the 1980s BSE crisis, resulting from inclusion of rendered small ruminants in cattle feed [3]. Furthermore, archival ASc isolates reveal ASc was present in the United Kingdom years before BSE [11]. Another study found oral transmission of ASc into sheep has resulted in a phenotype shift to CH1641, a classical scrapie strain showing an immunoblot profile similar to bovine BSE. Although CH1641 has not been diagnosed in Portuguese sheep as of yet, it is prudent to maintain vigilant systematic analysis of lesion profiles, PrPSc immunolabelling types and patterns, as well as PrPSc electrophoretic profiles in natural hosts for evidence of any phenotypic shift and strain conversion. Such surveillance is particularly relevant in a country such as Portugal, where, in contrast to other EU countries, ASc was first diagnosed in the absence of previous CSc cases.
The emergence of classical BSE from atypical/Nor98 scrapie
Alvina Huor, View ORCID ProfileJuan Carlos Espinosa, View ORCID ProfileEnric Vidal, Hervé Cassard, View ORCID ProfileJean-Yves Douet, Séverine Lugan, Naima Aron, View ORCID ProfileAlba Marín-Moreno, Patricia Lorenzo, Patricia Aguilar-Calvo, Juan Badiola, Rosa Bolea, Martí Pumarola, Sylvie L. Benestad, Leonore Orge, Alana M. Thackray, Raymond Bujdoso, View ORCID ProfileJuan-Maria Torres, and View ORCID ProfileOlivier Andreoletti
aUMR Institut National de la Recherche Agronomique (INRA)/École Nationale Vétérinaire de Toulouse (ENVT) 1225, Interactions Hôtes Agents Pathogènes, 31076 Toulouse, France;
bCentro de Investigación en Sanidad Animal–Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, 28130 Madrid, Spain;
cCentre de Recerca en Sanitat Animal, Universitat Autònoma de Barcelona (UAB)–Institut de Recerca i Tecnologia Agroalimentàries, Barcelona, Spain;
dCentro de Encefalopatías y Enfermedades Transmisibles Emergentes, Universidad de Zaragoza, 50013 Zaragoza, Spain;
eUnit of Murine and Comparative Pathology, UAB, 08193 Barcelona, Spain;
fNorwegian Veterinary Institute, N-0106 Oslo, Norway;
gLaboratory of Pathology, National Institute for Agrarian and Veterinary Research, 2780-157 Oeiras, Portugal;
hDepartment of Veterinary Medicine, University of Cambridge, Cambridge CB3 0ES, United Kingdom
PNAS December 26, 2019 116 (52) 26853-26862; first published December 16, 2019; https://doi.org/10.1073/pnas.1915737116
Edited by Michael B. A. Oldstone, Scripps Research Institute, La Jolla, CA, and approved November 15, 2019 (received for review September 11, 2019)
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Significance
The origin of transmissible BSE in cattle remains unestablished. Sheep scrapie is a potential source of this known zoonotic. Here we investigated the capacity of sheep scrapie to propagate in bovine PrP transgenic mice. Unexpectedly, transmission of atypical but not classical scrapie in bovine PrP mice resulted in propagation of classical BSE prions. Detection of prion seeding activity by in vitro protein misfolding cyclic amplification demonstrated BSE prions in the original atypical scrapie isolates. BSE prion seeding activity was also detected in ovine PrP mice inoculated with limiting dilutions of atypical scrapie. Our data demonstrate that classical BSE prions can emerge during intra- and interspecies passage of atypical scrapie and provide an unprecedented insight into the evolution of mammalian prions.
Abstract
Atypical/Nor98 scrapie (AS) is a prion disease of small ruminants. Currently there are no efficient measures to control this form of prion disease, and, importantly, the zoonotic potential and the risk that AS might represent for other farmed animal species remains largely unknown. In this study, we investigated the capacity of AS to propagate in bovine PrP transgenic mice. Unexpectedly, the transmission of AS isolates originating from 5 different European countries to bovine PrP mice resulted in the propagation of the classical BSE (c-BSE) agent. Detection of prion seeding activity in vitro by protein misfolding cyclic amplification (PMCA) demonstrated that low levels of the c-BSE agent were present in the original AS isolates. C-BSE prion seeding activity was also detected in brain tissue of ovine PrP mice inoculated with limiting dilutions (endpoint titration) of ovine AS isolates. These results are consistent with the emergence and replication of c-BSE prions during the in vivo propagation of AS isolates in the natural host. These data also indicate that c-BSE prions, a known zonotic agent in humans, can emerge as a dominant prion strain during passage of AS between different species. These findings provide an unprecedented insight into the evolution of mammalian prion strain properties triggered by intra- and interspecies passage. From a public health perspective, the presence of c-BSE in AS isolates suggest that cattle exposure to small ruminant tissues and products could lead to new occurrences of c-BSE.
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With the decline of the c-BSE epizootic in cattle and the combined increase in pressure from industry, EU authorities have begun to consider discontinuing certain TSE control measures. The abrogation of the SRM measures for small ruminants and the partial reauthorization of the use of processed animal protein, formerly known as MBM, in animal feed are part of the EU authorities’ agenda. Our observation of the presence of the c-BSE agent in AS-infected small ruminants suggests that modification of the TSE control measures could result in an increased risk of exposure to c-BSE prions for both animals and humans. Whether or not this exposure will result in further c-BSE transmission in cattle and/or humans remains an open and important question.
Classical BSE prions emerge from asymptomatic pigs challenged with atypical/Nor98 scrapie
Belén Marín1,7, Alicia Otero1,7*, Séverine Lugan2 , Juan Carlos Espinosa3 , Alba Marín‑Moreno3 , EnricVidal4 , Carlos Hedman1 , Antonio Romero5 , Martí Pumarola6 , Juan J. Badiola1 , Juan MaríaTorres3 , OlivierAndréoletti2 & Rosa Bolea1
Pigs are susceptible to infection with the classical bovine spongiform encephalopathy (C-BSE) agent following experimental inoculation, and PrPSc accumulation was detected in porcine tissues after the inoculation of certain scrapie and chronic wasting disease isolates. However, a robust transmission barrier has been described in this species and, although they were exposed to C-BSE agent in many European countries, no cases of natural transmissible spongiform encephalopathies (TSE) infections have been reported in pigs. Transmission of atypical scrapie to bovinized mice resulted in the emergence of C-BSE prions. Here, we conducted a study to determine if pigs are susceptible to atypical scrapie. To this end, 12, 8–9-month-old minipigs were intracerebrally inoculated with two atypical scrapie sources. Animals were euthanized between 22- and 72-months post inoculation without clinical signs of TSE. All pigs tested negative for PrPSc accumulation by enzyme immunoassay, immunohistochemistry, western blotting and bioassay in porcine PrP mice. Surprisingly, in vitro protein misfolding cyclic amplification demonstrated the presence of C-BSE prions in different brain areas from seven pigs inoculated with both atypical scrapie isolates. Our results suggest that pigs exposed to atypical scrapie prions could become a reservoir for C-BSE and corroborate that C-BSE prions emerge during interspecies passage of atypical scrapie.
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Discussion
The outbreak of C-BSE was followed by the appearance of TSE in species that had never been diagnosed with prion diseases and the emergence in humans of vCJD16–18. However, no natural prion disease has been described in pigs, even though they were exposed to C-BSE contaminated feed12. Posterior experimental challenges in pigs and mice expressing porcine PrP have demonstrated that, although they are not completely resistant, pigs present a robust transmission barrier for C-BSE prions4,14,19.
However, the possible transmission of a TSE to swine is a public health concern due to the wide use of pork as a source of human food, and the increasing use of pigs as tissue donors, being reported a case of vCJD in a human patient receiving a swine dura mater graf20. Although pigs are apparently non-susceptible to C-BSE after oral challenge4,5,21, infectivity has been detected in tissues from pigs orally inoculated with classical scrapie or CWD10,11. In addition, these positive orally inoculated pigs are often subclinical, what could represent a public health concern, considering that these animals could reach the slaughterhouse without showing signs suggestive of prion disease.
In the present study, we evaluated the transmissibility of atypical scrapie to pigs. Pigs were euthanized between 22- and 72-months post inoculation (mpi), and their tissues tested for PrPSc accumulation and infectivity. We did not find evidence of transmission of atypical scrapie to any of the animals by EIA (Table 2), western blotting, or mouse bioassay (Table 3). PrPSc accumulation can be detected in BSE-challenged pigs at 34 mpi4 , and at 22 mpi when inoculated with SBSE7 . Although scrapie or CWD-inoculated pigs do not show clinical signs, PrPSc presence can be found in scrapie-challenged animals at 51 mpi11 and as early as 6 mpi in the case of CWD10.
Our main goal was to test the ability of atypical scrapie/Nor98 strain to propagate in swine, given that mice expressing porcine PrP (PoPrP-Tg001/tgPo mice) showed to be susceptible to atypical scrapie inoculation. One atypical scrapie isolate adapted to this transgenic line, reaching a 100% attack rate and rapid incubation periods in serial passages13, a similar adaptation to that observed with the C-BSE agent19. However, when this atypical scrapie isolate was tested for propagation in tgPo mice again, together with other atypical scrapie isolates, no positive results were obtained, in vitro nor in vivo14. These results, together with the negative transmissions showed in the present study, reinforce the conclusion that porcine species is highly resistant to atypical scrapie. However, we only performed one passage in tgPo mice, and further passages in this line and/or PMCA analysis of tgPo brains to detect any possible prion replication would be of interest.
However, it was demonstrated that C-BSE prions can be present as a minor variant in ovine atypical scrapie isolates and that C-BSE can emerge during the passage of these isolates to bovine PrP mice15. Considering that the aforementioned atypical scrapie isolate also acquired BSE-like properties when transmitted to tgPo mice13, and that C-BSE is the only prion that efficiently propagates in swine PrP4,7,14, we decided to investigate whether C-BSE prions could emerge from atypical scrapie during the ovine-porcine interspecies transmission.
Interestingly, PMCA reactions seeded with brain material from 7 pigs propagated in tgBov substrate showing PrPres with identical biochemical characteristics to those of C-BSE (Fig. 1). Positive C-BSE amplification was detected in the brain of pigs inoculated with either the PS152 or TOA3 atypical scrapie isolates, at minimum incubation periods of 28- and 35-months post inoculation, respectively. From each animal, positive reactions were not obtained from all brain areas tested (Supplementary table 1). Although PrPres amplified from the pigs showed C-BSE biochemical characteristics, further bioassays in tgBov mice are required to know whether these prions replicate the neuropathological features of C-BSE.
Altogether, our results and data obtained from transmission studies of prions to pigs, tgPo mice and in vitro studies using porcine substrate have shown that pig PrP has a very limited ability to sustain prion replication. No significant polymorphisms have been described for pig PRNP22, and it has been suggested that the conformational flexibility of pig PrP sequence is very low, limiting the number of PrPSc conformations able to produce misfolding14. No differences have been found between pig and minipig PrP sequences either23, suggesting that the conclusions obtained here could be extrapolated to domestic, non-experimental pigs. However, using tgBov substrate, we have demonstrated in vitro the presence of C-BSE seeding activity in some pig brain areas, suggesting that C-BSE prions emerged during the transmission of ovine atypical scrapie prions to pigs. Interestingly, C-BSE prions did not emerge from brain material of all the pigs, and, of those from which it did emerge, it was not detected in all brain areas tested. No correlation between time after inoculation and BSE emergence was found either. When the emergence of C-BSE from atypical scrapie in PMCA was described, it was associated to low levels of C-BSE prions that were present in the original atypical scrapie isolates15. It is possible that this result is related to the great resistance that pigs present to prion diseases, making the penetrance of the BSE prions that could be present in the original inoculum incomplete. In addition, considering that the amount of C-BSE conformers in the atypical scrapie inocula is probably very reduced and perhaps not homogeneously distributed throughout the isolate, it is also possible that not all the pigs received a sufficient amount of C-BSE conformers capable of being detected by PMCA. Finally, we should consider that PMCA amplification of prions is sometimes a stochastic phenomenon, which could explain why no C-BSE propagation was obtained from some of the pigs. It could be also discussed that C-BSE emergence from the pig brains could be related to persistence of the original atypical scrapie inoculum. However, C-BSE amplification was not obtained from all of the pigs and, in some of them (i.e. P-1217 and P-1231) C-BSE propagation was detected in caudal regions of the brain (cerebellum or occipital cortex) but not in more rostral areas (such as parietal cortex). If C-BSE amplification from pig brain samples were associated to inoculum persistence and not bona fide propagation of C-BSE prions it would be expected that such amplification would be detected mainly in the most rostral areas of the brain. Finally, even though the titer generated was not enough to produce disease in the pigs, these results evidence again the issue that pigs could act as subclinical reservoirs for prion diseases as observed with scrapie and CWD, and that the presence of prions can be detected in pigs short after exposure to prions7,10,11.
In conclusion, our findings suggest that, although pigs present a strong transmission barrier against the propagation of atypical scrapie, they can propagate low levels of C-BSE prions. The prevalence of atypical scrapie and the presence of infectivity in tissues from atypical scrapie infected sheep are underestimated24,25. Given that pigs have demonstrated being susceptible to other prion diseases, and to propagate prions without showing signs of disease, the measures implemented to ban the inclusion of ruminant proteins in livestock feed must not be interrupted.
Research Project: Pathobiology, Genetics, and Detection of Transmissible Spongiform Encephalopathies Location: Virus and Prion Research
Title: Experimental transmission of the chronic wasting disease agent to swine after oral or intracranial inoculation
Author item MOORE, SARAH - Orise Fellow item WEST GREENLEE, M - Iowa State University item KONDRU, NAVEEN - Iowa State University item MANNE, SIREESHA - Iowa State University item Smith, Jodi item Kunkle, Robert item KANTHASAMY, ANUMANTHA - Iowa State University item Greenlee, Justin Submitted to: Journal of Virology Publication Type: Peer Reviewed Journal Publication Acceptance Date: 7/6/2017 Publication Date: 9/12/2017
Citation: Moore, S.J., West Greenlee, M.H., Kondru, N., Manne, S., Smith, J.D., Kunkle, R.A., Kanthasamy, A., Greenlee, J.J. 2017. Experimental transmission of the chronic wasting disease agent to swine after oral or intracranial inoculation. Journal of Virology. 91(19):e00926-17. https://doi.org/10.1128/JVI.00926-17.
Interpretive Summary: Chronic wasting disease (CWD) is a fatal disease of wild and captive deer and elk that causes damaging changes in the brain. The infectious agent is an abnormal protein called a prion that has misfolded from its normal state. Whether CWD can transmit to swine is unknown. This study evaluated the potential of pigs to develop CWD after either intracranial or oral inoculation. Our data indicates that swine do accumulate the abnormal prion protein associated with CWD after intracranial or oral inoculation. Further, there was evidence of abnormal prion protein accumulation in lymph nodes. Currently, swine rations in the U.S. could contain animal derived components including materials from deer or elk. In addition, feral swine could be exposed to infected carcasses in areas where CWD is present in wildlife populations. This information is useful to wildlife managers and individuals in the swine and captive cervid industries. These findings could impact future regulations for the disposal of offal from deer and elk slaughtered in commercial operations. U.S. regulators should carefully consider the new information from this study before relaxing feed ban standards designed to control potentially feed borne prion diseases.
Technical Abstract: Chronic wasting disease (CWD) is a naturally occurring, fatal neurodegenerative disease of cervids. The potential for swine to serve as a host for the agent of chronic wasting disease is unknown. The purpose of this study was to investigate the susceptibility of swine to the CWD agent following oral or intracranial experimental inoculation. Crossbred piglets were assigned to one of three groups: intracranially inoculated (n=20), orally inoculated (n=19), or non-inoculated (n=9). At approximately the age at which commercial pigs reach market weight, half of the pigs in each group were culled ('market weight' groups). The remaining pigs ('aged' groups) were allowed to incubate for up to 73 months post inoculation (MPI). Tissues collected at necropsy were examined for disease-associated prion protein (PrPSc) by western blotting (WB), antigen-capture immunoassay (EIA), immunohistochemistry (IHC) and in vitro real-time quaking induced conversion (RT-QuIC). Brain samples from selected pigs were also bioassayed in mice expressing porcine prion protein. Four intracranially inoculated aged pigs and one orally inoculated aged pig were positive by EIA, IHC and/or WB. Using RT-QuIC, PrPSc was detected in lymphoid and/or brain tissue from pigs in all inoculated groups. Bioassay was positive in 4 out of 5 pigs assayed. This study demonstrates that pigs can serve as hosts for CWD, though with scant PrPSc accumulation requiring sensitive detection methods. Detection of infectivity in orally inoculated pigs using mouse bioassay raises the possibility that naturally exposed pigs could act as a reservoir of CWD infectivity.
12 September 2017
Experimental Transmission of the Chronic Wasting Disease Agent to Swine after Oral or Intracranial Inoculation
Authors: S. Jo Moore, M. Heather West Greenlee, Naveen Kondru, Sireesha Manne, Jodi D. Smith, Robert A. Kunkle, Anumantha Kanthasamy, and Justin J. Greenlee
AUTHORS INFO & AFFILIATIONS
Volume 91, Number 19
1 October 2017
ABSTRACT
ABSTRACT
Chronic wasting disease (CWD) is a naturally occurring, fatal neurodegenerative disease of cervids. The potential for swine to serve as hosts for the agent of CWD is unknown. The purpose of this study was to investigate the susceptibility of swine to the CWD agent following experimental oral or intracranial inoculation. Crossbred piglets were assigned to three groups, intracranially inoculated (n = 20), orally inoculated (n = 19), and noninoculated (n = 9). At approximately the age at which commercial pigs reach market weight, half of the pigs in each group were culled (“market weight” groups). The remaining pigs (“aged” groups) were allowed to incubate for up to 73 months postinoculation (mpi). Tissues collected at necropsy were examined for disease-associated prion protein (PrPSc) by Western blotting (WB), antigen capture enzyme immunoassay (EIA), immunohistochemistry (IHC), and in vitro real-time quaking-induced conversion (RT-QuIC). Brain samples from selected pigs were also bioassayed in mice expressing porcine prion protein. Four intracranially inoculated aged pigs and one orally inoculated aged pig were positive by EIA, IHC, and/or WB. By RT-QuIC, PrPSc was detected in lymphoid and/or brain tissue from one or more pigs in each inoculated group. The bioassay was positive in four out of five pigs assayed. This study demonstrates that pigs can support low-level amplification of CWD prions, although the species barrier to CWD infection is relatively high. However, detection of infectivity in orally inoculated pigs with a mouse bioassay raises the possibility that naturally exposed pigs could act as a reservoir of CWD infectivity. IMPORTANCE We challenged domestic swine with the chronic wasting disease agent by inoculation directly into the brain (intracranially) or by oral gavage (orally). Disease-associated prion protein (PrPSc) was detected in brain and lymphoid tissues from intracranially and orally inoculated pigs as early as 8 months of age (6 months postinoculation). Only one pig developed clinical neurologic signs suggestive of prion disease. The amount of PrPSc in the brains and lymphoid tissues of positive pigs was small, especially in orally inoculated pigs. Regardless, positive results obtained with orally inoculated pigs suggest that it may be possible for swine to serve as a reservoir for prion disease under natural conditions.
Research Project: TRANSMISSION, DIFFERENTIATION, AND PATHOBIOLOGY OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES Location: Virus and Prion Research
Title: Disease-associated prion protein detected in lymphoid tissues from pigs challenged with the agent of chronic wasting disease
Conclusions: This study demonstrates that PrPSc accumulates in lymphoid tissues from pigs challenged intracranially or orally with the CWD agent, and can be detected as early as 4 months after challenge. CWD-infected pigs rarely develop clinical disease and if they do, they do so after a long incubation period. This raises the possibility that CWD-infected pigs could shed prions into their environment long before they develop clinical disease. Furthermore, lymphoid tissues from CWD-infected pigs could present a potential source of CWD infectivity in the animal and human food chains.
Research Project: Pathobiology, Genetics, and Detection of Transmissible Spongiform Encephalopathies Location: Virus and Prion Research
Title: The agent of chronic wasting disease from pigs is infectious in transgenic mice expressing human PRNP
Author item MOORE, S - Orise Fellow item Kokemuller, Robyn item WEST-GREENLEE, M - Iowa State University item BALKEMA-BUSCHMANN, ANNE - Friedrich-Loeffler-institut item GROSCHUP, MARTIN - Friedrich-Loeffler-institut item Greenlee, Justin Submitted to: Prion Publication Type: Abstract Only Publication Acceptance Date: 5/10/2018 Publication Date: 5/22/2018 Citation: Moore, S.J., Kokemuller, R.D., West-Greenlee, M.H., Balkema-Buschmann, A., Groschup, M.H., Greenlee, J.J. 2018. The agent of chronic wasting disease from pigs is infectious in transgenic mice expressing human PRNP. Prion 2018, Santiago de Compostela, Spain, May 22-25, 2018. Paper No. WA15, page 44.
Interpretive Summary:
The successful transmission of pig-passaged CWD to Tg40 mice reported here suggests that passage of the CWD agent through pigs results in a change of the transmission characteristics which reduces the transmission barrier of Tg40 mice to the CWD agent. If this biological behavior is recapitulated in the original host species, passage of the CWD agent through pigs could potentially lead to increased pathogenicity of the CWD agent in humans.
cwd scrapie pigs oral routes
***> However, at 51 months of incubation or greater, 5 animals were positive by one or more diagnostic methods. Furthermore, positive bioassay results were obtained from all inoculated groups (oral and intracranial; market weight and end of study) suggesting that swine are potential hosts for the agent of scrapie. <***
>*** Although the current U.S. feed ban is based on keeping tissues from TSE infected cattle from contaminating animal feed, swine rations in the U.S. could contain animal derived components including materials from scrapie infected sheep and goats. These results indicating the susceptibility of pigs to sheep scrapie, coupled with the limitations of the current feed ban, indicates that a revision of the feed ban may be necessary to protect swine production and potentially human health. <***
***> Results: PrPSc was not detected by EIA and IHC in any RPLNs. All tonsils and MLNs were negative by IHC, though the MLN from one pig in the oral <6 month group was positive by EIA. PrPSc was detected by QuIC in at least one of the lymphoid tissues examined in 5/6 pigs in the intracranial <6 months group, 6/7 intracranial >6 months group, 5/6 pigs in the oral <6 months group, and 4/6 oral >6 months group. Overall, the MLN was positive in 14/19 (74%) of samples examined, the RPLN in 8/18 (44%), and the tonsil in 10/25 (40%).
***> Conclusions: This study demonstrates that PrPSc accumulates in lymphoid tissues from pigs challenged intracranially or orally with the CWD agent, and can be detected as early as 4 months after challenge. CWD-infected pigs rarely develop clinical disease and if they do, they do so after a long incubation period. This raises the possibility that CWD-infected pigs could shed prions into their environment long before they develop clinical disease. Furthermore, lymphoid tissues from CWD-infected pigs could present a potential source of CWD infectivity in the animal and human food chains.
CONFIDENTIAL
EXPERIMENTAL PORCINE SPONGIFORM ENCEPHALOPATHY
LINE TO TAKE
3. If questions on pharmaceuticals are raised at the Press conference, the suggested line to take is as follows:-
"There are no medicinal products licensed for use on the market which make use of UK-derived porcine tissues with which any hypothetical “high risk" ‘might be associated. The results of the recent experimental work at the CSM will be carefully examined by the CSM‘s Working Group on spongiform encephalopathy at its next meeting.
DO Hagger RM 1533 MT Ext 3201
While this clearly is a cause for concern we should not jump to the conclusion that this means that pigs will necessarily be infected by bone and meat meal fed by the oral route as is the case with cattle. ...
we cannot rule out the possibility that unrecognised subclinical spongiform encephalopathy could be present in British pigs though there is no evidence for this: only with parenteral/implantable pharmaceuticals/devices is the theoretical risk to humans of sufficient concern to consider any action.
May I, at the outset, reiterate that we should avoid dissemination of papers relating to this experimental finding to prevent premature release of the information. ...
3. It is particularly important that this information is not passed outside the Department, until Ministers have decided how they wish it to be handled. ...
But it would be easier for us if pharmaceuticals/devices are not directly mentioned at all. ...
Our records show that while some use is made of porcine materials in medicinal products, the only products which would appear to be in a hypothetically ''higher risk'' area are the adrenocorticotrophic hormone for which the source material comes from outside the United Kingdom, namely America China Sweden France and Germany. The products are manufactured by Ferring and Armour. A further product, ''Zenoderm Corium implant'' manufactured by Ethicon, makes use of porcine skin - which is not considered to be a ''high risk'' tissue, but one of its uses is described in the data sheet as ''in dural replacement''. This product is sourced from the United Kingdom.....
Emerg Infect Dis. 2009 Aug; 15(8): 1214–1221. doi: 10.3201/eid1508.081218 PMCID: PMC2815954 PMID: 19751582
Transgenic Mice Expressing Porcine Prion Protein Resistant to Classical Scrapie but Susceptible to Sheep Bovine Spongiform Encephalopathy and Atypical Scrapie
Juan-Carlos Espinosa, 1 María-Eugenia Herva, 1 Olivier Andréoletti, Danielle Padilla, Caroline Lacroux, Hervé Cassard, Isabelle Lantier, Joaquin Castilla, and Juan-María Torres corresponding author
Abstract
How susceptible pigs are to infection with sheep prions is unknown. We show, through transmission experiments in transgenic mice expressing porcine prion protein (PrP), that the susceptibility of this mouse model to bovine spongiform encephalopathy (BSE) can be enhanced after its passage in ARQ sheep, indicating that the pathogenicity of the BSE agent is modified after passage in sheep. Transgenic mice expressing porcine PrP were, nevertheless, completely resistant to infection with a broad panel of classical scrapie isolates from different sheep PrP genotypes and with different biochemical characteristics. The atypical (Nor98 like) isolate (SC-PS152) was the only scrapie isolate capable of transmission in these mice, although with a marked transmission barrier. Unexpectedly, the atypical scrapie agent appeared to undergo a strain phenotype shift upon transmission to porcine-PrP transgenic mice and acquired new strain properties, suggesting that atypical scrapie agent may exhibit different phenotypes depending on the host cellular PrP or other genetic factors.
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Discussion In this study, transgenic mice expressing porcine PrP (8) were used to assess the transmission capacity of a wide range of TSE agents from sheep. Our results indicated that none of the classical scrapie isolates tested was transmitted to our porcine PrP mouse model after intracerebral inoculation (Table), suggesting a highly (if not completely) resistance to the classical scrapie strains tested independently of their origin and biochemical signature. The absence of successful transmission of the SC-PS48 isolates with an unglycosylated bands of 19 kDa-like BSE suggests a BSE-unrelated origin for these BSE-like scrapie strains.
The atypical isolate SC-PS152 was the only scrapie isolate able to infect the Po-PrP mouse model after intracerebral inoculation (Table), albeit with a low efficiency of infection in the first passage (attack rate 16%). These results suggest the potential ability of atypical scrapie prions to infect pigs, although with a strong transmission barrier. Given the increasing number of atypical scrapie cases found in Europe and in North America, the potential ability of atypical scrapie to adapt to the pig becoming more easily transmitted could raise concerns about the potential danger of feeding ruminant meat and bone meal to swine.
In our transmission experiments, an obviously shorter survival period (458 ± 11 dpi) and an increased attack rate (100%) were observed in PoPrP-Tg001 mice inoculated with sheep BSE (Table) compared with those inoculated with the original cattle BSE (>650 dpi, 19%). These last figures correlate well with those reported for other cattle BSE isolates (Table). Differences in survival times were maintained after subsequent passages in this mouse model (Table), suggesting that the increased infectivity of sheep BSE cannot be linked to a higher infectious titer in the initial inoculum but must be the outcome of a modification in the pathogenicity of the agent. We can also rule out that the primary amino acid sequence of the ovine PrPSC leads to more efficient conversion of porcine PrPC because scrapie isolates from sheep with the same ARQ-PrP genotype were not able to infect these mice (Table). Taken together, the increased infectivity of sheep BSE in the porcine PrP mouse model must be considered as increased pathogenicity of the agent attributable to its passage in sheep. These features support previous results indicating that the BSE agent modifies its biological properties after passage in sheep, with the result that its pathogenicity increases in transgenic mice expressing bovine PrP (24). An increased pathogenicity of ovine BSE was also reported in conventional RIII mice when compared with retrospective cattle BSE experiments (36). In other prion strains, passage through an intermediate species has also been noted to alter host susceptibility (37).
The enhanced infectivity of the BSE agent after its passage in ARQ sheep raises concern about its potential danger for other species, including humans. This question, as well as others related to the infectivity of the new porcine prion generated in this study, is currently being addressed in transmission experiments using transgenic mice expressing human PrP.
Upon passages in porcine PrP transgenic mice, the BSE agent retained most of its biochemical properties, except for its PrPres glycoprofile in which some differences were appreciable. Our comparative analysis of cattle BSE and sheep BSE upon transmission in porcine PrP transgenic mice showed that both agents exhibit similar molecular (Figure 2) and neuropathologic properties (Figure 4). These features were preserved after subsequent passages. These results suggest that, despite their modified pathogenicity, the 2 porcine prions generated share the same biochemical and neuropathologic properties, regardless of whether the BSE agent used to inoculate the mice was obtained from ARQ sheep or cows. In agreement with these results, the increased infectivity of sheep BSE previously observed upon transmission in bovine PrP transgenic mice was not reflected in its molecular or neuropathologic properties (24).
The atypical scrapie (SC-PS152) agent appeared to undergo a strain phenotype shift upon transmission to porcine PrP transgenic mice. Surprisingly, this novel strain phenotype was similar to that of sheep BSE propagated in the same mice in terms of several features: 1) survival times observed after stabilization in PoPrP-Tg001 mice (second passages) were similar (Table); 2) PrPres molecular profiles of the 2 agents in porcine PrP mice were indistinguishable (Figure 3); and 3) vacuolation profiles observed in second passages largely overlapped (Figure 4).
These findings could reflect the evolutionary potential of prion agents upon transmission to a foreign host able to promote strain shift and emergence of new properties (38,39). The converging molecular, neuropathologic, and biological properties of atypical scrapie and sheep BSE upon propagation in porcine transgenic mice could be the consequence of a restriction imposed by the porcine PrPC, which might only admit a few options as it changes its conformation to PrPSC.
Our results could also suggest a common origin for sheep BSE and atypical scrapie agents, which may exhibit different phenotypes depending on the host PrPC or other host factors. Although this last explanation seems to be less likely, so far we cannot draw any definitive conclusion on this issue. Whichever the case, the ability of an atypical scrapie to infect other species and its potential capacity to undergo a strain phenotype shift in the new host prompts new concerns about the possible spread of this uncommon TSE in other species as a masked prion undistinguishable from other strains.
Transmission of the atypical/Nor98 scrapie agent to Suffolk sheep with VRQ/ARQ, ARQ/ARQ, and ARQ/ARR genotypes
Eric D. Cassmann,Najiba Mammadova,S. Jo Moore,Sylvie Benestad,Justin J. Greenlee Published: February 11, 2021
Abstract
Scrapie is a transmissible spongiform encephalopathy that occurs in sheep. Atypical/Nor98 scrapie occurs in sheep that tend to be resistant to classical scrapie and it is thought to occur spontaneously. The purpose of this study was to test the transmission of the Atypical/Nor98 scrapie agent in three genotypes of Suffolk sheep and characterize the distribution of misfolded prion protein (PrPSc). Ten sheep were intracranially inoculated with brain homogenate from a sheep with Atypical/Nor98 scrapie. All sheep with the ARQ/ARQ and ARQ/ARR genotypes developed Atypical/Nor98 scrapie confirmed by immunohistochemistry, and one sheep with the VRQ/ARQ genotype had detectable PrPSc consistent with Atypical/Nor98 scrapie at the experimental endpoint of 8 years. Sheep with mild early accumulations of PrPSc in the cerebellum had concomitant retinal PrPSc. Accordingly, large amounts of retinal PrPSc were identified in clinically affected sheep and sheep with dense accumulations of PrPSc in the cerebellum.
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Results and discussion All three genotypes of sheep, VRQ/ARQ, ARQ/ARQ, and ARQ/ARR, were susceptible to the AS agent after intracranial inoculation of donor brain homogenate. The diagnosis of AS was confirmed by enzyme immunoassay (EIA) and immunohistochemistry (IHC) with the latter being confirmative. Previous studies have demonstrated experimental transmission of AS to AHQ/AHQ [14, 15] and ARQ/ARQ [16] genotype sheep after intracerebral transmission. Another study showed a phenotypic shift from AS to CH1641-like classical scrapie in a sheep with the AHQ/AHQ genotype [18]. In this study, sheep with the ARQ/ARR genotype had the shortest incubation period ranging from 4.9 years to the experimental endpoint of 8 years (Table 1), and the attack rate was 100% (5/5). Clinical signs were observed in all ARQ/ARR sheep except for a single wether that was culled early to help establish experimental endpoints. Three ARQ/ARR genotype sheep were euthanized due to clinical neurologic disease 4.9–6.7 years post-inoculation. Out of the three genotypes examined, only the ARQ/ARR genotype sheep developed clinical neurologic disease within the eight-year incubation period. In clinically neurologic sheep, we observed stiff legged and hypermetric ataxia (dysmetria), abnormal rear stance, generalized tremors, tremors of the lips, weight loss, and generalized malaise. The spectrum of clinical signs was comparable to other reports of experimental AS in sheep [14, 15]. Three ARQ/ARR genotype sheep (804, 927 and 948) with the most severe dysmetria also had the greatest amount of cerebellar PrPSc. Since dysmetria is typical of animals with cerebellar disease [20], the tendency to observe this as the most consistent and severe neurologic sign is likely related to the characteristic cerebellar accumulation of PrPSc in sheep with AS. The ARQ/ARQ genotype had a long incubation period and remained clinically asymptomatic, as also reported by Okada et al. [16].
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This experiment demonstrated the transmission of atypical scrapie to three genotypes of sheep after intracranially inoculation, and it is the first study demonstrating experimental transmission to sheep with a VRQ/ARQ PRNP genotype. Additionally, atypical scrapie is further characterized by demonstrating early accumulation of PrPSc in the retina of experimentally inoculated sheep.
Experimental Oral Transmission of Atypical Scrapie to Sheep
Marion M. Simmons, S. Jo Moore,1 Timm Konold, Lisa Thurston, Linda A. Terry, Leigh Thorne, Richard Lockey, Chris Vickery, Stephen A.C. Hawkins, Melanie J. Chaplin, and John Spiropoulos
To investigate the possibility of oral transmission of atypical scrapie in sheep and determine the distribution of infectivity in the animals’ peripheral tissues, we challenged neonatal lambs orally with atypical scrapie; they were then killed at 12 or 24 months. Screening test results were negative for disease-specifi c prion protein in all but 2 recipients; they had positive results for examination of brain, but negative for peripheral tissues. Infectivity of brain, distal ileum, and spleen from all animals was assessed in mouse bioassays; positive results were obtained from tissues that had negative results on screening. These fi ndings demonstrate that atypical scrapie can be transmitted orally and indicate that it has the potential for natural transmission and iatrogenic spread through animal feed. Detection of infectivity in tissues negative by current surveillance methods indicates that diagnostic sensitivity is suboptimal for atypical scrapie, and potentially infectious material may be able to pass into the human food chain.
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Although all TSEs are transmissible after intracerebral challenge to a susceptible host, only some are infectious under natural conditions. Therefore, it was important from a pathogenesis and disease control perspective to establish whether or not oral transmission can be successful. However, the challenge model in this study exposed animals as neonates, when the esophageal groove is operational and the lambs are physiologically monogastric. Exposure of 3-month-old ruminating animals to similar amounts of positive brain by the oral route have so far not resulted in any clinical disease, with all animals still alive >1,500 days post challenge (M.M. Simmons, unpub. data), but most natural cases have been recorded in animals older than this, so these animals may still progress to disease in the next few years. Since this challenge study in older animals has no time-kill component, and no losses caused by unrelated disease have occurred, whether any of these sheep are in a preclinical phase of disease is unknown. Unfortunately, the absence of detectable PrPSc in lymphoreticular tissues of sheep with atypical scrapie precludes the use of biopsies to ascertain early infection in these animals.
Transmission may be more effi cient in newborn animals; the incubation periods of sheep orally infected with classical scrapie were signifi cantly shorter in sheep challenged at 14 days of age than those challenged at 6 months of age (31). If, however, oral transmission is only effective in such young animals, then fi eld exposure would most likely have to be through milk, which is known to be a highly effective route of transmission for classical scrapie (32). No data are currently available on the potential infectivity of milk from animals with atypical scrapie.
Successful oral transmission also raises questions regarding the pathogenesis of this form of disease. There must be passage of the infectious agent from the alimentary canal to the brain through one of several possible routes, most likely those that have been suggested and discussed in detail for other TSEs, for example, retrograde neuronal transportation either directly (33–35) or through lymphoid structures or hematogenously (36). Infectivity in the absence of readily demonstrable PrPSc has been reported (37–39), and although the mouse bioassay may detect evidence of disease in other tissues, these data may not be available for at least another 2 years. More protease-sensitive forms of PrPSc may be broken down more effi ciently within cells and thus do not accumulate in peripheral tissues (19), enabling atypical PrPSc to transit the digestive tract and disseminate through other systems in small amounts before accumulating detectably in the central nervous system.
Although we do not have epidemiologic evidence that supports the effi cient spread of disease in the fi eld, these data imply that disease is potentially transmissible under fi eld situations and that spread through animal feed may be possible if the current feed restrictions were to be relaxed.
Additionally, almost no data are available on the potential for atypical scrapie to transmit to other food animal species, certainly by the oral route. However, work with transgenic mice has demonstrated the potential susceptibility of pigs, with the disturbing fi nding that the biochemical properties of the resulting PrPSc have changed on transmission (40). The implications of this observation for subsequent transmission and host target range are currently unknown.
How reassuring is this absence of detectable PrPSc from a public health perspective? The bioassays performed in this study are not titrations, so the infectious load of the positive gut tissues cannot be quantified, although infectivity has been shown unequivocally. No experimental data are currently available on the zoonotic potential of atypical scrapie, either through experimental challenge of humanized mice or any meaningful epidemiologic correlation with human forms of TSE. However, the detection of infectivity in the distal ileum of animals as young as 12 months, in which all the tissues tested were negative for PrPSc by the currently available screening and
confirmatory diagnostic tests, indicates that the diagnostic sensitivity of current surveillance methods is suboptimal for detecting atypical scrapie and that potentially infectious material may be able to pass into the human food chain undetected.
EFSA
***> AS is considered more likely (subjective probability range 50–66%) that AS is a non-contagious, rather than a contagious, disease.
2.3.2. New evidence on the zoonotic potential of atypical BSE and atypical scrapie prion strains
PLEASE NOTE;
2.3.2. New evidence on the zoonotic potential of atypical BSE and atypical scrapie prion strainsNo
Olivier Andreoletti, INRA Research Director, Institut National de la Recherche Agronomique (INRA) – École Nationale Vétérinaire de Toulouse (ENVT), invited speaker, presented the results of two recently published scientific articles of interest, of which he is co-author: ‘Radical Change in Zoonotic Abilities of Atypical BSE Prion Strains as Evidenced by Crossing of Sheep Species Barrier in Transgenic Mice’ (MarinMoreno et al., 2020) and ‘The emergence of classical BSE from atypical/Nor98 scrapie’ (Huor et al., 2019).
In the first experimental study, H-type and L-type BSE were inoculated into transgenic mice expressing all three genotypes of the human PRNP at codon 129 and into adapted into ARQ and VRQ transgenic sheep mice. The results showed the alterations of the capacities to cross the human barrier species (mouse model) and emergence of sporadic CJD agents in Hu PrP expressing mice: type 2 sCJD in homozygous TgVal129 VRQ-passaged L-BSE, and type 1 sCJD in homozygous TgVal 129 and TgMet129 VRQ-passaged H-BSE.
Previous work has shown that the Stetsonville, WI outbreak of TME could have been precipitated by feeding mink a downer cow with atypical BSE; therefore, it very well may have originated from a cow with L-BSE. The agent of TME appears to remain stable, and it has a high transmission efficiency after a sequence of interspecies transmission events. Although C-BSE is the archetypal foodborne TSE, our findings indicate that L-BSE and bTME have greater transmission efficiencies in bovinized mice. Previous work has demonstrated that L-BSE also is more virulent than C-BSE in mice expressing the human prion protein [46, 55]. Although the documented incidence of L-BSE is low, the propensity of L-BSE and the TME agent to cross species barriers support the continued monitoring for atypical BSE.
***>This work supports the ideas that L-BSE is a possible source for TME in mink and that the practice of feeding cattle with neurologic disease to mink should be avoided. This information is important to farmers who raise cattle, sheep, or mink.<***
1985
Evidence That Transmissible Mink Encephalopathy Results from Feeding Infected Cattle Over the next 8-10 weeks, approximately 40% of all the adult mink on the farm died from TME.
snip...
The rancher was a ''dead stock'' feeder using mostly (>95%) downer or dead dairy cattle...
57 The experiment which might have determined whether BSE and scrapie were caused by the same agent (ie, the feeding of natural scrapie to cattle) was never undertaken in the UK.
It was, however, performed in the USA in 1979, when it was shown that cattle inoculated with the scrapie agent endemic in the flock of Suffolk sheep at the United States Department of Agriculture in Mission, Texas, developed a TSE quite unlike BSE. 32
The findings of the initial transmission, though not of the clinical or neurohistological examination, were communicated in October 1988 to Dr Watson, Director of the CVL, following a visit by Dr Wrathall, one of the project leaders in the Pathology Department of the CVL, to the United States Department of Agriculture. 33
The results were not published at this point, since the attempted transmission to mice from the experimental cow brain had been inconclusive.
The results of the clinical and histological differences between scrapie-affected sheep and cattle were published in 1995.
Similar studies in which cattle were inoculated intracerebrally with scrapie inocula derived from a number of scrapie-affected sheep of different breeds and from different States, were carried out at the US National Animal Disease Centre. 34
The results, published in 1994, showed that this source of scrapie agent, though pathogenic for cattle, did not produce the same clinical signs of brain lesions characteristic of BSE..
32 Clark, W., Hourrigan, J. and Hadlow, W. (1995) Encephalopathy in Cattle Experimentally Infected with the Scrapie Agent, American Journal of Veterinary Research, 56, 606-12
33 YB88/10.00/1.1
Technical Abstract:
Prion strains may vary in their ability to transmit to humans and animals. Few experimental studies have been done to provide evidence of differences between U.S. strains of scrapie, which can be distinguished by incubation times in inbred mice, microscopic lesions, immunoreactivity to various antibodies, or molecular profile (electrophoretic mobility and glycoform ratio). Recent work on two U.S. isolates of sheep scrapie supports that at least two distinct strains exist based on differences in incubation time and genotype of sheep affected. One isolate (No. 13-7) inoculated intracerebrally caused scrapie in sheep AA at codon 136 (AA136) and QQ at codon 171 (QQ171) of the prion protein in an average of 19 months post-inoculation (PI) whereas a second isolate (No. x124) caused disease in less than 12 months after oral inoculation in AV136/QQ171 sheep. Striking differences were evident when further strain analysis was done in R111, VM, C57Bl6, and C57Bl6xVM (F1) mice. No. 13-7 did not induce disease in any mouse strain at any time post-inoculation (PI) nor were brain tissues positive by western blot (WB). Positive WB results were obtained from mice inoculated with isolate No. x124 starting at day 380 PI. Incubation times averaged 508, 559, 601, and 633 days PI for RIII, C57Bl6, VM, and F1 mice, respectively. Further passage will be required to characterize these scrapie strains in mice.
***>This work provides evidence that multiple scrapie strains exist in U.S. sheep.
One of these isolates (TR316211) behaved like the CH1641 isolate, with PrPres features in mice similar to those in the sheep brain. From two other isolates (O100 and O104), two distinct PrPres phenotypes were identified in mouse brains, with either high (h-type) or low (l-type) apparent molecular masses of unglycosylated PrPres, the latter being similar to that observed with CH1641, TR316211, or BSE. Both phenotypes could be found in variable proportions in the brains of the individual mice. In contrast with BSE, l-type PrPres from "CH1641-like" isolates showed lower levels of diglycosylated PrPres. From one of these cases (O104), a second passage in mice was performed for two mice with distinct PrPres profiles. This showed a partial selection of the l-type phenotype in mice infected with a mouse brain with predominant l-type PrPres, and it was accompanied by a significant increase in the proportions of the diglycosylated band. These results are discussed in relation to the diversity of scrapie and BSE strains.
***> In the US, scrapie is reported primarily in sheep homozygous for 136A/171Q (AAQQ) and the disease phenotype is similar to that seen with experimental strain CH1641.
- 59-
P-088 Transmission of experimental CH1641-like scrapie to bovine PrP overexpression mice
Kohtaro Miyazawa1, Kentaro Masujin1, Hiroyuki Okada1, Yuichi Matsuura1, Takashi Yokoyama2
1Influenza and Prion Disease Research Center, National Institute of Animal Health, NARO, Japan; 2Department of Planning and General Administration, National Institute of Animal Health, NARO
Introduction: Scrapie is a prion disease in sheep and goats. CH1641-lke scrapie is characterized by a lower molecular mass of the unglycosylated form of abnormal prion protein (PrpSc) compared to that of classical scrapie. It is worthy of attention because of the biochemical similarities of the Prpsc from CH1641-like and BSE affected sheep. We have reported that experimental CH1641-like scrapie is transmissible to bovine PrP overexpression (TgBoPrP) mice (Yokoyama et al. 2010). We report here the further details of this transmission study and compare the biological and biochemical properties to those of classical scrapie affected TgBoPrP mice.
Methods: The details of sheep brain homogenates used in this study are described in our previous report (Yokoyama et al. 2010). TgBoPrP mice were intracerebrally inoculated with a 10% brain homogenate of each scrapie strain. The brains of mice were subjected to histopathological and biochemical analyses.
Results: Prpsc banding pattern of CH1641-like scrapie affected TgBoPrP mice was similar to that of classical scrapie affected mice. Mean survival period of CH1641-like scrapie affected TgBoPrP mice was 170 days at the 3rd passage and it was significantly shorter than that of classical scrapie affected mice (439 days). Lesion profiles and Prpsc distributions in the brains also differed between CH1641-like and classical scrapie affected mice.
Conclusion: We succeeded in stable transmission of CH1641-like scrapie to TgBoPrP mice. Our transmission study demonstrates that CH 1641-like scrapie is likely to be more virulent than classical scrapie in cattle.
Research Project: TRANSMISSION, DIFFERENTIATION, AND PATHOBIOLOGY OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES
Title: Comparison of two US sheep scrapie isolates supports identification as separate strains
Authors
item Moore, Sarah - item Smith, Jodi item West Greenlee, Mary - item Nicholson, Eric item Richt, Juergen item Greenlee, Justin
Submitted to: Veterinary Pathology Publication Type: Peer Reviewed Journal Publication Acceptance Date: December 22, 2015 Publication Date: N/A
Interpretive Summary: Scrapie is a fatal disease of sheep and goats that causes damaging changes in the brain. The infectious agent is an abnormal protein called a prion that has misfolded from its normal state. Whether or not a sheep will get scrapie is determined primarily by their genetics. Furthermore, different scrapie strains exist that may result in a different expression of disease such as shorter incubation periods, unusual clinical signs, or unique patterns of lesions within the brain. This study evaluated two U.S. scrapie isolates in groups of sheep with varying susceptibilities to scrapie. Our data indicates that there are differences in incubation periods, sheep genotype susceptibilities, and lesion profiles that support designating these scrapie isolates as unique strains. The identification of a new scrapie strain in the United States means that control measures, methods of decontamination, and the potential for transmission to other species may need to be reevaluated. This information is useful to sheep farmers and breeders that are selectively breeding animals with genotypes resistant to the most prevalent strain of scrapie and could impact future regulations for the control of scrapie in the United States. Technical Abstract: Scrapie is a naturally occurring transmissible spongiform encephalopathy (TSE) of sheep and goats. There are different strains of sheep scrapie that are associated with unique molecular, transmission, and phenotype characteristics, but very little is known about the potential presence of scrapie strains within sheep in the US. Scrapie strain and PRNP genotype could both affect susceptibility, potential for transmission, incubation period, and control measures required for eliminating scrapie from a flock. Here we evaluate two US scrapie isolates, No. 13-7 and x124, after intranasal inoculation to compare clinical signs, incubation periods (IP), spongiform lesions, and patterns of PrPSc deposition in sheep with scrapie-susceptible PRNP genotypes (QQ171). After inoculation with x124, susceptibility and IP were associated with valine at codon 136 (V136) of the prion protein: VV136 had short IPs (6.9 months), AV136 sheep were 11.9 months, and AA136 sheep did not develop scrapie. All No.13-7 inoculated sheep developed scrapie with IP’s of 20.1 months for AA136 sheep, 22.8 months for AV136 sheep, and 26.7 months for VV136 sheep. Patterns of immunoreactivity in the brain were influenced by challenge isolate and host genotype. Differences in PrPSc profiles versus isolate were most striking when examining brains from sheep with the VV136 genotype. In summary, intranasal inoculation with isolates x124 and No. 13-7 resulted in differences in IP, sheep genotype susceptibility, and PrPSc profile that support designation as separate strains.
Last Modified: 6/6/2016
31
Appendix I VISIT TO USA - OR A E WRATHALL — INFO ON BSE AND SCRAPIE
Dr Clark lately of the scrapie Research Unit, Mission Texas has
successfully transmitted ovine and caprine scrapie to cattle. The
experimental results have not been published but there are plans to do
this. This work was initiated in 1978. A summary of it is:-
Expt A 6 Her x Jer calves born in 1978 were inoculated as follows with
a 2nd Suffolk scrapie passage:-
i/c 1ml; i/m, 5ml; s/c 5ml; oral 30ml.
1/6 went down after 48 months with a scrapie/BSE-like disease.
Expt B 6 Her or Jer or HxJ calves were inoculated with angora Goat
virus 2/6 went down similarly after 36 months.
Expt C Mice inoculated from brains of calves/cattle in expts A & B were resistant, only 1/20 going down with scrapie and this was the reason given for not publishing.
Diagnosis in A, B, C was by histopath. No reports on SAF were given.
Dr Warren Foote indicated success so far in eliminating scrapie in offspring from experimentally— (and naturally) infected sheep by ET. He had found difficulty in obtaining embryos from naturally infected sheep (cf SPA).
Prof. A Robertson gave a brief accout of BSE. The us approach was to
32
accord it a very low profile indeed. Dr A Thiermann showed the picture in the "Independent" with cattle being incinerated and thought this was a fanatical incident to be avoided in the US at all costs.
BSE was not reported in USA.
4. Scrapie incidents (ie affected flocks) have shown a dramatic increase since 1978. In 1953 when the National Control scheme was started there were 10-14 incidents, in 1978 - 1 and in 1988 so far 60.
5. Scrapie agent was reported to have been isolated from a solitary fetus.
6. A western blotting diagnostic technique (? on PrP) shows some promise.
7. Results of a questionnaire sent to 33 states on the subject of the national sheep scrapie programme survey indicated
17/33 wished to drop it
6/33 wished to develop it
8/33 had few sheep and were neutral
Information obtained from Dr Wrathall‘s notes of a meeting of the u.s.
Animal Health Association at Little Rock, Arkansas Nov. 1988.
33
In Confidence - Perceptions of unconventional slow virus diseases of animals in the USA - APRIL-MAY 1989 - G A H Wells
3. Prof. A. Robertson gave a brief account of BSE. The US approach was to accord it a very low profile indeed. Dr. A Thiermann showed the picture in the ''Independent'' with cattle being incinerated and thought this was a fanatical incident to be avoided in the US at all costs. ...
also see hand written notes ;
Scrapie Field Trial Experiments Mission, Texas, The Moore Air Force Base Scrapie Experiment 1964
How Did CWD Get Way Down In Medina County, Texas?
Confucius ponders...
Could the Scrapie experiments back around 1964 at Moore Air Force near Mission, Texas, could this area have been ground zero for CWD TSE Prion (besides the CWD cases that have waltzed across the Texas, New Mexico border near WSMR Trans Pecos region since around 2001)?
Epidemiology of Scrapie in the United States 1977
snip...
Scrapie Field Trial Experiments Mission, Texas A Scrapie Field Trial was developed at Mission, Texas, to provide additional information for the eradication program on the epidemiology of natural scrapie. The Mission Field Trial Station is located on 450 acres of pastureland, part of the former Moore Air Force Base, near Mission, Texas.
It was designed to bring previously exposed, and later also unexposed, sheep or goats to the Station and maintain and breed them under close observation for extended periods to determine which animals would develop scrapie and define more closely the natural spread and other epidemiological aspects of the disease.
The 547 previously exposed sheep brought to the Mission Station beginning in 1964 were of the Cheviot, Hampshire, Montadale, or Suffolk breeds.
They were purchased as field outbreaks occurred, and represented 21 bloodlines in which scrapie had been diagnosed.
Upon arrival at the Station, the sheep were maintained on pasture, with supplemental feeding as necessary.
The station was divided into 2 areas:
(1) a series of pastures and-pens occupied by male animals only, and
(2) a series of pastures and pens occupied by females and young progeny of both sexes.
... snip...
see full text ;
IBNC BSE TSE Prion mad cow disease
***however in 1 C-type challenged animal, Prion 2015 Poster Abstracts S67 PrPsc was not detected using rapid tests for BSE.
***Subsequent testing resulted in the detection of pathologic lesion in unusual brain location and PrPsc detection by PMCA only.
*** IBNC Tauopathy or TSE Prion disease, it appears, no one is sure ***
Posted by Terry S. Singeltary Sr. on 03 Jul 2015 at 16:53 GMT
Incomplete inactivation of atypical scrapie following recommended autoclave decontamination procedures.
Published on Sep 1, 2019in Transboundary and Emerging Diseases
5.005 · DOI :10.1111/TBED.13247 Copy DOI John Spiropoulos 21 (Animal and Plant Health Agency), Richard Lockey 14 (Animal and Plant Health Agency)+ 7 AuthorsLinda A. Terry 19 (Animal and Plant Health Agency)
Abstract : Prions are highly resistant to the decontamination procedures normally used to inactivate conventional pathogens. This is a challenging problem not only in the medical and veterinary fields for minimizing the risk of transmission from potentially infective sources but also for ensuring the safe disposal or subsequent use of animal by-products. Specific pressure autoclaving protocols were developed for this purpose, but different strains of prions have been reported to have differing resistance patterns to established prion decontamination procedures, and as additional TSE strains are identified it is necessary to determine the effectiveness of such procedures. In this study we assessed the efficacy of sterilization using the EU recommended autoclave procedure for prions (133°C, 3 Bar for 20 min) on the atypical or Nor98 (AS/Nor98) scrapie strain of sheep and goats. Using a highly sensitive murine mouse model (tg338) that overexpresses ovine PrPC , we determined that this method of decontamination reduced the infectivity titre by 1010 . Infectivity was nonetheless still detected after applying the recommended autoclaving protocol. This shows that AS/Nor98 can survive the designated legislative decontamination conditions, albeit with a significant decrease in titre. The infectivity of a classical scrapie isolate subjected to the same decontamination conditions was reduced by 106 suggesting that the AS/Nor98 isolate is less sensitive to decontamination than the classical scrapie source.
MONDAY, NOVEMBER 29, 2021
Experimental Oronasal Transmission of Chronic Wasting Disease Agent from White-Tailed Deer to Suffolk Sheep Volume 27, Number 12—December 2021 Dispatch
OIE Conclusions on transmissibility of atypical BSE among cattle
Given that cattle have been successfully infected by the oral route, at least for L-BSE, it is reasonable to conclude that atypical BSE is potentially capable of being recycled in a cattle population if cattle are exposed to contaminated feed. In addition, based on reports of atypical BSE from several countries that have not had C-BSE, it appears likely that atypical BSE would arise as a spontaneous disease in any country, albeit at a very low incidence in old cattle. In the presence of livestock industry practices that would allow it to be recycled in the cattle feed chain, it is likely that some level of exposure and transmission may occur. As a result, since atypical BSE can be reasonably considered to pose a potential background level of risk for any country with cattle, the recycling of both classical and atypical strains in the cattle and broader ruminant populations should be avoided.
Annex 7 (contd) AHG on BSE risk assessment and surveillance/March 2019
34 Scientific Commission/September 2019
3. Atypical BSE
The Group discussed and endorsed with minor revisions an overview of relevant literature on the risk of atypical BSE being recycled in a cattle population and its zoonotic potential that had been prepared ahead of the meeting by one expert from the Group. This overview is provided as Appendix IV and its main conclusions are outlined below. With regard to the risk of recycling of atypical BSE, recently published research confirmed that the L-type BSE prion (a type of atypical BSE prion) may be orally transmitted to calves1 . In light of this evidence, and the likelihood that atypical BSE could arise as a spontaneous disease in any country, albeit at a very low incidence, the Group was of the opinion that it would be reasonable to conclude that atypical BSE is potentially capable of being recycled in a cattle population if cattle were to be exposed to contaminated feed. Therefore, the recycling of atypical strains in cattle and broader ruminant populations should be avoided.
The Group acknowledged the challenges in demonstrating the zoonotic transmission of atypical strains of BSE in natural exposure scenarios. Overall, the Group was of the opinion that, at this stage, it would be premature to reach a conclusion other than that atypical BSE poses a potential zoonotic risk that may be different between atypical strains.
4. Definitions of meat-and-bone meal (MBM) and greaves
snip...
REFERENCES
SNIP...END SEE FULL TEXT;
***Moreover, sporadic disease has never been observed in breeding colonies or primate research laboratories, most notably among hundreds of animals over several decades of study at the National Institutes of Health25, and in nearly twenty older animals continuously housed in our own facility.***
Even if the prevailing view is that sporadic CJD is due to the spontaneous formation of CJD prions, it remains possible that its apparent sporadic nature may, at least in part, result from our limited capacity to identify an environmental origin.
Atypical L-type BSE
Emerg Infect Dis. 2017 Feb; 23(2): 284–287. doi: 10.3201/eid2302.161416 PMCID: PMC5324790 PMID: 28098532
Oral Transmission of L-Type Bovine Spongiform Encephalopathy Agent among Cattle
Our study clearly confirms, experimentally, the potential risk for interspecies oral transmission of the agent of L-BSE. In our model, this risk appears higher than that for the agent of classical BSE, which could only be transmitted to mouse lemurs after a first passage in macaques (14). We report oral transmission of the L-BSE agent in young and adult primates. Transmission by the IC route has also been reported in young macaques (6,7). A previous study of L-BSE in transgenic mice expressing human PrP suggested an absence of any transmission barrier between cattle and humans for this particular strain of the agent of BSE, in contrast to findings for the agent of classical BSE (9). Thus, it is imperative to maintain measures that prevent the entry of tissues from cattle possibly infected with the agent of L-BSE into the food chain.
Atypical H-type BSE
Research Project: Pathobiology, Genetics, and Detection of Transmissible Spongiform Encephalopathies Location: Virus and Prion Research
Title: The agent of H-type bovine spongiform encephalopathy associated with E211K prion protein polymorphism transmits after oronasal challenge
This study demonstrates that the H-type BSE agent is transmissible by the oronasal route.
These results reinforce the need for ongoing surveillance for classical and atypical BSE to minimize the risk of potentially infectious tissues entering the animal or human food chains.
P98 The agent of H-type bovine spongiform encephalopathy associated with E211K prion protein polymorphism transmits after oronasal challenge
Greenlee JJ (1), Moore SJ (1), and West Greenlee MH (2) (1) United States Department of Agriculture, Agricultural Research Service, National Animal Disease Center, Virus and Prion Research Unit, Ames, IA, United States (2) Department of Biomedical Sciences, Iowa State University College of Veterinary Medicine, Ames, IA, United States.
With the experiment currently at 55 months post-inoculation, no other cattle in this study have developed clinical signs suggestive of prion disease. This study demonstrates that the H-type BSE agent is transmissible by the oronasal route.
These results reinforce the need for ongoing surveillance for classical and atypical BSE to minimize the risk of potentially infectious tissues entering the animal or human food chains.
PRION CONFERENCE 2018 CONFERENCE ABSTRACT
Published: 23 June 2011
Experimental H-type bovine spongiform encephalopathy characterized by plaques and glial- and stellate-type prion protein deposits
The present study demonstrated successful intraspecies transmission of H-type BSE to cattle and the distribution and immunolabeling patterns of PrPSc in the brain of the H-type BSE-challenged cattle. TSE agent virulence can be minimally defined by oral transmission of different TSE agents (C-type, L-type, and H-type BSE agents) [59]. Oral transmission studies with H-type BSE-infected cattle have been initiated and are underway to provide information regarding the extent of similarity in the immunohistochemical and molecular features before and after transmission. In addition, the present data will support risk assessments in some peripheral tissues derived from cattle affected with H-type BSE.
References...END
223. Scrapie in white-tailed deer: a strain of the CWD agent that efficiently transmits to sheep?
Justin J. Greenleea, Robyn D. Kokemullera, S. Jo Moorea and Heather West Greenleeb
aVirus and Prion Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, Ames, IA, USA; bDepartment of Biomedical Sciences, Iowa State University College of Veterinary Medicine, Ames, IA, USA
CONTACT Justin J. Greenlee Justin.Greenlee@ars.usda.gov
ABSTRACT
Scrapie is a transmissible spongiform encephalopathy of sheep and goats that is associated with widespread accumulation of abnormal prion protein (PrPSc) in the central nervous and lymphoid tissues. Chronic wasting disease (CWD) is the natural prion disease of cervid species, and the tissue distribution of PrPSc in affected cervids is similar to scrapie in sheep. There are several lines of evidence that suggest that multiple strains of CWD exist, which may affect the agent’s potential to transmit to hosts of the same or different species. We inoculated white-tailed deer with the scrapie agent from ARQ/ARQ sheep, which resulted in 100% attack rates by either the intracranial or oronasal route of inoculation. When examining tissues from the brainstems or lymphoid tissues by traditional diagnostic methods such as immunohistochemistry or western blots, it is difficult to differentiate tissues from deer infected with scrapie from those infected with CWD. However, there are several important differences between tissues from scrapie-infected white-tailed deer (WTD scrapie) and those infected with CWD (WTD CWD). First, there are different patterns of PrPSc deposition in the brains of infected deer: brain tissues from deer with WTD scrapie had predominantly particulate and stellate immunoreactivity whereas those from deer with WTD-CWD had large aggregates and plaque-like deposits. Secondly, the incubation periods of WTD scrapie isolates are longer than CWD isolates in mice expressing cervid prion protein. Most notably, the transmission potential of these two isolates back to sheep is distinctly different. Attempts to transmit various CWD isolates to sheep by the oral or oronasal routes have been unsuccessful despite observation periods of up to 7 years. However, WTD scrapie efficiently transmitted back to sheep by the oronasal route. Upon transmission back to sheep, the WTD scrapie isolate exhibited different phenotypic properties when compared to the sheep receiving the original sheep scrapie inoculum including different genotype susceptibilities, distinct PrPSc deposition patterns, and much more rapid incubation periods in transgenic mice expressing the ovine prion protein. The scrapie agent readily transmits between sheep and deer after oronasal exposure. This could confound the identification of CWD strains in deer and the eradication of scrapie from sheep.
CONFIDENTIAL
EXPERIMENTAL PORCINE SPONGIFORM ENCEPHALOPATHY
LINE TO TAKE
3. If questions on pharmaceuticals are raised at the Press conference, the suggested line to take is as follows:-
"There are no medicinal products licensed for use on the market which make use of UK-derived porcine tissues with which any hypothetical “high risk" ‘might be associated. The results of the recent experimental work at the CSM will be carefully examined by the CSM‘s Working Group on spongiform encephalopathy at its next meeting.
DO Hagger RM 1533 MT Ext 3201
While this clearly is a cause for concern we should not jump to the conclusion that this means that pigs will necessarily be infected by bone and meat meal fed by the oral route as is the case with cattle. ...
we cannot rule out the possibility that unrecognised subclinical spongiform encephalopathy could be present in British pigs though there is no evidence for this: only with parenteral/implantable pharmaceuticals/devices is the theoretical risk to humans of sufficient concern to consider any action.
May I, at the outset, reiterate that we should avoid dissemination of papers relating to this experimental finding to prevent premature release of the information. ...
3. It is particularly important that this information is not passed outside the Department, until Ministers have decided how they wish it to be handled. ...
But it would be easier for us if pharmaceuticals/devices are not directly mentioned at all. ...
Our records show that while some use is made of porcine materials in medicinal products, the only products which would appear to be in a hypothetically ''higher risk'' area are the adrenocorticotrophic hormone for which the source material comes from outside the United Kingdom, namely America China Sweden France and Germany. The products are manufactured by Ferring and Armour. A further product, ''Zenoderm Corium implant'' manufactured by Ethicon, makes use of porcine skin - which is not considered to be a ''high risk'' tissue, but one of its uses is described in the data sheet as ''in dural replacement''. This product is sourced from the United Kingdom.....
***Moreover, sporadic disease has never been observed in breeding colonies or primate research laboratories, most notably among hundreds of animals over several decades of study at the National Institutes of Health25, and in nearly twenty older animals continuously housed in our own facility.***
Even if the prevailing view is that sporadic CJD is due to the spontaneous formation of CJD prions, it remains possible that its apparent sporadic nature may, at least in part, result from our limited capacity to identify an environmental origin.
https://www.nature.com/articles/srep11573
O.05: Transmission of prions to primates after extended silent incubation periods: Implications for BSE and scrapie risk assessment in human populations
Emmanuel Comoy, Jacqueline Mikol, Valerie Durand, Sophie Luccantoni, Evelyne Correia, Nathalie Lescoutra, Capucine Dehen, and Jean-Philippe Deslys Atomic Energy Commission; Fontenay-aux-Roses, France
Prion diseases (PD) are the unique neurodegenerative proteinopathies reputed to be transmissible under field conditions since decades. The transmission of Bovine Spongiform Encephalopathy (BSE) to humans evidenced that an animal PD might be zoonotic under appropriate conditions. Contrarily, in the absence of obvious (epidemiological or experimental) elements supporting a transmission or genetic predispositions, PD, like the other proteinopathies, are reputed to occur spontaneously (atpical animal prion strains, sporadic CJD summing 80% of human prion cases).
Non-human primate models provided the first evidences supporting the transmissibiity of human prion strains and the zoonotic potential of BSE. Among them, cynomolgus macaques brought major information for BSE risk assessment for human health (Chen, 2014), according to their phylogenetic proximity to humans and extended lifetime. We used this model to assess the zoonotic potential of other animal PD from bovine, ovine and cervid origins even after very long silent incubation periods.
*** We recently observed the direct transmission of a natural classical scrapie isolate to macaque after a 10-year silent incubation period,
***with features similar to some reported for human cases of sporadic CJD, albeit requiring fourfold long incubation than BSE. Scrapie, as recently evoked in humanized mice (Cassard, 2014),
***is the third potentially zoonotic PD (with BSE and L-type BSE),
***thus questioning the origin of human sporadic cases.
We will present an updated panorama of our different transmission studies and discuss the implications of such extended incubation periods on risk assessment of animal PD for human health.
===============
***thus questioning the origin of human sporadic cases***
===============
***our findings suggest that possible transmission risk of H-type BSE to sheep and human. Bioassay will be required to determine whether the PMCA products are infectious to these animals.
==============
https://prion2015.files.wordpress.com/2015/05/prion2015abstracts.pdf
***Transmission data also revealed that several scrapie prions propagate in HuPrP-Tg mice with efficiency comparable to that of cattle BSE. While the efficiency of transmission at primary passage was low, subsequent passages resulted in a highly virulent prion disease in both Met129 and Val129 mice.
***Transmission of the different scrapie isolates in these mice leads to the emergence of prion strain phenotypes that showed similar characteristics to those displayed by MM1 or VV2 sCJD prion.
***These results demonstrate that scrapie prions have a zoonotic potential and raise new questions about the possible link between animal and human prions.
http://www.tandfonline.com/doi/abs/10.1080/19336896.2016.1163048?journalCode=kprn20
Prion diseases (PD) are the unique neurodegenerative proteinopathies reputed to be transmissible under field conditions since decades. The transmission of Bovine Spongiform Encephalopathy (BSE) to humans evidenced that an animal PD might be zoonotic under appropriate conditions. Contrarily, in the absence of obvious (epidemiological or experimental) elements supporting a transmission or genetic predispositions, PD, like the other proteinopathies, are reputed to occur spontaneously (atpical animal prion strains, sporadic CJD summing 80% of human prion cases).
Non-human primate models provided the first evidences supporting the transmissibiity of human prion strains and the zoonotic potential of BSE. Among them, cynomolgus macaques brought major information for BSE risk assessment for human health (Chen, 2014), according to their phylogenetic proximity to humans and extended lifetime. We used this model to assess the zoonotic potential of other animal PD from bovine, ovine and cervid origins even after very long silent incubation periods.
*** We recently observed the direct transmission of a natural classical scrapie isolate to macaque after a 10-year silent incubation period,
***with features similar to some reported for human cases of sporadic CJD, albeit requiring fourfold long incubation than BSE. Scrapie, as recently evoked in humanized mice (Cassard, 2014),
***is the third potentially zoonotic PD (with BSE and L-type BSE),
***thus questioning the origin of human sporadic cases.
We will present an updated panorama of our different transmission studies and discuss the implications of such extended incubation periods on risk assessment of animal PD for human health.
===============
***thus questioning the origin of human sporadic cases***
===============
***our findings suggest that possible transmission risk of H-type BSE to sheep and human. Bioassay will be required to determine whether the PMCA products are infectious to these animals.
==============
https://prion2015.files.wordpress.com/2015/05/prion2015abstracts.pdf
***Transmission data also revealed that several scrapie prions propagate in HuPrP-Tg mice with efficiency comparable to that of cattle BSE. While the efficiency of transmission at primary passage was low, subsequent passages resulted in a highly virulent prion disease in both Met129 and Val129 mice.
***Transmission of the different scrapie isolates in these mice leads to the emergence of prion strain phenotypes that showed similar characteristics to those displayed by MM1 or VV2 sCJD prion.
***These results demonstrate that scrapie prions have a zoonotic potential and raise new questions about the possible link between animal and human prions.
http://www.tandfonline.com/doi/abs/10.1080/19336896.2016.1163048?journalCode=kprn20
PRION 2016 TOKYO
Saturday, April 23, 2016
SCRAPIE WS-01: Prion diseases in animals and zoonotic potential 2016
Prion. 10:S15-S21. 2016 ISSN: 1933-6896 printl 1933-690X online
Taylor & Francis
Prion 2016 Animal Prion Disease Workshop Abstracts
WS-01: Prion diseases in animals and zoonotic potential
Transmission of the different scrapie isolates in these mice leads to the emergence of prion strain phenotypes that showed similar characteristics to those displayed by MM1 or VV2 sCJD prion.
These results demonstrate that scrapie prions have a zoonotic potential and raise new questions about the possible link between animal and human prions.
http://www.tandfonline.com/doi/abs/10.1080/19336896.2016.1163048?journalCode=kprn20
Title: Transmission of scrapie prions to primate after an extended silent incubation period)
*** In complement to the recent demonstration that humanized mice are susceptible to scrapie, we report here the first observation of direct transmission of a natural classical scrapie isolate to a macaque after a 10-year incubation period. Neuropathologic examination revealed all of the features of a prion disease: spongiform change, neuronal loss, and accumulation of PrPres throughout the CNS.
*** This observation strengthens the questioning of the harmlessness of scrapie to humans, at a time when protective measures for human and animal health are being dismantled and reduced as c-BSE is considered controlled and being eradicated.
*** Our results underscore the importance of precautionary and protective measures and the necessity for long-term experimental transmission studies to assess the zoonotic potential of other animal prion strains.
http://www.ars.usda.gov/research/publications/publications.htm?SEQ_NO_115=313160
1: J Infect Dis 1980 Aug;142(2):205-8
Oral transmission of kuru, Creutzfeldt-Jakob disease, and scrapie to nonhuman primates.
Gibbs CJ Jr, Amyx HL, Bacote A, Masters CL, Gajdusek DC.
Kuru and Creutzfeldt-Jakob disease of humans and scrapie disease of sheep and goats were transmitted to squirrel monkeys (Saimiri sciureus) that were exposed to the infectious agents only by their nonforced consumption of known infectious tissues. The asymptomatic incubation period in the one monkey exposed to the virus of kuru was 36 months; that in the two monkeys exposed to the virus of Creutzfeldt-Jakob disease was 23 and 27 months, respectively; and that in the two monkeys exposed to the virus of scrapie was 25 and 32 months, respectively. Careful physical examination of the buccal cavities of all of the monkeys failed to reveal signs or oral lesions. One additional monkey similarly exposed to kuru has remained asymptomatic during the 39 months that it has been under observation.
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The successful transmission of kuru, Creutzfeldt-Jakob disease, and scrapie by natural feeding to squirrel monkeys that we have reported provides further grounds for concern that scrapie-infected meat may occasionally give rise in humans to Creutzfeldt-Jakob disease.
PMID: 6997404
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6997404&dopt=Abstract
Recently the question has again been brought up as to whether scrapie is transmissible to man. This has followed reports that the disease has been transmitted to primates. One particularly lurid speculation (Gajdusek 1977) conjectures that the agents of scrapie, kuru, Creutzfeldt-Jakob disease and transmissible encephalopathy of mink are varieties of a single "virus". The U.S. Department of Agriculture concluded that it could "no longer justify or permit scrapie-blood line and scrapie-exposed sheep and goats to be processed for human or animal food at slaughter or rendering plants" (ARC 84/77)" The problem is emphasised by the finding that some strains of scrapie produce lesions identical to the once which characterise the human dementias"
Whether true or not. the hypothesis that these agents might be transmissible to man raises two considerations. First, the safety of laboratory personnel requires prompt attention. Second, action such as the "scorched meat" policy of USDA makes the solution of the acrapie problem urgent if the sheep industry is not to suffer grievously.
snip...
76/10.12/4.6
Nature. 1972 Mar 10;236(5341):73-4.
Transmission of scrapie to the cynomolgus monkey (Macaca fascicularis).
Gibbs CJ Jr, Gajdusek DC.
Nature 236, 73 - 74 (10 March 1972); doi:10.1038/236073a0
Transmission of Scrapie to the Cynomolgus Monkey (Macaca fascicularis)
C. J. GIBBS jun. & D. C. GAJDUSEK
National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland
SCRAPIE has been transmitted to the cynomolgus, or crab-eating, monkey (Macaca fascicularis) with an incubation period of more than 5 yr from the time of intracerebral inoculation of scrapie-infected mouse brain. The animal developed a chronic central nervous system degeneration, with ataxia, tremor and myoclonus with associated severe scrapie-like pathology of intensive astroglial hypertrophy and proliferation, neuronal vacuolation and status spongiosus of grey matter. The strain of scrapie virus used was the eighth passage in Swiss mice (NIH) of a Compton strain of scrapie obtained as ninth intracerebral passage of the agent in goat brain, from Dr R. L. Chandler (ARC, Compton, Berkshire).
P03.141
Aspects of the Cerebellar Neuropathology in Nor98
Gavier-Widén, D1; Benestad, SL2; Ottander, L1; Westergren, E1 1National Veterinary Insitute, Sweden; 2National Veterinary Institute,
Norway Nor98 is a prion disease of old sheep and goats. This atypical form of scrapie was first described in Norway in 1998. Several features of Nor98 were shown to be different from classical scrapie including the distribution of disease associated prion protein (PrPd) accumulation in the brain. The cerebellum is generally the most affected brain area in Nor98. The study here presented aimed at adding information on the neuropathology in the cerebellum of Nor98 naturally affected sheep of various genotypes in Sweden and Norway. A panel of histochemical and immunohistochemical (IHC) stainings such as IHC for PrPd, synaptophysin, glial fibrillary acidic protein, amyloid, and cell markers for phagocytic cells were conducted. The type of histological lesions and tissue reactions were evaluated. The types of PrPd deposition were characterized. The cerebellar cortex was regularly affected, even though there was a variation in the severity of the lesions from case to case. Neuropil vacuolation was more marked in the molecular layer, but affected also the granular cell layer. There was a loss of granule cells. Punctate deposition of PrPd was characteristic. It was morphologically and in distribution identical with that of synaptophysin, suggesting that PrPd accumulates in the synaptic structures. PrPd was also observed in the granule cell layer and in the white matter. The pathology features of Nor98 in the cerebellum of the affected sheep showed similarities with those of sporadic Creutzfeldt-Jakob disease in humans.
***The pathology features of Nor98 in the cerebellum of the affected sheep showed similarities with those of sporadic Creutzfeldt-Jakob disease in humans.
PR-26
NOR98 SHOWS MOLECULAR FEATURES REMINISCENT OF GSS
R. Nonno1, E. Esposito1, G. Vaccari1, E. Bandino2, M. Conte1, B. Chiappini1, S. Marcon1, M. Di Bari1, S.L. Benestad3, U. Agrimi1 1 Istituto Superiore di Sanità, Department of Food Safety and Veterinary Public Health, Rome, Italy (romolo.nonno@iss.it); 2 Istituto Zooprofilattico della Sardegna, Sassari, Italy; 3 National Veterinary Institute, Department of Pathology, Oslo, Norway
Molecular variants of PrPSc are being increasingly investigated in sheep scrapie and are generally referred to as "atypical" scrapie, as opposed to "classical scrapie". Among the atypical group, Nor98 seems to be the best identified. We studied the molecular properties of Italian and Norwegian Nor98 samples by WB analysis of brain homogenates, either untreated, digested with different concentrations of proteinase K, or subjected to enzymatic deglycosylation. The identity of PrP fragments was inferred by means of antibodies spanning the full PrP sequence. We found that undigested brain homogenates contain a Nor98-specific PrP fragment migrating at 11 kDa (PrP11), truncated at both the C-terminus and the N-terminus, and not N-glycosylated. After mild PK digestion, Nor98 displayed full-length PrP (FL-PrP) and N-glycosylated C-terminal fragments (CTF), along with increased levels of PrP11. Proteinase K digestion curves (0,006-6,4 mg/ml) showed that FL-PrP and CTF are mainly digested above 0,01 mg/ml, while PrP11 is not entirely digested even at the highest concentrations, similarly to PrP27-30 associated with classical scrapie. Above 0,2 mg/ml PK, most Nor98 samples showed only PrP11 and a fragment of 17 kDa with the same properties of PrP11, that was tentatively identified as a dimer of PrP11. Detergent solubility studies showed that PrP11 is insoluble in 2% sodium laurylsorcosine and is mainly produced from detergentsoluble, full-length PrPSc. Furthermore, among Italian scrapie isolates, we found that a sample with molecular and pathological properties consistent with Nor98 showed plaque-like deposits of PrPSc in the thalamus when the brain was analysed by PrPSc immunohistochemistry. Taken together, our results show that the distinctive pathological feature of Nor98 is a PrP fragment spanning amino acids ~ 90-155. This fragment is produced by successive N-terminal and C-terminal cleavages from a full-length and largely detergent-soluble PrPSc, is produced in vivo and is extremely resistant to PK digestion.
*** Intriguingly, these conclusions suggest that some pathological features of Nor98 are reminiscent of Gerstmann-Sträussler-Scheinker disease.
119
A newly identified type of scrapie agent can naturally infect sheep with resistant PrP genotypes
Annick Le Dur*,?, Vincent Béringue*,?, Olivier Andréoletti?, Fabienne Reine*, Thanh Lan Laï*, Thierry Baron§, Bjørn Bratberg¶, Jean-Luc Vilotte?, Pierre Sarradin**, Sylvie L. Benestad¶, and Hubert Laude*,? +Author Affiliations
*Virologie Immunologie Moléculaires and ?Génétique Biochimique et Cytogénétique, Institut National de la Recherche Agronomique, 78350 Jouy-en-Josas, France; ?Unité Mixte de Recherche, Institut National de la Recherche Agronomique-Ecole Nationale Vétérinaire de Toulouse, Interactions Hôte Agent Pathogène, 31066 Toulouse, France; §Agence Française de Sécurité Sanitaire des Aliments, Unité Agents Transmissibles Non Conventionnels, 69364 Lyon, France; **Pathologie Infectieuse et Immunologie, Institut National de la Recherche Agronomique, 37380 Nouzilly, France; and ¶Department of Pathology, National Veterinary Institute, 0033 Oslo, Norway
***Edited by Stanley B. Prusiner, University of California, San Francisco, CA (received for review March 21, 2005)
Abstract Scrapie in small ruminants belongs to transmissible spongiform encephalopathies (TSEs), or prion diseases, a family of fatal neurodegenerative disorders that affect humans and animals and can transmit within and between species by ingestion or inoculation. Conversion of the host-encoded prion protein (PrP), normal cellular PrP (PrPc), into a misfolded form, abnormal PrP (PrPSc), plays a key role in TSE transmission and pathogenesis. The intensified surveillance of scrapie in the European Union, together with the improvement of PrPSc detection techniques, has led to the discovery of a growing number of so-called atypical scrapie cases. These include clinical Nor98 cases first identified in Norwegian sheep on the basis of unusual pathological and PrPSc molecular features and "cases" that produced discordant responses in the rapid tests currently applied to the large-scale random screening of slaughtered or fallen animals. Worryingly, a substantial proportion of such cases involved sheep with PrP genotypes known until now to confer natural resistance to conventional scrapie. Here we report that both Nor98 and discordant cases, including three sheep homozygous for the resistant PrPARR allele (A136R154R171), efficiently transmitted the disease to transgenic mice expressing ovine PrP, and that they shared unique biological and biochemical features upon propagation in mice. *** These observations support the view that a truly infectious TSE agent, unrecognized until recently, infects sheep and goat flocks and may have important implications in terms of scrapie control and public health.
Monday, December 1, 2008
When Atypical Scrapie cross species barriers
Authors
Andreoletti O., Herva M. H., Cassard H., Espinosa J. C., Lacroux C., Simon S., Padilla D., Benestad S. L., Lantier F., Schelcher F., Grassi J., Torres, J. M., UMR INRA ENVT 1225, Ecole Nationale Veterinaire de Toulouse.France; ICISA-INlA, Madrid, Spain; CEA, IBiTec-5, DSV, CEA/Saclay, Gif sur Yvette cedex, France; National Veterinary Institute, Postboks 750 Sentrum, 0106 Oslo, Norway, INRA IASP, Centre INRA de Tours, 3738O Nouzilly, France.
Content
Atypical scrapie is a TSE occurring in small ruminants and harbouring peculiar clinical, epidemiological and biochemical properties. Currently this form of disease is identified in a large number of countries. In this study we report the transmission of an atypical scrapie isolate through different species barriers as modeled by transgenic mice (Tg) expressing different species PRP sequence.
The donor isolate was collected in 1995 in a French commercial sheep flock. inoculation into AHQ/AHQ sheep induced a disease which had all neuro-pathological and biochemical characteristics of atypical scrapie. Transmitted into Transgenic mice expressing either ovine or PrPc, the isolate retained all the described characteristics of atypical scrapie.
Surprisingly the TSE agent characteristics were dramatically different v/hen passaged into Tg bovine mice. The recovered TSE agent had biological and biochemical characteristics similar to those of atypical BSE L in the same mouse model. Moreover, whereas no other TSE agent than BSE were shown to transmit into Tg porcine mice, atypical scrapie was able to develop into this model, albeit with low attack rate on first passage.
Furthermore, after adaptation in the porcine mouse model this prion showed similar biological and biochemical characteristics than BSE adapted to this porcine mouse model. Altogether these data indicate.
(i) the unsuspected potential abilities of atypical scrapie to cross species barriers
(ii) the possible capacity of this agent to acquire new characteristics when crossing species barrier
These findings raise some interrogation on the concept of TSE strain and on the origin of the diversity of the TSE agents and could have consequences on field TSE control measures.
WEDNESDAY, JUNE 10, 2020
Radical Change in Zoonotic Abilities of Atypical BSE Prion Strains as Evidenced by Crossing of Sheep Species Barrier in Transgenic Mice
Atypical BSE prions showed a modification in their zoonotic ability after adaptation to sheep-PrP producing agents able to infect TgMet129 and TgVal129, bearing features that make them indistinguishable of sporadic Creutzfeldt-Jakob disease prions.
our results clearly indicate that atypical BSE adaptation to an ovine-PrP sequence could modify the prion agent to potentially infect humans, showing strain features indistinguishable from those of classic sCJD prions, even though they might or might not be different agents.
However, the expanding range of TSE agents displaying the capacity to transmit in human-PrP–expressing hosts warrants the continuation of the ban on meat and bone meal recycling and underscores the ongoing need for active surveillance
Atypical L-type BSE
Emerg Infect Dis. 2017 Feb; 23(2): 284–287. doi: 10.3201/eid2302.161416 PMCID: PMC5324790 PMID: 28098532
Oral Transmission of L-Type Bovine Spongiform Encephalopathy Agent among Cattle
Our study clearly confirms, experimentally, the potential risk for interspecies oral transmission of the agent of L-BSE. In our model, this risk appears higher than that for the agent of classical BSE, which could only be transmitted to mouse lemurs after a first passage in macaques (14). We report oral transmission of the L-BSE agent in young and adult primates. Transmission by the IC route has also been reported in young macaques (6,7). A previous study of L-BSE in transgenic mice expressing human PrP suggested an absence of any transmission barrier between cattle and humans for this particular strain of the agent of BSE, in contrast to findings for the agent of classical BSE (9). Thus, it is imperative to maintain measures that prevent the entry of tissues from cattle possibly infected with the agent of L-BSE into the food chain.
Atypical H-type BSE
Research Project: Pathobiology, Genetics, and Detection of Transmissible Spongiform Encephalopathies Location: Virus and Prion Research
Title: The agent of H-type bovine spongiform encephalopathy associated with E211K prion protein polymorphism transmits after oronasal challenge
This study demonstrates that the H-type BSE agent is transmissible by the oronasal route.
These results reinforce the need for ongoing surveillance for classical and atypical BSE to minimize the risk of potentially infectious tissues entering the animal or human food chains.
P98 The agent of H-type bovine spongiform encephalopathy associated with E211K prion protein polymorphism transmits after oronasal challenge
Greenlee JJ (1), Moore SJ (1), and West Greenlee MH (2) (1) United States Department of Agriculture, Agricultural Research Service, National Animal Disease Center, Virus and Prion Research Unit, Ames, IA, United States (2) Department of Biomedical Sciences, Iowa State University College of Veterinary Medicine, Ames, IA, United States.
With the experiment currently at 55 months post-inoculation, no other cattle in this study have developed clinical signs suggestive of prion disease. This study demonstrates that the H-type BSE agent is transmissible by the oronasal route.
These results reinforce the need for ongoing surveillance for classical and atypical BSE to minimize the risk of potentially infectious tissues entering the animal or human food chains.
PRION CONFERENCE 2018 CONFERENCE ABSTRACT
Published: 23 June 2011
Experimental H-type bovine spongiform encephalopathy characterized by plaques and glial- and stellate-type prion protein deposits
The present study demonstrated successful intraspecies transmission of H-type BSE to cattle and the distribution and immunolabeling patterns of PrPSc in the brain of the H-type BSE-challenged cattle. TSE agent virulence can be minimally defined by oral transmission of different TSE agents (C-type, L-type, and H-type BSE agents) [59]. Oral transmission studies with H-type BSE-infected cattle have been initiated and are underway to provide information regarding the extent of similarity in the immunohistochemical and molecular features before and after transmission. In addition, the present data will support risk assessments in some peripheral tissues derived from cattle affected with H-type BSE.
References...END
Prion Infectivity and PrPBSE in the Peripheral and Central Nervous System of Cattle 8 Months Post Oral BSE Challenge
Prion Infectivity and PrPBSE in the Peripheral and Central Nervous System of Cattle 8 Months Post Oral BSE Challenge
Ivett Ackermann 1 , Reiner Ulrich 2 , Kerstin Tauscher 3 , Olanrewaju I. Fatola 1,4 , Markus Keller 1 , James C. Shawulu 1,5, Mark Arnold 6 , Stefanie Czub 7 , Martin H. Groschup 1 and Anne Balkema-Buschmann 1,*
1 Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institute, 17493 Greifswald-Insel Riems, Germany; Ivett.Ackermann@fli.de (I.A.); fatolan@yahoo.com (O.I.F.); Markus.Keller@fli.de (M.K.); james.shawulu@ymail.com (J.C.S.); Martin.Groschup@fli.de (M.H.G.) 2 Institute of Veterinary Pathology, Faculty of Veterinary Medicine, Leipzig University, 04103 Leipzig, Germany; reiner.ulrich@vetmed.uni-leipzig.de 3 Department of Experimental Animal Facilities and Biorisk Management, Friedrich-Loeffler-Institute, 17493 Greifswald-Insel Riems, Germany; Kerstin_Tauscher@gmx.de 4 Neuroscience Unit, Department of Veterinary Anatomy, Faculty of Veterinary Medicine, University of Ibadan, Ibadan 200284, Nigeria 5 Department of Veterinary Anatomy, Faculty of Veterinary Medicine, University of Abuja, Abuja 900105, Nigeria 6 Animal and Plant Health Agency Sutton Bonington, Sutton Bonington, Leicestershire LE12 5RB, UK; Mark.Arnold@apha.gov.uk 7 Canadian Food Inspection Agency, Lethbridge Laboratory, Lethbridge, AB T1J 3Z4, Canada; stefanie.czub37@gmail.com * Correspondence: anne.buschmann@fli.de
Abstract: After oral exposure of cattle with classical bovine spongiform encephalopathy (C-BSE), the infectious agent ascends from the gut to the central nervous system (CNS) primarily via the autonomic nervous system. However, the timeline of this progression has thus far remained widely undetermined. Previous studies were focused on later time points after oral exposure of animals that were already 4 to 6 months old when challenged. In contrast, in this present study, we have orally inoculated 4 to 6 weeks old unweaned calves with high doses of BSE to identify any possible BSE infectivity and/or PrPBSE in peripheral nervous tissues during the first eight months postinoculation (mpi). For the detection of BSE infectivity, we used a bovine PrP transgenic mouse bioassay, while PrPBSE depositions were analyzed by immunohistochemistry (IHC) and by protein misfolding cyclic amplification (PMCA). We were able to show that as early as 8 mpi the thoracic spinal cord as well as the parasympathetic nodal ganglion of these animals contained PrPBSE and BSE infectivity. This shows that the centripetal prion spread starts early after challenge at least in this age group, which represents an essential piece of information for the risk assessments for food, feed, and pharmaceutical products produced from young calves.
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5. Conclusions
In summary, we detected PrPBSE and BSE infectivity as early as 8 mpi in the nodal ganglion as well as in the thoracic spinal cord from one calf challenged before weaning in this study and also at eight mpi in the thoracic spinal cord sampled from cattle challenged at 4 to 6 months of age during an earlier pathogenesis study [5,20]. This current study considerably expands the existing data on the early C-BSE pathogenesis by demonstrating that after challenge with an unnaturally high dose of 100 g BSE-positive brainstem tissue, parts of the peripheral and central nervous system from cattle may already contain PrPBSE and BSE infectivity after short time periods up to 8 months after oral infection, which should be considered relevant information for risk assessments for food and pharmaceutical products.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ijms222111310/s1 .
Keywords: prion protein; BSE; infectivity; PrPBSE; cattle; peripheral and central nervous system; protein misfolding cyclic amplification (PMCA)
O.4.3
Spread of BSE prions in cynomolgus monkeys (Macaca fascicularis) after oral transmission
Edgar Holznagel1, Walter Schulz-Schaeffer2, Barbara Yutzy1, Gerhard Hunsmann3, Johannes Loewer1 1Paul-Ehrlich-Institut, Federal Institute for Sera and Vaccines, Germany; 2Department of Neuropathology, Georg-August University, Göttingen, Germany, 3Department of Virology and Immunology, German Primate Centre, Göttingen, Germany
Background: BSE-infected cynomolgus monkeys represent a relevant animal model to study the pathogenesis of variant Creutzfeldt-Jacob disease (vCJD).
Objectives: To study the spread of BSE prions during the asymptomatic phase of infection in a simian animal model.
Methods: Orally BSE-dosed macaques (n=10) were sacrificed at defined time points during the incubation period and 7 orally BSE-dosed macaques were sacrificed after the onset of clinical signs. Neuronal and non-neuronal tissues were tested for the presence of proteinase-K-resistant prion protein (PrPres) by western immunoblot and by paraffin-embedded tissue (PET) blot technique.
Results: In clinically diseased macaques (5 years p.i. + 6 mo.), PrPres deposits were widely spread in neuronal tissues (including the peripheral sympathetic and parasympathetic nervous system) and in lymphoid tissues including tonsils. In asymptomatic disease carriers, PrPres deposits could be detected in intestinal lymph nodes as early as 1 year p.i., but CNS tissues were negative until 3 – 4 years p.i. Lumbal/sacral segments of the spinal cord and medulla oblongata were PrPres positive as early as 4.1 years p.i., whereas sympathetic trunk and all thoracic/cervical segments of the spinal cord were still negative for PrPres. However, tonsil samples were negative in all asymptomatic cases.
Discussion: There is evidence for an early spread of BSE to the CNS via autonomic fibres of the splanchnic and vagus nerves indicating that trans-synaptical spread may be a time-limiting factor for neuroinvasion. Tonsils were predominantly negative during the main part of the incubation period indicating that epidemiological vCJD screening results based on the detection of PrPres in tonsil biopsies may mostly tend to underestimate the prevalence of vCJD among humans.
P.4.23
Transmission of atypical BSE in humanized mouse models
Liuting Qing1, Wenquan Zou1, Cristina Casalone2, Martin Groschup3, Miroslaw Polak4, Maria Caramelli2, Pierluigi Gambetti1, Juergen Richt5, Qingzhong Kong1 1Case Western Reserve University, USA; 2Instituto Zooprofilattico Sperimentale, Italy; 3Friedrich-Loeffler-Institut, Germany; 4National Veterinary Research Institute, Poland; 5Kansas State University (Previously at USDA National Animal Disease Center), USA
Background: Classical BSE is a world-wide prion disease in cattle, and the classical BSE strain (BSE-C) has led to over 200 cases of clinical human infection (variant CJD). Atypical BSE cases have been discovered in three continents since 2004; they include the L-type (also named BASE), the H-type, and the first reported case of naturally occurring BSE with mutated bovine PRNP (termed BSE-M). The public health risks posed by atypical BSE were largely undefined.
Objectives: To investigate these atypical BSE types in terms of their transmissibility and phenotypes in humanized mice. Methods: Transgenic mice expressing human PrP were inoculated with several classical (C-type) and atypical (L-, H-, or Mtype) BSE isolates, and the transmission rate, incubation time, characteristics and distribution of PrPSc, symptoms, and histopathology were or will be examined and compared.
Results: Sixty percent of BASE-inoculated humanized mice became infected with minimal spongiosis and an average incubation time of 20-22 months, whereas only one of the C-type BSE-inoculated mice developed prion disease after more than 2 years. Protease-resistant PrPSc in BASE-infected humanized Tg mouse brains was biochemically different from bovine BASE or sCJD. PrPSc was also detected in the spleen of 22% of BASE-infected humanized mice, but not in those infected with sCJD. Secondary transmission of BASE in the humanized mice led to a small reduction in incubation time. The atypical BSE-H strain is also transmissible with distinct phenotypes in the humanized mice, but no BSE-M transmission has been observed so far.
Discussion: Our results demonstrate that BASE is more virulent than classical BSE, has a lymphotropic phenotype, and displays a modest transmission barrier in our humanized mice.
BSE-H is also transmissible in our humanized Tg mice.
The possibility of more than two atypical BSE strains will be discussed.
Supported by NINDS NS052319, NIA AG14359, and NIH AI 77774.
P03.137
Transmission of BSE to Cynomolgus Macaque, a Non-human Primate; Development of Clinical Symptoms and Tissue Distribution of PrPSC
Yamakawa, Y1; Ono, F2; Tase, N3; Terao, K3; Tannno, J3; Wada, N4; Tobiume, M5; Sato, Y5; Okemoto-Nakamura, Y1; Hagiwara, K1; Sata, T5 1National Institure of Infectious diseases, Cell biology and Biochemistry, Japan; 2Corporation for Production and Research Laboratory Primates., Japan; 3National Institure of Biomedical Innovation, Tsukuba Primate Reserch Center, Japan; 4Yamauchi Univ., Veterinary Medicine, Japan; 5National Institure of Infectious diseases, Pathology, Japan
Two of three cynomolgus monkeys developed abnormal neuronal behavioral signs at 30-(#7) and 28-(#10) months after intracerebral inoculation of 200ul of 10% brain homogenates of BSE affected cattle (BSE/JP6). Around 30 months post inoculation (mpi), they developed sporadic anorexia and hyperekplexia with squeal against environmental stimulations such as light and sound. Tremor, myoclonic jerk and paralysis became conspicuous during 32 to 33-mpi, and symptoms become worsened according to the disease progression. Finally, one monkey (#7) fell into total paralysis at 36-mpi. This monkey was sacrificed at 10 days after intensive veterinary care including infusion and per oral supply of liquid food. The other monkey (#10) had to grasp the cage bars to keep an upright posture caused by the sever ataxia. This monkey was sacrificed at 35-mpi. EEG of both monkeys showed diffuse slowing. PSD characteristic for sporadic CJD was not observed in both monkeys. The result of forearm movement test showed the hypofunction that was observed at onset of clinical symptoms. Their cognitive function determined by finger maze test was maintained at the early stage of sideration. However, it was rapidly impaired followed by the disease progression. Their autopsied tissues were immunochemically investigated for the tissue distribution of PrPSc. Severe spongiform change in the brain together with heavy accumulation of PrPSc having the type 2B/4 glycoform profile confirmed successful transmission of BSE to Cynomolgus macaques. Granular and linear deposition of PrPSC was detected by IHC in the CNS of both monkeys. At cerebral cortex, PrPSC was prominently accumulated in the large plaques. Sparse accumulation of PrPSc was detected in several peripheral nerves of #7 but not in #10 monkey, upon the WB analysis. Neither #7 nor #10 monkey accumulated detectable amounts of PrPSc in their lymphatic organs such as tonsil, spleen, adrenal grands and thymus although PrPSc was barely detected in the submandibular lymph node of #7 monkey. Such confined tissue distribution of PrPSc after intracerebral infection with BSE agent is not compatible to that reported on the Cynomolgus macaques infected with BSE by oral or intra-venous (intra-peritoneal) routs, in which PrPSc was accumulated at not only CNS but also widely distributed lymphatic tissues.
P04.27
Experimental BSE Infection of Non-human Primates: Efficacy of the Oral Route
Holznagel, E1; Yutzy, B1; Deslys, J-P2; Lasmézas, C2; Pocchiari, M3; Ingrosso, L3; Bierke, P4; Schulz-Schaeffer, W5; Motzkus, D6; Hunsmann, G6; Löwer, J1 1Paul-Ehrlich-Institut, Germany; 2Commissariat à l´Energie Atomique, France; 3Instituto Superiore di Sanità, Italy; 4Swedish Institute for Infectious Disease control, Sweden; 5Georg August University, Germany; 6German Primate Center, Germany
Background: In 2001, a study was initiated in primates to assess the risk for humans to contract BSE through contaminated food. For this purpose, BSE brain was titrated in cynomolgus monkeys.
Aims: The primary objective is the determination of the minimal infectious dose (MID50) for oral exposure to BSE in a simian model, and, by in doing this, to assess the risk for humans. Secondly, we aimed at examining the course of the disease to identify possible biomarkers.
Methods: Groups with six monkeys each were orally dosed with lowering amounts of BSE brain: 16g, 5g, 0.5g, 0.05g, and 0.005g. In a second titration study, animals were intracerebrally (i.c.) dosed (50, 5, 0.5, 0.05, and 0.005 mg).
Results: In an ongoing study, a considerable number of high-dosed macaques already developed simian vCJD upon oral or intracerebral exposure or are at the onset of the clinical phase. However, there are differences in the clinical course between orally and intracerebrally infected animals that may influence the detection of biomarkers.
Conclusions: Simian vCJD can be easily triggered in cynomolgus monkeys on the oral route using less than 5 g BSE brain homogenate. The difference in the incubation period between 5 g oral and 5 mg i.c. is only 1 year (5 years versus 4 years). However, there are rapid progressors among orally dosed monkeys that develop simian vCJD as fast as intracerebrally inoculated animals.
The work referenced was performed in partial fulfilment of the study “BSE in primates“ supported by the EU (QLK1-2002-01096).
Bovine spongiform encephalopathy: the effect of oral exposure dose on attack rate and incubation period in cattle
G. A. H. Wells,1 T. Konold,1 M. E. Arnold,1 A. R. Austin,1 3 S. A. C. Hawkins,1 M. Stack,1 M. M. Simmons,1 Y. H. Lee,2 D. Gavier-Wide´n,3 M. Dawson1 4 and J. W. Wilesmith1 1 Correspondence G. A. H. Wells
1 Veterinary Laboratories Agency, Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, UK
2 National Veterinary Research and Quarantine Service, Anyang, Republic of Korea
3 National Veterinary Institute (SVA), SE-75189 Uppsala, Sweden
Received 27 July 2006
Accepted 18 November 2006
The dose–response of cattle exposed to the bovine spongiform encephalopathy (BSE) agent is an important component of modelling exposure risks for animals and humans and thereby, the modulation of surveillance and control strategies for BSE. In two experiments calves were dosed orally with a range of amounts of a pool of brainstems from BSE-affected cattle. Infectivity in the pool was determined by end-point titration in mice. Recipient cattle were monitored for clinical disease and, from the incidence of pathologically confirmed cases and their incubation periods (IPs), the attack rate and IP distribution according to dose were estimated. The dose at which 50 % of cattle would be clinically affected was estimated at 0.20 g brain material used in the experiment, with 95 % confidence intervals of 0.04–1.00 g. The IP was highly variable across all dose groups and followed a log-normal distribution, with decreasing mean as dose increased. There was no evidence of a threshold dose at which the probability of infection became vanishingly small, with 1/15 (7 %) of animals affected at the lowest dose (1 mg).
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DISCUSSION
The study has demonstrated that disease in cattle can be produced by oral exposure to as little as 1 mg brain homogenate (¡100.4 RIII mouse i.c./i.p. ID50 units) from clinically affected field cases of BSE and that the limiting dose for infection of calves is lower than this exposure...
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P04.27
Experimental BSE Infection of Non-human Primates: Efficacy of the Oral Route
Holznagel, E1; Yutzy, B1; Deslys, J-P2; Lasm�zas, C2; Pocchiari, M3; Ingrosso, L3; Bierke, P4; Schulz-Schaeffer, W5; Motzkus, D6; Hunsmann, G6; L�wer, J1 1Paul-Ehrlich-Institut, Germany; 2Commissariat � l�Energie Atomique, France; 3Instituto Superiore di Sanit�, Italy; 4Swedish Institute for Infectious Disease control, Sweden; 5Georg August University, Germany; 6German Primate Center, Germany
Background:
In 2001, a study was initiated in primates to assess the risk for humans to contract BSE through contaminated food. For this purpose, BSE brain was titrated in cynomolgus monkeys.
Aims:
The primary objective is the determination of the minimal infectious dose (MID50) for oral exposure to BSE in a simian model, and, by in doing this, to assess the risk for humans. Secondly, we aimed at examining the course of the disease to identify possible biomarkers.
Methods:
Groups with six monkeys each were orally dosed with lowering amounts of BSE brain: 16g, 5g, 0.5g, 0.05g, and 0.005g. In a second titration study, animals were intracerebrally (i.c.) dosed (50, 5, 0.5, 0.05, and 0.005 mg).
Results:
In an ongoing study, a considerable number of high-dosed macaques already developed simian vCJD upon oral or intracerebral exposure or are at the onset of the clinical phase. However, there are differences in the clinical course between orally and intracerebrally infected animals that may influence the detection of biomarkers.
Conclusions:
Simian vCJD can be easily triggered in cynomolgus monkeys on the oral route using less than 5 g BSE brain homogenate. The difference in the incubation period between 5 g oral and 5 mg i.c. is only 1 year (5 years versus 4 years). However, there are rapid progressors among orally dosed monkeys that develop simian vCJD as fast as intracerebrally inoculated animals.
The work referenced was performed in partial fulfilment of the study �BSE in primates� supported by the EU (QLK1-2002-01096).
look at the table and you'll see that as little as 1 mg (or 0.001 gm) caused 7% (1 of 14) of the cows to come down with BSE;
Risk of oral infection with bovine spongiform encephalopathy agent in primates
Corinne Ida Lasm�zas, Emmanuel Comoy, Stephen Hawkins, Christian Herzog, Franck Mouthon, Timm Konold, Fr�d�ric Auvr�, Evelyne Correia, Nathalie Lescoutra-Etchegaray, Nicole Sal�s, Gerald Wells, Paul Brown, Jean-Philippe Deslys Summary The uncertain extent of human exposure to bovine spongiform encephalopathy (BSE)--which can lead to variant Creutzfeldt-Jakob disease (vCJD)--is compounded by incomplete knowledge about the efficiency of oral infection and the magnitude of any bovine-to-human biological barrier to transmission. We therefore investigated oral transmission of BSE to non-human primates. We gave two macaques a 5 g oral dose of brain homogenate from a BSE-infected cow. One macaque developed vCJD-like neurological disease 60 months after exposure, whereas the other remained free of disease at 76 months. On the basis of these findings and data from other studies, we made a preliminary estimate of the food exposure risk for man, which provides additional assurance that existing public health measures can prevent transmission of BSE to man.
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BSE bovine brain inoculum
100 g 10 g 5 g 1 g 100 mg 10 mg 1 mg 0�1 mg 0�01 mg
Primate (oral route)* 1/2 (50%)
Cattle (oral route)* 10/10 (100%) 7/9 (78%) 7/10 (70%) 3/15 (20%) 1/15 (7%) 1/15 (7%)
RIII mice (ic ip route)* 17/18 (94%) 15/17 (88%) 1/14 (7%)
PrPres biochemical detection
The comparison is made on the basis of calibration of the bovine inoculum used in our study with primates against a bovine brain inoculum with a similar PrPres concentration that was
inoculated into mice and cattle.8 *Data are number of animals positive/number of animals surviving at the time of clinical onset of disease in the first positive animal (%). The accuracy of
bioassays is generally judged to be about plus or minus 1 log. ic ip=intracerebral and int****ritoneal.
Table 1: Comparison of transmission rates in primates and cattle infected orally with similar BSE brain inocula
Published online January 27, 2005
It is clear that the designing scientists must
also have shared Mr Bradley's surprise at the results because all the dose
levels right down to 1 gram triggered infection.
6. It also appears to me that Mr Bradley's answer (that it would take less than say 100 grams) was probably given with the benefit of hindsight; particularly if one considers that later in the same answer Mr Bradley expresses his surprise that it could take as little of 1 gram of brain to cause BSE by the oral route within the same species. This information did not become available until the "attack rate" experiment had been completed in 1995/96. This was a titration experiment designed to ascertain the infective dose. A range of dosages was used to ensure that the actual result was within both a lower and an upper limit within the study and the designing scientists would not have expected all the dose levels to trigger infection. The dose ranges chosen by the most informed scientists at that time ranged from 1 gram to three times one hundred grams. It is clear that the designing scientists must have also shared Mr Bradley's surprise at the results because all the dose levels right down to 1 gram triggered infection.
RESEARCH ARTICLE
Very low oral exposure to prions of brain or saliva origin can transmit chronic wasting disease
Nathaniel D. Denkers1☯, Clare E. Hoover2☯, Kristen A. DavenportID3, Davin M. Henderson1, Erin E. McNultyID1, Amy V. Nalls1, Candace K. Mathiason1, Edward A. HooverID1*
1 Department of Microbiology, Immunology, and Pathology, Prion Research Center, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado, United States of America, 2 AstraZeneca Inc., Waltham, Massachusetts, United States of America, 3 Department of Biochemistry, School of Medicine, University of Utah, Salt Lake City, Utah, United States of America ☯ These authors contributed equally to this work. * Edward.hoover@colostate.edu
Abstract
The minimum infectious dose required to induce CWD infection in cervids remains unknown, as does whether peripherally shed prions and/or multiple low dose exposures are important factors in CWD transmission. With the goal of better understand CWD infection in nature, we studied oral exposures of deer to very low doses of CWD prions and also examined whether the frequency of exposure or prion source may influence infection and pathogene- sis. We orally inoculated white-tailed deer with either single or multiple divided doses of pri- ons of brain or saliva origin and monitored infection by serial longitudinal tissue biopsies spanning over two years. We report that oral exposure to as little as 300 nanograms (ng) of CWD-positive brain or to saliva containing seeding activity equivalent to 300 ng of CWD- positive brain, were sufficient to transmit CWD disease. This was true whether the inoculum was administered as a single bolus or divided as three weekly 100 ng exposures. However, when the 300 ng total dose was apportioned as 10, 30 ng doses delivered over 12 weeks, no infection occurred. While low-dose exposures to prions of brain or saliva origin prolonged the time from inoculation to first detection of infection, once infection was established, we observed no differences in disease pathogenesis. These studies suggest that the CWD min- imum infectious dose approximates 100 to 300 ng CWD-positive brain (or saliva equivalent), and that CWD infection appears to conform more with a threshold than a cumulative dose dynamic.
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Discussion
As CWD expands across North America and Scandinavia, how this disease is transmitted so efficiently remains unclear, given the low concentrations of prions shed in secretions and excretions [13, 14]. The present studies demonstrated that a single oral exposure to as little as 300nmg of CWD-positive brain or equivalent saliva can initiate infection in 100% of exposed white-tailed deer. However, distributing this dose as 10, 30 ng exposures failed to induce infec- tion. Overall, these results suggest that the minimum oral infectious exposure approaches 100 to 300 ng of CWD-positive brain equivalent. These dynamics also invite speculation as to whether potential infection co-factors, such as particle binding [46, 47] or compromises in mucosal integrity may influence infection susceptibility, as suggested from two studies in rodent models [48, 49].
Few studies in rodent models have explored oral infection with murine or hamster adapted scrapie by assessing the same total dose administered as a single bolus vs. the same bolus divided into fractional, sequential exposures [50–52]. The results reported by Diringer et al. [50] and Jacquemot et al. [52] have indicated that divided-dose exposures were as effective as a single bolus only if the interval between doses was short (1–2 days). In deer, we likewise found that when a total dose of 300 ng of brain was administered as 10 doses divided doses over 12 weeks this exposure failed to induce CWD infection, whereas three weekly 100 ng doses (300 ng total) induced infection. While this latter outcome may have involved an additive dynamic, we cannot exclude that a dose 100 ng alone also may have been sufficient to establish infection. Our conclusions here are unfortunately limited by the absence of a single 100 ng dose group. Additional experiments are needed to further directly compare single vs. divided exposures to strengthen the tenet that establishment of CWD infection is more a threshold than cumulative dose phenomenon.
We also sought to examine a relatively unexamined possibility that prions emanating from different tissues and/or cells may possess different capacities to establish infections by mucosal routes. Our results indicated that brain and saliva inocula containing similar levels of prion seeding activity, also had similar infectivity, which did not support our hypothesis that saliva prions may be more infectious by mucosal routes. There are of course, several caveats bearing on this conclusion. These could include: the inherent limits in using an in vitro seeding assay as a surrogate to equate in vivo infectivity, the likelihood that small differences in prion suscep- tibility among deer may be more significant at very low exposure doses, and the greater varia- tion of inoculum uptake and routing through mucosal surfaces associated with the oral route of exposure.
The chief correlate we observed between magnitude of infectious dose and disease course was in time from exposure to first detected amplification of prions in tonsil, an event which is closely followed by or concurrent with detection in pharyngeal lymph nodes [41]. Once a threshold dose was established, the subsequent pathogenesis of infection and disease appeared to vary little.
In addition to potential cofactors that could influence CWD infectivity, such as particle binding [47] and compromised mucosal integrity [48, 53], there is PRNP genotype, in which polymorphisms at codon 96 of the white-tailed deer are known to affect the temporal dynam- ics of CWD infections [23, 41, 45]. In the present studies, most cohorts of 96GG deer became CWD-positive before 96GS animals in the same exposure group [cohorts 1, 2, 4, 6]. Thus, the low dose studies are consistent with the current concept of delayed conversion rate in PRNP 96GS vs. 96GG white-tailed deer [44].
In conclusion, we have attempted to model and better understand CWD infection relative to natural exposure. The results demonstrate: (a) that the minimum CWD oral infectious dose is vastly lower than historical studies used to establish infection; (b) that a direct relationship exists between dose and incubation time to first prion replication detection in tonsils, irrespec- tive of genotype; (c) that a difference was not discernible between brain vs. saliva source prions in ability to establish infection or in resultant disease course; and (d) that the CWD infection process appears to conform more to a threshold dose than an accumulative dose dynamic.
America BSE 589.2001 FEED REGULATIONS, BSE SURVEILLANCE, BSE TESTING, and CJD TSE Prion
so far, we have been lucky. to date, with the science at hand, no cwd transmitted to cattle, that has been documented, TO DATE, WITH THE SCIENCE AT HAND, it's not to say it has not already happened, just like with zoonosis of cwd i.e. molecular transmission studies have shown that cwd transmission to humans would look like sporadic cjd, NOT nvCJD or what they call now vCJD. the other thing is virulence and or horizontal transmission. this is very concerning with the recent fact of what seems to be a large outbreak of a new tse prion disease in camels in Africa. there is much concern now with hay, straw, grains, and such, with the cwd tse prion endemic countries USA, Canada. what is of greatest concern is the different strains of cwd, and the virulence there from? this thing (cwd) keeps mutating to different strains, and to different species, the bigger the chance of one of these strains that WILL TRANSMIT TO CATTLE OR HUMANS, and that it is documented (i believe both has already occured imo with scienct to date). with that said, a few things to ponder, and i am still very concerned with, the animal feed. we now know from transmission studies that cwd and scrapie will transmit to pigs by oral routes. the atypical bse strains will transmit by oral routes. i don't mean to keep kicking a mad cow, just look at the science;
***> cattle, pigs, sheep, cwd, tse, prion, oh my!
***> In contrast, cattle are highly susceptible to white-tailed deer CWD and mule deer CWD in experimental conditions but no natural CWD infections in cattle have been reported (Sigurdson, 2008; Hamir et al., 2006).
Sheep and cattle may be exposed to CWD via common grazing areas with affected deer but so far, appear to be poorly susceptible to mule deer CWD (Sigurdson, 2008). In contrast, cattle are highly susceptible to white-tailed deer CWD and mule deer CWD in experimental conditions but no natural CWD infections in cattle have been reported (Sigurdson, 2008; Hamir et al., 2006). It is not known how susceptible humans are to CWD but given that the prion can be present in muscle, it is likely that humans have been exposed to the agent via consumption of venison (Sigurdson, 2008). Initial experimental research suggests that human susceptibility to CWD is low and there may be a robust species barrier for CWD transmission to humans (Sigurdson, 2008), however the risk appetite for a public health threat may still find this level unacceptable.
Research Project: TRANSMISSION, DIFFERENTIATION, AND PATHOBIOLOGY OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES Location: Virus and Prion Research
Title: Limited amplification of chronic wasting disease prions in the peripheral tissues of intracerebrally inoculated cattle
Author item HALEY, NICHOLAS - Kansas State University item SIEPKER, CHRISTOPHER - Kansas State University item Greenlee, Justin item RICHT, JÜRGEN - Kansas State University Submitted to: Journal of General Virology Publication Type: Peer Reviewed Journal Publication Acceptance Date: 3/30/2016 Publication Date: 1/7/2016
Citation: Haley, N.J., Siepker, C., Greenlee, J.J., Richt, J.A. 2016. Limited amplification of chronic wasting disease prions in the peripheral tissues of intracerebrally inoculated cattle. Journal of General Virology. 97:1720-1724.
Interpretive Summary: Chronic Wasting Disease (CWD), a fatal neurodegenerative disease that occurs in farmed and wild cervids (deer and elk) of North America, is a transmissible spongiform encephalopathy (TSE). TSEs are caused by infectious proteins called prions that are resistant to various methods of decontamination and environmental degradation. Cattle could be exposed to chronic wasting disease (CWD) by contact with infected farmed or free-ranging cervids. The purpose of this study was to use an in vitro amplification method called real time quaking induced conversion (RT-QuIC) to assess tissues from cattle inoculated with CWD for low levels of prions not detected by traditional diagnostic methods such as western blot and immunohistochemistry. This study reports that prions were identified by RT-QuIC only in cattle that were confirmed positive by traditional methods. However, prions were rarely identified in some peripheral tissues such as mesenteric lymph node, tonsil, or nasal turbinate that were not considered positive by traditional methods. These results suggest that cattle experimentally inoculated with CWD may have some limited amount of prion infectivity outside of the brain and spinal cord that may represent a previously unrecognized risk for transmission. This information could have an impact on regulatory officials developing plans to reduce or eliminate TSEs and farmers with concerns about ranging cattle on areas where CWD may be present.
Technical Abstract: Chronic wasting disease (CWD) is a fatal neurodegenerative disease, classified as a prion disease or transmissible spongiform encephalopathy (TSE) similar to bovine spongiform encephalopathy (BSE). Cervids affected by CWD accumulate an abnormal protease resistant prion protein throughout the central nervous system (CNS), as well as in both lymphatic and excretory tissues – an aspect of prion disease pathogenesis not observed in cattle with BSE. Using seeded amplification through real time quaking induced conversion (RT-QuIC), we investigated whether the bovine host or prion agent was responsible for this aspect of TSE pathogenesis. We blindly examined numerous central and peripheral tissues from cattle inoculated with CWD for prion seeding activity. Seeded amplification was readily detected in the CNS, though rarely observed in peripheral tissues, with a limited distribution similar to that of BSE prions in cattle. This seems to indicate that prion peripheralization in cattle is a host-driven characteristic of TSE infection.
Title: Experimental transmission of transmissible spongiform encephalopathies (scrapie, chronic wasting disease, transmissible mink encephalopathy) to cattle and their differentiation from bovine spongiform encephalopathy
Author item Hamir, Amirali item CUTLIP, RANDALL item MILLER, JANICE item Kunkle, Robert item Richt, Juergen item Greenlee, Justin item Nicholson, Eric item Kehrli Jr, Marcus Submitted to: World Association of Veterinary Laboratory Diagnosticians Publication Type: Proceedings
Publication Acceptance Date: 8/10/2007 Publication Date: 11/11/2007
Citation: Hamir, A.N., Cutlip, R.C., Miller, J.M., Kunkle, R.A., Richt, J.A., Greenlee, J.J., Nicholson, E.M., Kehrli, Jr., M.E. 2007. Experimental transmission of transmissible spongiform encephalopathies (scrapie, chronic wasting disease, transmissible mink encephalopathy) to cattle and their differentiation from bovine spongiform encephalopathy. In: Proceedings of the World Association of Veterinary Laboratory Diagnosticians 13th International Symposium, November 11-14, 2007, Melbourne, Australia. p. 29. Interpretive Summary:
Technical Abstract: Introduction: Experimental cross-species transmission of TSE agents provides valuable information for identification of potential host ranges of known TSEs. This report provides a synopsis of TSE (scrapie, CWD, TME) transmission studies that have been conducted in cattle and compares these findings to those seen in animals with BSE. Materials & Methods: Generally 6-month-old bull calves were obtained and assigned to inoculated and control groups. Inoculated calves were housed in a Biosafety Level 2 isolation barn at the National Animal Disease Center (NADC), Ames, Iowa. Calves were inoculated intracerebrally with 1 ml of a 10% TSE brain inoculum. Results: Results of various TSE cattle experiments with intracerebral inoculation of scrapie, CWD and TME are shown in tabular form (Table 1). Table 1. Comparison of experimental scrapie, chronic wasting disease (CWD) and transmissible mink encephalopathy (TME) in cattle inoculated by the intracerebral route during first passage of the inocula. Abnormal CNS signs: Scrapie. Anorexia, weight loss, leg and back stiffness. Some showed incoordination and posterior weakness. Eventual severe lethargy. CWD. Anorexia, weight loss, occasional aimless circling, listlessness and excited by loud noises. TME. Variable hyperexcitability with occasional falling to the ground. Some showing circling and aggressive behavior. Incubation (survival) time: Scrapie. 14 – 18 months. CWD. 23 – 63 months. TME. 13 – 16 months. Attack rate: Scrapie. 100%. CWD. CWD from mule deer: 38%. CWD from elk: 86%. TME. 100% Histopatholgic lesions: Scrapie. Some vacuolation and central of chromatolysis of neurons. CWD. Isolated vacuolated neurons, a few degenerate axons, and a mild astrocytosis. TME. Extensive vacuolation of neuronal perikarya and neuropil. Presence of mild multifocal gliosis. Western blot (brainstem): Scrapie. All three isoforms of PrP**res present. CWD. All three isoforms of PrP**res seen. TME. All three isoforms of PrP**res seen. Immunohistochemistry: PrP**res in lymphoreticular tissues: Scrapie. Not present. CWD. Not present. TME. Not present. PrP**res in CNS: Scrapie. Present within perikaryon and processes of neurons. CWD. Multifocal distribution with labeling primarily in glial cells (astrocytes). TME. Diffusely present and usually evenly distributed in neuropil. Conclusions: 1. All three TSEs agents (scrapie, CWD and TME) are capable of propagating in cattle tissues when administered intracerebrally. 2. All three TSEs can be distinguished from each other and from BSE when inoculated intracerebrally by histopathology, immunohistochemistry and Western blot techniques.
Title: EXPERIMENTAL SECOND PASSAGE OF CHRONIC WASTING DISEASE (CWD(MULE DEER)) AGENT TO CATTLE
Author item Hamir, Amirali item Kunkle, Robert item MILLER, JANICE item Greenlee, Justin item Richt, Juergen
Submitted to: Journal of Comparative Pathology Publication Type: Peer Reviewed Journal Publication Acceptance Date: 7/25/2005 Publication Date: 1/1/2006
Citation: Hamir, A.N., Kunkle, R.A., Miller, J.M., Greenlee, J.J., Richt, J.A. 2006. Experimental second passage of chronic wasting disease (CWD(mule deer)) agent to cattle. Journal of Comparative Pathology. 134(1):63-69.
Interpretive Summary: To compare the findings of experimental first and second passage of chronic wasting disease (CWD) in cattle, 6 calves were inoculated into the brain with CWD-mule deer agent previously (first) passaged in cattle. Two other uninoculated calves served as controls. Beginning 10-12 months post inoculation (PI), all inoculates lost appetite and weight. Five animals subsequently developed clinical signs of central nervous system (CNS) abnormality. By 16.5 months PI, all cattle had been euthanized because of poor prognosis. None of the animals showed microscopic lesions of spongiform encephalopathy (SE) but the CWD agent was detected in their CNS tissues by 2 laboratory techniques (IHC and WB). These findings demonstrate that inoculated cattle amplify CWD agent but also develop clinical CNS signs without manifestation of microscopic lesions of SE. This situation has also been shown to occur following inoculation of cattle with another TSE agent, namely, sheep scrapie. The current study confirms previous work that indicates that the diagnostic tests currently used for confirmation of bovine spongiform encephalopathy (BSE) in the U.S. would detect CWD in cattle, should it occur naturally. Furthermore, it raises the possibility of distinguishing CWD from BSE in cattle due to the absence of microscopic lesions and a unique multifocal distribution of PrPres, as demonstrated by IHC, which in this study, appears to be more sensitive than the WB.
Technical Abstract: To compare clinicopathological findings of first and second passage of chronic wasting disease (CWD) in cattle, a group of calves (n=6) were intracerebrally inoculated with CWD-mule deer agent previously (first) passaged in cattle. Two other uninoculated calves served as controls. Beginning 10-12 months post inoculation (PI), all inoculates lost appetite and lost weight. Five animals subsequently developed clinical signs of central nervous system (CNS) abnormality. By 16.5 months PI, all cattle had been euthanized because of poor prognosis. None of the animals showed microscopic lesions of spongiform encephalopathy (SE) but PrPres was detected in their CNS tissues by immunohistochemistry (IHC) and Western blot (WB) techniques. These findings demonstrate that intracerebrally inoculated cattle not only amplify CWD PrPres but also develop clinical CNS signs without manifestation of morphologic lesions of SE. This situation has also been shown to occur following inoculation of cattle with another TSE agent, scrapie. The current study confirms previous work that indicates the diagnostic techniques currently used for confirmation of bovine spongiform encephalopathy (BSE) in the U.S. would detect CWD in cattle, should it occur naturally. Furthermore, it raises the possibility of distinguishing CWD from BSE in cattle due to the absence of neuropathologic lesions and a unique multifocal distribution of PrPres, as demonstrated by IHC, which in this study, appears to be more sensitive than the WB.
FRIDAY, AUGUST 27, 2021
Cattle are highly susceptible to white-tailed deer CWD and mule deer CWD in experimental conditions
Friday, December 14, 2012
DEFRA U.K. What is the risk of Chronic Wasting Disease CWD being introduced into Great Britain? A Qualitative Risk Assessment October 2012
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In the USA, under the Food and Drug Administration's BSE Feed Regulation (21 CFR 589.2000) most material (exceptions include milk, tallow, and gelatin) from deer and elk is prohibited for use in feed for ruminant animals. With regards to feed for non-ruminant animals, under FDA law, CWD positive deer may not be used for any animal feed or feed ingredients. For elk and deer considered at high risk for CWD, the FDA recommends that these animals do not enter the animal feed system. However, this recommendation is guidance and not a requirement by law. Animals considered at high risk for CWD include:
1) animals from areas declared to be endemic for CWD and/or to be CWD eradication zones and
2) deer and elk that at some time during the 60-month period prior to slaughter were in a captive herd that contained a CWD-positive animal.
Therefore, in the USA, materials from cervids other than CWD positive animals may be used in animal feed and feed ingredients for non-ruminants.
The amount of animal PAP that is of deer and/or elk origin imported from the USA to GB can not be determined, however, as it is not specified in TRACES.
It may constitute a small percentage of the 8412 kilos of non-fish origin processed animal proteins that were imported from US into GB in 2011.
Overall, therefore, it is considered there is a __greater than negligible risk___ that (nonruminant) animal feed and pet food containing deer and/or elk protein is imported into GB.
There is uncertainty associated with this estimate given the lack of data on the amount of deer and/or elk protein possibly being imported in these products.
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36% in 2007 (Almberg et al., 2011). In such areas, population declines of deer of up to 30 to 50% have been observed (Almberg et al., 2011). In areas of Colorado, the prevalence can be as high as 30% (EFSA, 2011). The clinical signs of CWD in affected adults are weight loss and behavioural changes that can span weeks or months (Williams, 2005). In addition, signs might include excessive salivation, behavioural alterations including a fixed stare and changes in interaction with other animals in the herd, and an altered stance (Williams, 2005). These signs are indistinguishable from cervids experimentally infected with bovine spongiform encephalopathy (BSE). Given this, if CWD was to be introduced into countries with BSE such as GB, for example, infected deer populations would need to be tested to differentiate if they were infected with CWD or BSE to minimise the risk of BSE entering the human food-chain via affected venison. snip..... The rate of transmission of CWD has been reported to be as high as 30% and can approach 100% among captive animals in endemic areas (Safar et al., 2008).
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In summary, in endemic areas, there is a medium probability that the soil and surrounding environment is contaminated with CWD prions and in a bioavailable form. In rural areas where CWD has not been reported and deer are present, there is a greater than negligible risk the soil is contaminated with CWD prion. snip..... In summary, given the volume of tourists, hunters and servicemen moving between GB and North America, the probability of at least one person travelling to/from a CWD affected area and, in doing so, contaminating their clothing, footwear and/or equipment prior to arriving in GB is greater than negligible... For deer hunters, specifically, the risk is likely to be greater given the increased contact with deer and their environment. However, there is significant uncertainty associated with these estimates.
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Therefore, it is considered that farmed and park deer may have a higher probability of exposure to CWD transferred to the environment than wild deer given the restricted habitat range and higher frequency of contact with tourists and returning GB residents.
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TUESDAY, SEPTEMBER 07, 2021
Atypical Bovine Spongiform Encephalopathy BSE OIE, FDA 589.2001 FEED REGULATIONS, and Ingestion Therefrom
FRIDAY, DECEMBER 10, 2021
Scrapie at Abattoir: Monitoring, Control, and Differential Diagnosis of Wasting Conditions during Meat Inspection
WEDNESDAY, JANUARY 12, 2022
Bovine Spongiform Encephalopathy BSE TSE Prion Origin USA, what if?
THURSDAY, AUGUST 19, 2021
TME to cattle equal atypical L-type BSE USA, madcow origin, what if?
MONDAY, FEBRUARY 14, 2022
Atypical Nor98 Scrapie, Atypical BSE, CWD, Can Emerge As Different TSE PrP In Cross Species Transmission, A Volatile Situation For Human and Animal Health
WEDNESDAY, OCTOBER 6, 2021
Classical BSE prions emerge from asymptomatic pigs challenged with atypical/Nor98 scrapie
Sunday, April 17, 2022
Phenotypic Heterogeneity of Variably Protease-Sensitive Prionopathy: A Report of Three Cases Carrying Different Genotypes at PRNP Codon 129
FRIDAY, DECEMBER 24, 2021
Creutzfeldt Jakob Disease CJD TSE Prion Update December 25, 2021
Diagnosis and Reporting of Creutzfeldt-Jakob Disease
Singeltary, Sr et al. JAMA.2001; 285: 733-734. Vol. 285 No. 6, February 14, 2001 JAMA
Diagnosis and Reporting of Creutzfeldt-Jakob Disease
To the Editor: In their Research Letter, Dr Gibbons and colleagues1 reported that the annual US death rate due to Creutzfeldt-Jakob disease (CJD) has been stable since 1985. These estimates, however, are based only on reported cases, and do not include misdiagnosed or preclinical cases. It seems to me that misdiagnosis alone would drastically change these figures. An unknown number of persons with a diagnosis of Alzheimer disease in fact may have CJD, although only a small number of these patients receive the postmortem examination necessary to make this diagnosis. Furthermore, only a few states have made CJD reportable. Human and animal transmissible spongiform encephalopathies should be reportable nationwide and internationally.
Terry S. Singeltary, Sr Bacliff, Tex
1. Gibbons RV, Holman RC, Belay ED, Schonberger LB. Creutzfeldt-Jakob disease in the United States: 1979-1998. JAMA. 2000;284:2322-2323.
Terry S. Singeltary Sr.
