A C-Terminal Protease-Resistant Prion Fragment Distinguishes Ovine “CH1641-Like” Scrapie from Bovine Classical and L-Type BSE in Ovine Transgenic Mice
Thierry Baron*, Anna Bencsik, Johann Vulin, Anne-Gaëlle Biacabe, Eric Morignat, Jérémy Verchere, Dominique Betemps
Agence Française de Sécurité Sanitaire des Aliments–Lyon, Unité ATNC, Lyon, France
Abstract The protease-resistant prion protein (PrPres) of a few natural scrapie isolates identified in sheep, reminiscent of the experimental isolate CH1641 derived from a British natural scrapie case, showed partial molecular similarities to ovine bovine spongiform encephalopathy (BSE). Recent discovery of an atypical form of BSE in cattle, L-type BSE or BASE, suggests that also this form of BSE might have been transmitted to sheep. We studied by Western blot the molecular features of PrPres in four “CH1641-like” natural scrapie isolates after transmission in an ovine transgenic model (TgOvPrP4), to see if “CH1641-like” isolates might be linked to L-type BSE. We found less diglycosylated PrPres than in classical BSE, but similar glycoform proportions and apparent molecular masses of the usual PrPres form (PrPres #1) to L-type BSE. However, the “CH1641-like” isolates differed from both L-type and classical BSE by an abundant, C-terminally cleaved PrPres product (PrPres #2) specifically recognised by a C-terminal antibody (SAF84). Differential immunoprecipitation of PrPres #1 and PrPres #2 resulted in enrichment in PrPres #2, and demonstrated the presence of mono- and diglycosylated PrPres products. PrPres #2 could not be obtained from several experimental scrapie sources (SSBP1, 79A, Chandler, C506M3) in TgOvPrP4 mice, but was identified in the 87V scrapie strain and, in lower and variable proportions, in 5 of 5 natural scrapie isolates with different molecular features to CH1641. PrPres #2 identification provides an additional method for the molecular discrimination of prion strains, and demonstrates differences between “CH1641-like” ovine scrapie and bovine L-type BSE transmitted in an ovine transgenic mouse model.
Author Summary The origin of the transmissible agent involved in the food-borne epidemic of bovine spongiform encephalopathy (BSE) remains a mystery. It has recently been proposed that this could have been the result of the recycling of an atypical, more probably sporadic, form of BSE (called bovine amyloidotic spongiform encephalopathy, or L-type BSE) in an intermediate host, such as sheep. In this study we analyzed the molecular features of the disease-associated protease-resistant prion protein (PrPres) found in the brain of transgenic mice overexpressing the ovine prion protein after experimental infection with prions from bovine classical and L-type BSEs or from ovine scrapie. Scrapie cases included rare “CH1641-like” isolates, which share some PrPres molecular features with classical BSE and L-type BSE. Scrapie isolates induced in transgenic mouse brains the production of a C-terminally cleaved form of PrPres, which was particularly abundant from “CH1641-like” cases. In contrast, this C-terminal prion protein product was undetectable in ovine transgenic mice infected with bovine prions from both classical and L-type BSE. These findings add a novel approach for the discrimination of prions that may help to understand their possible changes during cross-species transmissions.
Discussion This study describes the molecular analyses of PrPres after transmission into TgOvPrP4 ovine transgenic mice from 4 natural ovine scrapie isolates whose PrPres features in sheep were similar to those previously described for the experimental CH1641 scrapie isolate . Two of these previously unreported isolates (05-825 and 06-017) behaved as previously described for CH1641 and another natural isolate (TR316211) during the first passage in TgOvPrP4 mice, showing low molecular mass PrPres (l-type PrPres) in all mice ,. In contrast, all the TgOvPrP4 mice receiving 5 natural scrapie isolates characterized by high PrPres molecular masses (h-type PrPres) in the sheep brain, showed PrPres of high molecular mass. Detailed analyses showed, as previously described in the CH1641 isolate in sheep  and in TgOvPrP4 mice , a slightly lower PrPres molecular mass in TgOvPrP4 mice from the “CH1641-like” isolates than from ovine BSE, although the resolution of small gels made discrimination difficult. Our results are quite consistent with previous studies of the CH1641 isolate by the immunohistochemical “peptide mapping” method, which revealed that PrPd in the CH1641 isolate was truncated further upstream in the N terminus than from experimental BSE . The biochemical PrPres features of these scrapie isolates differ from BSE mainly in their moderately high proportions of di-glycosylated PrPres (50%–60%), whereas ovine BSE is characterized by higher proportions of di-glycosylated PrPres ,,. Molecular discrimination of strains based on the relative proportions of glycoforms is however less reliable than that of PrPres molecular masses, given the large measurement variations and poor standardization of analytical methods , , –. Furthermore glycoforms proportions of BSE in sheep have only been determined from a very limited number of sources. A recent study of classical BSE in cattle showed large individual variations (~20%) in the proportions of di-glycosylated PrPres .
The question of a possible transmission of BSE in small ruminants now needs to be re-examined considering the recent identification of atypical cases of BSE (H-type or L-type) in cattle –. Recent studies have indeed hypothesized that cross-species transmission of such rare atypical cases could be at the origin of the BSE epidemic in cattle ,,. The first experimental support for this hypothesis was obtained following the discovery of a BSE-like phenotype in mice following transmission of L-type BSE in wild-type mice (C57Bl, SJL)  or in an ovine transgenic (tg338) mouse line . However, unlike tg338, which expressed 8- to 10-fold levels of V136 R154 Q171 ovine PrP, the phenotype of the L-type BSE remained distinct from classical BSE during at least two passages in TgOvPrP4 mice that expressed 2- to 4-fold levels of the A136 R154 Q171 ovine PrP . It is noteworthy that, in cattle, the essential difference between L-type BSE and classical BSE is the slightly lower apparent molecular mass and the lower proportions of diglycosylated PrPres –, reminiscent of the differences between CH1641 and classical BSE experimentally transmitted to sheep ,,. The phenotypic features of L-type BSE have not yet been reported in sheep. In this study we showed that the PrPres molecular masses and glycoform proportions between “CH1641-like” scrapie isolates and L-type BSE transmitted into TgOvPrP4 mice were indistinguishable, in addition to survival periods in the same range at second passage.
However our study revealed that a highly sensitive C-terminal antibody (SAF84) recognised an abundant PrPres product (PrPres #2) in TgOvPrP4 mice infected with “CH1641-like” isolates, the unglycosylated form of which migrates at ~14 kDa, in addition to the usual PrPres product (PrPres #1) which migrates at ~19 kDa in its unglycosylated form. The presence of mono- and di-glycosylated forms derived from this PrPres cleavage product was confirmed by differential immunoprecipitation of PrPres #1 and PrPres #2. Depletion of PrPres #1 using N-terminal antibodies allowed the samples to be enriched in C-terminally cleaved PrPres #2, which then appeared in a 3-band pattern between 14 and 22 kDa. Such experiments also confirm that PrPres #2 is only faintly recognized by Sha 31 antibody, which recognizes the 148–155 region of the ovine PrP protein, suggesting that this region is absent from most of the PrPres #2 fragments. PNGase deglycosylation also facilitated the identification of PrPres #2, and permitted quantification of the respective proportions of PrPres #2 and PrPres #1. Whereas PrPres #2 was abundant in TgOvPrP4 mice infected with “CH1641-like” isolates, lower levels of PrPres #2 could also be detected from 5 natural isolates with h-type PrPres transmitted into TgOvPrP4 mice. C-terminally cleaved PrPres products have previously been described in sporadic or genetic Creutzfeldt-Jakob disease in humans . Although the presence of low levels of PrPres #2 in BSE and L-type BSE cannot be fully excluded, this PrPres form remained undetected in our experiments with these BSE forms, even after differential immunoprecipitation. This was also the case in classical BSE transmitted in a variety of different species. Interestingly, similar results were obtained in TgOvPrP4 mice infected with an isolate from cattle experimentally infected with transmissible mink encephalopathy (TME), consistent with previous studies showing similarities with L-type BSE . Our results thus reinforce the molecular discrimination of “CH1641-like” scrapie isolates from classical BSE, but also indicate a clear molecular difference with L-type BSE transmitted from cattle to ovine transgenic mice. However, further comparisons including those of biological and histopathological features during serial passages in this mouse model will be required, as well as transmission studies performed from L-type BSE experimentally transmitted to sheep.
We have also recently described the identification of a C-terminally cleaved PrPres #2 form in H-type BSE, in cattle and after transmission to C57Bl/6 mice . However, a relationship between “CH1641-like” scrapie isolates and H-type BSE seems unlikely. H-type BSE is indeed characterized by a high PrPres molecular mass comparable to most natural scrapie cases, in contrast to the low PrPres molecular mass, which is the hallmark of “CH1641-like” isolates. Although the transmission of H-type BSE in sheep has not yet been reported, a high PrPres molecular mass was maintained upon transmission in tg338 ovine transgenic mice . Unfortunately, direct comparisons with H-type BSE in TgOvPrP4 mice were not possible since we were unable to transmit the disease from several cattle H-type isolates to these mice, at least at first passage . As these same H-type isolates were transmitted in tg338 expressing higher levels of the V136 R154 Q171 ovine PrP protein , this could suggest a high species and/or strain barrier for H-type BSE in sheep. Conversely, both classical and L-type BSEs were readily transmitted in TgOvPrP4 mice ,.
The presence of PrPres #2 within the different scrapie sources, was preferentially associated with PrPres #1 of low molecular mass. When several experimental scrapie sources were analysed, PrPres #2 was only detected in the 87V strain, characterized by l-type PrPres, but not in C506M3, Chandler or 79A strains or in the SSBP/1 isolate with h-type PrPres, still emphasizing the need of further comparisons between 87V and “CH1641-like” isolates . Although PrPres #2 could also be detected after the transmission of natural scrapie isolates with high molecular mass, the levels were consistently lower than in “CH1641-like” isolates. It might be that the presence of low levels of PrPres #2 in scrapie isolates with h-type PrPres indicates a mixture of PrPres phenotypes in these scrapie sources, with the levels of l-type PrPres undetectable. This possibility should be considered in the light of certain observations. (1) A scrapie case with both h-type and l-type PrPres has recently been described in the UK, each PrPres phenotype originating from two different brain areas . (2) Our recent transmission studies of two “CH1641-like” isolates (O100 and O104) from the same flock into TgOvPrP4 showed the presence of h-type PrPres in some of the mice suggesting a possible mixture of the two PrPres phenotypes in the initial ovine scrapie isolates; these two PrPres phenotypes might be selected, at least in part, during the second passage in TgOvPrP4 mice . Studies of the initial ovine brain samples by immunohistochemistry indeed revealed the presence of differently cleaved PrPres forms in different brain nuclei . (3) Transmission of scrapie in cattle from a brain pool (British source) with h-type PrPres produced two cows with l-typePrPres . h-type PrPres was detected in a second brain sample from one of the two animals. (4) Similar results were observed in a bovine transgenic mouse line, the mobility in mice being faster than in the original scrapie isolate (Irish source) . All together, these data suggest that l-type PrPres could be present in a number of scrapie sources. The identification of “CH1641-like” isolates might be the fortuitous and rare result of analysing samples in which the l-type PrPres of low molecular mass is more abundant.
Further characterization of the biological properties of scrapie sources with l-type PrPres will be required firstly to establish whether these correspond to a single strain of infectious agent or involve a variety of distinct scrapie strains, and secondly to better understand the characteristics of their transmission.
12/10/76 AGRICULTURAL RESEARCH COUNCIL REPORT OF THE ADVISORY COMMITTE ON SCRAPIE Office Note CHAIRMAN: PROFESSOR PETER WILDY
A The Present Position with respect to Scrapie The Problem
Scrapie is a natural disease of sheep and goats. It is a slow and inexorably progressive degenerative disorder of the nervous system and it is fatal. It is enzootic in the United Kingdom but not in all countries.
The field problem has been reviewed by a MAFF working group (ARC 35/77). It is difficult to assess the incidence in Britain for a variety of reasons but the disease causes serious financial loss; it is estimated that it cost Swaledale breeders alone $l.7 M during the five years 1971-1975. A further inestimable loss arises from the closure of certain export markets, in particular those of the United States, to British sheep.
It is clear that scrapie in sheep is important commercially and for that reason alone effective measures to control it should be devised as quickly as possible.
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 emphasized by the finding that some strains of scrapie produce lesions identical to the once which characterize 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 scrapie problem urgent if the sheep industry is not to suffer grievously.
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.
EVIDENCE OF SCRAPIE IN SHEEP AS A RESULT OF FOOD BORNE EXPOSURE
This is provided by the statistically significant increase in the incidence of sheep scrape from 1985, as determined from analyses of the submissions made to VI Centres, and from individual case and flock incident studies. ........
Like lambs to the slaughter
31 March 2001 Debora MacKenzie Magazine issue 2284
What if you can catch old-fashioned CJD by eating meat from a sheep infectedwith scrapie?FOUR years ago, Terry Singeltary watched his mother die horribly from adegenerative brain disease. Doctors told him it was Alzheimer's, butSingeltary was suspicious. The diagnosis didn't fit her violent symptoms,and he demanded an autopsy. It showed she had died of sporadicCreutzfeldt-Jakob disease.
Most doctors believe that sCJD is caused by a prion protein deforming bychance into a killer. But Singeltary thinks otherwise. He is one of a numberof campaigners who say that some sCJD, like the variant CJD related to BSE,is caused by eating meat from infected animals. Their suspicions havefocused on sheep carrying scrapie, a BSE-like disease that is widespread inflocks across Europe and North America.
Now scientists in France have stumbled across new evidence that adds weightto the campaigners' fears. To their complete surprise, the researchers foundthat one strain of scrapie causes the same brain damage in ...
The complete article is 889 words long.
1: Neuroepidemiology. 1985;4(4):240-9.
Sheep consumption: a possible source of spongiform encephalopathy in humans.
Davanipour Z, Alter M, Sobel E, Callahan M.
A fatal spongiform encephalopathy of sheep and goats (scrapie) shares many characteristics with Creutzfeldt-Jakob disease (CJD), a similar dementing illness of humans. To investigate the possibility that CJD is acquired by ingestion of contaminated sheep products, we collected information on production, slaughtering practices, and marketing of sheep in Pennsylvania. The study revealed that sheep were usually marketed before central nervous system signs of scrapie are expected to appear; breeds known to be susceptible to the disease were the most common breeds raised in the area; sheep were imported from other states including those with a high frequency of scrapie; use of veterinary services on the sheep farms investigated and, hence, opportunities to detect the disease were limited; sheep producers in the area knew little about scrapie despite the fact that the disease has been reported in the area, and animal organs including sheep organs were sometimes included in processed food. Therefore, it was concluded that in Pennsylvania there are some 'weak links' through which scrapie-infected animals could contaminate human food, and that consumption of these foods could perhaps account for spongiform encephalopathy in humans. The weak links observed are probably not unique to Pennsylvania.
Date: September 26, 2007 at 4:06 pm PST
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.
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.
Edited by Stanley B. Prusiner, University of California, San Francisco, CA, and approved September 12, 2005 (received for review March 21, 2005)
Tuesday, June 3, 2008 SCRAPIE USA UPDATE JUNE 2008 NOR-98 REPORTED PA
In FY 2007, 331 scrapie cases have been confirmed and reported by the National Veterinary Services Laboratories (NVSL), including 59* Regulatory Scrapie Slaughter Surveillance (RSSS) cases (Figure 5 and Slide 16). In FY 2007, two field cases, one validation case, and two RSSS cases were consistent with Nor-98 scrapie. The Nor98-like cases originated from flocks in California, Minnesota, Colorado, Wyoming and Indiana respectively. Nineteen cases of scrapie in goats have been reported since 1990 (Figure 6). The last goat case was reported in September 2007.
see full report here ;
ANIMALS SAMPLED FOR SCRAPIE TESTING
As of April 30, 2008, 26,703 animals have been sampled for scrapie testing: 23,378 RSSS, 1,517 goats for the CSPS study, 1,466 regulatory field cases, 270 regulatory third eyelid biopsies, and 72 regulatory rectal biopsies (chart 8).
TESTING OF LYMPHOID TISSUE OBTAINED BY RECTAL BIOPSY WAS APPROVED BY USDA AS AN OFFICIAL LIVE-ANIMAL TEST ON JANUARY 11, 2008. ...
PLEASE NOTE, (FIGURE 6), Scrapie Confirmed Cases in FY 2008 MAP, PA 3, 1**, Two cases-state of ID UNKNOWN, 1 case Nor98-like**
Sunday, June 15, 2008
A descriptive study of the prevalence of atypical and classical scrapie in sheep in 20 European countries
Thursday, April 24, 2008 RE-FOIA OF DECLARATION OF EXTRAORDINARY EMERGENCY BECAUSE OF AN ATYPICAL T.S.E. (PRION DISEASE) OF FOREIGN ORIGIN IN THE UNITED STATES [Docket No. 00-072-1]
Wednesday, August 20, 2008
Bovine Spongiform Encephalopathy Mad Cow Disease typical and atypical strains, was there a cover-up ?
August 20, 2008
Atypical BSE (BASE) Transmitted from Asymptomatic Aging Cattle to a Primate
Sunday, March 16, 2008
MAD COW DISEASE terminology UK c-BSE (typical), atypical BSE H or L, and or Italian L-BASE
HUMAN and ANIMAL TSE Classifications i.e. mad cow disease and the UKBSEnvCJD only theory JUNE 2008
Tissue infectivity and strain typing of the many variants Manuscript of the human and animal TSEs are paramount in all variants of all TSE. There must be a proper classification that will differentiate between all these human TSE in order to do this. With the CDI and other more sensitive testing coming about, I only hope that my proposal will some day be taken seriously. ...