Monday, January 2, 2017

Bovine Spongiform Encephalopathy Induces Misfolding of Alleged Prion-Resistant Species Cellular Prion Protein without Altering Its Pathobiological Features

Articles, Neurobiology of Disease

Bovine Spongiform Encephalopathy Induces Misfolding of Alleged Prion-Resistant Species Cellular Prion Protein without Altering Its Pathobiological Features

Enric Vidal, Natalia Fernández-Borges, Belén Pintado, Montserrat Ordóñez, Mercedes Márquez, Dolors Fondevila, Juan María Torres, Martí Pumarola and Joaquín Castilla

Journal of Neuroscience 1 May 2013, 33 (18) 7778-7786; DOI:

Enric Vidal

3Centre de Recerca en Sanitat Animal, Campus de la Universitat Autònoma de Barcelona (UAB)-IRTA, 08193 Bellaterra, Barcelona, Spain,


Bovine spongiform encephalopathy (BSE) prions were responsible for an unforeseen epizootic in cattle which had a vast social, economic, and public health impact. This was primarily because BSE prions were found to be transmissible to humans. Other species were also susceptible to BSE either by natural infection (e.g., felids, caprids) or in experimental settings (e.g., sheep, mice). However, certain species closely related to humans, such as canids and leporids, were apparently resistant to BSE. In vitro prion amplification techniques (saPMCA) were used to successfully misfold the cellular prion protein (PrPc) of these allegedly resistant species into a BSE-type prion protein. The biochemical and biological properties of the new prions generated in vitro after seeding rabbit and dog brain homogenates with classical BSE were studied. Pathobiological features of the resultant prion strains were determined after their inoculation into transgenic mice expressing bovine and human PrPC. Strain characteristics of the in vitro-adapted rabbit and dog BSE agent remained invariable with respect to the original cattle BSE prion, suggesting that the naturally low susceptibility of rabbits and dogs to prion infections should not alter their zoonotic potential if these animals became infected with BSE. This study provides a sound basis for risk assessment regarding prion diseases in purportedly resistant species.



Prion diseases have been known for a long time, especially scrapie in sheep and goats, and some diseases affecting humans, including Kuru and Creutzfeldt-Jakob disease. Yet the occurrence and identification of the BSE epizootic was a remarkable milestone in the history of prion disease as cattle had never before been affected by TSE and so were presumed resistant. The recognition that BSE was zoonotic turned prion diseases of animals into a serious threat to public health, which resulted in an unprecedented crisis of confidence in consumers. Vast amounts of money were spent in numerous countries, not just to eliminate hundreds of thousands of infected animals, but also to establish radical changes related to disease control and surveillance, i.e., diagnostic tests, disposal of specified risk material, which could no longer be used in the food industry, etc. However, as prion diseases became the subject of profound scientific study, previously unknown prion strains have been discovered, many with unknown species susceptibilities and zoonotic potential. Thus, the susceptibility of different species to prions, particularly those that may come into close contact with humans, is a matter of continuous debate and study in the scientific community and great concern when determining health and safety policies.

Detailed study of naturally occurring prions is not enough to understand the behavior of these proteins in certain species. Before 1985, no one could have foreseen the massive cattle BSE epizootic. Hence, the scientific community is now particularly cautious when assessing the risk associated with host susceptibilities of various prions. Nevertheless, certain species display a limited or apparently null susceptibility to prion disease. Therefore, could one assume that such species would be resistant to all existing prion strains? If only one prion strain should be able to infect a new species, it might adapt to the new host and become easily transmissible. To address this we used all the tools available (i.e., in vitro amplification and transgenic mouse models) to evaluate the behavior of BSE prions in two historically considered prion disease resistant species: dogs and rabbits. BSE was chosen due to its zoonotic properties and because, as mentioned above, it is a promiscuous strain that has demonstrated infectivity in a wider range of species than most other prions. Even though these experimental conditions are far from modeling natural scenarios, they may enable us to predict the outcome of potential unforeseen species susceptibilities and epizootics as occurred with BSE (mad cow disease). During the last decade, in vitro replication studies endorsed PMCA as one of the most powerful technologies to overcome transmission barriers (Castilla et al., 2008; Green et al., 2008; Fernández-Borges and Castilla, 2010; Barria et al., 2011; Kurt et al., 2011; Yoshioka et al., 2011; Chianini et al., 2012). Thus, dog and rabbit normal brain homogenates were seeded with BSE prions and submitted to in vitro amplification (saPMCA) to try to overcome the species transmission barrier. Interestingly, rabbit PrPC could be misfolded with relative ease by any of the prion strains used TSE (Barlow and Rennie, 1976; Fernández-Borges et al., 2009; Chianini et al., 2012). This was rather surprising since this species had, until then, been considered resistant to TSE (Barlow and Rennie, 1976; Fernández-Borges et al., 2009; Chianini et al., 2012). However, dog brain homogenate could not be misfolded until the initial BSE prion seed concentration was raised considerably, and even with this modification only BSE was able to misfold dog PrPC, which suggested a strong resistance of this species to BSE.

Comparison of the original cattle BSE prions with those generated by seeding rabbit and dog brain homogenate with BSE followed by in vitro saPMCA demonstrated that the biochemical strain properties of BSE were maintained in the new species, i.e., glycoform proportion and molecular weight after PK digestion. This suggests that the BSE prion's conformation is reliably transmitted to new PrPC species in vitro. However, to ensure that the BSE strain's pathobiological properties was also transmitted, particularly those regarding infectivity and the ability to cross certain species barriers, an in vivo approach was mandatory. The transgenic mouse model expressing the bovine PRNP gene was chosen as it had previously demonstrated to reproduce reliably BSE strain features. This model was key in determining that BSE acquired a different behavior regarding the length of the incubation period upon its passage through sheep, while maintaining other strain features, such as Western blot electrophoretical profile, molecular weight, and PK resistance (Espinosa et al., 2007). As predicted by the in vitro experiments (once initially converted, BSE-DoPrPres and BSE-RaPrPres easily amplified further dog and rabbit normal brain homogenates in vitro), both rabbit and dog adapted BSE efficiently infected tgBov mice with attack rates of 100%. Initially the incubation period was slightly longer for both inocula than that of cattle BSE, probably due to a lack of adaptation of the in vitro generated prions to the new host (bovine) prion. However, on second passage the incubation periods were significantly shortened. Interestingly the behavior of BSE-DoPrPres was similar to that observed in sheep BSE where the biochemical, lesion, and immunohistochemical features were unchanged but with a reduction in the incubation period (Espinosa et al., 2007). The neuropathology and molecular signature of PrPres found in tgBov mice showed no significant differences between cattle BSE and the two in vitro-generated prions, suggesting that the strain features had not changed upon passage through rabbit and dog brain homogenates.

The data shown here prove that the difficulties found in misfolding certain species of PrPC molecules do not imply a loss of the pathobiological features encoded in the BSE structure. Compared with cat PrPC (data not shown), dog PrP was tremendously difficult to misfold in vitro. It could only be converted when a relatively large dose of BSE prion was used as seed. However, the similarity of dog PrP primary sequence, differing principally in just three residues (positions: 163, 181, and 189) with cat PrP (Lysek et al., 2005; Stewart et al., 2012), would predict a similar behavior to feline spongiform encephalopathy (FSE). In fact, FSE was one of the first nonbovine prions shown to maintain BSE strain features (Lezmi et al., 2006). Accordingly, once the bovine–canine barrier was surpassed, BSE pathobiological features remained stable in BSE-DoPrPres.

However, the results obtained with BSE cannot be used to predict the behavior of other prion strains when adapted to these species in vivo or in vitro. Thus, instead of a species transmission barrier a strain transmission barrier should be considered as suggested by Scott and coworkers (Scott et al., 2005). Prion strains might be classified according to their ability to transmit to different species and also in relation to their adaptability to new hosts and their permissiveness to change. Therefore, BSE would be a strain with a low (or null) permissiveness to change and yet high adaptability to different environments. As such, the zoonotic behavior of BSE toward humans can be predicted regardless of the host infected with the BSE prion. Any species derivation of BSE, be it rabbit-BSE, dog-BSE, or any other version, is likely to encode a structure capable of misfolding human PrPC. Second passage experiments in transgenic mice expressing human PrP (Tg340 mice) would have been useful to determine whether silent infection was present in mice inoculated with cattle BSE and BSE-DoPrPres which seems to be a plausible assumption according to experiments published in this model (Padilla et al., 2011).

Our in vitro and in vivo results predict that, hypothetically, if BSE infected canids and leporids (considered, so far, prion resistant) it would maintain its pathobiological features; including zoonotic potential. This is particularly relevant with respect to rabbit amplified BSE as rabbits are eaten by humans. In conclusion, it is strongly recommended that no mammalian species be fed with animal protein potentially contaminated with BSE to prevent a new epizootic and zoonosis of unknown consequences.

Given the difficulties of performing infectivity studies in many natural hosts (without previous in vitro replication), transgenic mice have been generated expressing rabbit and dog PRNP gene which have been inoculated with cattle BSE, among other strains of interest, to test the in vivo susceptibility of these PRNP sequences. In addition, a detailed study of the primary amino acid sequences of bovine PrP compared with rabbit and dog PrP, focusing on the structural peculiarities of each amino acid change, is ongoing to explain the different PrPC to PrPd conversion abilities of each species.


  • This work was financially supported by two national grants from Spain (AGL2009-11553-C02-01 and AGL2008-05296-C02), Basque Government Grant PI2010-18, and Etortek Research Programs 2011/2013. We thank the IKERBasque Foundation for their support. We thank CIC bioGUNE, Sierra Espinar, Marta Valle, Mariano Moreno, and Paola Marco for the providing the vivarium and maintenance; the CReSA Biocontainment Unit staff for care and maintenance of the animals; Tomás Mayoral for the bovine spongiform encephalopathy brain tissue samples; and Mark Daeglish for his critical revision of the paper.
  • The authors declare no competing financial interests.
  • Correspondence should be addressed to Dr. Joaquín Castilla, CIC bioGUNE, Parque tecnológico de Bizkaia, Derio 48160, Bizkaia, Spain.

EP-021 Canine Prions: A New Form of Prion Disease

Mourad Tayebi1, Monique A David2, Brian Summers3

1 University of Melbourne, Veterinary Sciences, Australia; 2Ausbiologics, Sydney, Australia; 3Royal Veterinary College, London, UK

The origin of bovine spongiform encephalopathy (BSE), which rapidly evolved into a major epidemic remains unresolved and was initially widely attributed to transmission of sheep scrapie to cattle with contaminated feed prepared from rendered sheep carcasses. Alternative transmission hypotheses also include feed contaminated with unrecognized subclinical case(s) of bovine prion disease or with prion-infected human remains. However, following the demonstration of a BSE case exhibiting the novel mutation E211 K, similar to the E200K mutation associated with most genetic CJD in humans, support for a genetic origin of prion disease in cattle is gaining momentum. In contrast to other animal species such as feline, the canine species seems to be resistant to prion disease as no canine prion cases were previously reported.

We describe here three cases of Rottweiler puppy (called RWD cases) with neurological deficits and spongiform change. We used animal bioassays and in vitro studies to show efficient interspecies transmission of this novel canidae prion isolate to other species.

Biochemical studies revealed the presence of partially proteinase K (PK)-resistant fragment and immunohistochemistry displayed staining for PrPSc in the cerebral cortex. Importantly, interspecies transmission of canine PrPSc derived from RWD3 brain homogenates following inoculation of hamsters led to signs of prion disease and replication of PrPSc in brains, spinal cords and spleens of these animals.

These findings if confirmed by further cases of prion disease in canidae and regardless of the origin of the disease would have a major impact on animal and public health.



DEFRA Department for Environment, Food & Rural Affairs

Area 307, London, SW1P 4PQ Telephone: 0207 904 6000 Direct line: 0207 904 6287 E-mail:


Mr T S Singeltary P.O. Box 42 Bacliff Texas USA 77518

21 November 2001

Dear Mr Singeltary


Thank you for e-mail regarding the hounds survey. I am sorry for the long delay in responding.

As you note, the hound survey remains unpublished. However the Spongiform Encephalopathy Advisory Committee (SEAC), the UK Government's independent Advisory Committee on all aspects related to BSE-like disease, gave the hound study detailed consideration at their meeting in January 1994. As a summary of this meeting published in the BSE inquiry noted, the Committee were clearly concerned about the work that had been carried out, concluding that there had clearly been problems with it, particularly the control on the histology, and that it was more or less inconclusive. However was agreed that there should be a re-evaluation of the pathological material in the study.

Later, at their meeting in June 95, The Committee re-evaluated the hound study to see if any useful results could be gained from it. The Chairman concluded that there were varying opinions within the Committee on further work. It did not suggest any further transmission studies and thought that the lack of clinical data was a major weakness.

Overall, it is clear that SEAC had major concerns about the survey as conducted. As a result it is likely that the authors felt that it would not stand up to r~eer review and hence it was never published. As noted above, and in the detailed minutes of the SEAC meeting in June 95, SEAC considered whether additional work should be performed to examine dogs for evidence of TSE infection. Although the Committee had mixed views about the merits of conducting further work, the Chairman noted that when the Southwood Committee made their recommendation to complete an assessment of possible spongiform disease in dogs, no TSEs had been identified in other species and hence dogs were perceived as a high risk population and worthy of study. However subsequent to the original recommendation, made in 1990, a number of other species had been identified with TSE ( e.g. cats) so a study in hounds was less

critical. For more details see- http://www.bseinquiry, .pdf

As this study remains unpublished, my understanding is that the ownership of the data essentially remains with the original researchers. Thus unfortunately, I am unable to help with your request to supply information on the hound survey directly. My only suggestion is that you contact one of the researchers originally involved in the project, such as Gerald Wells. He can be contacted at the following address.

Dr Gerald Wells, Veterinary Laboratories Agency, New Haw, Addlestone, Surrey, KT 15 3NB, UK

You may also wish to be aware that since November 1994 all suspected cases of spongiform encephalopathy in animals and poultry were made notifiable. Hence since that date there has been a requirement for vets to report any suspect SE in dogs for further investigation. To date there has never been positive identification of a TSE in a dog.

I hope this is helpful

Yours sincerely 4




I am sorry, but I really could have been a co-signatory of Gerald's minute.

I do NOT think that we can justify devoting any resources to this study, especially as larger and more important projects such as the pathogenesis study will be quite demanding.

If there is a POLITICAL need to continue with the examination of hound brains then it should be passed entirely to the VI Service.

J W WILESMITH Epidemiology Unit 18 October 1991

Mr. R Bradley

cc: Mr. G A H Wells

3.3. Mr R J Higgins in conjunction with Mr G A Wells and Mr A C Scott would by the end of the year, indentify the three brains that were from the ''POSITIVE'' end of the lesion spectrum.

TSE in dogs have not been documented simply because OF THE ONLY STUDY, those brain tissue samples were screwed up too. see my investigation of this here, and to follow, later follow up, a letter from defra, AND SEE SUSPICIOUS BRAIN TISSUE SAF's. ...TSS


GAH WELLS (very important statement here...TSS)


AS implied in the Inset 25 we must not _ASSUME_ that transmission of BSE to other species will invariably present pathology typical of a scrapie-like disease.


76 pages on hound study;


The spongiform changes were not pathognomonic (ie. conclusive proof) for prion disease, as they were atypical, being largely present in white matter rather than grey matter in the brain and spinal cord. However, Tony Scott, then head of electron microscopy work on TSEs, had no doubt that these SAFs were genuine and that these hounds therefore must have had a scrapie-like disease. I reviewed all the sections myself (original notes appended) and although the pathology was not typical, I could not exclude the possibility that this was a scrapie-like disorder, as white matter vacuolation is seen in TSEs and Wallerian degeneration was also present in the white matter of the hounds, another feature of scrapie.

38.I reviewed the literature on hound neuropathology, and discovered that micrographs and descriptive neuropathology from papers on 'hound ataxia' mirrored those in material from Robert Higgins' hound survey. Dr Tony Palmer (Cambridge) had done much of this work, and I obtained original sections from hound ataxia cases from him. This enabled me provisionally to conclude that Robert Higgins had in all probability detected hound ataxia, but also that hound ataxia itself was possibly a TSE. Gerald Wells confirmed in 'blind' examination of single restricted microscopic fields that there was no distinction between the white matter vacuolation present in BSE and scrapie cases, and that occurring in hound ataxia and the hound survey cases.

39.Hound ataxia had reportedly been occurring since the 1930's, and a known risk factor for its development was the feeding to hounds of downer cows, and particularly bovine offal. Circumstantial evidence suggests that bovine offal may also be causal in FSE, and TME in mink. Despite the inconclusive nature of the neuropathology, it was clearly evident that this putative canine spongiform encephalopathy merited further investigation.

40.The inconclusive results in hounds were never confirmed, nor was the link with hound ataxia pursued. I telephoned Robert Higgins six years after he first sent the slides to CVL. I was informed that despite his submitting a yearly report to the CVO including the suggestion that the hound work be continued, no further work had been done since 1991. This was surprising, to say the very least.

41.The hound work could have provided valuable evidence that a scrapie-like agent may have been present in cattle offal long before the BSE epidemic was recognised. The MAFF hound survey remains unpublished.

Histopathological support to various other published MAFF experiments

42.These included neuropathological examination of material from experiments studying the attempted transmission of BSE to chickens and pigs (CVL 1991) and to mice (RVC 1994).

It was thought likely that at least some, and probably all, of the cases in zoo animals were caused by the BSE agent. Strong support for this hypothesis came from the findings of Bruce and others (1994) ( Bruce, M.E., Chree, A., McConnell, I., Foster, J., Pearson, G. & Fraser, H. (1994) Transmission of bovine spongiform encephalopathy and scrapie to mice: strain variation and species barrier. Philosophical Transactions of the Royal Society B 343, 405-411: J/PTRSL/343/405 ), who demonstrated that the pattern of variation in incubation period and lesion profile in six strains of mice inoculated with brain homogenates from an affected kudu and the nyala, was similar to that seen when this panel of mouse strains was inoculated with brain from cattle with BSE. The affected zoo bovids were all from herds that were exposed to feeds that were likely to have contained contaminated ruminant-derived protein and the zoo felids had been exposed, if only occasionally in some cases, to tissues from cattle unfit for human consumption.



Friday, March 8, 2013

Dogs may have been used to make Petfood and animal feed

Monday, March 26, 2012


Monday, February 14, 2011



Journal of Wildlife Diseases, 47(1), 2011, pp. 78-93 © Wildlife Disease Association 2011

Monday, March 8, 2010

Canine Spongiform Encephalopathy aka MAD DOG DISEASE

snip...see full text ;

Friday, May 27, 2016

Canine Prions: A New Form of Prion Disease EP-021 PRION 2016 TOKYO

Monday, August 8, 2011

Susceptibility of Domestic Cats to CWD Infection

Monday, August 8, 2011

Susceptibility of Domestic Cats to CWD Infection

Oral.29: Susceptibility of Domestic Cats to CWD Infection

Amy Nalls, Nicholas J. Haley, Jeanette Hayes-Klug, Kelly Anderson, Davis M. Seelig, Dan S. Bucy, Susan L. Kraft, Edward A. Hoover and Candace K. Mathiason†

Colorado State University; Fort Collins, CO USA†Presenting author; Email:

Domestic and non-domestic cats have been shown to be susceptible to one prion disease, feline spongiform encephalopathy (FSE), thought to be transmitted through consumption of bovine spongiform encephalopathy (BSE) contaminated meat. Because domestic and free ranging felids scavenge cervid carcasses, including those in CWD affected areas, we evaluated the susceptibility of domestic cats to CWD infection experimentally. Groups of n = 5 cats each were inoculated either intracerebrally (IC) or orally (PO) with CWD deer brain homogenate. Between 40–43 months following IC inoculation, two cats developed mild but progressive symptoms including weight loss, anorexia, polydipsia, patterned motor behaviors and ataxia—ultimately mandating euthanasia. Magnetic resonance imaging (MRI) on the brain of one of these animals (vs. two age-matched controls) performed just before euthanasia revealed increased ventricular system volume, more prominent sulci, and T2 hyperintensity deep in the white matter of the frontal hemisphere and in cortical grey distributed through the brain, likely representing inflammation or gliosis. PrPRES and widely distributed peri-neuronal vacuoles were demonstrated in the brains of both animals by immunodetection assays. No clinical signs of TSE have been detected in the remaining primary passage cats after 80 months pi. Feline-adapted CWD was sub-passaged into groups (n=4 or 5) of cats by IC, PO, and IP/SQ routes. Currently, at 22 months pi, all five IC inoculated cats are demonstrating abnormal behavior including increasing aggressiveness, pacing, and hyper responsiveness. Two of these cats have developed rear limb ataxia. Although the limited data from this ongoing study must be considered preliminary, they raise the potential for cervid-to-feline transmission in nature.

Hunting and diet

A successful generalist predator, the cougar will eat any animal it can catch, from insects to large ungulates (over 500 kg). Like all cats, it is an obligate carnivore, feeding only on meat. The mean weight of vertebrate prey (MWVP) was positively correlated (r=0.875) with puma body weight and inversely correlated (r=-0.836) with food niche breadth in all America. In general, MWVP was lower in areas closer to the Equator.[3] Its most important prey species are various deer species, particularly in North America; mule deer, white-tailed deer, elk, and even large moose are taken by the cat. Other species such as Bighorn Sheep, wild horses of Arizona, domestic horses, and domestic livestock such as cattle and sheep are also primary food bases in many areas.[38] A survey of North America research found 68% of prey items were ungulates, especially deer. Only the Florida Panther showed variation, often preferring feral hogs and armadillos.[3]

Shown eating. Cougars are ambush predators, feeding mostly on deer and other mammals. Investigation in Yellowstone National Park showed that elk, followed by mule deer, were the cougar's primary targets; the prey base is shared with the park's gray wolves, with whom the cougar competes for resources.[39] Another study on winter kills (November–April) in Alberta showed that ungulates accounted for greater than 99% of the cougar diet. Learned, individual prey recognition was observed, as some cougars rarely killed bighorn sheep, while others relied heavily on the species.[40]


Transmission and Pathogenesis of Chronic Wasting Disease in Cervid and Non-Cervid Species

Edward Hoover,† Candace K. Mathiason, Nicholas J. Haley, Timothy D. Kurt, Davis M. Seelig, Amy V. Nalls, Mark D. Zabel, Glenn C. Telling Department of Microbiology, Immunology, and Pathology; Colorado State University; Fort Collins, CO; Department of Microbiology, Immunology and Molecular Genetics and Neurology; University of Kentucky Medical Center; Lexington, KY USA †Presenting author

Now recognized in 18 states in the US, two Canadian provinces, and one Asian country, efficient horizontal transmission is a signature trait of chronic wasting disease (CWD) of cervids. The facile spread of CWD appears linked to the prion/host relationship facilitating efficient mucosal uptake, peripheral lymphoreticular amplification, and horizontal dissemination exploiting excretory tissues and their products. In addition, recent studies suggest the likelihood of early life mother to offspring transmission. Growing evidence from studies of cervid CWD exposure by natural routes indicate that the incubation period for overt infection detection and disease onset (if any) may be much longer than originally thought. Whether non-cervid species (including humans) may be susceptible to CWD infection and/or act as reservoirs for infection in nature remains unknown. In vitro and in vivo studies of the CWD species barrier indicate the potential for a host range extending beyond cervid species, although no evidence for this has thus far been detected in nature. Interestingly, rodent and mustelid species sympatric with free ranging cervids have been shown susceptible to CWD prions and such trans-species infection broadens the host range/strain characteristics of CWD prions. While the origins of CWD remain unknown, the relationship between sheep scrapie and CWD and the existence of multiple CWD prion strains/quasispecies remain interesting and merit further investigation.


Monday, August 8, 2011

Susceptibility of Domestic Cats to CWD Infection


Wednesday, September 08, 2010


Sunday, November 01, 2009

American crows (Corvus brachyrhynchos) and potential spreading of CWD through feces of digested infectious carcases

Monday, July 13, 2009

Deer Carcass Decomposition and Potential Scavenger Exposure to Chronic Wasting Disease


Wednesday, December 21, 2016





Thursday, December 08, 2016


USDA APHIS National Scrapie Eradication Program October 2016 Monthly Report Fiscal Year 2017 atypical NOR-98 Scrapie



Chronic Wasting Disease CWD TSE Prion Cervid



Creutzfeldt Jakob Disease CJD  



kind regards, terry

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