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Molecular Method for Prion Strain Analysis

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Well-known member
Sep 3, 2005
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From: TSS ()
Subject: Molecular Method for Prion Strain Analysis
Date: December 8, 2005 at 7:40 pm PST


Research Project: Molecular Method for Prion Strain Analysis
Location: Foodborne Contaminants Research

Project Number: 5325-32000-003-02
Project Type: Specific C/A

Start Date: Aug 01, 2004
End Date: Sep 30, 2006

The goal of the proposed research is to develop molecular methods to distinguish and thereby analyze different prion strains. If successful, these methods could be used to address the following question: Is the December, 2003 North American BSE case the same strain as the United Kingdom BSE strain, or is it analogous to rare atypical BSE cases such as those seen in Italy and Japan? The UK strain has been linked to feed contaminated with BSE, wheras the atypical cases are hypothesized to be of sporadic origin. The answer to this question has obvious implications for 1) the scientific basis of regulations that are designed to prevent future BSE cases caused by feed contamination and 2) the explanation as to the cause of future BSE cases which may still arise despite 100% compliance of feed ban regulations.

Transmissible spongiform encephalophies (TSEs) affect humans and domesticated animals such as sheep (scrapie) and cattle (BSE). TSEs can be genetic (inherited mutations in the prion gene), infectious (dietary or accidental exposure to prions as in iatrogenic cases or consumption of prion-infected food) or sporadic v.g. sporadic Cruetzfeld-Jacob Disease (CJD). Prions have properties that are maintained upon transmission from one host to the next, allowing different 'strains' to be distinguished. Strains cause specific phenotypes, such as different symptoms, incubation time, and tissue distribution of PrPSc. Differentiation of strains is of paramount importance: as an example, the strain of sheep PrPSc that causes scrapie is not transmissible to humans, while the strain that causes ovine BSE presumably is. By SDS-PAGE analysis, PrPSc from different strains maintain specific ratios of non-, mono-, and di-glycosylated glycoforms and different size of the proteinase K (PK) resistent core. However, these methods have significant limitations. Some strains exhibit similar glycoform patterns, and prions of a given strain isolated from different regions of the brain show differences in glycoform patterns, leading to uncertainty. Examination of the molecular weight of PrPSc after proteolysis by SDS-PAGE can only distinguish gross molecular weight differences. We propose to develop new methods to differentiate prion strains based on mass spectrometric analysis. Specifically, we will use tandem mass spectrometry to identify and quantitate peptides of different molecular weights after treatment of the PK-resistant core with trypsin. We will inoculate Syrian hamsters with different prion strains in BL-2 facilities. PrPSc will be isolated from their brains, digested with PK, and denatured. Inactivated prions can then be safely shipped, cleaved with trypsin and analyzed by nanoLC-ESI-MSMS at the WRRC. The scope of this work is to provide proof of principle in a well characterized animal model. If sucessful, future effort will focus on adaptation from animal models to BSE. Documents SCA with U. of Compostela Santiago.


> 1: J Infect Dis. 1994 Apr;169(4):814-20.
> Intracerebral transmission of scrapie to cattle.
> Cutlip RC, Miller JM, Race RE, Jenny AL, Katz JB, Lehmkuhl HD, DeBey BM,
> Robinson MM.
> USDA, Agriculture Research Service, National Animal Disease Center, Ames,
> 50010.
> To determine if sheep scrapie agent(s) in the United States would induce a
> disease in cattle resembling bovine spongiform encephalopathy, 18 newborn
> calves were inoculated intracerebrally with a pooled suspension of brain
> from 9 sheep with scrapie. Half of the calves were euthanatized 1 year
> inoculation. All calves kept longer than 1 year became severely lethargic
> and demonstrated clinical signs of motor neuron dysfunction that were
> manifest as progressive stiffness, posterior paresis, general weakness,
> permanent recumbency. The incubation period was 14-18 months, and the
> clinical course was 1-5 months. The brain from each calf was examined for
> lesions and for protease-resistant prion protein. Lesions were subtle, but
> disease-specific isoform of the prion protein was present in the brain of
> all calves. Neither signs nor lesions were characteristic of those for
> bovine spongiform encephalopathy.
> MeSH Terms:
> Animals
> Brain/microbiology*
> Brain/pathology
> Cattle
> Cattle Diseases/etiology*
> Cattle Diseases/pathology
> Encephalopathy, Bovine Spongiform/etiology*
> Encephalopathy, Bovine Spongiform/pathology
> Immunoblotting/veterinary
> Immunohistochemistry
> Male
> Motor Neurons/physiology
> Prions/analysis
> Scrapie/pathology
> Scrapie/transmission*
> Sheep
> Sleep Stages
> Time Factors
> Substances:
> Prions
> ds=8133096&dopt=Citation
> Intracerebral transmission of scrapie to cattle FULL TEXT PDF;
> SNIP...
> Discussion
> WE conclude that American sources of sheep scrapie are transmissible to
> cattle by direct intracerebral inoculation but the disease induced is NOT
> identical to BSE as seen in the United Kingdom. While there were
> similarities in clinical signs between this experimental disease and BSE,
> there was no evidence of aggressiveness, hyperexcitability, hyperesthesia
> (tactile or auditory), or hyperemetria of limbs as has been reported for
> (9). Neither were there extensive neurologic lesions, which are primary
> BSE, such as severe vacuolation of neurons and neuropil or neuronal
> and gliosis. Although some vacuolation of neuropil, chromotolysis in
> neurons, and gliosis were seen in the brains of some affected calves,
> were industinguishable from those of controls. Vacuolated neurons in the
> nucleus of both challenged and normal calves were considered normal for
> bovines as previously described (50).
> the amount of PrP-res positively related to the length of the incubation.
> ...
> snip...
> WE also conclude from these studies that scrapie in cattle MIGHT NOT BE
> THAT DETECTION OF PrP-res by immunohistochemistry or immunoblotting is
> necessary to make a definitive diagnosis. THUS, undiagnosed scrapie
> infection could contribute to the ''DOWNER-COW'' syndrome and could be
> responsible for some outbreaks of transmissible mink encephalopathy
> by Burger and Hartsough (8) and Marsh and harsough (52). ...
> snip...
> Multiple sources of sheep affected with scrapie and two breeds of cattle
> from several sources were used inthe current study in an effort to avoid a
> single strain of either agent or host. Preliminary results from mouse
> inoculations indicate multiple strains of the agent were present in the
> pooled inoculum (unpublished data). ...
> Transmission of the sheep scrapie to cattle was attempted in 1979 by using
> intracerebral, intramuscular, subcutaneous, and oral routes of inoculation
> of 5, 8- to 11-month old cattlw with a homologous mixture of brain from 1
> affected sheep (61, 62). ONE of the 5 cattle develped neurologic signs 48
> months after inoculation. Signs were disorientation, incoordination, a
> stiff-legged stilted gait, progressive difficulty in rising, and finally
> terminal recumbency. The clinical course was 2.5 months. TWO of the 5
> similarly inoculated with brain tissue from a goat with scrapie exhibited
> similar signs 27 and 36 months after incoluation. Clinical courses were 43
> an 44 days. Brain lesions of mild gliosis and vacuolation and mouse
> inoculation data were insufficient to confirm a diagnosis of scrapie. This
> work remained controversial until recent examination of the brains
> PrP-res in all 3 cattle with neurologic disease but in none of the
> unaffected cattle (62). Results of these studies are similar to ours and
> underscore the necessity of methods other than histopathology to diagnose
> scrapie infection in cattle. We believe that immunologic techniques for
> detecting PrP-res currently provide the most sensitive and reliable way to
> make a definitive diagnosis...
> http://www.bseinquiry.gov.uk/files/sc/seac17/tab03.pdf
> Visit to USA ... info on BSE and Scrapie
> http://www.bseinquiry.gov.uk/files/yb/1988/10/00001001.pdf
> 85
> 12/10/76
> Office Note
> snip...
> A The Present Position with respect to Scrapie
> A] 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 ia 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 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
> http://www.bseinquiry.gov.uk/files/yb/1976/10/12004001.pdf


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