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From: TSS ()
Subject: Different prion disease phenotypes result from inoculation of cattle with two temporally separated sources of sheep scrapie from Great Britain
Date: October 24, 2006 at 7:53 am PST

Different prion disease phenotypes result from inoculation of cattle with two temporally separated sources of sheep scrapie from Great Britain

BMC Veterinary Research 2006, 2:31 doi:10.1186/1746-6148-2-31

Timm Konold (

Yoon Hee Lee (

Michael J Stack (

Claire Horrocks (

Robert B Green (

Melanie Chaplin (

Marion M Simmons (

Steve A C Hawkins (

Richard Lockey (

John Spiropoulos (

John W Wilesmith (john.wilesmith@DEFRA.GSI.GOV.UK)

Gerald A H Wells (

ISSN 1746-6148

Article type Research article

Submission date 20 June 2006

Acceptance date 17 October 2006

Publication date 17 October 2006

Article URL




Given the theoretical proposal that bovine spongiform encephalopathy (BSE)

could have originated from sheep scrapie, this study investigated the

pathogenicity for cattle, by intracerebral (i.c.) inoculation, of two pools of

scrapie agents sourced in Great Britain before and during the BSE epidemic.

Two groups of ten cattle were each inoculated with pools of brain material

from sheep scrapie cases collected prior to 1975 and after 1990. Control

groups comprised five cattle inoculated with sheep brain free from scrapie,

five cattle inoculated with saline, and for comparison with BSE, naturally

infected cattle and cattle i.c. inoculated with BSE brainstem homogenate from

a parallel study. Phenotypic characterisation of the disease forms transmitted

to cattle was conducted by morphological, immunohistochemical, biochemical

and biological methods.


Disease occurred in 16 cattle, nine inoculated with the pre-1975 inoculum and

seven inoculated with the post-1990 inoculum, with four cattle still alive at 83

months post challenge (as at June 2006). The different inocula produced

predominantly two different disease phenotypes as determined by

histopathological, immunohistochemical and Western immunoblotting

methods and biological characterisation on transmission to mice, neither of

which was identical to BSE. Whilst the disease presentation was uniform in all

scrapie-affected cattle of the pre-1975 group, the post-1990 inoculum

produced a more variable disease, with two animals sharing

immunohistochemical and molecular profile characteristics with animals in the

pre-1975 group.


The study has demonstrated that cattle inoculated with different pooled

scrapie sources can develop different prion disease phenotypes, which were

not consistent with the phenotype of BSE of cattle and whose isolates did not

have the strain typing characteristics of the BSE agent on transmission to



Bovine spongiform encephalopathy (BSE) is a progressive and fatal

neurological disease of cattle, which was first discovered in Great Britain (GB)

in 1986 [1]. Epidemiological studies support the view that the cattle population

was exposed to a scrapie-like agent through the feeding of meat and bone

meal after changes in rendering practices. Recycling of the agent increased

exposure of cattle to a scrapie-like agent, resulting in the large epidemic of

BSE. It is hypothesised that the cattle-adapted BSE agent originated either

from sheep scrapie, the transmission resulting directly in clinical disease, or

from cattle with a scrapie-like disease that was present endemically in cattle

at a low level prior to the epidemic [2].

Strain typing of transmissible spongiform encephalopathy (TSE) isolates in

mice from cattle, exotic ruminant species and domestic cats, also of isolates

of experimental BSE in sheep, goats and pigs [3,4] indicate the unique

singularity of the BSE agent in GB, its apparent dissimilarity to isolates of

natural scrapie of sheep, and its stability on natural or experimental single

passage in the several species. Neuropathological studies profiling vacuolar

changes in BSE [5,6] have also provided evidence of the single phenotype of

BSE in GB and allow comparisons to be made with possible other TSE

phenotypes in cattle caused by different agent strains.

As well as vacuolation within the brain, all TSEs are characterised by the

accumulation of abnormal prion protein (PrPSc) in the central nervous system.

Synthetic forms of this protein may transmit disease phenotypes without

utilising a nucleic acid genome by acting as a template, binding, and changing

the conformation of the normal cellular prion protein (PrPC) to replicates of

PrPSc [7]. Although both have the same amino-acid sequence, PrPSc, unlike

PrPC, is relatively resistant to proteolysis and insoluble in mild detergents.

These different physico-chemical properties between normal and diseaseassociated

PrP can be used to study the pathology and biochemistry of the

disease group by the detection of PrPSc by immunohistochemistry (IHC), and

the protease-resistant core of PrPSc (PrPres) by Western immunoblotting (WB).

WB methods have recently been used to characterise different TSE strains,

based on molecular masses and ratio of the resultant glycoforms after

digestion with proteinase K [8]. These have shown similarities in different BSE

isolates [8-10], which further suggests that BSE is caused by a single strain.

Experimental studies conducted in the USA have demonstrated that natural

ovine scrapie can be transmitted to cattle after parenteral inoculation resulting

in a prion disease with clinical and pathological findings unlike those of BSE

[11,12] and that, on passage in cattle, the disease phenotype does not

change substantially [13,14]. The results cannot be extrapolated to the

situation in GB because of the potential difference between scrapie strains in

the USA and in GB. To provide some understanding of the strain (or strains)

of scrapie in sheep in GB, their possible changed properties on passage in

cattle and the dynamics of a selection process which might result in BSE, a

large series of studies were initiated in the United Kingdom in the mid 1990s.

One of these, the present study, investigated the pathogenicity for cattle, by

intracerebral (i.c.) inoculation, of two pools of scrapie agents sourced in GB

before and during the BSE epidemic. The transmitted disease forms in

recipient cattle were characterised by clinical, pathological, biochemical and

biological strain typing approaches and compared with i.c. induced and

naturally infected cases of BSE in cattle. This study design could, within the

limitations of the source materials, potentially identify an endemic form of

scrapie pathogenic for cattle and/ or the occurrence of the BSE agent in the

GB sheep population.


At the time of writing (June 2006), nine out of ten cattle from the pre-1975

group and seven out of ten cattle from the post-1990 group have succumbed

to experimental challenge. The remaining live animals are currently at 83

months post inoculation (mpi, this is based on the calendar month and always

rounded down).



Cattle infected with sheep scrapie agents may develop a disease that is

different from BSE in its clinical, pathological and biochemical features and

variable disease presentations may exist. The process by which scrapie

strains might produce a BSE-like disease in cattle was not evident from this

primary transmission experiment. However, it is diagnostically significant that

despite differences in the biochemical and immunohistochemical

characteristics within both scrapie-infected groups in this study, and between

the ‘bovine scrapie’ phenotypes and BSE, a prion disease was nevertheless

diagnosed by IHC and WB examination of brainstem samples, consistent with

internationally approved prion disease diagnostic methods in ruminant




[Docket No. FSIS-2006-0011] FSIS Harvard Risk Assessment of Bovine
Spongiform Encephalopathy (BSE)


4. WHAT does USDA/FDA ET AL intend to do about the risks of atypical BSE/TSE in cattle now that infectivity

shows in tissue samples other than CNS in Japan, the fact now that the last Texas mad cow and that last mad cow in

Alabama were indeed of the atypical strain, the fact that the studies long ago in Mission, Texas of USA sheep scrapie

transmission to the USA bovine, which proved an 'atypical tse' in the USA bovine, the fact also that USDA/FDA are

still floundering on the last SRM regulations, but with the BASE strain now in cattle that is not similar to nvCJD, but

very similar to the sporadic CJD, and sporadic CJD has tripled in the last few years in the USA. WHAT do you plan to

do to protect human health from these atypical strains of TSE, in relations to SRMs ?


> 1985 The Stetsonville outbreak (farmer's name: Brecke). In addition to the
> downer cows and horses Brecke's mink recieved a cereal supplement.
> Hartsough's view was that this would contain bone meal and would be from a
> commercial source. If this were so and it was contaminated with a TME
> why were no other ranches affected?
> Many mink ranches now feed a commerical pelleted diet. Brecke was equipped
> snip...
> Dead mink go for rendering but are used only in poultry feed.
> A commercial mink ranch was visited. This was Johny Werth's, Capitol Fur
> Farm comprising 1400 breeding females. The feed is bought in from a
> commercial supplier in the form of frozen packs of ''poultry'', ''fish'',
> ''dried egg'' or ''tripe''. A commercial mink cereal supplement is used
> contains ''animal meat meal'' which was said to contain material mainly
> poultry or fish origin but OCCASIONALLY FROM BEEF SOURCES. the partially
> thawed packs were tipped into an augur mixer which has a fully loaded
> capacity of 6000lb and this would be approximately 15000 mink per day.
> In the fall at pelting time the skinned carcasses of the mink are placed
> large barrels which are left in the open to freeze. When full, a renderer
> collects ''for use in poultry feeds''.
> Sections from the brains of the two Brecke TME inoculated cattle were
> examined and Marsh provided all the blocks from the 2nd steer for study at
> CVL and comparison with BSE. In general the vacuolar changes were more
> severe than in most cases of BSE but very similar in distribution.
> Unfortunately material aken fro histopathology from those anials omitted
> representaion of most of the brain stem. ...........
> Wilbur Clarke (reference the Mission, Texas scrapie transmission
> transmission to cattle study) is now the State Veterinarian for Montana
> based at Helena.
> I was given confidential access to sections from the Clarke scrapie-cattle
> transmission experiment. Details of the experimental design were as
> previously by Dr. Wrathall (copy of relevant information appended). Only 3
> animals (2 inoculated with 2nd pass Suffolk scrapie and 1 inoculated with
> Angora goat passaged scrapie) showed clinical signs. Clinical signs were
> characterised by weakness, ''a stilted hindlimb gait'', disorientation,
> ataxia and, terminally, lateral recumbency. The two cattle from which I
> examined material were inocluated at 8 months of age and developed signs
> months pi (goat scrapie inoculum) and 49 months pi (one of the Suffolk
> scrapie inoculated) respectively. This latter animal was killed at 58
> of age and so the clinical duration was only 1 month. The neuropathology
> somewhat different from BSE or the Stetsonville TME in cattle. Vacuolar
> changes were minimal, to the extent that detection REQUIRED CAREFUL
> SEARCHING. Conversely astrocyte hypertrophy was a widespread and prominent
> Transmission Studies
> Mule deer transmissions of CWD were by intracerebral inoculation and
> compared with natural cases
> {the following was written but with a single line marked through it
> passage (by this route)}...TSS
> resulted in a more rapidly progressive clinical disease with repeated
> episodes of synocopy ending in coma. One control animal became affected,
> is believed through contamination of inoculum (?saline). Further CWD
> transmissions were carried out by Dick Marsh into ferret, mink and
> monkey. Transmission occurred in ALL of these species with the shortest
> incubation period in the ferret.
> snip...
> Appendix 3
> 1. 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 SAT were given.
> 2. 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).
> 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.
> 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
> end...TSS
> >> Differences in tissue distribution could require new regulations
> >> regarding specific risk material (SRM) removal.
> snip...end
> full text 33 PAGES ;
> It was, however, performed in the USA in 1979, when it was shown that
> 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
> 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
> 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.
> 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
> 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...
> Visit to USA ... info on BSE and Scrapie



Terry S. Singeltary Sr.

P.O. Box 42

Bacliff, Texas USA 77518

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