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From: TSS (216-119-136-88.ipset16.wt.net)
Subject: Re: Identification of a second BASE: Molecular similarities with sporadic Creutzfeldt–Jakob disease (FULL TEXT)
Date: March 1, 2004 at 8:01 pm PST
-------- Original Message --------
Subject: Re: Identification of a second bovine amyloidotic spongiform encephalopathy: Molecular similarities with sporadic Creutzfeldt–Jakob disease (FULL TEXT)
Date: Mon, 1 Mar 2004 21:13:58 -0600
From: "Terry S. Singeltary Sr."
Reply-To: Bovine Spongiform Encephalopathy
To: BSE-L@uni-karlsruhe.de
References: <40327436.4010900@wt.net> <403A63BE.5050407@wt.net>
######## Bovine Spongiform Encephalopathy #########
Greetings List Members, here is full text of this man and cat study to compare...TSS [Image] Research letters Volume 352, Number 9134 [Image] 3 October
1998
[Previous] [Next] [Image][Image]Simultaneous occurrence of spongiform encephalopathy in a man
and his cat in Italy
[Image] Gianluigi Zanusso, Ettore Nardelli, Anna Rosati, GianMaria Fabrizi, Sergio
Ferrari, Antonella Carteri, Franco De Simone, Nicola Rizzuto, Salvatore
Monaco Transmissible spongiform encephalopathies (TSE) encompass inherited,
acquired, and sporadic mammalian neurological disorders, and are
characterised by the conversion of the cellular prion protein (PrP) in an
insoluble and protease-resistant isoform (PrPres). In human TSE, four types
of PrPres have been identified according to size and glycoform ratios, which
may represent different prion strains. Type-1 and type-2 PrPres are
associated with sporadic Creutzfeldt-Jakob disease (CJD), type 3 with
iatrogenic CJD, and type 4 with variant CJD.1,2 There is evidence that
variant CJD is caused by the bovine spongiform encephalopathy (BSE)-prion
strain.2-4 The BSE strain has been identified in three cats with feline
spongiform encephalopathy (FSE), a prion disease which appeared in 1990 in
the UK.5 We report the simultaneous occurrence of sporadic CJD in a man and
a new variety of FSE in his cat. A 60-year-old man, with no unusual dietary habits, was admitted in November,
1993, because of dysarthria, cerebellar ataxic gait, visual agnosia, and
myoclonus. An electroencephalogram (EEG) showed diffuse theta-delta
activity. A brain magnetic resonance imaging scan was unremarkable. 10 days
later, he was speechless and able to follow only simple commands. Repeat
EEGs showed periodic triphasic complexes. 2 weeks after admission, he was
mute, akinetic, and unable to swallow. He died in early January, 1994. His 7-year-old, neutered, female shorthaired cat presented in November,
1993, with episodes of frenzy, twitching of its body, and hyperaesthesia.
The cat was usually fed on canned food and slept on its owner's bed. No
bites from the cat were recalled. In the next few days, the cat became
ataxic, with hindquarter locomotor dysfunction; the ataxia got worse and
there was diffuse myoclonus. The cat was killed in mid-January, 1994. No pathogenic mutations in the patient's PrP gene were found. The patient
and the cat were methionine homozygous at codon 129. Histology of the
patient's brain showed neocortical and cerebellar neuronal loss,
astrocytosis, and spongiosis (figure A). PrP immunoreactivity showed a
punctate pattern and paralleled spongiform changes (figure B). The cat's
brain showed mild and focal spongiosis in deeper cortical layers of all four
lobes (figure C), vacuolated cortical neurons (figure D), and mild
astrogliosis. The cerebellar cortex and the dentate nucleus were gliosed.
Immunoreactive PrP showed a punctate pattern in neocortex, allocortex, and
caudate nucleus (figure E). Western blot analysis of control and affected
human and cat brain homogenates showed 3 PrP bands of 27-35 kDa. After
digestion with proteinase K and deglycosylation, only samples from the
affected patient and cat showed type-1 PrPres, with PrP glycoform ratios
comparable to those observed in sporadic CJD1 (details available from
author). [Image] Microscopic sections of patient and cat brains A: Occipital cortex of the patient showing moderate spongiform
degeneration and neuronal loss (haematoxylin and eosin) and B: punctate
perineuronal pattern of PrP immunoreactivity; peroxidase
immunohistochemistry with monoclonal antibody 3F4. C: cat parietal cortex
showing mild spongiform degeneration (haematoxylin and eosin).D:
vacuolated neurons (arrow, haematoxylin and eosin), E: peroxidase
immunohistochemistry with antibody 3F4 shows punctate perineuronal
deposition of PrP in temporal cortex. This study shows a spatio-temporal association between human and feline
prion diseases. The clinical features of the cat were different from
previously reported cases of FSE which were characterised by gradual onset
of behavioural changes preceding locomotor dysfunction and ataxia.5
Neuropathological changes were also at variance with the diffuse spongiosis
and vacuolation of brainstem neurons, seen in FSE.5 The synaptic pattern of
PrP deposition, similar in the cat and in the patient, was atypical for a
BSE-related condition. Evidence of a new type of FSE was further provided by
the detection of a type-1 PrPres, other than the BSE-associated type 4.2
Taken together, our data suggest that the same agent strain of sporadic CJD
was involved in the patient and in his cat. It is unknown whether these TSE occurred as the result of horizontal
transmission in either direction, infection from an unknown common source,
or the chance occurrence of two sporadic forms. 1 Parchi P, Castellani R, Capellari S, et al. Molecular basis of phenotypic
variablity in sporadic Creutzfeldt-Jakob disease. Ann Neurol 1996; 39:
767-78 [PubMed]. 2 Collinge J, Sidle KCL, Meads J, Ironside J, Hill AF. Molecular analysis of
prion strain variation and the aetiology of 'new variant' CJD. Nature 1996;
383: 685-90 [PubMed]. 3 Bruce ME, Will RG, Ironside JW, et al. Transmissions to mice indicate that
'new variant' CJD is caused by the BSE agent. Nature 1997; 389: 498-501
[PubMed]. 4 Hill AF, Desbruslais M, Joiner S, et al. The same prion strain causes vCJD
and BSE. Nature 1997; 389: 448-50 [PubMed]. 5 Pearson GR, Wyatt JM, Henderson JP, Gruffydd-Jones TJ. Feline spongiform
encephalopathy: a review. Vet Annual 1993; 33: 1-10. ------------------------------------------------------------------------
Sezione di Neurologie Clinica, Dipartimento di Scienze Neurologiche e della
Visione, Università di Verona, Policlinico Borgo Roma, 37134 Verona, Italy
(S Monaco; e mail rizzuto@Gorgorna.univr.it); and Istituto Zooprofilattico
Sperimentale della Lombardia e dell' Emilia, Brescia Terry S. Singeltary Sr. wrote: > ######## Bovine Spongiform Encephalopathy
> #########
>
> Greetings list members,
>
> ODD that some FELINE in Italy seem to have this same or maybe very
> similar
> phenotype of TSE;
>
> In October 1998 the simultaneous occurrence of spongiform encephalopathy
> in a man and his pet cat was reported. The report from Italy noted that
> the cat did not display the same clinical features as FSE cases
> previously seen. Indeed, the presence of a new type of FSE was
> suggested. The man was diagnosed as having sporadic CJD, and neither
> case (man nor cat) appeared to be affected by a BSE-related condition.
>
> http://www.defra.gov.uk/animalh/bse/bse-science/level-4-othertses.html
>
> some data i have filed away on FSE and pet food industry;
>
> http://www.vegsource.com/talk/madcow/messages/91634.html
>
> TSS
>
> Terry S. Singeltary Sr. wrote:
>
>> ######## Bovine Spongiform Encephalopathy
>> #########
>>
>> Identification of a second bovine amyloidotic
>> spongiform encephalopathy: Molecular similarities
>> with sporadic Creutzfeldt–Jakob disease
>> Cristina Casalone*†, Gianluigi Zanusso†‡, Pierluigi Acutis*, Sergio
>> Ferrari‡, Lorenzo Capucci§, Fabrizio Tagliavini¶,
>> Salvatore Monaco‡, and Maria Caramelli*
>> *Centro di Referenza Nazionale per le Encefalopatie Animali, Istituto
>> Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Via
>> Bologna, 148,
>> 10195 Turin, Italy; ‡Department of Neurological and Visual Science,
>> Section of Clinical Neurology, Policlinico G.B. Rossi, Piazzale L.A.
>> Scuro, 10, 37134
>> Verona, Italy; §Istituto Zooprofilattico Sperimentale della Lombardia ed
>> Emilia Romagna, Via Bianchi, 9, 25124 Brescia, Italy; and ¶Istituto
>> Nazionale
>> Neurologico ‘‘Carlo Besta,’’ Via Celoria 11, 20133 Milan, Italy
>> Edited by Stanley B. Prusiner, University of California, San Francisco,
>> CA, and approved December 23, 2003 (received for review September 9,
>> 2003)
>> Transmissible spongiform encephalopathies (TSEs), or prion diseases,
>> are mammalian neurodegenerative disorders characterized
>> by a posttranslational conversion and brain accumulation of an
>> insoluble, protease-resistant isoform (PrPSc) of the host-encoded
>> cellular prion protein (PrPC). Human and animal TSE agents exist as
>> different phenotypes that can be biochemically differentiated on
>> the basis of the molecular mass of the protease-resistant PrPSc
>> fragments and the degree of glycosylation. Epidemiological, molecular,
>> and transmission studies strongly suggest that the single
>> strain of agent responsible for bovine spongiform encephalopathy
>> (BSE) has infected humans, causing variant Creutzfeldt–Jakob disease.
>> The unprecedented biological properties of the BSE agent,
>> which circumvents the so-called ‘‘species barrier’’ between cattle
>> and humans and adapts to different mammalian species, has raised
>> considerable concern for human health. To date, it is unknown
>> whether more than one strain might be responsible for cattle TSE
>> or whether the BSE agent undergoes phenotypic variation after
>> natural transmission. Here we provide evidence of a second cattle
>> TSE. The disorder was pathologically characterized by the presence
>> of PrP-immunopositive amyloid plaques, as opposed to the lack of
>> amyloid deposition in typical BSE cases, and by a different pattern
>> of regional distribution and topology of brain PrPSc accumulation.
>> In addition, Western blot analysis showed a PrPSc type with
>> predominance of the low molecular mass glycoform and a protease-
>> resistant fragment of lower molecular mass than BSE-PrPSc.
>> Strikingly, the molecular signature of this previously undescribed
>> bovine PrPSc was similar to that encountered in a distinct subtype
>> of sporadic Creutzfeldt–Jakob disease.
>> The transmissible spongiform encephalopathies (TSEs), or
>> prion diseases (1), encompass a group of progressive neurodegenerative
>> disorders, including Creutzfeldt–Jakob disease
>> (CJD) in humans, scrapie in sheep, and bovine spongiform
>> encephalopathy (BSE) (1–4). These disorders are characterized
>> by brain deposition of an insoluble, protease-resistant isoform of
>> the host-encoded cellular prion protein (PrPC), named PrPSc (1,
>> 4, 5) In different TSE phenotypes, PrPSc exhibits disease-specific
>> properties, including distinctive cleavage sites after proteolytic
>> treatment, ratio of glycoforms, and deposition patterns, all
>> features useful in providing a means of strain identification
>> (6–10).
>> Although not contagious, TSEs are potentially infective, and
>> in humans may present as sporadic, inherited, and acquired
>> diseases. Human-to-human transmission of TSE is well documented
>> and has occurred either through oral or mucocutaneous
>> route of infection, as in kuru (11), or after medical and surgical
>> procedures, as in iatrogenic CJD (12). Recently, animal-tohuman
>> transmission has also occurred. Epidemiological (13),
>> experimental transmission (14), and biochemical PrPSc typing
>> (8) have provided strong evidence that the single prion strain
>> responsible for BSE has infected humans, causing variant CJD
>> (vCJD), in addition to several animal species. In BSE and
>> BSE-related disorders, including vCJD, the molecular typing of
>> disease-associated PrPSc shows identical PrP fragment sizes and
>> predominance of the high molecular mass glycoform both in
>> natural hosts and in experimentally inoculated animals. To date,
>> at variance with CJD in humans and scrapie in sheep, only a
>> single strain and a single PrPSc type have been detected in BSE.
>> The spreading of the BSE agent across mammalian species
>> barriers has aroused considerable concern for the following
>> reasons: (i) the possible existence of new or previously unrecognized
>> cattle TSE strains, potentially pathogenic for humans;
>> and (ii) the occurrence of phenotypic variation of the BSE strain,
>> with propagation of a new agent encoding distinctive molecular
>> and biological properties.
>> In Italy, an active surveillance system on BSE in cattle was
>> started in January 2001, and by August 2003 a total of 103 BSE
>> cases had been diagnosed of 1,638,275 statutory tested brainstem
>> samples. Confirmatory positive results have been obtained in all
>> cases by immunohistochemical and Western immunoblot demonstration
>> of disease-specific protease-resistant PrPSc.
>> To assess molecular and neuropathological characteristics in
>> Italian BSE cases, we have over the last few months collected
>> whole brains of eight Italian cattle that were PrPSc-positive in
>> Western immunoblots. In two cattle, older than other affected
>> bovines, the PrPSc glycotype was clearly different from the
>> BSE-associated PrPSc molecule, and widespread PrP-amyloid
>> plaques were seen in supratentorial brain regions. Unlike typical
>> BSE, the brainstem was less involved and no PrP deposition was
>> detected in the dorsal nucleus of the vagus nerve. Given the
>> biochemical and pathological similarities with sporadic CJD
>> (sCJD) cases linked to type-2 PrPSc (9) and methioninevaline
>> (MV) polymorphism at codon 129 in the prion protein gene
>> (PRNP), these findings have prompted ongoing strain typing in
>> inbred mice. Although the present findings dictate caution, here
>> we show that a PrPSc type associated with sCJD and the
>> previously undescribed bovine PrPSc show convergent molecular
>> signatures.
>> Materials and Methods
>> Tissue Collection and Processing. Whole brains were collected from
>> four Friesian, three Bruna Alpina, and one Piemontese cattle
>> between 5 and 15 years old. All these animals were routinely
>> slaughtered and resulted positive to the statutory rapid TSE test
>> This paper was submitted directly (Track II) to the PNAS office.
>> Abbreviations: TSE, transmissible spongiform encephalopathy, BSE, bovine
>> spongiform
>> encephalopathy; CJD, Creutzfeldt–Jakob disease; vCJD, variant CJD; sCJD,
>> sporadic CJD; PrP,
>> prion protein, PrPSc pathological PrP; BASE, bovine amyloidotic
>> spongiform encephalopathy.
>> †C.C. and G.Z. contributed equally to this work.
>> To whom correspondence should be addressed. E-mail:
>> salvatore.monaco@mail.univr.it.
>> © 2004 by The National Academy of Sciences of the USA
>> www.pnas.orgcgidoi10.1073pnas.0305777101 PNAS March 2, 2004 vol.
>> 101 no. 9 3065–3070
>> MEDICAL SCIENCES
>> (Prionics, Zurich), which is based on the immunobiochemical
>> detection of bovine PrPSc in brain samples. Brains were longitudinally
>> cut into two halves; the left hemibrain was frozen and
>> stored at 80°C until biochemical studies, whereas the right part
>> was fixed in 10% buffered formaldehyde solution and dissected
>> in 5-mm-thick sections that were embedded in paraffin after
>> decontamination with 96% formic acid for 1 h. The paraffinembedded
>> blocks selected for the study included coronal sections
>> at the level of the olfactory bulb, the frontal, parietal, and
>> occipital cortices, the pyriform lobus, hippocampus, striatum,
>> thalamus, brainstem, and sagittal sections through the cerebellum.
>> Brains were also obtained from three routinely slaughtered
>> cattle free of neurological disorders. Tissues from patients with
>> CJD were obtained as described (15).
>> Bovine PrP Gene Determination. Genomic DNA was isolated from
>> frozen brain tissues by using a QIAamp DNA Mini Kit (Qiagen).
>> PCR amplification of the PrP gene was performed in 50-l
>> reaction volumes containing 0.5–1 g of genomic DNA, 25 mM
>> TrisHCl at pH 8.7, 200 M each dNTP, 1.5 mM MgCl2, 1 unit
>> of Taq DNA polymerase, and 1 M each primer, modified p78
>> () (5-TAAGTGGGCATATGATGCTC-3) and p9 () (5-
>> CTGGGATTCTCTCTGGTACT-3), according to previously
>> described procedures (16). Amplification reactions were performed
>> in a Gene Amp PCR system 9700 (Applied Biosystems)
>> for 41 cycles of 1 min at 94°C, 1.5 min at 56°C, and 1 min at 72°C.
>> PrP polymorphisms were detected by DNA sequencing on both
>> strands of the PCR products in an ABI 310 capillary system
>> (Applied Biosystems). To determine the number of copies of the
>> octapeptide repeats, PCR was carried out by using as primers
>> modified p78 () and p60 () (5-GATAGTAACGGTCCTCATAG-
>> 3). PCR amplification products were examined in
>> ethidium bromide-stained 3% agarose gels.
>> Neuropathology and PrP Immunohistochemistry. Histological sections
>> obtained from each sampled specimen were deparaffinized,
>> rehydrated, and stained with hematoxylin and eosin for
>> evaluation of pathological changes; additional sections were
>> stained with thioflavin-S. For the immunohistochemical study,
>> after rehydration, sections were treated with 96% formic acid for
>> 20 min at room temperature, followed by autoclaving at 121°C
>> for 30 min. After rinsing, sections were incubated overnight at
>> 4°C with anti-PrP monoclonal antibody F9997.6.1 (17) diluted
>> to 11,000. Subsequent antibody detection was carried out by
>> using a biotinylated goat anti-mouse secondary antibody diluted
>> to 1200 for 20 min (Vector Laboratories, Burlingame, CA) at
>> room temperature, followed by the avidin-biotin-peroxidase
>> complex (Vectastain ABC kit, Vector Laboratories) according
>> to manufacturer’s protocol. Immunoreactivity was visualized by
>> using 3,3-diaminobenzidine as chromogen.
>> For electron microscopic study, formalin-fixed specimens of
>> brain tissues were extensively washed in PBS, fixed in 2.5%
>> glutaraldehyde in 0.1Msodium cacodylate buffer, and postfixed
>> with 2% osmium tetroxide for 2 h. After dehydration in graded
>> acetone solutions, tissues were embedded in Spurr’s resin.
>> Subsequently, the sections were prepared for electron microscopy
>> and observed with a Zeiss EM 109 electron microscope.
>> Immunoblot Analysis. From each central nervous system sample,
>> 100 mg of tissue was homogenized in 9 vol of lysis buffer (100
>> mM sodium chloride10 mM EDTA0.5% Nonidet P-400.5%
>> sodium deoxycholate10 mM TrisHCl, pH 7.4) and digested
>> with 50 gml proteinase K (Boehringer Mannheim) for 1 h at
>> 37°C. Digestion was blocked by the addition of phenylmethylsulfonyl
>> fluoride at 2 mM. For deglycosylation, proteinase
>> K-digested samples were deglycosylated with recombinant peptide
>> N-glycosidase F (PNGase F) according to the supplier’s
>> instructions (Boehringer Mannheim). Samples, equivalent to 400
>> g of wet tissue, were resolved by electrophoresis on 13%
>> polyacrylamide gels and then transferred onto PVDF membrane
>> (Immobilon P; Millipore) for 2 h at 60 V. Membranes were
>> blocked with 1% nonfat dry milk in TBST (10 mM TrisHCl150
>> mMsodium chloride0.1% Tween 20, pH 7.5) for 1 h at 37°C and
>> incubated overnight at 4°C with anti-PrP monoclonal antibody
>> 6H4 (Prionics) diluted to 15,000. Blots were developed by using
>> the Amersham Pharmacia enhanced chemiluminescence (ECL)
>> system, as described by the supplier and visualized on an
>> autoradiography film. Films were scanned by using a densitometer
>> (GS-710; Bio-Rad). The relative amounts of PrPSc distribution
>> were calculated as previously described (18).
>> Results
>> Genetic Analysis. In four cattle a silent mutation at codon 70
>> (CAG 3 CAA) was found. As to the number of octapeptide
>> repeats, a common cattle polymorphism, five animals were
>> homozygous for PrP genotype with six copies and one for
>> seven copies, whereas two Bruna Alpina cattle were heterozygous,
>> having fiveseven and sixseven repeats, respectively.
>> Genetic, pathological and biochemical findings are summarized
>> in Table 1.
>> Neuropathology and Immunohistochemistry.Although the presence
>> of early autolysis precluded an accurate pathological assessment
>> in some brain areas, in all animals spongiosis was not consistently
>> found in the brainstem, at the level of the obex or in more rostral
>> areas. The frontal, parietal, and occipital cortices were apparently
>> spared, and no vacuolation was detected in the olfactory
>> bulb, piriform cortex, and hippocampus. Mild spongiform
>> changes of the neuropil were observed only in two Friesian cattle
>> at the level of the thalamus. However, after PrP immunohisto-
>> Table 1. Epidemiological, neuropathological, and biochemical findings in
>> examined cattle
>> Code Breed Age, yr
>> Alleles,
>> octapeptide
>> repeats Genetic variation
>> PrP-amyloid
>> plaques
>> Prevailing
>> PrPSc glycoform
>> on Western blot
>> 1088 Piemontese 15 66 Wild type Low molecular mass
>> 109655 Bruna Alpina 5 57 Wild type 0 High molecular mass
>> 102417 Friesian 9 66 Wild type 0 High molecular mass
>> 141387 Bruna Alpina 11 67 Codon 70 CAGCAA, encodes QQ Low molecular
>> mass
>> 78437 Bruna Alpina 5 77 Codon 70 CAACAA, encodes QQ 0 High
>> molecular mass
>> 16193 Friesian 5 66 Codon 70 CAGCAA, encodes QQ 0 High molecular mass
>> 128204 Friesian 7 66 Wild type 0 High molecular mass
>> 72797 Friesian 8 66 Codon 70 CAGCAA, encodes QQ 0 High molecular mass
>> The presence of amyloid plaques was assessed after thioflavin-S
>> staining, PrP immunohistochemistry, and ultrastructural examination.
>> Codon 70 in control cattle and other affected animals was CAGCAG,
>> encoding QQ.
>> 3066 www.pnas.orgcgidoi10.1073pnas.0305777101 Casalone et al.
>> chemistry, two groups of animals were readily distinguished,
>> because of striking differences in patterns and topography of PrP
>> deposition (Fig. 1). Group 1, comprising six Friesian and Bruna
>> Alpina cattle, including the two cases with thalamic spongiosis,
>> matched the typical phenotype of BSE, characterized by the
>> occurrence of PrP deposits of granular type (in the neuronal
>> cytoplasm or in gray matter neuropil), linear type (thick, threadlike
>> profiles), and glial type, which confers a star-like appearance
>> (Fig. 1 a, c, e, and g). By contrast, group 2, one each Piemontese
>> and Bruna Alpina cattle 15 and 11 years old, respectively, was
>> characterized by the presence of PrP-amyloid plaque-like deposits,
>> kuru-like plaques, and granular extracellular and glial
>> deposits (Fig. 1 b, d, f, and h). The kuru-like plaques appeared
>> as dense unicentric (Fig. 2a), or less frequently multicentric,
>> round structures up to 25 m in diameter (Fig. 2b), with a pale
>> core and a dark radial periphery. PrP-positive plaques were also
>> fluorescent after thioflavin-S and were ultrastructurally composed
>> of bundles of straight, unbranched fibrils with a diameter
>> of 7 nm (Fig. 2 c and d). The two groups of cattle also showed
>> remarkable differences in brain regional distribution of PrP
>> deposits. In group 1, large amounts of granular PrP deposits were
>> observed in brainstem (Fig. 1a) and thalamus (Fig. 1c), whereas
>> the lobus piriformis (Fig. 1e), the olfactory bulb (Fig. 1g), and
>> cerebral cortexes were less involved. By contrast, the brainstem
>> showed only a weak PrP positivity in group 2, and the dorsal
>> motor nucleus of the vagus was unstained (Fig. 1b); PrP-amyloid
>> Fig. 1. PrP deposition in the brains of group 1 and group 2 cattle.
>> Immunohistochemistry showing the glial and granular patterns of PrP
>> deposition observed
>> in the dorsal nucleus of vagus nerve (a, 210), thalamus (c, 210),
>> pyriform cortex (e, 220), and olfactory bulb (g, 150) of an animal
>> representative of group
>> 1. In group 2 cattle, the dorsal nucleus of the vagus nerve is unstained
>> (b, 210), whereas PrP-positive plaques are observed in the thalamus
>> (d, 210), pyriform
>> cortex ( f, 210), and olfactory bulb (h, 80).
>> Casalone et al. PNAS March 2, 2004 vol. 101 no. 9 3067
>> MEDICAL SCIENCES
>> plaques were seen in the thalamus (Fig. 1d), subcortical white
>> matter and deeper layers of cerebral cortexes (Fig. 1f ), and
>> olfactory bulb (Fig. 1h). Finally, the molecular layer of the
>> cerebellum exhibited PrP deposits of the glial type in group 1,
>> whereas some amyloid plaques were observed in group 2.
>> Biochemical Characterization and Regional Distribution of PrPSc.
>> Western immunoblots of proteinase K-treated brain homogenates,
>> obtained from different cortical and subcortical regions of
>> group 1 and group 2 animals, showed the presence of two distinct
>> PrPSc types, which were distinguishable on the basis of the
>> molecular mass of their unglycosylated fragments and the ratio
>> of differently glycosylated forms. The typical molecular ‘‘BSE
>> signature,’’ characterized by overrepresentation of the high
>> molecular mass glycoform, was detected in group 1 animals (Fig.
>> 3a, odd lanes). In contrast, the Piemontese and Bruna Alpina
>> cattle (group 2) showed a predominance of the low molecular
>> mass glycoform and a protease-resistant fragment with a faster
>> electrophoretic mobility (Fig. 3a, even lanes).
>> In prion diseases, distinct PrPSc types usually result in different
>> patterns of deposition and brain regional distribution of the
>> abnormal protein. In group 1 animals, the highest amounts of
>> PrPSc were recovered, as expected, in the brainstem, hypothalamus,
>> and thalamus, and very low PrPSc levels were found in the
>> olfactory bulb and pyriform cortex (Fig. 3b). Conversely, the
>> distribution of PrPSc in group 2 cattle was more widespread than
>> in typical BSE cases, and the largest amounts of PrPSc were
>> detected in the thalamus, olfactory bulb, hippocampus, and
>> olfactory cortex, whereas lower PrPSc levels were recovered in
>> the brainstem (Fig. 3c). On the basis of the neuropathological
>> phenotype and the PrPSc distribution and glycotype, group 2
>> cattle were reminiscent of the sCJD phenotype seen in subjects
>> with MV at PRNP codon 129 and type 2 PrPSc (MV2) (9, 19).
>> Therefore, we compared proteinase K-treated brain homogenates
>> from group 1 and group 2 cattle with sCJD with different
>> molecular types of PrPSc, either homozygous or heterozygous at
>> PRNP codon 129. Remarkably, the PrPSc type detected in
>> TSE-affected cattle from group 2 had fragment size (Fig. 4a) and
>> glycoform ratios (Fig. 4b) similar to a PrPSc type encountered in
>> sCJD MV2 (9, 19).
>> Discussion
>> In natural and experimental TSEs, PrPSc deposition represents
>> an early event that occurs weeks to months before the development
>> of spongiform changes (20, 21). As a consequence, the
>> detection of PrPSc by Western immunoblot provides a unique
>> opportunity in the diagnosis of BSE early in the incubation
>> period and, therefore, in presymptomatic animals. The identification
>> of the present cattle by postmortem biochemical tests, in
>> the absence of clear neurological involvement, suggests that the
>> disorder was detected at early stages, and this may also explain
>> the lack of widespread vacuolar changes.
>> Previous pathological studies in clinically suspect cases of BSE
>> in Great Britain have provided evidence for a uniform pattern
>> in the severity and distribution of vacuolar lesions in affected
>> animals, with medulla oblongata nuclei being the most involved
>> (22). While confirming that the BSE epidemic has been sustained
>> by a single agent, these studies have assessed the validity
>> of statutory criteria for the diagnosis of BSE, which is currently
>> based on both histopathological and immunobiochemical exam-
>> Fig. 2. PrP-positive amyloid plaques in group 2 animals.
>> PrP-immunostaining
>> of the pyriform cortex from group 2 cattle, showing the presence of
>> kuru-like
>> amyloid plaques (a and b, 450). At ultrastructural examination amyloid
>> deposits are composed of aggregates and bundles of unbranched fibrils
>> (c,
>> 12,550; d, 60,000).
>> Fig. 3. Biochemical analysis and regional distribution of PrPSc in group
>> 1 and
>> group 2 cattle. (a) Immunoblot with 6H4 monoclonal antibody of
>> proteinase
>> K-treated brain homogenates from the thalamus of group 1 (odd lanes) and
>> group 2 animals (even lanes), before (lanes 1–4) and after (lanes 5–8)
>> enzymatic
>> deglycosylation. (b and c) Regional distribution of brain PrPSc in group
>> 1 (b) and group 2 (c) cattle; values of PrPSc are reported below each
>> gel as the
>> percentage of the highest value obtained. Molecular size markers are
>> shown
>> on the right as Mr 103.
>> 3068 www.pnas.orgcgidoi10.1073pnas.0305777101 Casalone et al.
>> ination of the medulla. However, the prevailing involvement of
>> cortical regions in the cattle with amyloid deposition suggests
>> that postmortem brain sampling should not be limited to the
>> obex. In addition, a careful analysis of PrPSc glycoform profiles
>> at the confirmatory Western immunoblot may provide a molecular
>> means of identifying atypical cases of bovine TSE.
>> Bovine Amyloidotic Spongiform Encephalopathy (BASE): A Second
>> Bovine TSE. The present findings show that a previously undescribed
>> pathological and immunohistochemical phenotype, associated
>> with cattle TSE, is related to the presence of a PrPSc type
>> with biochemical properties, including the gel mobility of the
>> protease-resistant fragment and glycoform ratios, different from
>> those encountered in cattle BSE. Brain deposition of this
>> pathological isoform of cattle PrP correlates with the formation
>> of PrP-amyloid plaques, as opposed to typical BSE cases. Although
>> in several natural and experimental recipients of the
>> BSE agent, including humans (13), neuropathological changes
>> are characterized by the presence of PrP-positive amyloid
>> deposits with surrounding vacuolation, cattle BSE is not associated
>> with PrP-amyloid plaque formation. On the basis of the
>> above features, we propose to name the disease described here
>> BASE. Although observed in only two cattle, the BASE phenotype
>> could be more common than expected. In previous
>> studies, amyloid congophilic plaques were found in 1 of 20 BSE
>> cases examined systematically for amyloid (23), and it was reported
>> that focal cerebral amyloidosis is present in a small proportion of
>> BSE cases (24). Although no biochemical analysis of PrPSc glycotype
>> is available for these animals with ‘‘atypical BSE phenotype,’’
>> our present results underscore the importance of performing
>> a strain-typing in bovine TSE with amyloid deposition.
>> In sCJD, the neuropathological phenotype largely correlates
>> with the molecular type of PrPSc and distinct polymorphic sites
>> of PRNP (9, 19). This is in contrast with the situation in cattle,
>> where different genotypes have been reported based on the
>> variable numbers of octapeptide repeats in each allele, but no
>> evidence for single-codon polymorphisms in the PrP gene has
>> been established (25, 26). Because the present animals shared a
>> similar genetic background and breed, differences in disease
>> phenotypes between cattle with BSE and BASE can be tentatively
>> related only to distinct PrPSc types or alternative routes of
>> infection and spread of prion pathology. Accordingly, the lack of
>> involvement of the motor dorsal nucleus of the vagus and the
>> slight involvement of the brainstem in BASE, suggests a route for
>> spreading of the agent other than the alimentary tract. Therefore,
>> unless the BASE agent propagates throughout the olfactory
>> pathway or other peripheral routes, it is possible that this
>> disorder represents a sporadic form of cattle TSE, which would
>> also explain the difference in ages between the two groups of
>> affected animals.
>> Phenotypic Similarities Between BASE and sCJD. The transmissibility
>> of CJD brains was initially demonstrated in primates (27), and
>> classification of atypical cases as CJD was based on this property
>> (28). To date, no systematic studies of strain typing in sCJD have
>> been provided, and classification of different subtypes is based
>> on clinical, neuropathological, and molecular features (the polymorphic
>> PRNP codon 129 and the PrPSc glycotype) (8, 9, 15, 19).
>> The importance of molecular PrPSc characterization in assessing
>> the identity of TSE strains is underscored by several studies,
>> showing that the stability of given disease-specific PrPSc types is
>> maintained upon experimental propagation of sCJD, familial
>> CJD, and vCJD isolates in transgenic PrP-humanized mice (8,
>> 29). Similarly, biochemical properties of BSE- and vCJDassociated
>> PrPSc molecules remain stable after passage to mice
>> expressing bovine PrP (30). Recently, however, it has been
>> reported that PrP-humanized mice inoculated with BSE tissues
>> may also propagate a distinctive PrPSc type, with a ‘‘monoglycosylated-
>> dominant’’ pattern and electrophoretic mobility of the
>> unglycosylated fragment slower than that of vCJD and BSE (31).
>> Strikingly, this PrPSc type shares its molecular properties with the
>> a PrPSc molecule found in classical sCJD. This observation is at
>> variance with the PrPSc type found in MV2 sCJD cases and in
>> cattle BASE, showing a monoglycosylated-dominant pattern but
>> faster electrophoretic mobility of the protease-resistant fragment
>> as compared with BSE. In addition to molecular properties
>> of PrPSc, BASE and MV2 sCJD share a distinctive pattern of
>> intracerebral PrP deposition, which occurs as plaque-like and
>> amyloid-kuru plaques. Differences were, however, observed in
>> the regional distribution of PrPSc. While inMV2 sCJD cases the
>> largest amounts of PrPSc were detected in the cerebellum,
>> brainstem, and striatum, in cattle BASE these areas were less
>> involved and the highest levels of PrPSc were recovered from the
>> thalamus and olfactory regions.
>> In conclusion, decoding the biochemical PrPSc signature of
>> individual human and animal TSE strains may allow the identification
>> of potential risk factors for human disorders with
>> unknown etiology, such as sCJD. However, although BASE and
>> sCJD share several characteristics, caution is dictated in assessing
>> a link between conditions affecting two different mammalian
>> species, based on convergent biochemical properties of diseaseassociated
>> PrPSc types. Strains of TSE agents may be better
>> characterized upon passage to transgenic mice. In the interim
>> until this is accomplished, our present findings suggest a strict
>> epidemiological surveillance of cattle TSE and sCJD based on
>> molecular criteria.
>> We are grateful to Giuseppe Ru (Centro di Referenza Nazionale per le
>> Encefalopatie Animali, Istituto Zooprofilattico Sperimentale di Torino)
>> for the provision of surveillance data. We also thank Diana Bazan for
>> preparing material for transmission electron microscopy, and Ines
>> Fig. 4. Electrophoretic analysis of PrPSc in cattle TSE and sCJD. (a)
>> Western
>> blot detection of PrPSc in brains of group 1 animals (lanes 1 and 5);
>> subject with
>> sCJD and type 1 PrPSc, methioninemethionine at codon 129 (lane 2);
>> subject
>> with sCJD and type 2 PrPSc, methioninevaline at codon 129 (lane 3); and
>> group
>> 2 cattle (lane 4). (b) Relative proportions of the three PrPSc
>> glycoforms in group
>> 1 and group 2 cattle compared with glycoform profiles obtained in nine
>> sCJD
>> patients, methioninevaline at codon 129 and with type 2 PrPSc. Mean
>> standard deviation is shown. Upper band, diglycosylated form; middle
>> band,
>> monoglycosylated form; and lower band, unglycosylated form.
>> Casalone et al. PNAS March 2, 2004 vol. 101 no. 9 3069
>> MEDICAL SCIENCES
>> Crescio, Cristiano Corona, Cristiano Longo, Michele Fiorini, Alessia
>> Farinazzo, and Matteo Gelati for technical assistance. This work was
>> supported by a grant from the Italian Ministry of Health (IZS PLV
>> 00401 to M.C. and S.M.), a grant from Fondazione Cariverona (2002-
>> Malattie neurodegenerative to S.M.), and in part by the Italian Ministry
>> of Health (RF 2001.96 to F.T.).
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>> 3070 www.pnas.orgcgidoi10.1073pnas.0305777101 Casalone et al.
>>
>> 17 February 2004; Vol. 101, No. 7
>>
>> URL: http://www.pnas.org/content/vol101/issue7/index.shtml?etoc
>>
>>
>>
>> Washington AP article Paul Brown states;
>>
>>
>>> Italian scientists discover new form of mad cow disease WASHINGTON
>>> (AP) 2/16/04
>>
>>
>>
>>
>> snip...
>>
>>
>>
>>> Dr. Paul Brown of the National Institutes of Health said the finding
>>> does not indicate an increased threat to humans.
>>>
>>> If a new form of the disease were affecting humans there should be an
>>> increase in the incidence of CJD, said Brown, who was not part of the
>>> research team.
>>>
>>> However, scientists in Europe have studied all cases of sporadic CJD
>>> for
>>> the last decade and the incidence has not changed, said Brown, an
>>> expert
>>> in the disease, who works at the National Institute of Neurological
>>> Disease and Stroke.
>>
>>
>>
>>
>> what part of upward trend does Dr. Paul Brown not understand?
>> let us look at the _ upward trend_ of other sporadic CJD cases
>> in other BSE documented countries, some with atypical BSE;
>>
>>
>> Mouse model sheds new light on human prion disease
>>
>> snip...
>>
>>
>> Professor John Collinge said We are not saying that all or even most
>> cases of sporadic CJD are as a result of BSE exposure, but some more
>> recent cases may be the incidence of sporadic CJD has shown an upward
>> trend in the UK over the last decade. While most of this apparent
>> increase may be because doctors are now more aware of CJD and better at
>> diagnosing it, serious consideration should be given to a proportion of
>> this rise being BSE-related. Switzerland, which has had a substantial
>> BSE epidemic, has noted a sharp recent increase in sporadic CJD.
>>
>> snip...
>>
>> http://www.mrc.ac.uk/txt/index/public-interest/public-news-4/public-news_archive/public-news-archive_nov_dec_02/public-bse_and_sporadic_cjd.htm
>>
>>
>>
>>
>> from 1993 to 2002 France went from 35 to 108 sCJD cases. Italy went from
>> 27 to 80 sCJD cases. Germany 21 to 102. besides the increase noted above
>> in both the UK and Switzerland by John Collinge;
>>
>> http://www.eurocjd.ed.ac.uk/sporadic.htm
>>
>> The CDC has no idea of CJD/TSE in humans in the USA with the existing
>> TSE surveillance unit.
>> They have no CJD questionnaire that ask questions pertaining to route
>> and source...TSS
>>
>>
>> Asante/Collinge et al, that BSE transmission to the 129-methionine
>> genotype can lead to an alternate phenotype that is indistinguishable
>> from type 2 PrPSc, the commonest _sporadic_ CJD;
>>
>>
>> http://www.fda.gov/ohrms/dockets/ac/03/slides/3923s1_OPH.htm
>>
>>
>> Terry S. Singeltary Neurology Online, 27 Jan 2003
>>
>> RE-Monitoring the occurrence of emerging forms of Creutzfeldt-Jakob
>>
>> disease in the United States 26 March 2003
>>
>> Next Post-Publication Peer Review Top Terry S. Singeltary,
>>
>> retired (medically)
>>
>> CJD WATCH
>>
>>
>> Send Post-Publication Peer Review to journal:
>>
>> Re: RE-Monitoring the occurrence of emerging forms of Creutzfeldt-Jakob
>>
>> disease in the United States
>>
>>
>> Email Terry S. Singeltary:
>>
>> flounder@wt.net
>>
>>
>> I lost my mother to hvCJD (Heidenhain Variant CJD). I would like to
>>
>> comment on the CDC's attempts to monitor the occurrence of emerging
>>
>> forms of CJD. Asante, Collinge et al [1] have reported that BSE
>>
>> transmission to the 129-methionine genotype can lead to an alternate
>>
>> phenotype that is indistinguishable from type 2 PrPSc, the commonest
>>
>> sporadic CJD. However, CJD and all human TSEs are not reportable
>>
>> nationally. CJD and all human TSEs must be made reportable in every
>>
>> state and internationally. I hope that the CDC does not continue to
>>
>> expect us to still believe that the 85%+ of all CJD cases which are
>>
>> sporadic are all spontaneous, without route/source. We have many TSEs in
>>
>> the USA in both animal and man. CWD in deer/elk is spreading rapidly and
>>
>> CWD does transmit to mink, ferret, cattle, and squirrel monkey by
>>
>> intracerebral inoculation. With the known incubation periods in other
>>
>> TSEs, oral transmission studies of CWD may take much longer. Every
>>
>> victim/family of CJD/TSEs should be asked about route and source of this
>>
>> agent. To prolong this will only spread the agent and needlessly expose
>>
>> others. In light of the findings of Asante and Collinge et al, there
>>
>> should be drastic measures to safeguard the medical and surgical arena
>>
>> from sporadic CJDs and all human TSEs. I only ponder how many sporadic
>>
>> CJDs in the USA are type 2 PrPSc?
>>
>>
>> http://www.neurology.org/cgi/eletters/60/2/176#535
>>
>> Diagnosis and Reporting of Creutzfeldt-Jakob Disease Singeltary, Sr et
>> al. JAMA.2001; 285: 733-734.
>>
>> http://jama.ama-assn.org/cgi/content/full/285/6/733?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&fulltext=dignosing+and+reporting+creutzfeldt+jakob+disease&searchid=1048865596978_1528&stored_search=&FIRSTINDEX=0&journalcode=jama
>>
>>
>>
>>
>> Tracking Spongiform Encephalopathies in North America (Lancet Infectious
>> Disease Volume 3, Number 8 01 August 2003)
>>
>> http://infection.thelancet.com/journal/vol3/iss8/contents
>>
>> BRITISH MEDICAL JOURNAL
>>
>> BMJ
>>
>> http://www.bmj.com/cgi/eletters/319/7220/1312/b#EL2
>>
>> BMJ
>>
>> http://www.bmj.com/cgi/eletters/320/7226/8/b#EL1
>>
>> suppressed peer review of Harvard study October 31, 2002
>>
>> http://www.fsis.usda.gov/oa/topics/BSE_Peer_Review.pdf
>>
>> Terry S. Singeltary Sr.
>> P.O. Box 42
>> Bacliff, Texas USA 77518
>>
>> ########### http://mailhost.rz.uni-karlsruhe.de/warc/bse-l.html
>> ############
>>
>
> ########### http://mailhost.rz.uni-karlsruhe.de/warc/bse-l.html
> ############
> ########### http://mailhost.rz.uni-karlsruhe.de/warc/bse-l.html ############
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