Wednesday, July 28, 2010

Atypical prion proteins and IBNC in cattle DEFRA project code SE1796 FOIA Final report

Atypical prion proteins and IBNC in cattle DEFRA project code SE1796 FOIA Final report

Executive Summary

Studies of Bovine Spongiform Encephalopathy (BSE), carried out in the UK, showed it to be a single strain of prion disease based on histopathological (Simmons et al., 1996) and transmission data (Bruce et al., 1992 ). First reported in the 1980s (Wells et al., 1987) there appears to have been little change in the characteristics of the disease throughout the epidemic and BSE maintains a distinct molecular profile even following cross species transmission. However, during surveillance programmes in Europe and in North America two other distinct isolates of bovine prion disease have come to light, H and L type, so-called to reflect their unique molecular profiles (Yamakawa et al., 2003; Biacabe et al., 2004).

Reports were also emerging of atypical forms of scrapie that were distinct from classical scrapie isolates and were less easily recognised by the then current diagnostic tests (Benestad et al., 2003; Buschman et al., 2004). This led to concerns that cattle could also harbour a prion disease that was not detected by the current diagnostic tests for BSE. Importantly, approximately 15-20% of the clinical cases submitted for investigation were indeed negative and this proportion of negative cattle did not appear to vary despite increasing awareness of BSE clinical signs by the farming and veterinary community. While there maybe other explanations for this discrepancy (McGill et al., 1993), another underlying undiagnosed prion disease of cattle distinct from classical BSE could not be ruled out.

The study reported here investigated a small number of these BSE negative clinical cases by using more sensitive and modified diagnostic tests for abnormal PrP.

The majority of the cases that we studied were negative by all the tests employed and based on this observation we conclude that there was not a simultaneous epidemic of another form of bovine prion disease. However, we observed a number of classical cases that were missed prior to the advent of sensitive and rapid diagnostic tests and this provides an estimate of the number of cattle that were mis-diagnosed before 2000. In addition, we observed a few rare cases where the diagnostic tests were not in agreement and these cases were investigated further. One of these unusual samples emerged as a case of idiopathic bovine neuronal chromatolysis (IBNC).

During the study we also reported the first H-type BSE case in the UK (Terry et al., 2007).

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Scientific Objectives as prescribed in the project:

All of the objectives have been met and are described in detail below. Three annexes accompany this report, one with the figures for the results below and two papers for submission to peer-reviewed journals.

Objective 1:

To determine the variation of PK sensitivity of bovine PrPc from uninfected cattle brains and compare with bovine PrPsc from classical cases of BSE in order to set thresholds for negative, weak and strong positive values in commercially available rapid diagnostic tests.

Objective 2:

Determine whether there are a greater proportion of bovine brain samples positive for the rapid diagnostic tests (hereby called reactors) in the clinically-suspect, negative subset of cattle than in healthy negative cattle. (True positives will be determined on the basis of evaluation by IHC but should be strongly positive in both the rapid diagnostic tests).

Objective 3:

Determine whether the phenotypic and molecular characteristics of PrP from cattle identified in 2 are distinct from normal PrPc and from bovine PrPsc normally associated with classical BSE.

Studies of Bovine Spongiform Encephalopathy (BSE), carried out in the UK, showed it to be a single strain of prion disease based on histopathological (Simmons et al., 1996) and transmission data (Bruce et al., 1992). First reported in the 1980s (Wells et al., 1987) there appears to have been little change in the these characteristics of the disease throughout the epidemic; BSE also appear to maintain a distinct molecular profile in cattle and even when experimentally (or naturally) transmitted to other species such as humans and cats. However, during surveillance programmes in Europe, Japan and in North America, two other distinct isolates of bovine prion disease have come to light, H and L type, so-called to reflect their unique molecular profiles (Yamakawa et al., 2003; Biacabe et al., 2004).

In the late 1990’s, a novel prion disease was discovered in sheep (Benestad et al., 2003; Buschman et al., 2004); this Nor98 or atypical scrapie is widespread in Europe but had previously been missed by histopathological or immunohistological examination. This led to concerns that cattle could also harbour a prion disease that, unlike H- and L-type BSE, was not detected by the current diagnostic tests for BSE. Importantly, approximately 15-20% of the clinical cases submitted for investigation were indeed negative and this proportion of negative cattle did not appear to vary despite increasing awareness of BSE clinical signs by the farming and veterinary community. While there maybe other explanations for this discrepancy (McGill et al., 1993), another underlying undiagnosed prion disease of cattle distinct from classical BSE could not be ruled out.

The study reported here investigated a small number of these BSE negative clinical cases by using more sensitive and modified diagnostic tests for abnormal PrP. The majority of the cases that we studied were negative by all the tests employed and based on this observation we conclude that there was not a simultaneous epidemic of another form of bovine prion disease. However, we observed a number of cases of BSE in this “BSE negative” sub-set that were missed prior to the advent of more sensitive and rapid diagnostic tests and this provides an estimate of the number of cattle that were mis-diagnosed before 2000. In addition, we observed a few rare cases where the diagnostic tests were not in agreement and these cases were investigated further. One of these unusual samples emerged as a case of idiopathic bovine neuronal chromatolysis (IBNC) (Jeffrey & Wilesmith, 1992; 1996; Jeffrey et al., 2009).

During the study we also reported the first H-type BSE case in the UK (Terry et al., 2007).

Materials and Methods

Tissue samples.

Test samples: Frozen brain stem from 501 bovine BSE suspects with neurological signs, a) that were negative at the level of the obex for vacuolation by standard histopathological techniques from years 1991-1999 and b) by IHC and diagnostic Bio-Rad PlateliaTM from 2000 onwards. These tissues have been stored at the VLA at –80oC since submission.

Negative controls: Frozen brain stem from 90 cattle investigated as part of the active surveillance programme. These samples were submitted in 2006 to LGC for rapid testing by Bio-Rad TeSeE diagnostic ELISA and were negative. These samples were stored at –80oC prior to testing and were stored for a maximum of 36 months and therefore considerably less time than all experimental samples under investigation.

Cattle with suppurative encephalitis: 10 additional cattle samples were retrieved from the VLA Archive that were negative for BSE but showed signs of suppurative encephalitis and inflammation (lymphocyte cuffing and gliosis). These signs were consistent with listeria infection.

Tests for disease-associated PrP

IDEXX BSE Herdchek BSE antigen test kit

All samples were assayed using the IDEXX Herdchek Bovine Spongiform Encephalopathy (BSE) Antigen Test Kit, EIA according to the manufacturer’s instructions and without modification. Briefly, brains were homogenised in the buffer provided by the manufacturer and diluted prior to adding to the seprion (polyanion) coated multiwell plate and incubated prior to washing. The samples were then treated with a conditioning buffer to expose the antigen epitopes. PrPsc was detected by PrP specific antibodies conjugated to horseradish peroxidase and visualised with TMB substrate. Samples were read using a microtitre plate reader (Victor-Perkin-Elmer). The method has no Proteinase K digestion step and has only a mild trypsin treatment that is not required for specificity but aids in the epitope exposure step. The normal curve of negative control samples is provided by the manufacturer and shows the diagnostic cut off value is set higher than most negative controls. The amount of brain added to a single well is approximately 20 mg.

Diagnostic Bio-Rad TeSeE EIA

Sample extraction and detection was carried out according to the manufacturer’s instructions for the Bio-Rad TeSeE BSE ELISA. Briefly brain samples were homogenised in buffer provided by the manufacturer and then treated for 10 mins with Proteinase K at 37oC . The PK concentration is not provided by the manufacturer so we refer to it as 4 ul/ml which is the quantity of stock PK to final solution directed by Bio-Rad. A comparison with sigma PK indicated that the concentration is approximately 40 ug/ml. The samples were then precipitated and concentrated by centrifugation. Pellets were reconstituted and diluted in the buffers provided by the manufacturer. The PrPsc was then detected by a sandwich ELISA provided by the manufacturer. Details of the antibodies are not provided. Samples are read using a microtitre plate as above. Cut off values for the ELISA are calculated using the mean of four negative control ODs. The manufacturers indicate that a value of 0.14 should be added to the negative control mean and samples equal to or greater than this value should be further analysed. The amount of brain added to a single well is approximately 65 mg.

BioRad TeSeE EIA with reduced PK digestion (0.3 Bio-Rad TeSeE ELISA)

The PrPsc associated with atypical scrapie is believed to be less PK resistant than classical scrapie (Everest et al., 2006). In order to investigate whether an atypical form of BSE in cattle exists biochemically similar to atypical scrapie a modified version of the Bio-Rad TeSeE protocol, using sub-diagnostic levels of Proteinase K (0.3ul/ml), was used. This quantity of PK was arrived at by titration of PK and digestion of PrPc from 47 cattle brains negative for TSEs.

The Bio-Rad TeSeE BSE diagnostic test was used as directed by the manufacturer with the addition of DNAse prior to the Proteinase K (0.3 ul/ml PK) treatment and Pefabloc was added alongside the kit PK stopping solution. The PK dilution for these assays was prepared from a Sigma stock solution and 0.3 units/ml was the equivalent activity as 0.3 ul/ml of Biorad PK.

Bio-Rad TeSeE Western Blot

Sample extraction was carried out according to the manufacturer’s instructions (Bio-Rad TeSeE Western Blot) with several modifications. In brief, brain tissue was ribolysed to give 20 % (w/v) homogenate and subsequently incubated with DNAse. The samples were then digested with 0.3, 1, 4 or 20 units/ml PK (Sigma; where units/ml is an in-house nomenclature and 0.3 units/ml is equivalent to 0.3 µl of the Bio-Rad test PK in terms of activity as compared using the TAME test -Pierce) and the reaction stopped with Pefabloc. Following precipitation and centrifugation at 15,000 g for 7 minutes, in accordance with the Bio-Rad TeSeE Western blot protocol, the pellets were re-suspended in Laemmli sample buffer.

For analysis, the supernatants were heated at 100oC for 5 minutes, loaded on a 12% Criterion XT Bis-tris SDS gel (Bio-Rad) and subjected to electrophoresis in XT-MOPS running buffer (Bio-Rad) at 200 V for 50 minutes. Proteins were transferred to a PVDF membrane (Bio-Rad) at 115 V, 60 min using Tris/CAPS transfer buffer (Bio-Rad).

Blots to be evaluated using the Sha31 (Bio-Rad) antibody were incubated for one hour with the blocking solution provided by the manufacturers; and antibodies SAF84 (aa 175-180), P4 (aa 89-104) and FH11 (aa 55-65) using a 5% milk powder in PBS supplemented with Tween 20 (PBST). The membranes were incubated for one hour with the primary antibody and then with goat anti-mouse IgG antibody conjugated to horseradish peroxidase (Bio-Rad) prior to visualization by chemiluminescence (ECL; Amersham).

Immunohistochemical analysis

Formalin-fixed, paraffin wax-embedded tissue blocks were sectioned at 4mm, collected onto frosted charged slides (GmbH) and melted on at 60°C overnight to improve adhesion. Sections were de-waxed in xylene and alcohol and washing in water. They were subsequently put into 98% formic acid (Merck) for 30 minutes, washed in running tap water for 15 minutes and then fully immersed into citrate buffer (200mM trisodium citrate dehydrate (Sigma), 30mM citric acid (Sigma), pH 6.1) prior to being autoclaved for 30 minutes at 121°C. Endogenous peroxidase activity was quenched using 3% hydrogen peroxide (Sigma) and the sections immersed in purified water and stored at 4°C overnight. After warming to room temperature, non-specific antibody binding sites were blocked using normal goat serum (Vector Laboratories) for 20 minutes. Rat monoclonal anti-PrP R145 (VLA) was diluted to 2mg/ml and applied for one hour at ambient (19°C-24°C) temperature. Biotinylated rabbit-anti-mouse IgG (Vector Laboratories) was diluted appropriately and applied for 30 minutes at ambient temperature. Elite ABC (Vector Laboratories) was prepared according to the manufacturers’ directions and applied for 20 minutes at ambient temperature. Sections were washed between each stage using 5mM tris buffered saline supplemented with Tween-20 (5mM tris, 0.85% NaCl, 0.05% tween-20 (all from Sigma), pH 7.6). Diaminobenzidine tablets (Sigma) were prepared in McIlvanes buffer (200mM disodium hydrogen orthophosphate, 100mM citric acid (both from Sigma), pH 6.4) and applied for 10 minutes at ambient temperature. Sections were counterstained in Mayer’s haematoxylin and “blued” in running tap water, before being dehydrated through three changes each of absolute alcohol and xylene for three minutes each and finally mounted in DPX (Sigma).

Definition of terms

Disease associated isoforms of PrP may be distinguished from normal PrP by its increased resistance to Proteinase digestion in immunoblotting or ELISA tests (PrPres), binding to polyanions or labelling with PrP specific antibodies in fixed and treated paraffin-embedded section (PrPd). Included within the operational definition of PrPd are all those detection systems that do not use Proteinase K digestion. The correlation between prion infectivity and PrPres or PrPd is inexact, and infectivity has been dissociated from PrPres or PrPd in several experiments, putatively this is because only a fraction of abnormal PrP isoforms are infectious. We will therefore use operational definitions for detected abnormal PrP forms and PrPsc for the hypothetical infectious sub-population of PrP isoforms detected by bioassay.

Results

Brains from cattle previously diagnosed as negative for BSE based on histopathological examination were investigated in this study for evidence of unusual prion diseases. The majority of the cattle investigated were submitted to the VLA as BSE suspect during the years 1997-2005 and were reported to have clinical signs similar to BSE. We applied a combination of modified and previously unused diagnostic tests to this subgroup of cattle including lower concentrations of PK for protein digestion, tests that do not use PK for PrPsc detection and standard Western blot (WB) procedures with Mabs reactive with different regions of the PrP glycoprotein. A flow chart detailing the sequence for the investigation of potential unusual prion diseases of cattle are shown in Figure 1.

1) Determination of the lowest PK concentration that digests PrPc from brains of cattle

The minimum concentration of PK required for the elimination of PrPc in the majority of non-exposed control cattle samples, resulting in a negative value in the Bio-Rad TeSeE ELISA, was determined. PK titrations were performed on BSE positive and negative control reference material (CRM) and subsequently on 47 individual confirmed negative brainstems. The brainstems had previously tested negative with the diagnostic Bio-Rad TeSeE ELISA by LGC and were obtained from active surveillance and therefore unlikely to have had clinical signs of disease. An amount of 0.3 µl/ml PK was selected for use in the adapted Bio-Rad TeSeE ELISA (0.3 Bio-Rad) (Table 1).

2) Determination of threshold values for the IDEXX HerdChek and 0.3 modified Bio-Rad rapid tests

The diagnostic tests have cut-off values that are set by the manufacturers. For the 0.3 Bio-Rad ELISA new cut-off values were determined to take account of the modifications. While no modifications were made to the IDEXX HerdChek assay cut-off values were calculated using the same test samples for consistency. 90 confirmed BSE negative brainstems were assayed and threshold values calculated as 3 standard deviations above the mean (Table 2). Threshold values of 0.166 Absorbance Units (AU) and 0.137 AU were set for the 0.3 Bio-Rad TeSeE and IDEXX Herdchek EIAs respectively. A single confirmed negative sample gave a value above the IDEXX threshold limit (0.240AU) on first assay. However, when repeated this sample was negative (0.016AU).

3) Results of assays applied to the test BSE cattle population

The assays described above and mapped in Figure 1 were then applied to the brains from 501 clinically suspect cattle. Following analysis the cattle were divided into five groups and these are described below. The results are summarised in Table 3.

Group1: Confirmed negative diagnosis of clinically suspect cattle

Brainstems from 501 cattle submitted to the VLA for BSE diagnosis between the years 1991 and 2005 that were subsequently diagnosed as negative by the tests used at time of slaughter, were assayed using the IDEXX and 0.3 Bio-Rad immunoassays for detection of abnormal PrP. 436 (87%) were negative by both tests. All of the samples submitted after 1999 were confirmed negative (see below) (Figure 8). By these criteria we were unable to detect abnormal PrP in the brainstems of these cattle and this subset of clinically suspect cattle is unlikely to harbour a prion disease. However, we were unable to test other areas of the brain from these cattle and PrPsc distribution patterns distinct from classical BSE cannot be ruled out. In addition to the 501 brainstems we also tested 191 cerebella by the same methods, all of which were negative by standard tests.

Group 2: Confirmed positive for BSE by all diagnostic tests

Sixty five samples remained that were positive in either the IDEXX or the 0.3 Bio-Rad assays or in both of these tests. Of these, 40 were positive by both tests (modified as above) and following retesting were positive using diagnostic concentrations of PK for the Bio-Rad TeSeE (figure 2). Immunohistochemical evaluation of abnormal PrP in the obex demonstrated normal distribution of PrPsc deposits similar to those observed for classical BSE (Figure 8).

To confirm that the PK resistant glycoproteins of abnormal PrP resembled the molecular profile of classical BSE, all 40 cases were immunoblotted using SHa31 MAb (figure 3). In all cases a signature 3 glycoprotein banding pattern was observed with relative mass and glycoprotein ratios indistinguishable from classical BSE. These animals ranged in age from 5 years to 12 years, with a mean age of 6 years, 10 months. All 40 animals were female and comprised 32 Friesians, 2 Holsteins, 2 Herefords, 1 Limousin/Friesian Cross, 2 Holstein/Friesian Cross and 1 Simmental.

The 40 confirmed positive samples were from cattle slaughtered between the years 1997 and 1999. We tested a total of 285 from this period and this represents 14.0% of the clinical suspects that were confirmed negative for BSE at this time. If this is representative of the entire clinical suspect unconfirmed cattle (total 2,426) during this period (1997-1999 inclusive) a total of 340 BSE positive cattle would have been missed. This under-diagnosis is likely to be a result of the diagnostic tests applied at the time. Up until the year 2000, all BSE cases were diagnosed by detection of vacuolation and gliosis in the obex. It is clear that this method is not 100% sensitive for prion diseases either because not all cases present with vacuolation or that vacuolation is a late onset phenomenon during clinical disease (Arnold et al., 2007). Our data showed that there were no additional cases of under-diagnosis after more sensitive diagnostic tests were introduced in 2000. During the years 1997-1999, a total of 12,171 clinical cases were submitted for BSE diagnosis of which 9,745 (80.1%) were confirmed positive with an estimated 2.8% of the total suspects submitted under-diagnosed by our calculations.

Assuming no other factors influenced the levels of correct diagnosis and that the numbers estimated for 1997 to 1999 were a true representation of the potential under-diagnosis of the entire epidemic up until 1999, then the total number of missed cases positive for BSE could have been in the region of 5,500.

A draft version of this manuscript has been prepared.

Group 3: Confirmed positive for BSE by all rapid diagnostic tests but negative by IHC

2 of the 501 negative subset brainstem tested were positive by standard biochemical, diagnostic tests (Table 4) but abnormal PrP deposits were not observed in the obex when evaluated by IHC (Figure 8). This is clearly an unusual finding and both cases were rigorously audited prior to further investigation to determine that the sample for biochemistry was identical to the paraffin-embedded sample. As far as can be determined no errors in sampling and dispatch occurred for these two samples. Further DNA profiling and matching frozen sample to histology processed sample would confirm this. There was insufficient sample to perform any further analysis on one case, but the other case was further investigated using the modified TeSeE Western blot protocol described above – at the diagnostic standard PK concentration of 4 µl/ml for PrPsc digestion. Western blotting of abnormal PrP from this sample confirmed the ELISA data with intense labelling of PK resistant PrP using the PrP-specific antibodies Sha31 and SAF84 (Figure 4a and 4b). The glycoprofile and molecular mass of the PrP bands were indistinguishable from classical BSE A band was labelled strongly with FH11 Mab (that recognises an N terminal PrP epitope) and is therefore likely to represent undigested PrP (Figure 4c). In addition, at 4 µl/ml PK, strong reactivity is also observed with the P4 mAb (Figure 4d). Molecular comparison of this case with classical BSE and with scrapie – using different levels of PK, different dilutions of positive sample and different PrP-specific antibodies, indicates that there is no discernible difference of the test sample with classical BSE. Both cases were extensively followed up by IHC using Mabs to different regions of the PrP molecule but were negative in all cases (data not shown).

Why the PrPsc could not be detected by IHC is unclear. Further analysis by transmission to rodent models of prion disease may shed further light on the characteristics of this sample. Indeed, murine models of prion disease have been reported where PrPsc cannot be detected in the brains but these studies confirmed the lack of PrPsc by all assays including Western Blot.

Group 4: IDEXX Herdchek positive, 0.3 Bio-Rad negative, IHC positive.

Two brainstem samples (98/00819; 98/02316) were positive by the IDEXX Herdchek EIA (Table 5) but Bio-Rad test negative even following PK digestion at sub-optimal concentrations. Both of these samples demonstrated abnormal PrP deposition in the obex by IHC evaluation (Figure 8). Western blot analysis of PK resistant PrP glycoprotein from sample 98/2316 indicated that low amounts of PrPres could be detected using Sha31 and SAF84 Mabs. From these blots and taking into account the low levels of PrPres detected the banding patterns appeared indistinguishable from classical BSE (Figure 5a and 5b). No further sample was available for 98/00819.

The sample contained very low levels of PrPres as shown by the WB data and this is likely to be the reason for lack of signal in the Bio-Rad ELISA. At these levels of abnormal PrP we are at the threshold of detection. The IDEXX HerdChek assay has consistently shown a higher analytical sensitivity for classical scrapie in our hands than the Bio-Rad assays. The values for the IDEXX HerdChek were in the region of 0.15-0.88 and these values are much lower than any of the other samples we have tested in this study. These data suggest that the IDEXX assay is more analytically sensitive than the Bio-Rad TeSeE for BSE.

However, there are alternative explanations for the discordance in test results. The Bio-Rad TeSeE ELISA detects PrPres with Mabs that detect 2 regions of the molecule. Any changes in PrP sequence in the region of Mab binding could alter analytical sensitivity. Therefore the bovine PrP open reading frame from 98/02316 was compared with that of two classical BSE samples, all three samples were 6:6 with respect to the octapeptide repeat. The only mutation seen in this unusual sample was at codon 78 and this is a “silent” mutation in that it does not affect the PrP protein sequence (glutamine, Q78). The Western blot results suggest that the PK cleavage sites of sample 98/02316 were not different from classical cases of BSE. Therefore we conclude that PrPres concentration in this sample was low, as indicated by the control BSE positive brain homogenate, when diluted to a level of 1/250, still producing bands of a far greater density than the test sample when assayed neat.

Group 5: Diagnostic Bio-Rad and IDEXX negative, IHC negative but 0.3 Bio-Rad positive

Twenty-one of the clinical suspect brainstems tested by 0.3 Bio-Rad modified protocol had OD values above the calculated cut off point (range 0.166 to 0.857) (Figure 6) but were IDEXX Herdchek negative and IHC negative (figure 8). The samples were also diagnostic Bio-Rad TeSeE negative and the cattle, all female, ranged in age from 3 years to 11.5 years. They comprised Friesian, Friesian/Holsten, Hereford Cross, Aberdeen Angus Cross, Simmental Cross and Limousin Cross breeds. These samples, where sufficient tissue was available, were analysed, for the presence of partially PK resistant PrP, using the Bio-Rad Western blot protocol with digestion carried out at 20 and 0.3 µl/ml of PK and detected using the SHa31 Mab. Following digestion of the samples with 20 µl/ml PK the samples were shown to be negative for the characteristic PrPsc banding patterns when compared to three individual BSE-negative samples and a classical BSE positive sample (Fig 7a). However, faint bands were observed at approximately 16 and 25 KDa for 4 of the samples (T5, T8-T10) but this faint banding is consistent with partially digested PrPc but could also be a result of variable amounts of protein loaded per lane. At 0.3 µl/ml PK, banding is observed for all test samples, with banding consistent with partially digested PrPC, as also observed for the three known BSE-negative samples. In contrast, the classical BSE-positive sample gave a distinct banding pattern, different from that observed for the BSE-negative samples (Fig.7b). Consistent with the above results samples T5 and T8-T10 demonstrated increased intensity of labelling that could result from an up-regulation or increase in PrPc and could also account for the high signals in the modified ELISA.

Variable banding intensity between lanes may also be a result of inconsistent loading of amounts of protein per lane. However, our previous experience of testing protein concentrations PRIOR to PK digestion in the individual samples showed that they were very consistent to within <5% of the total amount. In addition, although we add pefabloc to stop PK digestion it is also likely that there is variation in the PK digestion amongst samples. Both variables could account for the differences in intensities between lanes. However, we cannot exclude the possibilitity that a PK sensitive variant of abnormal PrP is present as demonstrated by Barron et al 2007 who also demonstrated a 22 KDa band following sub optimal PK digestion. The samples were further investigated as below. Encephalitis may up regulate PrPc One explanation for high values in the immunoassay following digestion with suboptimal concentrations of PK could be high levels of PrPc in the sample. Increased levels of PrPc may occur as a result of up-regulation of PrPc on tissue resident cells or from the influx of inflammatory cells into the site following infection. Differential diagnoses were available for 9 of the 21 animals and nine had confirmed encephalitic lesions and inflammation. Further to this observation we therefore analysed brainstems from 10 BSE negative cattle (but also clinical suspects) by both modified rapid tests that had confirmed encephalitis. The brainstems from 9 encephalitis cattle were negative by both the 0.3 Bio-Rad TeSeE and IDEXX assays. The brainstem from 1 animal was positive by the 0.3 Bio-Rad assay but negative by the IDEXX EIA. The result from this sample is similar to the 21 observed above in group 5. It is unclear therefore whether the high levels of PrP are a result of concurrent infection as there is not a 100% correlation. However, PrPc is more susceptible to endogenous proteases and a low signal could be partly explained by inappropriate handling of the tissue at post-mortem. Loss of PrP detection following retesting of group 5 samples. When all 21 samples were re-analysed from a fresh piece of tissue from the archive (likely to have been frozen and thawed by the archive staff) only one retested as positive (figure 6). Further analysis of this sample (sample number 99/00514) by Western blot has not shown any bands suggesting the presence of an atypical form of prion protein. Any PK sensitive PrP, whether PrPc or unusual prion disease-associated PrP, is likely to be affected by tissue handling techniques including freezing, thawing and the amount of time in storage. This could explain loss of signal. These samples may also represent a small number of outliers in the negative population. This is still higher than we would expect given that only 1/90 negative control samples were outliers in the original testing. Identification of Idiopathic Brainstem Neuronal Chromatolysis (IBNC) in group 5 samples One of the 21 samples identified in group 5 was shown to have IBNC following histological investigation (03/00002) (figure 8). Concurrently, we investigated the PrP distribution in known cases of IBNC (Jeffrey et al 2008; “Idiopathic Brainstem Neuronal Chromatolysis (IBNC): a novel prion protein related disorder of cattle?” BMC Vet Res. 2008 Sep 30;4:38. The IHC and histology profile of this case was very similar to that of the known IBNC cases. Investigation of the distribution and molecular characteristics of PrP from known IBNC See also: Idiopathic Brainstem Neuronal Chromatolysis (IBNC): a novel prion protein related disorder of cattle? Jeffrey M, Perez BB, Martin S, Terry L, González L. BMC Vet Res. 2008 Sep 30;4:38 Further investigations demonstrated that 57% the assays performed on the confirmed IBNC samples, using the 0.3 Bio-Rad TeSeE assay (n=42), gave values above those of the test kit control and also the BSE negative brain pool control. Half brains from six IBNC affected animals were retrieved from the TSE archive alongside the brainstem from a seventh animal. The cortex, brainstem, cerebellum and midbrain from these brains were sub-sampled and the adapted Bio-Rad TeSeE EIA, IDEXX Herdchek and Western Blot protocols applied to these tissues, in order to determine whether they could represent a form of atypical BSE. These samples had previously been found to be negative using the commercial Bio-Rad EIA and re-testing using this assay and the IDEXX Herdchek assay confirmed their negative status. When assayed using the adapted Bio-Rad protocol at 0.3µl/ml PK, 24/42 (57%) of the sample assays performed gave values above those of the test kit control and also the BSE negative brain pool control. Values above twice that of the calculated cut-off levels were found for each case but not for each brain site No PrPres was detected when Western blotting these samples at either 20 or 4µl/ml PK but a signal was detected on the gels when blotted at the 0.12 and 0.3µl/ml PK levels. At 0.12µl/ml PK the IBNC samples were indistinguishable from the negative controls but at the 0.3µl/ml level more PrPres was detected in the IBNC cases than in the controls with each of the antibodies tested (SHA31, F99, SAF84 and P4). Illustrations of the F99 blot are shown in the paper. Other data not shown. These data suggest that IBNC affected cattle abnormally express or accumulate PrP in brain and that the abnormal PrP is not strongly resistant to protease digestion. The results suggest that either the range of prion diseases is still wider than previously thought or that abnormalities of prion protein expression may be associated with brain lesions unconnected with prion disorders. Biochemical and transmission studies are planned in order to investigate further (under SE2014). First case of H-type BSE identified in GB During the course of this study, 1/5 frozen brainstem from bovine BSE cases when immunoblotted using the Bio-Rad TeSeE Western blot with antibodies P4, L42, 6H4, Sha31 and SAF84, was found to have a PrP profile indistinguishable from French H-type BSE. This sample was the first case of H-type BSE to be identified in GB. It was a fallen 13-year-old Galloway cow, first tested and confirmed as a case of BSE in November 2005. Due to autolysis its brain was unsuitable for further characterisation by IHC. Its age and reported absence of clinical signs are consistent with other cases of H-type BSE. When blotting the samples, mAbs Sha31 and 6H4 revealed, in this sample, an unglycosylated band with relative mobility less than BSE, and mAb P4, labelled the sample more strongly than the BSE samples hence supporting the observed similarities with the French H-type sample. Additionally, this study revealed in both this unusual sample and the French H-type a lower molecular weight band with relative mobility of between 6 and 10 kD labelled with the P4 and L42 mAbs. This band is not seen in BSE samples. This data was published in June 2007 (L. A. Terry et al. Veterinary record (2007) 160, 873-875). Discussion and Conclusions Here we report the investigation of 501 cattle samples that were submitted to the VLA for BSE diagnosis but subsequently confirmed as negative by the diagnostic test used at the time of submission. Prior to 2000 this was by histology alone and positive diagnosis was made solely upon the observation of vacuolation and gliosis in the relevant brain regions. As a result, using more sensitive diagnostic assays, we were able to diagnose BSE positive cattle from the years 1997-1999 inclusive that were originally negative by vacuolation. From these data we have estimated that approximately 3% of the total suspect cases submitted up until the year 1999 were mis-diagnosed. This is likely to be due to the relative sensitivities of the methods. In addition, it has been demonstrated in cattle that vacuolation occurs after PrPsc can be detected in the brain stem and that PrPsc is detected prior to clinical disease (Arnold et al, 2007). Thus these cattle may have suffering very early clinical signs. However, we have not ruled out the possibility that there may be a subset of BSE affected cattle where vacuolation at the obex does not occur. The two cattle that were positive by the rapid biochemical tests but negative by IHC is an unexplained observation. The samples both contained high amounts of abnormal prion protein as determined by the OD values from the rapid tests that according to our experience of confirmatory testing should have been easily detected by IHC. Furthermore, epitope mapping of the PK cleaved proteins demonstrated no unusual glycoform patterns and IHC evaluation with the same antibodies still did not reveal PrPd deposition in the wax embedded sections. Thus it is unlikely that lack of detection by IHC is the result of an unusual conformation of the PrPd that masks the epitope of R145, the antibody of choice for IHC evaluation at the VLA. The two cattle that were positive by all tests except Bio-Rad ELISA are easier to explain. Previously we have demonstrated that the IDEXX HerdChek scrapie antigen EIA is more analytically sensitive for scrapie than the Bio-Rad ELISA (project SE2007) and this also appears to be the case for bovine BSE. Indeed the two samples were positive by the Bio-Rad Western blot but with significantly reduced signals compared to a bovine positive control. Samples in group 5 were only positive in the Bio-Rad ELISA and only if sub-optimal concentrations of PK were used. Several explanations could account for this result. First, the samples may contain a subset of PrP molecules that have a slightly higher resistance to PK digestion than normal PrPc and that it is not sufficiently aggregated to be detected by the IDEXX assay; whether this is related to a prion disease or some other event that confers such properties on normal PrP remains unanswered. There are notable descriptions in the literature of TSE models where disease is not accompanied by the characteristic accumulation of PK resistant PrP or was found at extremely low levels (Piccardo et al., 2007; Barron et al 2007; Nazor et al., 2005). These findings together might suggest an additional family of neurodegenerative diseases where the infectious form of PrP is not readily detected by our current diagnostic tests. Second, the higher signal could be the result of an increase in the overall amount of PrPc in the samples as discussed in the results and related to up-regulation of PrP in cells resident in the brain or due to influx of inflammatory cells either as a result of damage or the presence of a non-prion related disease. Third, that the PrP in these samples is bound to an unidentified molecule that confers higher PK resistance, or fourth, inhibits proteinase K. IBNC is likely to represent a subset of this group of cattle. Based on these data, our overall conclusion is that a second type of BSE is unlikely to have co-existed at a high prevalence with the classical form in the cattle population during the UK epidemic.


snip...see full text ;


http://randd.defra.gov.uk/Document.aspx?Document=SE1796_8548_FIN.doc



IBNC BSE TSE update ;


Tuesday, November 17, 2009 SEAC NEW RESULTS ON IDIOPATHIC BRAINSTEM NEURONAL CHROMATOLYSIS (IBNC) FROM THE VETERINARY LABORATORIES AGENCY (VLA) SEAC 103/1


http://bse-atypical.blogspot.com/2009/11/seac-new-results-on-idiopathic.html


31 March 2009 - A summary of the 102nd SEAC meeting (35 KB) held on 4th March 2009


snip...


SEAC noted that IBNC appeared to be a rare disease that occurred in older cattle, predominantly as single cases, although it is possible that surveillance may not detect all cases. Biochemical studies suggested that the prion protein may play a role in the disease. However, it is unclear whether the normal form of the protein or an abnormal form is involved. Studies are required to determine whether IBNC is transmissible or not. SEAC concluded, noting that specified risk material controls are in place to prevent cattle brain from entering the food supply, that current data on IBNC do not suggest it presents a risk to human health.



http://www.seac.gov.uk/summaries/seac102_summary.pdf



>>> All of the 15 cattle tested showed that the brains had abnormally accumulated prion protein. <<<


Saturday, February 28, 2009

NEW RESULTS ON IDIOPATHIC BRAINSTEM NEURONAL CHROMATOLYSIS "All of the 15 cattle tested showed that the brains had abnormally accumulated PrP" 2009

SEAC 102/2

http://bse-atypical.blogspot.com/2009/02/new-results-on-idiopathic-brainstem.html



Wednesday, October 08, 2008

Idiopathic Brainstem Neuronal Chromatolysis (IBNC): a novel prion protein related disorder of cattle?

http://bse-atypical.blogspot.com/2008/10/idiopathic-brainstem-neuronal.html



8. I was in receipt of no extra funds beyond those provided by the NHS and the University of London to run my laboratories and pay my salary as a senior lecturer/honorary Consultant and I suffered no constraints over my publications, lectures to my students, or statements to the media. However, I became increasingly aware after 1988 that questioning official dogma about BSE brought difficulties to one’s career. I was myself about to retire from the Charing Cross Hospital, where I worked as a Consultant Neuropathologist, but I observed with horror that the good reputations of dissenting scientists in the field, not least Dr Stephen Dealler and especially Dr Harash Narang were systematically undermined.

http://collections.europarchive.org/tna/20080102135133/http://www.bseinquiry.gov.uk/files/ws/s410.pdf



THEY KNEW 2 DECADES AGO the damn BSE mad cow testing were not finding cases ;


BSE-NON-CONFIRMATION OF DISEASE

3. A question posed by Mr Whaley (para 2) is that classical lesions of BSE may not occur in all cases. Supposing we had a strain variant that produced it's lesions in the cerebrum these would not be detected by our current method. I think this would be unlikely but not impossible - another reason why at least a proportion of complete brains (or blocks) should be retained during the epidemic so if the problem Mr Whaley indicates escalates, it can be investigated.

snip...

5. IF you had the information what benefit would there be ? what would you do with it ?

CONCLUSION

I do not recommend any action. The situation should be accepted. I do not think the VIS can do more at present. The situation should be kept under review particularly if there is an escalation in numbers in this category.

R BRADLEY

15 MAY 1990

90/5.15/3.2

http://collections.europarchive.org/tna/20090505194948/http://bseinquiry.gov.uk/files/yb/1990/05/15003001.pdf



''THE LINE TO TAKE'' ON IBNC $$$ 1995 $$$

1995

page 9 of 14 ;

30. The Committee noted that the results were unusual. the questioned whether there could be coincidental BSE infection or contamination with scrapie. Dr. Tyrell noted that the feeling of the committee was that this did not represent a new agent but it was important to be prepared to say something publicly about these findings. A suggested line to take was that these were scientifically unpublishable results but in line with the policy of openness they would be made publicly available and further work done to test their validity. Since the BSE precautions were applied to IBNC cases, human health was protected. Further investigations should be carried out on isolations from brains of IBNC cases with removal of the brain and subsequent handling under strict conditions to avoid the risk of any contamination.

31. Mr. Bradley informed the Committee that the CVO had informed the CMO about the IBNC results and the transmission from retina and he, like the Committee was satisfied that the controls already in place or proposed were adequate. ...

snip... see full text ;


http://collections.europarchive.org/tna/20080102204938/http://www.bseinquiry.gov.uk/files/yb/1995/06/21005001.pdf




SEAC MINUTES OF THE 19TH MEETING HELD ON 21 JUNE 1995 AT THE CENTRAL VETERINARY LABORATORY

31. Mr Bradley informed the Committee that the CVO had informed the CMO about the IBNC results and the transmission from retina and he, like the Committee was satisfied that the controls already in place or proposed were adequate. ...

http://web.archive.org/web/20030327015011/http://www.bseinquiry.gov.uk/files/yb/1995/06/21005001.pdf




ALSO, please note, atypical BSE-H strain is also transmissible in the humanized transgenic mice with distinct phenotype ;


P26

TRANSMISSION OF ATYPICAL BOVINE SPONGIFORM ENCEPHALOPATHY (BSE) IN HUMANIZED MOUSE MODELS

Liuting Qing1, Fusong Chen1, Michael Payne1, Wenquan Zou1, Cristina Casalone2, Martin Groschup3, Miroslaw Polak4, Maria Caramelli2, Pierluigi Gambetti1, Juergen Richt5*, and Qingzhong Kong1 1Department of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA; 2CEA, Istituto Zooprofilattico Sperimentale, Italy; 3Friedrich-Loeffler-Institut, Germany; 4National Veterinary Research Institute, Poland; 5Kansas State University, Diagnostic Medicine/Pathobiology Department, Manhattan, KS 66506, USA. *Previous address: USDA National Animal Disease Center, Ames, IA 50010, USA

Classical BSE is a world-wide prion disease in cattle, and the classical BSE strain (BSE-C) has led to over 200 cases of clinical human infection (variant CJD). Two atypical BSE strains, BSE-L (also named BASE) and BSE-H, have been discovered in three continents since 2004. The first case of naturally occurring BSE with mutated bovine PrP gene (termed BSE-M) was also found in 2006 in the USA. The transmissibility and phenotypes of these atypical BSE strains/isolates in humans were unknown. We have inoculated humanized transgenic mice with classical and atypical BSE strains (BSE-C, BSE-L, BSE-H) and the BSE-M isolate. We have found that the atypical BSE-L strain is much more virulent than the classical BSE-C. The atypical BSE-H strain is also transmissible in the humanized transgenic mice with distinct phenotype, but no transmission has been observed for the BSE-M isolate so far.

III International Symposium on THE NEW PRION BIOLOGY: BASIC SCIENCE, DIAGNOSIS AND THERAPY 2 - 4 APRIL 2009, VENEZIA (ITALY)

http://www.istitutoveneto.it/prion_09/Abstracts_09.pdf



BUT yet, USDA scientist even managed to change that science around too, by having the only cow known in the world to date that is familial BSE $ PLEASE NOTE, this so-called BSE with mutated bovine PrP gene (termed BSE-M) that was found in 2006 in the USA. a supposedly new strain of familial BSE ? takes me back to the infamous sporadic FFI, that's not familial ? they don't have a clue, in my opinion. but yet the USDA officials will blame it on anything and everything, but the most likely cause i.e. MAD COW FEED IN COMMERCE. Instead, now we have another new strain of mad cow disease, only this is in the USA, and it is just a spontaneous old cow disease i.e. 'familial h-BSEalabama'.


''We hypothesize that the bovine Prnp E211K mutation most likely has caused BSE in “the approximately 10-year-old cow” carrying the E221K mutation.''


WHAT a hoot. They have now taken the same strain of mad cow disease (h-BSE), that Kong et al in 2009 showed was transmissible to humans via human TG mice, and termed it another new mad cow disease, termed ‘‘U.S. BSE Alabama’’ as being another spontaneous happening from nothing. I swear, this just get's better and better. what about IBNC BSE, no cases yet in the USA ? and just what is IBNC BSE ? (more on that later).


BSE Case Associated with Prion Protein Gene Mutation

Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy (TSE) of cattle and was first detected in 1986 in the United Kingdom. It is the most likely cause of variant Creutzfeldt-Jakob disease (CJD) in humans. The origin of BSE remains an enigma. Here we report an H-type BSE case associated with the novel mutation E211K within the prion protein gene (Prnp). Sequence analysis revealed that the animal with H-type BSE was heterozygous at Prnp nucleotides 631 through 633. An identical pathogenic mutation at the homologous codon position (E200K) in the human Prnp has been described as the most common cause of genetic CJD. This finding represents the first report of a confirmed case of BSE with a potential pathogenic mutation within the bovine Prnp gene. A recent epidemiological study revealed that the K211 allele was not detected in 6062 cattle from commercial beef processing plants and 42 cattle breeds, indicating an extremely low prevalence of the E211K variant (less than 1 in 2000) in cattle.

Author Summary Top

Bovine spongiform encephalopathy (BSE or Mad Cow Disease), a transmissible spongiform encephalopathy (TSE) or prion disease of cattle, was first discovered in the United Kingdom in 1986. BSE is most likely the cause of a human prion disease known as variant Creutzfeldt Jakob Disease (vCJD). In this study, we identified a novel mutation in the bovine prion protein gene (Prnp), called E211K, of a confirmed BSE positive cow from Alabama, United States of America. This mutation is identical to the E200K pathogenic mutation found in humans with a genetic form of CJD. This finding represents the first report of a confirmed case of BSE with a potential pathogenic mutation within the bovine Prnp gene. We hypothesize that the bovine Prnp E211K mutation most likely has caused BSE in “the approximately 10-year-old cow” carrying the E221K mutation.


http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1000156



http://www.plospathogens.org/article/fetchObjectAttachment.action?uri=info%3Adoi%2F10.1371%2Fjournal.ppat.1000156&representation=PDF



WHAT does this study show. IS it really a novel mutation in the bovine prion protein gene (Prnp), called E211K, of a confirmed BSE positive cow from Alabama, United States of America, the only one in the world ?

How can scientist around the globe continue to believe such junk science coming from the ARS and USDA, and not see through all this, and still continue to want to trade with them ?

maybe I am not suppose to understand all this, or maybe they just wish I couldn't (and I am not claiming to understand all of it). I just know that something is not computing anymore with the UKBSEnvCJD theory, the rest of different atypical TSE, and the USDA and OIE only theory we are suppose to believe in. I see now where Japan and the USA are going to swap out BSE risk for FMD risk, and it's all about trade once again $ I have seen the FDA claim that feeding 5.5 grams of banned mad cow feed at the Purina plant in Gonzales Texas to each cow was not enough of the TSE agent to worry about. I have seen the TDAH and or the USDA et al completely cover up a highly suspect stumbling and staggering mad cow, sent straight to be rendered without any test at all for mad cow disease and then just claim ignorance, but we knew it was ordered from higher up officials. then we watched another mad cow in Texas, that was confirmed by a secret test to be a positive test, but yet this cow was ordered to be negative, until it took an act of Congress, and 7 months later and scientist complaining from around the globe, that this cow should be re-tested. IT was and it was CONFIRMED 7 months later. I have seen, and it was proven that part of the infamous June 2004 enhanced BSE surveillance and testing program was a sham, they were getting healthy brains from cattle they knew did not have mad cow disease and submitting them for testing. The were busted for that too. It was proven to be true. Then another 9,200 cows in the same program used a test least likely to find mad cow disease i.e. the IHC. oh yea, they did everything they could to claim the Washington cow that was positive, was not a USA cow, and in the end it worked, the suspect mad cow that was suppose to be from the USA, well that was not the right cow, it was another one, this one from Canada. after the back to back h-familial-BSE in Alabama, and h-BSE(whatever they come up with later) in Texas, right after these two cows were documented, they saw the writing on the wall and shut the testing down to numbers so low, it's now mathematically impossible to detect a mad cow case in the USA. what I have seen in these 13+ years is politics manipulate science, and it's not pretty.


The OIE and USDA et al sold there soul to the devil, and in doing so, they sold yours too.


EU IBNC BSE, ANOTHER OLD COW DISEASE, is m-BSE i.e. h-familial-BSE in Alabama only, is this EU IBNC BSE ???

OR, is this just another case of mad cow disease officials are trying to pawn off as something else $$$

Let's look at this closer.

h-familial-BSE in Alabama case

Obvious lesions of spongiform encephalopathy diagnostic for BSE were not present in the brainstem, however it was positive for the presence of PrPd by IHC (Figure 1B). Distribution of PrPd in the brainstem of this animal was not as uniform or as intense as seen with the C-type U.S. BSE case from 2003 (Figure 1C) [4]

Immunohistochemistry

Brain tissue was placed in 10% buffered formalin and after a minimum of 4 days of fixation appropriate sections of brainstem in the obex region were put in cassettes and kept in fresh formalin until they were processed for routine paraffin embedding. The procedure was described in detail previously [4]. The IHC results were interpreted as follows: (i) positive for PrPd: pink to red and (ii) background and negative for PrPd: only blue background. As positive controls, slides from the brainstem of a BSE-positive cow, obtained from the United Kingdom and from the U.S. BSE Case 2003 were used. As negative controls, slides from brainstem material of BSE-negative cattle and scrapie-negative sheep were used.

http://www.plospathogens.org/article/fetchObjectAttachment.action?uri=info%3Adoi%2F10.1371%2Fjournal.ppat.1000156&representation=PDF



IBNC BSE, that's not BSE, but is another novel prion disease.

Atypical prion proteins in cattle DEFRA project code SE1796 FOIA Final report

snip...

Investigation of the distribution and molecular characteristics of PrP from known IBNC

See also: Idiopathic Brainstem Neuronal Chromatolysis (IBNC): a novel prion protein related disorder of cattle?

Jeffrey M, Perez BB, Martin S, Terry L, González L.

BMC Vet Res. 2008 Sep 30;4:38

Further investigations demonstrated that 57% the assays performed on the confirmed IBNC samples, using the 0.3 Bio-Rad TeSeE assay (n=42), gave values above those of the test kit control and also the BSE negative brain pool control.

Half brains from six IBNC affected animals were retrieved from the TSE archive alongside the brainstem from a seventh animal. The cortex, brainstem, cerebellum and midbrain from these brains were sub-sampled and the adapted Bio-Rad TeSeE EIA, IDEXX Herdchek and Western Blot protocols applied to these tissues, in order to determine whether they could represent a form of atypical BSE. These samples had previously been found to be negative using the commercial Bio-Rad EIA and re-testing using this assay and the IDEXX Herdchek assay confirmed their negative status. When assayed using the adapted Bio-Rad protocol at 0.3µl/ml PK, 24/42 (57%) of the sample assays performed gave values above those of the test kit control and also the BSE negative brain pool control. Values above twice that of the calculated cut-off levels were found for each case but not for each brain site

No PrPres was detected when Western blotting these samples at either 20 or 4µl/ml PK but a signal was detected on the gels when blotted at the 0.12 and 0.3µl/ml PK levels. At 0.12µl/ml PK the IBNC samples were indistinguishable from the negative controls but at the 0.3µl/ml level more PrPres was detected in the IBNC cases than in the controls with each of the antibodies tested (SHA31, F99, SAF84 and P4). Illustrations of the F99 blot are shown in the paper. Other data not shown.

These data suggest that IBNC affected cattle abnormally express or accumulate PrP in brain and that the abnormal PrP is not strongly resistant to protease digestion. The results suggest that either the range of prion diseases is still wider than previously thought or that abnormalities of prion protein expression may be associated with brain lesions unconnected with prion disorders. Biochemical and transmission studies are planned in order to investigate further (under SE2014).

http://randd.defra.gov.uk/Document.aspx?Document=SE1796_8548_FIN.doc



c-BSE, atypical l-BSE, atypical h-BSE, m-BSE and or atypical familial h-BSEalabama, IBNC

just what is this IBNC and or the m-BSE in the bovine, and or could they be the same ?

are they just another strain of BSE ?


TSS

Labels: , , , , ,

Friday, January 29, 2010

14th International Congress on Infectious Diseases H-type and L-type Atypical BSE January 2010 (special pre-congress edition)

----- Original Message -----
From: Terry S. Singeltary Sr.
To: TERRY SINGELTARY
Sent: Friday, January 29, 2010 3:23 PM
Subject: 14th International Congress on Infectious Diseases H-type and L-type Atypical BSE January 2010 (special pre-congress edition)


18.173 page 189

Experimental Challenge of Cattle with H-type and L-type Atypical BSE

A. Buschmann1, U. Ziegler1, M. Keller1, R. Rogers2, B. Hills3, M.H. Groschup1. 1Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany, 2Health Canada, Bureau of Microbial Hazards, Health Products & Food Branch, Ottawa, Canada, 3Health Canada, Transmissible Spongiform Encephalopathy Secretariat, Ottawa, Canada

Background: After the detection of two novel BSE forms designated H-type and L-type atypical BSE the question of the pathogenesis and the agent distribution of these two types in cattle was fully open. From initial studies of the brain pathology, it was already known that the anatomical distribution of L-type BSE differs from that of the classical type where the obex region in the brainstem always displays the highest PrPSc concentrations. In contrast in L-type BSE cases, the thalamus and frontal cortex regions showed the highest levels of the pathological prion protein, while the obex region was only weakly involved.

Methods:We performed intracranial inoculations of cattle (five and six per group) using 10%brainstemhomogenates of the two German H- and L-type atypical BSE isolates. The animals were inoculated under narcosis and then kept in a free-ranging stable under appropriate biosafety conditions.At least one animal per group was killed and sectioned in the preclinical stage and the remaining animals were kept until they developed clinical symptoms. The animals were examined for behavioural changes every four weeks throughout the experiment following a protocol that had been established during earlier BSE pathogenesis studies with classical BSE.

Results and Discussion: All animals of both groups developed clinical symptoms and had to be euthanized within 16 months. The clinical picture differed from that of classical BSE, as the earliest signs of illness were loss of body weight and depression. However, the animals later developed hind limb ataxia and hyperesthesia predominantly and the head. Analysis of brain samples from these animals confirmed the BSE infection and the atypical Western blot profile was maintained in all animals. Samples from these animals are now being examined in order to be able to describe the pathogenesis and agent distribution for these novel BSE types. Conclusions: A pilot study using a commercially avaialble BSE rapid test ELISA revealed an essential restriction of PrPSc to the central nervous system for both atypical BSE forms. A much more detailed analysis for PrPSc and infectivity is still ongoing.




http://www.isid.org/14th_icid/



http://ww2.isid.org/Downloads/IMED2009_AbstrAuth.pdf



http://www.isid.org/publications/ICID_Archive.shtml




From: xxxx
To: Terry Singeltary
Sent: Saturday, December 05, 2009 9:09 AM
Subject: 14th ICID - abstract accepted for 'International Scientific Exchange'

Your preliminary abstract number: 670

Dear Mr. Singeltary,

On behalf of the Scientific Committee, I am pleased to inform you that your abstract

'Transmissible Spongiform encephalopathy (TSE) animal and human TSE in North America update October 2009'

WAS accepted for inclusion in the INTERNATIONAL SCIENTIFIC EXCHANGE (ISE) section of the 14th International Congress on Infectious Diseases. Accordingly, your abstract will be included in the "Intl. Scientific Exchange abstract CD-rom" of the Congress which will be distributed to all participants.

Abstracts accepted for INTERNATIONAL SCIENTIFIC EXCHANGE are NOT PRESENTED in the oral OR poster sessions.

Your abstract below was accepted for: INTERNATIONAL SCIENTIFIC EXCHANGE

#0670: Transmissible Spongiform encephalopathy (TSE) animal and human TSE in North America update October 2009

Author: T. Singeltary; Bacliff, TX/US

Topic: Emerging Infectious Diseases Preferred type of presentation: International Scientific Exchange

This abstract has been ACCEPTED.

#0670: Transmissible Spongiform encephalopathy (TSE) animal and human TSE in North America update October 2009

Authors: T. Singeltary; Bacliff, TX/US

Title: Transmissible Spongiform encephalopathy (TSE) animal and human TSE in North America update October 2009

Body: Background

An update on atypical BSE and other TSE in North America. Please remember, the typical U.K. c-BSE, the atypical l-BSE (BASE), and h-BSE have all been documented in North America, along with the typical scrapie's, and atypical Nor-98 Scrapie, and to date, 2 different strains of CWD, and also TME. All these TSE in different species have been rendered and fed to food producing animals for humans and animals in North America (TSE in cats and dogs ?), and that the trading of these TSEs via animals and products via the USA and Canada has been immense over the years, decades.

Methods

12 years independent research of available data

Results

I propose that the current diagnostic criteria for human TSEs only enhances and helps the spreading of human TSE from the continued belief of the UKBSEnvCJD only theory in 2009. With all the science to date refuting it, to continue to validate this old myth, will only spread this TSE agent through a multitude of potential routes and sources i.e. consumption, medical i.e., surgical, blood, dental, endoscopy, optical, nutritional supplements, cosmetics etc.

Conclusion

I would like to submit a review of past CJD surveillance in the USA, and the urgent need to make all human TSE in the USA a reportable disease, in every state, of every age group, and to make this mandatory immediately without further delay. The ramifications of not doing so will only allow this agent to spread further in the medical, dental, surgical arena's. Restricting the reporting of CJD and or any human TSE is NOT scientific. Iatrogenic CJD knows NO age group, TSE knows no boundaries.

I propose as with Aguzzi, Asante, Collinge, Caughey, Deslys, Dormont, Gibbs, Gajdusek, Ironside, Manuelidis, Marsh, et al and many more, that the world of TSE Transmissible Spongiform Encephalopathy is far from an exact science, but there is enough proven science to date that this myth should be put to rest once and for all, and that we move forward with a new classification for human and animal TSE that would properly identify the infected species, the source species, and then the route.

Keywords: Transmissible Spongiform Encephalopathy Creutzfeldt Jakob Disease Prion



see page 114 :




http://ww2.isid.org/Downloads/14th_ICID_ISE_Abstracts.pdf





http://www.isid.org/14th_icid/



http://www.isid.org/publications/ICID_Archive.shtml



http://ww2.isid.org/Downloads/IMED2009_AbstrAuth.pdf




Monday, October 19, 2009


Atypical BSE, BSE, and other human and animal TSE in North America Update October 19, 2009


snip...


I ask Professor Kong ;

Thursday, December 04, 2008 3:37 PM Subject: RE: re--Chronic Wating Disease (CWD) and Bovine Spongiform Encephalopathies (BSE): Public Health Risk Assessment

''IS the h-BSE more virulent than typical BSE as well, or the same as cBSE, or less virulent than cBSE? just curious.....''

Professor Kong reply ;

.....snip

''As to the H-BSE, we do not have sufficient data to say one way or another, but we have found that H-BSE can infect humans. I hope we could publish these data once the study is complete.

Thanks for your interest.''

Best regards,

Qingzhong Kong, PhD Associate Professor Department of Pathology Case Western Reserve University Cleveland, OH 44106 USA

END...TSS

I look forward to further transmission studies, and a true ENHANCED BSE/atypical BSE surveillance program put forth testing all cattle for human and animal consumption for 5 years. a surveillance program that uses the most sensitive TSE testing, and has the personnel that knows how to use them, and can be trusted. I look forward to a stringent mad cow feed ban being put forth, and then strictly enforced. we need a forced, not voluntary feed ban, an enhanced feed ban at that, especially excluding blood. we need some sort of animal traceability. no more excuses about privacy. if somebody is putting out a product that is killing folks and or has the potential to kill you, then everybody needs to know who they are, and where that product came from. same with hospitals, i think medical incidents in all states should be recorded, and made public, when it comes to something like a potential accidental transmission exposure event. so if someone is out there looking at a place to go have surgery done, if you have several hospitals having these type 'accidental exposure events', than you can go some place else. it only makes sense. somewhere along the road, the consumer lost control, and just had to take whatever they were given, and then charged these astronomical prices. some where along the line the consumer just lost interest, especially on a long incubating disease such as mad cow disease i.e. Transmissible Spongiform Encephalopathy. like i said before, there is much more to the mad cow story than bovines and eating a hamburger, we must start focusing on all TSE in all species. ...TSS



http://bse-atypical.blogspot.com/2009/10/atypical-bse-bse-and-other-human-and.html




JOURNAL OF NEUROLOGY

MARCH 26, 2003

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


http://www.neurology.org/cgi/eletters/60/2/176#535




Newsdesk The Lancet Infectious Diseases, Volume 3, Issue 8, Page 463, August 2003 doi:10.1016/S1473-3099(03)00715-1Cite or Link Using DOI

Tracking spongiform encephalopathies in North America

Xavier Bosch

“My name is Terry S Singeltary Sr, and I live in Bacliff, Texas. I lost my mom to hvCJD (Heidenhain variant CJD) and have been searching for answers ever since. What I have found is that we have not been told the truth. CWD in deer and elk is a small portion of a much bigger problem.” 49-year-old Singeltary is one of a number of people who have remained largely unsatisfied after being told that a close relative died from a rapidly progressive dementia compatible with spontaneous Creutzfeldt-Jakob disease (CJD). So he decided to gather hundreds of documents on transmissible spongiform encephalopathies (TSE) and realised that if Britons could get variant CJD from bovine spongiform encephalopathy (BSE), Americans might get a similar disorder from chronic wasting disease (CWD)—the relative of mad cow disease seen among deer and elk in the USA. Although his feverish…


http://linkinghub.elsevier.com/retrieve/pii/S1473309903007151



http://www.thelancet.com/journals/laninf/article/PIIS1473-3099(03)00715-1/fulltext



http://www.mdconsult.com/das/article/body/180784492-2/jorg=journal&source=&sp=13979213&sid=0/N/368742/1.html?issn=14733099



Diagnosis and Reporting of Creutzfeldt-Jakob Disease Singeltary, Sr et al. JAMA.2001; 285: 733-734. Vol. 285 No. 6, February 14, 2001 JAMA

Diagnosis and Reporting of Creutzfeldt-Jakob Disease

To the Editor: In their Research Letter, Dr Gibbons and colleagues1 reported that the annual US death rate due to Creutzfeldt-Jakob disease (CJD) has been stable since 1985. These estimates, however, are based only on reported cases, and do not include misdiagnosed or preclinical cases. It seems to me that misdiagnosis alone would drastically change these figures. An unknown number of persons with a diagnosis of Alzheimer disease in fact may have CJD, although only a small number of these patients receive the postmortem examination necessary to make this diagnosis. Furthermore, only a few states have made CJD reportable. Human and animal transmissible spongiform encephalopathies should be reportable nationwide and internationally.

Terry S. Singeltary, Sr Bacliff, Tex

1. Gibbons RV, Holman RC, Belay ED, Schonberger LB. Creutzfeldt-Jakob disease in the United States: 1979-1998. JAMA. 2000;284:2322-2323. FREE FULL TEXT


http://jama.ama-assn.org/cgi/content/extract/285/6/733?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&fulltext=singeltary&searchid=1&FIRSTINDEX=0&resourcetype=HWCIT



http://jama.ama-assn.org/cgi/content/full/285/6/733?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&fulltext=singeltary&searchid=1&FIRSTINDEX=0&resourcetype=HWCIT



2 January 2000

British Medical Journal

U.S. Scientist should be concerned with a CJD epidemic in the U.S., as well


http://www.bmj.com/cgi/eletters/320/7226/8/b#6117




15 November 1999

British Medical Journal

vCJD in the USA * BSE in U.S.


http://www.bmj.com/cgi/eletters/319/7220/1312/b#5406




THE PATHOLOGICAL PROTEIN

BY Philip Yam

Yam Philip Yam News Editor Scientific American www.sciam.com

Answering critics like Terry Singeltary, who feels that the U.S. under- counts CJD, Schonberger conceded that the current surveillance system has errors but stated that most of the errors will be confined to the older population.

CHAPTER 14

Laying Odds

Are prion diseases more prevalent than we thought?

Researchers and government officials badly underestimated the threat that mad cow disease posed when it first appeared in Britain. They didn’t think bovine spongiform encephalopathy was a zoonosis—an animal disease that can sicken people. The 1996 news that BSE could infect humans with a new form of Creutzfeldt-Jakob disease stunned the world. It also got some biomedical researchers wondering whether sporadic CJD may really be a manifestation of a zoonotic sickness. Might it be caused by the ingestion of prions, as variant CJD is?

Revisiting Sporadic CJD

It’s not hard to get Terry Singeltary going. “I have my conspiracy theories,” admitted the 49-year-old Texan.1 Singeltary is probably the nation’s most relentless consumer advocate when it comes to issues in prion diseases. He has helped families learn about the sickness and coordinated efforts with support groups such as CJD Voice and the CJD Foundation. He has also connected with others who are critical of the American way of handling the threat of prion diseases. Such critics include Consumers Union’s Michael Hansen, journalist John Stauber, and Thomas Pringle, who used to run the voluminous www.madcow. org Web site. These three lend their expertise to newspaper and magazine stories about prion diseases, and they usually argue that prions represent more of a threat than people realize, and that the government has responded poorly to the dangers because it is more concerned about protecting the beef industry than people's health.

Singeltary has similar inclinations. ...


http://books.google.com/books?id=ePbrQNFrHtoC&pg=PA223&lpg=PA223&dq=the+pathological+protein+laying+odds+It%E2%80%99s+not+hard+to+get+Terry+Singeltary+going&source=bl&ots=um0PFAZSZD&sig=JWaGR7M7-1WeAr2qAXq8D6J_jak&hl=en&ei=MhtjS8jMJM2ztgeFoa2iBg&sa=X&oi=book_result&ct=result&resnum=1&ved=0CAcQ6AEwAA#v=onepage&q=&f=false



http://www.springerlink.com/content/r2k2622661473336/fulltext.pdf?page=1



http://www.thepathologicalprotein.com/




DER SPIEGEL (9/2001) - 24.02.2001 (9397 Zeichen) USA: Loch in der Mauer Die BSE-Angst erreicht Amerika: Trotz strikter Auflagen gelangte in Texas verbotenes Tiermehl ins Rinderfutter - die Kontrollen der Aufsichtsbehördensind lax.Link auf diesen Artikel im Archiv: http://service.spiegel.de/digas/find?DID=18578755


"Löcher wie in einem Schweizer Käse" hat auch Terry Singeltary im Regelwerk der FDA ausgemacht. Der Texaner kam auf einem tragischen Umweg zu dem Thema: Nachdem seine Mutter 1997 binnen weniger Wochen an der Creutzfeldt-Jakob-Krankheit gestorben war, versuchte er, die Ursachen der Infektion aufzuspüren. Er klagte auf die Herausgabe von Regierungsdokumenten und arbeitete sich durch Fachliteratur; heute ist er überzeugt, dass seine Mutter durch die stetige Einnahme von angeblich kräftigenden Mitteln erkrankte, in denen - völlig legal - Anteile aus Rinderprodukten enthalten sind.


Von der Fachwelt wurde Singeltary lange als versponnener Außenseiter belächelt. Doch mittlerweile sorgen sich auch Experten, dass ausgerechnet diese verschreibungsfreien Wundercocktails zur Stärkung von Intelligenz, Immunsystem oder Libido von den Importbeschränkungen ausgenommen sind. Dabei enthalten die Pillen und Ampullen, die in Supermärkten verkauft werden, exotische Mixturen aus Rinderaugen; dazu Extrakte von Hypophyse oder Kälberföten, Prostata, Lymphknoten und gefriergetrocknetem Schweinemagen. In die USA hereingelassen werden auch Blut, Fett, Gelatine und Samen. Diese Stoffe tauchen noch immer in US-Produkten auf, inklusive Medizin und Kosmetika. Selbst in Impfstoffen waren möglicherweise gefährliche Rinderprodukte enthalten. Zwar fordert die FDA schon seit acht Jahren die US-Pharmaindustrie auf, keine Stoffe aus Ländern zu benutzen, in denen die Gefahr einer BSE-Infizierung besteht. Aber erst kürzlich verpflichteten sich fünf Unternehmen, darunter Branchenführer wie GlaxoSmithKline, Aventis und American Home Products, ihre Seren nur noch aus unverdächtigem Material herzustellen.



"Its as full of holes as Swiss Cheese" says Terry Singeltary of the FDA regulations. ...


http://www.spiegel.de/spiegel/print/d-18578755.html




http://wissen.spiegel.de/wissen/image/show.html?did=18578755&aref=image024/E0108/SCSP200100901440145.pdf&thumb=false




http://service.spiegel.de/digas/servlet/find/DID=18578755





Suspect symptoms

What if you can catch old-fashioned CJD by eating meat from a sheep infected with scrapie?

28 Mar 01

Like lambs to the slaughter 31 March 2001 by Debora MacKenzie Magazine issue 2284. Subscribe and get 4 free issues. FOUR years ago, Terry Singeltary watched his mother die horribly from a degenerative brain disease. Doctors told him it was Alzheimer's, but Singeltary was suspicious. The diagnosis didn't fit her violent symptoms, and he demanded an autopsy. It showed she had died of sporadic Creutzfeldt-Jakob disease.

Most doctors believe that sCJD is caused by a prion protein deforming by chance into a killer. But Singeltary thinks otherwise. He is one of a number of campaigners who say that some sCJD, like the variant CJD related to BSE, is caused by eating meat from infected animals. Their suspicions have focused on sheep carrying scrapie, a BSE-like disease that is widespread in flocks across Europe and North America.

Now scientists in France have stumbled across new evidence that adds weight to the campaigners' fears. To their complete surprise, the researchers found that one strain of scrapie causes the same brain damage in mice as sCJD.

"This means we cannot rule out that at least some sCJD may be caused by some strains of scrapie," says team member Jean-Philippe Deslys of the French Atomic Energy Commission's medical research laboratory in Fontenay-aux-Roses, south-west of Paris. Hans Kretschmar of the University of Göttingen, who coordinates CJD surveillance in Germany, is so concerned by the findings that he now wants to trawl back through past sCJD cases to see if any might have been caused by eating infected mutton or lamb.

Scrapie has been around for centuries and until now there has been no evidence that it poses a risk to human health. But if the French finding means that scrapie can cause sCJD in people, countries around the world may have overlooked a CJD crisis to rival that caused by BSE.

Deslys and colleagues were originally studying vCJD, not sCJD. They injected the brains of macaque monkeys with brain from BSE cattle, and from French and British vCJD patients. The brain damage and clinical symptoms in the monkeys were the same for all three. Mice injected with the original sets of brain tissue or with infected monkey brain also developed the same symptoms.

As a control experiment, the team also injected mice with brain tissue from people and animals with other prion diseases: a French case of sCJD; a French patient who caught sCJD from human-derived growth hormone; sheep with a French strain of scrapie; and mice carrying a prion derived from an American scrapie strain. As expected, they all affected the brain in a different way from BSE and vCJD. But while the American strain of scrapie caused different damage from sCJD, the French strain produced exactly the same pathology.

"The main evidence that scrapie does not affect humans has been epidemiology," says Moira Bruce of the neuropathogenesis unit of the Institute for Animal Health in Edinburgh, who was a member of the same team as Deslys. "You see about the same incidence of the disease everywhere, whether or not there are many sheep, and in countries such as New Zealand with no scrapie." In the only previous comparisons of sCJD and scrapie in mice, Bruce found they were dissimilar.

But there are more than 20 strains of scrapie, and six of sCJD. "You would not necessarily see a relationship between the two with epidemiology if only some strains affect only some people," says Deslys. Bruce is cautious about the mouse results, but agrees they require further investigation. Other trials of scrapie and sCJD in mice, she says, are in progress.

People can have three different genetic variations of the human prion protein, and each type of protein can fold up two different ways. Kretschmar has found that these six combinations correspond to six clinical types of sCJD: each type of normal prion produces a particular pathology when it spontaneously deforms to produce sCJD.

But if these proteins deform because of infection with a disease-causing prion, the relationship between pathology and prion type should be different, as it is in vCJD. "If we look at brain samples from sporadic CJD cases and find some that do not fit the pattern," says Kretschmar, "that could mean they were caused by infection."

There are 250 deaths per year from sCJD in the US, and a similar incidence elsewhere. Singeltary and other US activists think that some of these people died after eating contaminated meat or "nutritional" pills containing dried animal brain. Governments will have a hard time facing activists like Singeltary if it turns out that some sCJD isn't as spontaneous as doctors have insisted.

Deslys's work on macaques also provides further proof that the human disease vCJD is caused by BSE. And the experiments showed that vCJD is much more virulent to primates than BSE, even when injected into the bloodstream rather than the brain. This, says Deslys, means that there is an even bigger risk than we thought that vCJD can be passed from one patient to another through contaminated blood transfusions and surgical instruments.


http://www.newscientist.com/article/mg16922840.300-like-lambs-to-the-slaughter.html




Sunday, August 09, 2009

CJD...Straight talk with...James Ironside...and...Terry Singeltary... 2009


http://creutzfeldt-jakob-disease.blogspot.com/2009/08/cjdstraight-talk-withjames.html






Tuesday, August 18, 2009

BSE-The Untold Story - joe gibbs and singeltary 1999 - 2009


http://madcowusda.blogspot.com/2009/08/bse-untold-story-joe-gibbs-and.html






Subject: BSE--U.S. 50 STATE CONFERENCE CALL Jan. 9, 2001
Date: Tue, 9 Jan 2001 16:49:00 -0800
From: "Terry S. Singeltary Sr."
Reply-To: Bovine Spongiform Encephalopathy
To: BSE-L@uni-karlsruhe.de



######### Bovine Spongiform Encephalopathy #########


Greetings List Members,


I was lucky enough to sit in on this BSE conference call today and even managed to ask a question. that is when the trouble started.

I submitted a version of my notes to Sandra Blakeslee of the New York Times, whom seemed very upset, and rightly so.

"They tell me it is a closed meeting and they will release whatever information they deem fit. Rather infuriating."

and i would have been doing just fine, until i asked my question. i was surprised my time to ask a question so quick.

(understand, these are taken from my notes for now. the spelling of names and such could be off.)

[host Richard Barns] and now a question from Terry S. Singeltary of CJD Watch.

[TSS] yes, thank you, U.S. cattle, what kind of guarantee can you give for serum or tissue donor herds?

[no answer, you could hear in the back ground, mumbling and 'we can't. have him ask the question again.]

[host Richard] could you repeat the question?

[TSS] U.S. cattle, what kind of guarantee can you give for serum or tissue donor herds?

[not sure whom ask this] what group are you with?

[TSS] CJD Watch, my Mom died from hvCJD and we are tracking CJD world-wide.

[not sure who is speaking] could you please disconnect Mr. Singeltary

[TSS] you are not going to answer my question?

[not sure whom speaking] NO

from this point, i was still connected, got to listen and tape the whole conference. at one point someone came on, a woman, and ask again;

[unknown woman] what group are you with?

[TSS] CJD Watch and my Mom died from hvCJD we are trying to tract down CJD and other human TSE's world wide. i was invited to sit in on this from someone inside the USDA/APHIS and that is why i am here. do you intend on banning me from this conference now?

at this point the conference was turned back up, and i got to finish listening. They never answered or even addressed my one question, or even addressed the issue. BUT, i will try and give you a run-down for now, of the conference.

IF i were another Country, I would take heed to my notes, BUT PLEASE do not depend on them. ask for transcript from;

RBARNS@ORA.FDA.GOV 301-827-6906

he would be glad to give you one ;-)

Rockville Maryland, Richard Barns Host

BSE issues in the U.S., How they were labelling ruminant feed? Revising issues.

The conference opened up with the explaining of the U.K. BSE epidemic winding down with about 30 cases a week.

although new cases in other countries were now appearing.

Look at Germany whom said NO BSE and now have BSE.

BSE increasing across Europe.

Because of Temporary Ban on certain rendered product, heightened interest in U.S.

A recent statement in Washington Post, said the New Administration (old GW) has a list of issues. BSE is one of the issues.

BSE Risk is still low, minimal in U.S. with a greater interest in MBM not to enter U.S.

HOWEVER, if BSE were to enter the U.S. it would be economically disastrous to the render, feed, cattle, industries, and for human health.

(human health-they just threw that in cause i was listening. I will now jot down some figures in which they told you, 'no need to write them down'. just hope i have them correct. hmmm, maybe i hope i don't ???)

80% inspection of rendering

*Problem-Complete coverage of rendering HAS NOT occurred.

sizeable number of 1st time FAILED INITIAL INSPECTION, have not been reinspected (70% to 80%).

Compliance critical, Compliance poor in U.K. and other European Firms.

Gloria Dunason Major Assignment 1998 goal TOTAL compliance. This _did not_ occur. Mixed level of compliance, depending on firm.

Rendering FDA license and NON FDA license

system in place for home rendering & feed 76% in compliance 79% cross contamination 21% DID NOT have system 92% record keeping less than 60% total compliance

279 inspectors 185 handling prohibited materials

Renderer at top of pyramid, significant part of compliance. 84% compliance

failed to have caution statement render 72% compliance & cross contamination caution statement on feed, 'DO NOT FEED TO CATTLE'

56 FIRMS NEVER INSPECTED

1240 FDA license feed mills 846 inspected

"close to 400 feed mills have not been inspected"

80% compliance for feed.

10% don't have system.

NON-FDA licensed mills There is NO inventory on non licensed mills. approximately 6000 to 8000 Firms ??? 4,344 ever inspected. "FDA does not have a lot of experience with"

40% do NOT have caution statement 'DO NOT FEED'.

74% Commingling compliance

"This industry needs a lot of work and only half gotten to"

"700 Firms that were falitive, and need to be re-inspected, in addition to the 8,000 Firms."

Quote to do BSE inspection in 19 states by end of January or 30 days, and other states 60 days. to change feed status??? Contract check and ask questions and pass info.

At this time, we will take questions.

[I was about the third or fourth to ask question. then all B.S.eee broke loose, and i lost my train of thought for a few minutes. picked back up here]

someone asking about nutritional supplements and sourcing, did not get name. something about inspectors not knowing of BSE risk??? the conference person assuring that Steve Follum? and the TSE advisory Committee were handling that.

Some other Dr. Vet, whom were asking questions that did not know what to do???

[Dennis Wilson] California Food Agr. Imports, are they looking at imports?

[Conference person] they are looking at imports, FDA issued imports Bulletin.

[Linda Singeltary ??? this was a another phone in question, not related i don't think] Why do we have non-licensed facilities?

(conference person) other feed mills do not handle as potent drugs???

Dennis Blank, Ken Jackson licensed 400 non FDA 4400 inspected of a total of 6000 to 8000,

(they really don't know how many non licensed Firms in U.S. they guess 6000 to 8000??? TSS)

Linda Detwiler asking everyone (me) not to use emergency BSE number, unless last resort. (i thought of calling them today, and reporting the whole damn U.S. cattle herd ;-) 'not'

Warren-Maryland Dept. Agr. Prudent to re-inspect after 3 years. concerned of Firms that have changed owners.

THE END

TSS

############ http://mailhost.rz.uni-karlsruhe.de/warc/bse-l.html ############


FROM New York TIMES

Subject: Re: BSE 50 STATE CONFERENCE CALL thread from BSE List and FDA Posting of cut version...
Date: Thu, 11 Jan 2001 22:02:47 -0700
From: "Sandy Blakeslee"
To: "Terry S. Singeltary Sr." References: 1


Hi terry -- thanks for all your help. I know it made a difference with the FDA getting out that release.


----- Original Message -----
From: "Terry S. Singeltary Sr."
To: Sent: Thursday, January 11, 2001 2:06 PM
Subject: BSE 50 STATE CONFERENCE CALL thread from BSE List and FDA Posting of cut version...

hi sandy,

From the New York Times NYTimes.com, January 11, 2001

Many Makers of Feed Fail to Heed Rules on Mad Cow Disease By SANDRA BLAKESLEE

Large numbers of companies involved in manufacturing animal feed are not complying with regulations meant to prevent the emergence and spread of mad cow disease in the United States, the Food and Drug Administration said yesterday.

The widespread failure of companies to follow the regulations, adopted in August 1997, does not mean that the American food supply is unsafe, Dr. Stephen Sundlof, director of the Center for Veterinary Medicine at the F.D.A., said in an interview.

But much more needs to be done to ensure that mad cow disease does not arise in this country, Dr. Sundlof said.

The regulations state that feed manufacturers and companies that render slaughtered animals into useful products generally may not feed mammals to cud-chewing animals, or ruminants, which can carry mad cow disease.

All products that contain rendered cattle or sheep must have a label that says, "Do not feed to ruminants," Dr. Sundlof said. Manufacturers must also have a system to prevent ruminant products from being commingled with other rendered material like that from chicken, fish or pork. Finally, all companies must keep records of where their products originated and where they were sold.

Under the regulations, F.D.A. district offices and state veterinary offices were required to inspect all rendering plants and feed mills to make sure companies complied. But results issued yesterday demonstrate that more than three years later, different segments of the feed industry show varying levels of compliance.

Among 180 large companies that render cattle and another ruminant, sheep, nearly a quarter were not properly labeling their products and did not have a system to prevent commingling, the F.D.A. said. And among 347 F.D.A.-licensed feed mills that handle ruminant materials - these tend to be large operators that mix drugs into their products - 20 percent were not using labels with the required caution statement, and 25 percent did not have a system to prevent commingling.

Then there are some 6,000 to 8,000 feed mills so small they do not require F.D.A. licenses. They are nonetheless subject to the regulations, and of 1,593 small feed producers that handle ruminant material and have been inspected, 40 percent were not using approved labels and 25 percent had no system in place to prevent commingling.

On the other hand, fewer than 10 percent of companies, big and small, were failing to comply with the record-keeping regulations.

The American Feed Industry Association in Arlington, Va., did not return phone calls seeking comment.

http://www.nytimes.com/2001/01/11/science/11COW.html


Subject: USDA/APHIS response to BSE-L--U.S. 50 STATE CONFERENCE CALL Jan. 9, 2001
Date: Wed, 10 Jan 2001 14:04:21 -0500
From: "Gomez, Thomas M."
Reply-To: Bovine Spongiform Encephalopathy To: BSE-L@uni-karlsruhe.de

######### Bovine Spongiform Encephalopathy #########

USDA/APHIS would like to provide clarification on the following point from Mr. Singeltary's 9 Jan posting regarding the 50 state conference call.

[Linda Detwiler asking everyone (me) not to use emergency BSE number, unless last resort. (i thought of calling them today, and reporting the whole damn U.S. cattle herd ;-) 'not']

Dr. Detwiler was responding to an announcement made during the call to use the FDA emergency number if anyone wanted to report a cow with signs suspect for BSE. Mr. Singeltary is correct that Dr. Detwiler asked participants to use the FDA emergency number as a last resort to report cattle suspect for BSE. What Mr. Singeltary failed to do was provide the List with Dr. Detwiler's entire statement. Surveillance for BSE in the United States is a cooperative effort between states, producers, private veterinarians, veterinary hospitals and the USDA. The system has been in place for over 10 years. Each state has a system in place wherein cases are reported to either the State Veterinarian, the federal Veterinarian in Charge or through the veterinary diagnostic laboratory system. The states also have provisions with emergency numbers. Dr. Detwiler asked participants to use the systems currently in place to avoid the possibility of a BSE-suspect report falling through the cracks. Use of the FDA emergency number has not been established as a means to report diseased cattle of any nature.

############ http://mailhost.rz.uni-karlsruhe.de/warc/bse-l.html ############


Subject: Re: USDA/APHIS response to BSE-L--U.S. 50 STATE CONFERENCE CALL Jan.9, 2001
Date: Wed, 10 Jan 2001 13:44:49 -0800
From: "Terry S. Singeltary Sr."
Reply-To: Bovine Spongiform Encephalopathy
To: BSE-L@uni-karlsruhe.de References: 1


######### Bovine Spongiform Encephalopathy #########


Hello Mr. Thomas,


> What Mr. Singeltary failed to do was provide the List with Dr. Detwiler's entire statement.


would you and the USDA/APHIS be so kind as to supply this list with a full text version of the conference call and or post on your web-site? if so when, and thank you. if not, why not?

The system has been in place for over 10 years.

that seems to be a very long time for a system to be in place, and only test 10,700 cattle from some 1.5 BILLION head (including calf crop). Especially since French are testing some 20,000 weekly and the E.U. as a whole, are testing many many more than the U.S., with less cattle, same risk of BSE/TSEs.

Why does the U.S. insist on not doing massive testing with the tests which the E.U. are using? Why is this, please explain?

Please tell me why my question was not answered?

U.S. cattle, what kind of guarantee can you give for serum or tissue donor herds?

It was a very simple question, a very important question, one that pertained to the topic of BSE/feed, and asked in a very diplomatic way. why was it not answered?

If all these years, we have been hearing that pharmaceutical grade bovines were raised for pharmaceuticals vaccines etc. But yet the USA cannot comply with feed regulations of the ruminant feed ban, PLUS cannot even comply with the proper labelling of the feed, cross contamination etc. Then how in the world can you Guarantee the feed fed to pharmaceutical grade bovine, were actually non ruminant feed?

Before i was ask to be 'disconnected', i did hear someone in the background say 'we can't'-- have him ask the question again.

could you please be so kind, as to answer these questions?

thank you, Terry S. Singeltary Sr. Bacliff, Texas USA

P.S. if you will also notice, i did not post that emergency phone number and do not intend on passing it on to anyone. I was joking when i said i should call and report the whole damn U.S. Herd. So please pass that on to Dr. Detwiler, so she can rest easily.

BUT, they should be reported, some are infected with TSE. The U.S. is just acting as stupid as Germany and other Countries that insist they are free of BSE.

TSS

Subject: Report on the assessment of the Georgraphical BSE-risk of the USA July 2000 (not good)
Date: Wed, 17 Jan 2001 21:23:51 -0800
From: "Terry S. Singeltary Sr."
Reply-To: Bovine Spongiform Encephalopathy To: BSE-L@uni-karlsruhe.de

######### Bovine Spongiform Encephalopathy #########


Greetings List Members and ALL EU Countries,

Because of this report, and the recent findings of the 50-state BSE Conference call, I respectfully seriously suggest that these Countries and the SSC re-evaluate the U.S.A. G.B.R. to a risk factor of #3.

I attempted to post this to list in full text, but would not accept...

thank you, kind regards, Terry S. Singeltary Sr., Bacliff, Texas USA

Report on the assessment of the Geographical BSE-risk of the USA July 2000

PART II

REPORT ON THE ASSESSMENT OF THE GEOGRAPHICAL BSE RISK OF THE UNITED STATES OF AMERICA

- 29 -

Report on the assessment of the Geographical BSE-risk of the USA July 2000

EXECUTIVE SUMMARY

OVERALL ASSESSMENT

The current geographical BSE-risk (GBR) level is II, i.e. it is unlikely but cannot be excluded that domestic cattle are (clinically or pre-clinically) infected with the BSE-agent.

Stability: Before 1990 the system was extremely unstable because feeding of MBM to cattle happened, rendering was inappropriate with regard to deactivation of the BSE-agent and SRM and fallen stock were rendered for feed. From 1990 to 1997 it improved to very unstable, thanks to efforts undertaken to trace imported animals and exclude them from the feed chain and intensive surveillance. In 1998 the system became neutrally stable after the RMBM-ban of 1997.

External challenges: A moderate external challenge occurred in the period before 1990 because of importation of live animals from BSE-affected countries, in particular from the UK and Ireland. It cannot be excluded that some BSE-infected animals have been imported by this route and did enter the US rendering and feed production system. The efforts undertaken since 1990 to trace back UK-imported cattle and to exclude them from the feed chain reduced the impact of the external challenge significantly.

Interaction of external challenges and stability: While extremely unstable, the US system was exposed to a moderate external challenge, mainly resulting from cattle imports from the UK. It can not be excluded that BSE-infectivity entered the country by this route and has been recycled to domestic cattle. The resulting domestic cases would have been processed while the system was still very unstable or unstable and would hence have initiated a number of second or third generation cases. However, the level of the possible domestic prevalence must be below the low detection level of the surveillance in place.

As long as there are no changes in stability or challenge the probability of cattle to be (pre-clinically or clinically) infected with the BSE-agent will remain at the current level.

JUSTIFICATION

1. DATA

The available information was suitable to carry out the GBR risk assessment.

- 30 -

Report on the assessment of the Geographical BSE-risk of the USA July 2000

2. STABILITY

2.1 Overall appreciation of the ability to identify BSE-cases and to eliminate animals at risk of being infected before they are processed

· Before 1989, the ability of the system to identify (and eliminate) BSE cases was limited. · Since 1990 this ability is significantly improved, thanks to a good BSE-surveillance and culling system (contingency plan). · Today the surveillance should be able to detect clinical BSE-cases within the limits set by an essential passive surveillance system, i.e. some cases might remain undetected.

2.2 Overall appreciation of the ability to avoid recycling BSE-infectivity, should it enter processing

· Before 1997 the US rendering and feed producing system would not have been able to avoid recycling of the BSE agent to any measurable extent. If the BSE-agent was introduced the feed chain, it could probably have reached cattle. · After the introduction of the RMBM-to-ruminants-ban in August 1997 the ability of the system to avoid recycling of BSE-infectivity was somewhat increased. It is still rather low due to the rendering system of ruminant material (including SRM and fallen stock) and the persisting potential for cross-contamination of cattle feed with other feeds and hence RMBM.

2.3 Overall assessment of the Stability

· Until 1990 the US BSE/cattle system was extremely unstable as RMBM was commonly fed to cattle, the rendering system was not able to reduce BSE-infectivity and SRM were rendered. This means that incoming BSE infectivity would have been most probably recycled to cattle and amplified and the disease propagated. · Between 1990 and 1995 improvements in the BSE surveillance and the efforts to trace back and remove imported cattle gradually improved the stability but the system remained very unstable. In 1998 the system became unstable because of an RMBM-ban introduced in 1997. After 1998 the ban was fully implemented and the system is regarded to be neutrally stable since 1998. The US system is therefore seen to neither be able to amplify nor to reduce circulating or incoming BSE-infectivity.

3. CHALLENGES

A moderate external challenge occurred in the period 1980-1989 because of importation of live animals from the UK. imports from other countries are regarded to have been negligible challenges. · As a consequence of this external challenge, infectivity could have entered the feed cycle and domestic animals could have been exposed to the agent. These domestic BSE-incubating animals might have again entered processing, leading to an internal challenge since 1991. · This internal challenge could have produced domestic cases of BSE, yet prevalence levels could have been below the detection limits of the surveillance system until now. (According to US calculations, the current surveillance

-31 -

Report on the assessment of the Geographical BSE-risk of the USA July 2000

system could detect clinical incidence of 1-3 cases per year per million adult cattle, i.e. in absolute numbers 43-129 cases per year). Between 1990 und 1995, with the exclusion of the imported animals from Europe from the feed chain, the effect of the external challenges decreased.

4. CONCLUSION ON THE RESULTING RISKS

4.1 Interaction of stability and challenqe

· In the late 80s, early 90s a moderate external challenges met an extremely unstable system. This would have amplified the incoming BSE-infectivity and propagated the disease. · With the exclusion of the imported animals from Europe from the feed chain between 1990 and 1995 the effect of the external challenge decreased. · Before 1998 an internal challenge, if it developed, would have met a still unstable system (inappropriate rendering, no SRM ban, RMBM ban only after 1997) and the BSE-infectivity could have been recycled and amplified. · After 1998 the neutrally stable system could still recycle the BSE-agent but due to the RMBM-ban of 1997 the BSE-infectivity circulating in the system would probably not be amplified.

4.2 Risk that BSE-infectivity enters processing

· A very low processing risk developed in the late 80s when the UK-imports were slaughtered or died. It increased until 1990 because of the higher risk to be infected with BSE of cattle imported from the UK in 1988/89, as these animals could have been processed prior to the back-tracing of the UK-imports in 1990. · From 1990 to 1995 a combination of surviving non-traced UK imports and some domestic (pre-)clinical cases could have arrived at processing resulting in an assumed constant low but non-negligible processing risk. · After 1995 any processing risk relates to assumed domestic cases arriving at processing. · The fact that no domestic cases have been shown-up in the BSE-surveillance is reassuring - it indicates that BSE is in fact not present in the country at levels above the detection limits of the country's surveillance system. This detection level has been calculated according to US-experts to be between 1 & 3 clinical cases per million adult cattle per year.

Note: The high turnover in parts of the dairy cattle population with a young age at slaughter makes it unlikely that fully developed clinical cases would occur (and could be detected) or enter processing. However, the theoretical infective load of the pre-clinical BSE-cases that under this scenario could be processed, can be assumed to remain relatively low.

4.3 Risk that BSE-infectivity is recycled and propagated

· During the period covered by this assessment (1980-1999) the US-system was not able to prevent propagation of BSE should it have entered, even if this ability was significantly improved with the MBM-ban of 1997. · However, since the likelihood that BSE-infectivity entered the system is regarded to be small but non-negligible, the risk that propagation of the disease took place is also small but not negligible.

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Report on the assessment of the Geographical BSE-risk of the USA July 2000

5. CONCLUSION ON THE GEOGRAPHICAL BSE-RISK

5.1 The current GBR

The current geographical BSE-risk (GBR) level is II, i.e. it is unlikely but cannot be excluded that domestic cattle are (clinically or pre-clinically) infected with the BSE-agent.

5.2 The expected development of the GBR

As long as there are no changes in stability or challenge the probability of cattle to be (pre-clinically or clinically) infected with the BSE-agent remains at the current level.

5.3 Recommendations for influencin.q the future GBR

· As long as the stability of the US system is not significantly enbanced above neutral levels it remains critically important to avoid any new external challenges. · All measures that would improve the stability of the system, in particular with regard to its ability to avoid recycling of the BSE-agent should it be present in the cattle population, would reduce, over time, the probability that cattle could be infected with the BSE-agent. Possible actions include: removal of SRMs and/or fallen stock from rendering, better rendering processes, improved compliance with the MBM-ban including control and reduction of cross-contamination. · Results from an improved intensive surveillance programme, targeting at risk sub-populations such as adult cattle in fallen stock or in emergency slaughter, could verify the current assessment.

snip...


http://www.regulations.gov/search/Regs/contentStreamer?objectId=09000064801f3413&disposition=attachment&contentType=msw8





The most recent assessments (and reassessments) were published in June 2005 (Table I; 18), and included the categorisation of Canada, the USA, and Mexico as GBR III. Although only Canada and the USA have reported cases, the historically open system of trade in North America suggests that it is likely that BSE is present also in Mexico.


http://www.oie.int/boutique/extrait/06heim937950.pdf





*** HISTORY OF THE MAD COW CJD BLUNDER IN THE U.S.A. AND THE FOOD, FEED SUPPLY ;



Tuesday, January 26, 2010


Establishing a Fully Integrated National Food Safety System with Strengthened Inspection, Laboratory and Response Capacity Draft 09/24/09


http://fdafailedus.blogspot.com/2010/01/establishing-fully-integrated-national.html





2009-2010 ***



Monday, December 14, 2009 R.I.P. MOM DOD

Similarities between Forms of Sheep Scrapie and Creutzfeldt-Jakob Disease Are Encoded by Distinct Prion Types


> Sporadic CJD type 1 and atypical/ Nor98 scrapie are characterized by fine (reticular) deposits,


http://nor-98.blogspot.com/2009/12/similarities-between-forms-of-sheep.html




see also ;


> All of the Heidenhain variants were of the methionine/ methionine type 1 molecular subtype.


http://cjdusa.blogspot.com/2009/09/co-existence-of-scrapie-prion-protein.html




Thursday, January 07, 2010

Scrapie and Nor-98 Scrapie November 2009 Monthly Report Fiscal Year 2010 and FISCAL YEAR 2008


http://scrapie-usa.blogspot.com/2010/01/scrapie-and-nor-98-scrapie-november.html




Monday, September 1, 2008

RE-FOIA OF DECLARATION OF EXTRAORDINARY EMERGENCY BECAUSE OF AN ATYPICAL T.S.E. (PRION DISEASE) OF FOREIGN ORIGIN IN THE UNITED STATES [No. 00-072-1] September 1, 2008


http://foiamadsheepmadrivervalley.blogspot.com/2008/09/re-foia-of-declaration-of-extraordinary.html




Monday, October 19, 2009

Atypical BSE, BSE, and other human and animal TSE in North America Update October 19, 2009

http://bse-atypical.blogspot.com/2009/10/atypical-bse-bse-and-other-human-and.html




2009 UPDATE ON ALABAMA AND TEXAS MAD COWS 2005 and 2006


http://bse-atypical.blogspot.com/2006/08/bse-atypical-texas-and-alabama-update.html




Monday, November 23, 2009

BSE GBR RISK ASSESSMENTS UPDATE NOVEMBER 23, 2009 COMMISSION OF THE EUROPEAN COMMUNITIES AND O.I.E.


http://docket-aphis-2006-0041.blogspot.com/2009/11/bse-gbr-risk-assessments-update.html




Tuesday, August 11, 2009

Characteristics of Established and Proposed Sporadic Creutzfeldt-Jakob Disease Variants

Brian S. Appleby, MD; Kristin K. Appleby, MD; Barbara J. Crain, MD, PhD; Chiadi U. Onyike, MD, MHS; Mitchell T. Wallin, MD, MPH; Peter V. Rabins, MD, MPH


http://creutzfeldt-jakob-disease.blogspot.com/2009/08/characteristics-of-established-and.html





Sunday, May 10, 2009

Meeting of the Transmissible Spongiform Encephalopathies Committee On June 12, 2009 (Singeltary submission) TO : william.freas@fda.hhs.gov

May 8, 2009

Greetings again Dr. Freas, TSEAC et al,


http://tseac.blogspot.com/2009/05/meeting-of-transmissible-spongiform.html




SEE 2001 SUBMISSION BELOW ;


Freas, William TSS SUBMISSION

Page 1. J Freas, William From: Sent: To: Subject: Terry S. Singeltary

Sr. [flounder@wt.net] Monday, January 08,200l 3:03 PM freas ...


http://www.fda.gov/ohrms/dockets/ac/01/slides/3681s2_09.pdf




Thursday, January 28, 2010

Multiorgan Detection and Characterization of Protease-Resistant Prion Protein in a Case of Variant CJD Examined in the United States


http://creutzfeldt-jakob-disease.blogspot.com/2010/01/multiorgan-detection-and.html




Saturday, June 13, 2009

Monitoring the occurrence of emerging forms of Creutzfeldt-Jakob disease in the United States 2003 revisited 2009


http://cjdusa.blogspot.com/2009/06/monitoring-occurrence-of-emerging-forms.html





*** CJD USA RISING, with UNKNOWN PHENOTYPE ;

5 Includes 41 cases in which the diagnosis is pending, and 17 inconclusive cases; 6 Includes 46 cases with type determination pending in which the diagnosis of vCJD has been excluded.



http://www.cjdsurveillance.com/pdf/case-table.pdf




Saturday, January 2, 2010

Human Prion Diseases in the United States January 1, 2010 ***FINAL***



http://prionunitusaupdate2008.blogspot.com/2010/01/human-prion-diseases-in-united-states.html




my comments to PLosone here ;



http://www.plosone.org/annotation/listThread.action?inReplyTo=info%3Adoi%2F10.1371%2Fannotation%2F04ce2b24-613d-46e6-9802-4131e2bfa6fd&root=info%3Adoi%2F10.1371%2Fannotation%2F04ce2b24-613d-46e6-9802-4131e2bfa6fd






Terry S. Singeltary Sr. P.O. Box 42 Bacliff, Texas USA 77518

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