BSE research project final report 2005 to 2008 SE1796 SID5
Subject: BSE research project final report 2005 to 2008General enquiries on this form should be made to: Defra, Science Directorate, Management Support and Finance Team, Telephone No. 020 7238 1612 E-mail: research.competitions@defra.gsi.gov.uk | ||
SID 5 | Research Project Final Report | |
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Project identification |
1. Defra Project code | SE1796 |
2. Project title
Atypical prion proteins in cattle |
3. Contractor organisation(s) | Molecular Pathogenesis and Genetics Department; Immunochemistry Group Veterinary Laboratories Agency New Haw Surrey KT15 3NB |
54. Total Defra project costs | £ 309,930 |
5. Project: start date.............. | 01 December 2005 |
end date............... | 31 December 2008 |
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Executive Summary |
7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.
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). |
Project Report to Defra |
8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include:
l the scientific objectives as set out in the contract;
l the extent to which the objectives set out in the contract have been met;
l details of methods used and the results obtained, including statistical analysis (if appropriate);
l a discussion of the results and their reliability;
l the main implications of the findings;
l possible future work; and
l any action resulting from the research (e.g. IP, Knowledge Transfer).
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?
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.
References to published material |
9. This section should be used to record links (hypertext links where possible) or references to other
published material generated by, or relating to this project.
published material generated by, or relating to this project.
Arnold, M. E., Ryan, J. B. M., Konold, T., Simmons, M. M., Spencer, Y. I., Wear, A., Chaplin, M., Stack, M., Czub, S., Mueller, R., Webb, P. R., Davis, A., Spiropoulos, J., Holdaway, J., Hawkins, S. A. H., Austin, A. R. and Wells, G. A. H. (2007). Estimating the temporal relationship between PrPsc detection and incubation period in experimental bovine spongiform encephalopathy of cattle. J. Gen Virol, 88: 3198-3208. Barron RM, Campbell SL, King D, Bellon A, Chapman KE, Williamson RA, Manson JC. (2007). High titres of TSE infectivity associated with extrememly low levels of PrPsc in vivo. J Biol Chem 282:35878-35886. Benestad, S. L., Sarradin, P., Thu, B., Schonheit, J., Tranulis, M. A. and Bratberg, B. (2003). Cases of scrapie with unusual features in Norway and designation of a new type, Nor98. Vet Rec 153 (7): 202-208. Biacabe, A. G., Laplanche, J. L., Ryder, S. and Baron, T. (2004). Distinct molecular phenotypes in bovine prion diseases. EMBO Rep. 5 (1): 110-115 Buschmann, A., Biacbe, A. G., Ziegler, U., Bencsik, A., Madec, J. Y., Erhardt, G., Luhken, G., Baron, T. and Groschup, M. H. (2004). Atypical scrapie cases in Germany and France are identified by discrepant reaction patterns in BSE rapid tests. J Virol Methods 117 (1): 27-36. Fraser, H., McConnell, I., Wells, G. A. H. and Dawson, M. (1988). Transmission of bovine spongiform encephalopathy to mice. Vet Rec, 123: 472. McGill, I. S. and Wells, G. A. H. (1993). Neuropathological findings in cattle with clinically suspect but histologically unconfirmed bovine spongiform encephalopathy (BSE). J Comp Path 108: 241-260. Nazor KE, Kuhn F, Seward T, Green M, Zwald D, Pürro M, Schmid J, Biffiger K, Power AM, Oesch B, Raeber AJ, Telling GC. (2005). Immunodetection of disease associated PrP which accelerates disease in GSS transgenic mice. EMBO J 24:2472-2477. Piccardo P, Manson JC, King D, Ghetti B, Barron RM. (2007). Accumulation of prion protein in the brain that is not associated with transmissible disease. PNAS 104:4712-4717. Simmons, M. M., Harris, P., Jeffrey, M., Meek, S. C., Blamire, I. W. and Wells, G. A. (1996). BSE in Great Britain: consistency of the neurohistopathological findings in two random annual samples of clinically suspect cases. Vet Rec, 138 (8): 175-177. Terry, L. A., Jenkins, R., Thorne, L., Everest, S. J., Chaplin, M. J., Davis, L. A. and Stack, M. J. (2007). First case of H-type bovine spongiform encephalopathy identified in Great Britain. Vet Rec 160; 873-875 Wells, G. A. H., Scott, A. C., Johnson, C. T., Gunning, R. F., Hancock, R. D., Jeffrey, M., Dawson, M. and Bradley, R (1987). A novel progressive spongiform encephalopathy in cattle. Vet Rec, 121: 419-420. Yamakawa, Y., Hagiwara, K., Nohtomi, K., Nakamura, Y., Nishijima, M., Higuchi, Y., Sato, Y., Sata, T.; Expert Committee for BSE Diagnosis, Ministry of Health, Labour and Welfare of Japan (2003). Atypical Proteinase K-resistant prion protein (PrPres) observed in an apparently healthy 23-month-old Holstein steer. Jpn J Infect Dis 56 (5-6): 221-222 |
***>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.
***>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.
YOU know, Confucius is confused again LOL, i seem to have remembered something in line with this here in the USA...
USDA did not test possible mad cows
By Steve Mitchell
United Press International
Published 6/8/2004 9:30 PM
WASHINGTON, June 8 (UPI) -- The U.S. Department of Agriculture claims ittested 500 cows with signs of a brain disorder for mad cow disease last year, but agency documents obtained by United Press International show the agency tested only half that number.
"These 9,200 cases were different because brain tissue samples were preserved with formalin, which makes them suitable for only one type of test--immunohistochemistry, or IHC."
THIS WAS DONE FOR A REASON!
THE IHC test has been proven to be the LEAST LIKELY to detect BSE/TSE in the bovine, and these were probably from the most high risk cattle pool, the ones the USDA et al, SHOULD have been testing. ...TSS
TEXAS 2ND MAD COW THAT WAS COVERED UP, AFTER AN ACT OF CONGRESS, AND CALLS FROM TSE PRION SCIENTIST AROUND THE GLOBE, THIS 2ND MAD COW IN TEXAS WAS CONFIRMED
THE USDA MAD COW FOLLIES POSITIVE TEST COVER UP
JOHANNS SECRET POSTIVE MAD COW TEST THAT WERE IGNORED
OIG AND THE HONORABLE FONG CONFIRMS TEXAS MAD AFTER AN ACT OF CONGRESS 7 MONTHS LATER
TEXAS MAD COW
THEY DID FINALLY TEST AFTER SITTING 7+ MONTHS ON A SHELF WHILE GW BORE THE BSE MRR POLICY, i.e. legal trading of all strains of TSE. now understand, i confirmed this case 7 months earlier to the TAHC, and then, only after i contacted the Honorable Phyllis Fong and after an act of Congress, this animal was finally confirmed ;
During the course of the investigation, USDA removed and tested a total of 67 animals of interest from the farm where the index animal's herd originated. All of these animals tested negative for BSE. 200 adult animals of interest were determined to have left the index farm. Of these 200, APHIS officials determined that 143 had gone to slaughter, two were found alive (one was determined not to be of interest because of its age and the other tested negative), 34 are presumed dead, one is known dead and 20 have been classified as untraceable. In addition to the adult animals, APHIS was looking for two calves born to the index animal. Due to record keeping and identification issues, APHIS had to trace 213 calves. Of these 213 calves, 208 entered feeding and slaughter channels, four are presumed to have entered feeding and slaughter channels and one calf was untraceable.
Executive Summary In June 2005, an inconclusive bovine spongiform encephalopathy (BSE) sample from November 2004, that had originally been classified as negative on the immunohistochemistry test, was confirmed positive on SAF immunoblot (Western blot). The U.S. Department of Agriculture (USDA) identified the herd of origin for the index cow in Texas; that identification was confirmed by DNA analysis. USDA, in close cooperation with the Texas Animal Health Commission (TAHC), established an incident command post (ICP) and began response activities according to USDA’s BSE Response Plan of September 2004. Response personnel removed at-risk cattle and cattle of interest (COI) from the index herd, euthanized them, and tested them for BSE; all were negative. USDA and the State extensively traced all at-risk cattle and COI that left the index herd. The majority of these animals entered rendering and/or slaughter channels well before the investigation began. USDA’s response to the Texas finding was thorough and effective.
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Trace Herd 3 The owner of Trace Herd 3 was identified as possibly having received an animal of interest. The herd was placed under hold order on 7/27/05. The herd inventory was conducted on 7/28/05. The animal of interest was not present within the herd, and the hold order was released on 7/28/05. The person who thought he sold the animal to the owner of Trace Herd 3 had no records and could not remember who else he might have sold the cow to. Additionally, a search of GDB for all cattle sold through the markets by that individual did not result in a match to the animal of interest. The animal of interest traced to this herd was classified as untraceable because all leads were exhausted.
Trace Herd 4 The owner of Trace Herd 4 was identified as having received one of the COI through an order buyer. Trace Herd 4 was placed under hold order on 7/29/05. A complete herd inventory was conducted on 8/22/05 and 8/23/05. There were 233 head of cattle that were examined individually by both State and Federal personnel for all man-made identification and brands. The animal of interest was not present within the herd. Several animals were reported to have died in the herd sometime after they arrived on the premises in April 2005. A final search of GDB records yielded no further results on the eartag of interest at either subsequent market sale or slaughter. With all leads having been exhausted, this animal of interest has been classified as untraceable. The hold order on Trace Herd 4 was released on 8/23/05.
Trace Herd 5 The owner of Trace Herd 5 was identified as having received two COI and was placed under hold order on 8/1/05. Trace Herd 5 is made up of 67 head of cattle in multiple pastures. During the course of the herd inventory, the owner located records that indicated that one of the COI, a known birth cohort, had been sold to Trace Herd 8 where she was subsequently found alive. Upon completion of the herd inventory, the other animal of interest was not found within the herd. A GDB search of all recorded herd tests conducted on Trace Herd 5 and all market sales by the owner failed to locate the identification tag of the animal of interest and she was subsequently classified as untraceable due to all leads having been exhausted. The hold order on Trace Herd 5 was released on 8/8/05.
Trace Herd 6 The owner of Trace Herd 6 was identified as possibly having received an animal of interest and was placed under hold order on 8/1/05. This herd is made up of 58 head of cattle on two pastures. A herd inventory was conducted and the animal of interest was not present within the herd. The owner of Trace Herd 6 had very limited records and was unable to provide further information on where the cow might have gone after he purchased her from the livestock market. A search of GDB for all cattle sold through the markets by that individual did not result in a match to the animal of interest. Additionally, many of the animals presented for sale by the owner of the herd had been re-tagged at the market effectually losing the traceability of the history of that animal prior to re-tagging. The animal of interest traced to this herd was classified as untraceable due to all leads having been exhausted. The hold order on Trace Herd 6 was released on 8/3/05.
Trace Herd 7 The owner of Trace Herd 7 was identified as having received an animal of interest and was placed under hold order on 8/1/05. Trace Herd 7 contains 487 head of cattle on multiple pastures in multiple parts of the State, including a unit kept on an island. The island location is a particularly rough place to keep cattle and the owner claimed to have lost 22 head on the island in 2004 due to liver flukes. Upon completion of the herd inventory, the animal of interest was not found present within Trace Herd 7. A GDB search of all recorded herd tests conducted on Trace Herd 7 and all market sales by the owner failed to locate the identification tag of the animal of interest. The cow was subsequently classified as untraceable. It is quite possible though that she may have died within the herd, especially if she belonged to the island unit. The hold order on Trace Herd 7 was released on 8/8/05.
Owner and Corporation Plead Guilty to Defrauding Bovine Spongiform Encephalopathy (BSE) Surveillance Program
An Arizona meat processing company and its owner pled guilty in February 2007 to charges of theft of Government funds, mail fraud, and wire fraud. The owner and his company defrauded the BSE Surveillance Program when they falsified BSE Surveillance Data Collection Forms and then submitted payment requests to USDA for the services. In addition to the targeted sample population (those cattle that were more than 30 months old or had other risk factors for BSE), the owner submitted to USDA, or caused to be submitted, BSE obex (brain stem) samples from healthy USDA-inspected cattle. As a result, the owner fraudulently received approximately $390,000. Sentencing is scheduled for May 2007.
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4 USDA OIG SEMIANNUAL REPORT TO CONGRESS FY 2007 1st Half
Audit Report Animal and Plant Health Inspection Service Bovine Spongiform Encephalopathy (BSE) Surveillance Program – Phase II and Food Safety and Inspection Service Controls Over BSE Sampling, Specified Risk Materials, and Advanced Meat Recovery Products - Phase III
UNITED STATES DEPARTMENT OF AGRICULTURE OFFICE OF INSPECTOR GENERAL Washington, D.C. 20250 January 25, 2006 REPLY TO ATTN OF: 50601-10-KC TO: W. Ron DeHaven Administrator Animal and Plant Health Inspection Service Barbara Masters Administrator Food Safety and Inspection Service ATTN: William J. Hudnall Deputy Administrator Marketing Regulatory Program Business Services William C. Smith Assistant Administrator Office of Program Evaluation, Enforcement, and Review FROM: Robert W. Young /s/ Assistant Inspector General for Audit SUBJECT: Animal and Plant Health Inspection Service - Bovine Spongiform Encephalopathy (BSE) Surveillance Program - Phase II and Food Safety and Inspection Service - Controls Over BSE Sampling, Specified Risk Materials, and Advanced Meat Recovery Products - Phase III This report presents the results of our audit of the enhanced BSE surveillance program and controls over specified risk materials and advanced meat recovery products. Your written response to the official draft report, dated January 20, 2006, is included as exhibit G with excerpts of the response and the Office of Inspector General’s (OIG) position incorporated into the Findings and Recommendations section of the report, where applicable. We accept the management decisions for all recommendations. Please follow your agency’s internal procedures in forwarding documentation for final action to the Office of the Chief Financial Officer (OCFO). We are providing a separate memorandum to the agencies and OCFO that provides specific information on the actions to be completed to achieve final action. We appreciate your timely response and the cooperation and assistance provided to our staff during the audit USDA/OIG-A/50601-10-KC/ Page i
Executive Summary
Animal and Plant Health Inspection Service - Bovine Spongiform Encephalopathy (BSE) Surveillance Program - Phase II and Food Safety and Inspection Service - Controls Over BSE Sampling, Specified Risk Materials, and Advanced Meat Recovery Products - Phase III
Results in Brief This report evaluates elements of the interlocking safeguards in place to protect United States (U.S.) beef from Bovine Spongiform Encephalopathy, widely known as BSE or "mad cow disease." Since 1990, the U.S. Department of Agriculture (USDA), Animal and Plant Health Inspection Service (APHIS), has led a multi-agency effort to monitor and prevent BSE from entering the food supply. After discovering a BSE-positive cow in December 2003, APHIS expanded its BSE surveillance program. To further protect the food supply, USDA banned materials identified as being at risk of carrying BSE (specified risk materials (SRM)), such as central nervous system tissue. As part of this effort, USDA’s Food Safety and Inspection Service (FSIS) required beef slaughter and processing facilities to incorporate controls for handling such materials into their operational plans. Onsite FSIS inspectors also inspect cattle for clinical signs in order to prevent diseased animals from being slaughtered for human consumption. To evaluate the effectiveness of the safeguards, we assessed APHIS’ implementation of the expanded surveillance program, as well as FSIS’ controls to prevent banned SRMs from entering the food supply.
In June 2004, APHIS implemented its expanded surveillance program; participation by industry in this surveillance program is voluntary. As of May 2005, over 350,000 animals were sampled and tested for BSE. To date, two animals tested positive for BSE; one tested positive after implementation of the expanded surveillance program.
USDA made significant efforts to implement the expanded BSE surveillance program. Much needed to be done in a short period of time to establish the necessary processes, controls, infrastructure, and networks to assist in this effort. In addition, extensive outreach and coordination was undertaken with other Federal, State, and local entities, private industry, and laboratory and veterinary networks. This report provides an assessment as to the progress USDA made in expanding its surveillance effort and the effectiveness of its controls and processes. This report also discusses the limitations of its program and data in assessing the prevalence of BSE in the U.S. herd.
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40 ELISA test procedures require two additional (duplicate) tests if the initial test is reactive, before final interpretation. If either of the duplicate tests is reactive, the test is deemed inconclusive.
41 Protocol for BSE Contract Laboratories to Receive and Test Bovine Brain Samples and Report Results for BSE Surveillance Standard Operating Procedure (SOP), dated October 26, 2004.
42 The NVSL conducted an ELISA test on the original material tested at the contract laboratory and on two new cuts from the sample tissue.
43 A visual examination of brain tissue by a microscope.
44 A localized pathological change in a bodily organ or tissue.
SNIP...
PLEASE SEE FLAMING EVIDENCE THAT THE USDA ET AL COVERED UP MAD COW DISEASE IN TEXAS ;
PAGE 43;
Section 2. Testing Protocols and Quality Assurance Controls
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FULL TEXT 130 PAGES
Comments on technical aspects of the risk assessment were then submitted to FSIS.
Comments were received from Food and Water Watch, Food Animal Concerns Trust (FACT), Farm Sanctuary, R-CALF USA, Linda A Detwiler, and Terry S. Singeltary.
This document provides itemized replies to the public comments received on the 2005 updated Harvard BSE risk assessment. Please bear the following points in mind:
Owens, Julie From: Terry S. Singeltary Sr. [flounder9@verizon.net]
Sent: Monday, July 24, 2006 1:09 PM To: FSIS RegulationsComments
Subject: [Docket No. FSIS-2006-0011] FSIS Harvard Risk Assessment of Bovine Spongiform Encephalopathy (BSE) Page 1 of 98 8/3/2006
Greetings FSIS, I would kindly like to comment on the following ;
Suppressed peer review of Harvard study October 31, 2002.
October 31, 2002 Review of the Evaluation of the Potential for Bovine Spongiform Encephalopathy in the United States Conducted by the Harvard Center for Risk Analysis, Harvard School of Public Health and Center for Computational Epidemiology, College of Veterinary Medicine, Tuskegee University Final Report Prepared for U.S. Department of Agriculture Food Safety and Inspection Service Office of Public Health and Science Prepared by RTI Health, Social, and Economics Research Research Triangle Park, NC 27709 RTI Project Number 07182.024
Sunday, February 14, 2010
[Docket No. FSIS-2006-0011] FSIS Harvard Risk Assessment of Bovine Spongiform Encephalopathy (BSE)
Thursday, April 9, 2009
Docket No. FDA2002N0031 (formerly Docket No. 2002N0273) RIN 0910AF46 Substances Prohibited From Use in Animal Food or Feed; Final Rule: Proposed Thursday, April 09, 2009
Docket No. FDA2002N0031 (formerly Docket No. 2002N0273) RIN 0910AF46 Substances Prohibited From Use in Animal Food or Feed; Final Rule: Proposed
Greetings FDA et al,
I kindly wish to comment on the following ;
[Docket No. FDA-2002-N-0031] (formerly Docket No. 2002N-0273) RIN 0910-AF46
[Federal Register: April 9, 2009 (Volume 74, Number 67)] [Proposed Rules] [Page 16160-16161] From the Federal Register Online via GPO Access [wais.access.gpo.gov] [DOCID:fr09ap09-18]
Tuesday, September 14, 2010
Transmissible Spongiform Encephalopathies Advisory Committee; Notice of Meeting October 28 and 29, 2010 (COMMENT SUBMISSION)
FULL TEXT OF GOA REPORT BELOW (takes a while to load)
2. Mad Cow Disease: Improvements in the Animal Feed Ban and Other Regulatory Areas Would Strengthen U.S. Prevention Efforts. GAO-02-183, January 25.
8 hr BSE confirmation turnaround took 7+ months to confirm this case, so the BSE MRR policy could be put into place. ...TSS
-------- Original Message --------
Subject: re-USDA's surveillance plan for BSE aka mad cow disease
Date: Mon, 02 May 2005 16:59:07 -0500
From: "Terry S. Singeltary Sr."
Greetings Honorable Paul Feeney, Keith Arnold, and William Busbyet al at OIG, ...............
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There will be several more emails of my research to follow. I respectfully request a full inquiry into the cover-up of TSEs in the United States of America over the past 30 years. I would be happy to testify...
Thank you, I am sincerely, Terry S. Singeltary Sr. P.O. Box , Bacliff, Texas USA 77518 xxx xxx xxxx
Date: June 14, 2005 at 1:46 pm PST
In Reply to:
Re: Transcript Ag. Secretary Mike Johanns and Dr. John Clifford, Regarding further analysis of BSE Inconclusive Test Results
posted by TSS on June 13, 2005 at 7:33 pm:
Secretary of Agriculture Ann M. Veneman resigns Nov 15 2004, three days later inclusive Mad Cow is announced. June 7th 2005 Bill Hawks Under Secretary for Marketing and Regulatory Programs resigns. Three days later same mad cow found in November turns out to be positive. Both resignation are unexpected. just pondering... TSS
*** 2009 UPDATE ON ALABAMA AND TEXAS MAD COWS 2005 and 2006 ***
THURSDAY, OCTOBER 22, 2015
Former Ag Secretary Ann Veneman talks women in agriculture and we talk mad cow disease USDA and what really happened
THURSDAY, FEBRUARY 23, 2012
EIGHT FORMER SECRETARIES OF AGRICULTURE SPEAKING AT USDA'S 2012 AGRICULTURE OUTLOOK FORUM INDUCTED INTO USA MAD COW HALL OF SHAME
2020 DECEMBER
WEDNESDAY, DECEMBER 9, 2020
Biden's pick Tom Vilsack Failed Terribly on Mad Cow BSE TSE Prion, why put him back as top Agriculture pick?
Transmissible Spongiform Encephalopathy TSE Prion End of Year Report
CJD FOUNDATION VIRTUAL CONFERENCE CJD Foundation Research Grant Recipient Reports Panel 2 Nov 3, 2020
zoonotic potential of PMCA-adapted CWD PrP 96SS inoculum
4 different CWD strains, and these 4 strains have different potential to induce any folding of the human prion protein.
***> PIGS, WILD BOAR, CWD <***
***> POPULATIONS OF WILD BOARS IN THE UNITED STATES INCREASING SUPSTANTUALLY AND IN MANY AREAS WE CAN SEE A HIGH DENSITY OF WILD BOARS AND HIGH INCIDENT OF CHRONIC WASTING DISEASE
HYPOTHOSIS AND SPECIFIC AIMS
HYPOTHOSIS
BSE, SCRAPIE, AND CWD, EXPOSED DOMESTIC PIGS ACCUMULATE DIFFERENT QUANTITIES AND STRAINS OF PRIONS IN PERIPHERAL TISSUES, EACH ONE OF THEM WITH PARTICULAR ZOONOTIC POTENTIALS
Final Report – CJD Foundation Grant Program A.
Project Title: Systematic evaluation of the zoonotic potential of different CWD isolates. Principal Investigator: Rodrigo Morales, PhD.
Systematic evaluation of the zoonotic potential of different CWD isolates. Rodrigo Morales, PhD Assistant Professor Protein Misfolding Disorders lab Mitchell Center for Alzheimer’s disease and Related Brain Disorders Department of Neurology University of Texas Health Science Center at Houston Washington DC. July 14th, 2018
Conclusions and Future Directions • We have developed a highly sensitive and specific CWD-PMCA platform to be used as a diagnostic tool. • Current PMCA set up allow us to mimic relevant prion inter-species transmission events. • Polymorphic changes at position 96 of the prion protein apparently alter strain properties and, consequently, the zoonotic potential of CWD isolates. • Inter-species and inter-polymorphic PrPC → PrPSc conversions further increase the spectrum of CWD isolates possibly present in nature. • CWD prions generated in 96SS PrPC substrate apparently have greater inter-species transmission potentials. • Future experiments will explore the zoonotic potential of CWD prions along different adaptation scenarios, including inter-species and inter-polymorphic.
Research Project: TRANSMISSION, DIFFERENTIATION, AND PATHOBIOLOGY OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES Location: Virus and Prion Research
Title: Disease-associated prion protein detected in lymphoid tissues from pigs challenged with the agent of chronic wasting disease
Author item MOORE, SARAH - Orise Fellow item Kunkle, Robert item KONDRU, NAVEEN - Iowa State University item MANNE, SIREESHA - Iowa State University item SMITH, JODI - Iowa State University item KANTHASAMY, ANUMANTHA - Iowa State University item WEST GREENLEE, M - Iowa State University item Greenlee, Justin Submitted to: Prion Publication Type: Abstract Only Publication Acceptance Date: 3/15/2017 Publication Date: N/A Citation: N/A Interpretive Summary:
Technical Abstract: Aims: Chronic wasting disease (CWD) is a naturally-occurring, fatal neurodegenerative disease of cervids. We previously demonstrated that disease-associated prion protein (PrPSc) can be detected in the brain and retina from pigs challenged intracranially or orally with the CWD agent. In that study, neurological signs consistent with prion disease were observed only in one pig: an intracranially challenged pig that was euthanized at 64 months post-challenge. The purpose of this study was to use an antigen-capture immunoassay (EIA) and real-time quaking-induced conversion (QuIC) to determine whether PrPSc is present in lymphoid tissues from pigs challenged with the CWD agent.
Methods: At two months of age, crossbred pigs were challenged by the intracranial route (n=20), oral route (n=19), or were left unchallenged (n=9). At approximately 6 months of age, the time at which commercial pigs reach market weight, half of the pigs in each group were culled (<6 month challenge groups). The remaining pigs (>6 month challenge groups) were allowed to incubate for up to 73 months post challenge (mpc). The retropharyngeal lymph node (RPLN) was screened for the presence of PrPSc by EIA and immunohistochemistry (IHC). The RPLN, palatine tonsil, and mesenteric lymph node (MLN) from 6-7 pigs per challenge group were also tested using EIA and QuIC.
Results: PrPSc was not detected by EIA and IHC in any RPLNs. All tonsils and MLNs were negative by IHC, though the MLN from one pig in the oral <6 month group was positive by EIA. PrPSc was detected by QuIC in at least one of the lymphoid tissues examined in 5/6 pigs in the intracranial <6 months group, 6/7 intracranial >6 months group, 5/6 pigs in the oral <6 months group, and 4/6 oral >6 months group. Overall, the MLN was positive in 14/19 (74%) of samples examined, the RPLN in 8/18 (44%), and the tonsil in 10/25 (40%).
Conclusions: This study demonstrates that PrPSc accumulates in lymphoid tissues from pigs challenged intracranially or orally with the CWD agent, and can be detected as early as 4 months after challenge. CWD-infected pigs rarely develop clinical disease and if they do, they do so after a long incubation period. This raises the possibility that CWD-infected pigs could shed prions into their environment long before they develop clinical disease. Furthermore, lymphoid tissues from CWD-infected pigs could present a potential source of CWD infectivity in the animal and human food chains.
Research Project: Pathobiology, Genetics, and Detection of Transmissible Spongiform Encephalopathies Location: Virus and Prion Research
Title: The agent of chronic wasting disease from pigs is infectious in transgenic mice expressing human PRNP
Author item MOORE, S - Orise Fellow item Kokemuller, Robyn item WEST-GREENLEE, M - Iowa State University item BALKEMA-BUSCHMANN, ANNE - Friedrich-Loeffler-institut item GROSCHUP, MARTIN - Friedrich-Loeffler-institut item Greenlee, Justin Submitted to: Prion Publication Type: Abstract Only Publication Acceptance Date: 5/10/2018 Publication Date: 5/22/2018 Citation: Moore, S.J., Kokemuller, R.D., West-Greenlee, M.H., Balkema-Buschmann, A., Groschup, M.H., Greenlee, J.J. 2018. The agent of chronic wasting disease from pigs is infectious in transgenic mice expressing human PRNP. Prion 2018, Santiago de Compostela, Spain, May 22-25, 2018. Paper No. WA15, page 44.
Interpretive Summary:
Technical Abstract: We have previously shown that the chronic wasting disease (CWD) agent from white-tailed deer can be transmitted to domestic pigs via intracranial or oral inoculation although with low attack rates and restricted PrPSc accumulation. The objective of this study was to assess the potential for cross-species transmission of pig-passaged CWD using bioassay in transgenic mice. Transgenic mice expressing human (Tg40), bovine (TgBovXV) or porcine (Tg002) PRNP were inoculated intracranially with 1% brain homogenate from a pig that had been intracranially inoculated with a pool of CWD from white-tailed deer. This pig developed neurological clinical signs, was euthanized at 64 months post-inoculation, and PrPSc was detected in the brain. Mice were monitored daily for clinical signs of disease until the end of the study. Mice were considered positive if PrPSc was detected in the brain using an enzyme immunoassay (EIA). In transgenic mice expressing porcine prion protein the average incubation period was 167 days post-inoculation (dpi) and 3/27 mice were EIA positive (attack rate = 11%). All 3 mice were found dead and clinical signs were not noted prior to death. One transgenic mouse expressing bovine prion protein was euthanized due to excessive scratching at 617 dpi and 2 mice culled at the end of the study at 700 dpi were EIA positive resulting in an overall attack rate of 3/16 (19%). None of the transgenic mice expressing human prion protein that died or were euthanized up to 769 dpi were EIA positive and at study end point at 800 dpi 2 mice had positive EIA results (overall attack rate = 2/20 = 10%). The EIA optical density (OD) readings for all positive mice were at the lower end of the reference range (positive mice range, OD = 0.266-0.438; test positive reference range, OD = 0.250-4.000). To the authors’ knowledge, cervid-derived CWD isolates have not been successfully transmitted to transgenic mice expressing human prion protein. The successful transmission of pig-passaged CWD to Tg40 mice reported here suggests that passage of the CWD agent through pigs results in a change of the transmission characteristics which reduces the transmission barrier of Tg40 mice to the CWD agent. If this biological behavior is recapitulated in the original host species, passage of the CWD agent through pigs could potentially lead to increased pathogenicity of the CWD agent in humans.
cwd scrapie pigs oral routes
***> However, at 51 months of incubation or greater, 5 animals were positive by one or more diagnostic methods. Furthermore, positive bioassay results were obtained from all inoculated groups (oral and intracranial; market weight and end of study) suggesting that swine are potential hosts for the agent of scrapie. <***
>*** Although the current U.S. feed ban is based on keeping tissues from TSE infected cattle from contaminating animal feed, swine rations in the U.S. could contain animal derived components including materials from scrapie infected sheep and goats. These results indicating the susceptibility of pigs to sheep scrapie, coupled with the limitations of the current feed ban, indicates that a revision of the feed ban may be necessary to protect swine production and potentially human health. <***
***> Results: PrPSc was not detected by EIA and IHC in any RPLNs. All tonsils and MLNs were negative by IHC, though the MLN from one pig in the oral <6 month group was positive by EIA. PrPSc was detected by QuIC in at least one of the lymphoid tissues examined in 5/6 pigs in the intracranial <6 months group, 6/7 intracranial >6 months group, 5/6 pigs in the oral <6 months group, and 4/6 oral >6 months group. Overall, the MLN was positive in 14/19 (74%) of samples examined, the RPLN in 8/18 (44%), and the tonsil in 10/25 (40%).
***> Conclusions: This study demonstrates that PrPSc accumulates in lymphoid tissues from pigs challenged intracranially or orally with the CWD agent, and can be detected as early as 4 months after challenge. CWD-infected pigs rarely develop clinical disease and if they do, they do so after a long incubation period. This raises the possibility that CWD-infected pigs could shed prions into their environment long before they develop clinical disease. Furthermore, lymphoid tissues from CWD-infected pigs could present a potential source of CWD infectivity in the animal and human food chains.
MONDAY, NOVEMBER 23, 2020
***> Chronic Wasting Disease CWD TSE Prion Cervid State by State and Global Update November 2020
MONDAY, NOVEMBER 30, 2020
***> REPORT OF THE MEETING OF THE OIE SCIENTIFIC COMMISSION FOR ANIMAL DISEASES Paris, 9–13 September 2019 BSE, TSE, PRION
see updated concerns with atypical BSE from feed and zoonosis...terry
Monday, November 30, 2020
Tunisia has become the second country after Algeria to detect a case of CPD within a year
TUESDAY, NOVEMBER 17, 2020
The European Union summary report on surveillance for the presence of transmissible spongiform encephalopathies (TSE) in 2019 First published 17 November 2020
WEDNESDAY, OCTOBER 28, 2020
EFSA Annual report of the Scientific Network on BSE-TSE 2020 Singeltary Submission
WEDNESDAY, DECEMBER 2, 2020
EFSA Evaluation of public and animal health risks in case of a delayed post-mortem inspection in ungulates EFSA Panel on Biological Hazards (BIOHAZ) ADOPTED: 21 October 2020
i wonder if a 7 month delay on a suspect BSE case in Texas is too long, on a 48 hour turnaround, asking for a friend???
I URGE EVERYONE TO READ IN FULL, THE OIE REPORT 2019 ABOUT ATYPICAL BSE TSE PRION, SRMs, SBOs, and feed...tss
''Experts could not rule out other causes due to the difficulty of investigating individual cases. Some constraints are the long incubation period of the disease and the lack of detailed information available from farms at the time of the trace-back investigation.''
Scientists investigate origin of isolated BSE cases
The European response to bovine spongiform encephalopathy (BSE) after the crisis of the 1980s has significantly reduced prevalence of the disease in cattle. However, isolated cases are still being reported in the EU and for this reason the European Commission asked EFSA to investigate their origin.
The key measure for controlling BSE in the EU is a ban on the use of animal proteins in livestock feed. This is because BSE can be transmitted to cattle through contaminated feed, mainly in the first year of life.
Sixty cases of classical BSE have been reported in cattle born after the EU ban was enforced in 2001. None of these animals entered the food chain. Classical BSE is the type of BSE transmissible to humans. The Commission asked EFSA to determine if these cases were caused by contaminated feed or whether they occurred spontaneously, i.e. without an apparent cause.
EFSA experts concluded that contaminated feed is the most likely source of infection. This is because the infectious agent that causes BSE has the ability to remain active for many years. Cattle may have been exposed to contaminated feed because the BSE infectious agent was present where feed was stored or handled. A second possibility is that contaminated feed ingredients may have been imported from non-EU countries.
Experts could not rule out other causes due to the difficulty of investigating individual cases. Some constraints are the long incubation period of the disease and the lack of detailed information available from farms at the time of the trace-back investigation.
EFSA experts made a series of recommendations to maintain and strengthen the EU monitoring and reporting system, and to evaluate new scientific data that become available.
The European response to BSE
The coordinated European response to BSE has succeeded in reducing the prevalence of the disease. Between 2005 and 2015 about 73,000,000 cattle were tested for BSE in the EU, out of which 60 born after the ban tested positive for classical BSE. The number of affected animals rises to 1,259 if cattle born before the ban are included. The number of classical BSE cases has dropped significantly in the EU over time, from 554 cases reported in 2005 to just two in 2015 (both animals born after the ban). Moreover the EU food safety system is designed to prevent the entry of BSE-contaminated meat into the food chain.
THURSDAY, SEPTEMBER 26, 2019
Veterinary Biologics Guideline 3.32E: Guideline for minimising the risk of introducing transmissible spongiform encephalopathy prions and other infectious agents through veterinary biologics
U.S.A. 50 STATE BSE MAD COW CONFERENCE CALL Jan. 9, 2001
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
snip...
[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
snip...see full archive and more of this;
THURSDAY, DECEMBER 17, 2020
THE MAD COW BSE TSE PRION THAT STOLE CHRISTMAS DECEMBER 2003, WHAT REALLY HAPPENED, A REVIEW 2020
WEDNESDAY, DECEMBER 23, 2020
Idiopathic Brainstem Neuronal Chromatolysis IBNC BSE TSE Prion a Review 2020
Transmissible Spongiform Encephalopathy TSE Prion End of Year Report
CJD FOUNDATION VIRTUAL CONFERENCE CJD Foundation Research Grant Recipient Reports Panel 2 Nov 3, 2020
zoonotic potential of PMCA-adapted CWD PrP 96SS inoculum
4 different CWD strains, and these 4 strains have different potential to induce any folding of the human prion protein.
***> PIGS, WILD BOAR, CWD <***
***> POPULATIONS OF WILD BOARS IN THE UNITED STATES INCREASING SUPSTANTUALLY AND IN MANY AREAS WE CAN SEE A HIGH DENSITY OF WILD BOARS AND HIGH INCIDENT OF CHRONIC WASTING DISEASE
HYPOTHOSIS AND SPECIFIC AIMS
HYPOTHOSIS
BSE, SCRAPIE, AND CWD, EXPOSED DOMESTIC PIGS ACCUMULATE DIFFERENT QUANTITIES AND STRAINS OF PRIONS IN PERIPHERAL TISSUES, EACH ONE OF THEM WITH PARTICULAR ZOONOTIC POTENTIALS
Final Report – CJD Foundation Grant Program A.
Project Title: Systematic evaluation of the zoonotic potential of different CWD isolates. Principal Investigator: Rodrigo Morales, PhD.
Systematic evaluation of the zoonotic potential of different CWD isolates. Rodrigo Morales, PhD Assistant Professor Protein Misfolding Disorders lab Mitchell Center for Alzheimer’s disease and Related Brain Disorders Department of Neurology University of Texas Health Science Center at Houston Washington DC. July 14th, 2018
Conclusions and Future Directions • We have developed a highly sensitive and specific CWD-PMCA platform to be used as a diagnostic tool. • Current PMCA set up allow us to mimic relevant prion inter-species transmission events. • Polymorphic changes at position 96 of the prion protein apparently alter strain properties and, consequently, the zoonotic potential of CWD isolates. • Inter-species and inter-polymorphic PrPC → PrPSc conversions further increase the spectrum of CWD isolates possibly present in nature. • CWD prions generated in 96SS PrPC substrate apparently have greater inter-species transmission potentials. • Future experiments will explore the zoonotic potential of CWD prions along different adaptation scenarios, including inter-species and inter-polymorphic.
Research Project: TRANSMISSION, DIFFERENTIATION, AND PATHOBIOLOGY OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES Location: Virus and Prion Research
Title: Disease-associated prion protein detected in lymphoid tissues from pigs challenged with the agent of chronic wasting disease
Author item MOORE, SARAH - Orise Fellow item Kunkle, Robert item KONDRU, NAVEEN - Iowa State University item MANNE, SIREESHA - Iowa State University item SMITH, JODI - Iowa State University item KANTHASAMY, ANUMANTHA - Iowa State University item WEST GREENLEE, M - Iowa State University item Greenlee, Justin Submitted to: Prion Publication Type: Abstract Only Publication Acceptance Date: 3/15/2017 Publication Date: N/A Citation: N/A Interpretive Summary:
Technical Abstract: Aims: Chronic wasting disease (CWD) is a naturally-occurring, fatal neurodegenerative disease of cervids. We previously demonstrated that disease-associated prion protein (PrPSc) can be detected in the brain and retina from pigs challenged intracranially or orally with the CWD agent. In that study, neurological signs consistent with prion disease were observed only in one pig: an intracranially challenged pig that was euthanized at 64 months post-challenge. The purpose of this study was to use an antigen-capture immunoassay (EIA) and real-time quaking-induced conversion (QuIC) to determine whether PrPSc is present in lymphoid tissues from pigs challenged with the CWD agent.
Methods: At two months of age, crossbred pigs were challenged by the intracranial route (n=20), oral route (n=19), or were left unchallenged (n=9). At approximately 6 months of age, the time at which commercial pigs reach market weight, half of the pigs in each group were culled (<6 month challenge groups). The remaining pigs (>6 month challenge groups) were allowed to incubate for up to 73 months post challenge (mpc). The retropharyngeal lymph node (RPLN) was screened for the presence of PrPSc by EIA and immunohistochemistry (IHC). The RPLN, palatine tonsil, and mesenteric lymph node (MLN) from 6-7 pigs per challenge group were also tested using EIA and QuIC.
Results: PrPSc was not detected by EIA and IHC in any RPLNs. All tonsils and MLNs were negative by IHC, though the MLN from one pig in the oral <6 month group was positive by EIA. PrPSc was detected by QuIC in at least one of the lymphoid tissues examined in 5/6 pigs in the intracranial <6 months group, 6/7 intracranial >6 months group, 5/6 pigs in the oral <6 months group, and 4/6 oral >6 months group. Overall, the MLN was positive in 14/19 (74%) of samples examined, the RPLN in 8/18 (44%), and the tonsil in 10/25 (40%).
Conclusions: This study demonstrates that PrPSc accumulates in lymphoid tissues from pigs challenged intracranially or orally with the CWD agent, and can be detected as early as 4 months after challenge. CWD-infected pigs rarely develop clinical disease and if they do, they do so after a long incubation period. This raises the possibility that CWD-infected pigs could shed prions into their environment long before they develop clinical disease. Furthermore, lymphoid tissues from CWD-infected pigs could present a potential source of CWD infectivity in the animal and human food chains.
Research Project: Pathobiology, Genetics, and Detection of Transmissible Spongiform Encephalopathies Location: Virus and Prion Research
Title: The agent of chronic wasting disease from pigs is infectious in transgenic mice expressing human PRNP
Author item MOORE, S - Orise Fellow item Kokemuller, Robyn item WEST-GREENLEE, M - Iowa State University item BALKEMA-BUSCHMANN, ANNE - Friedrich-Loeffler-institut item GROSCHUP, MARTIN - Friedrich-Loeffler-institut item Greenlee, Justin Submitted to: Prion Publication Type: Abstract Only Publication Acceptance Date: 5/10/2018 Publication Date: 5/22/2018 Citation: Moore, S.J., Kokemuller, R.D., West-Greenlee, M.H., Balkema-Buschmann, A., Groschup, M.H., Greenlee, J.J. 2018. The agent of chronic wasting disease from pigs is infectious in transgenic mice expressing human PRNP. Prion 2018, Santiago de Compostela, Spain, May 22-25, 2018. Paper No. WA15, page 44.
Interpretive Summary:
Technical Abstract: We have previously shown that the chronic wasting disease (CWD) agent from white-tailed deer can be transmitted to domestic pigs via intracranial or oral inoculation although with low attack rates and restricted PrPSc accumulation. The objective of this study was to assess the potential for cross-species transmission of pig-passaged CWD using bioassay in transgenic mice. Transgenic mice expressing human (Tg40), bovine (TgBovXV) or porcine (Tg002) PRNP were inoculated intracranially with 1% brain homogenate from a pig that had been intracranially inoculated with a pool of CWD from white-tailed deer. This pig developed neurological clinical signs, was euthanized at 64 months post-inoculation, and PrPSc was detected in the brain. Mice were monitored daily for clinical signs of disease until the end of the study. Mice were considered positive if PrPSc was detected in the brain using an enzyme immunoassay (EIA). In transgenic mice expressing porcine prion protein the average incubation period was 167 days post-inoculation (dpi) and 3/27 mice were EIA positive (attack rate = 11%). All 3 mice were found dead and clinical signs were not noted prior to death. One transgenic mouse expressing bovine prion protein was euthanized due to excessive scratching at 617 dpi and 2 mice culled at the end of the study at 700 dpi were EIA positive resulting in an overall attack rate of 3/16 (19%). None of the transgenic mice expressing human prion protein that died or were euthanized up to 769 dpi were EIA positive and at study end point at 800 dpi 2 mice had positive EIA results (overall attack rate = 2/20 = 10%). The EIA optical density (OD) readings for all positive mice were at the lower end of the reference range (positive mice range, OD = 0.266-0.438; test positive reference range, OD = 0.250-4.000). To the authors’ knowledge, cervid-derived CWD isolates have not been successfully transmitted to transgenic mice expressing human prion protein. The successful transmission of pig-passaged CWD to Tg40 mice reported here suggests that passage of the CWD agent through pigs results in a change of the transmission characteristics which reduces the transmission barrier of Tg40 mice to the CWD agent. If this biological behavior is recapitulated in the original host species, passage of the CWD agent through pigs could potentially lead to increased pathogenicity of the CWD agent in humans.
cwd scrapie pigs oral routes
***> However, at 51 months of incubation or greater, 5 animals were positive by one or more diagnostic methods. Furthermore, positive bioassay results were obtained from all inoculated groups (oral and intracranial; market weight and end of study) suggesting that swine are potential hosts for the agent of scrapie. <***
>*** Although the current U.S. feed ban is based on keeping tissues from TSE infected cattle from contaminating animal feed, swine rations in the U.S. could contain animal derived components including materials from scrapie infected sheep and goats. These results indicating the susceptibility of pigs to sheep scrapie, coupled with the limitations of the current feed ban, indicates that a revision of the feed ban may be necessary to protect swine production and potentially human health. <***
***> Results: PrPSc was not detected by EIA and IHC in any RPLNs. All tonsils and MLNs were negative by IHC, though the MLN from one pig in the oral <6 month group was positive by EIA. PrPSc was detected by QuIC in at least one of the lymphoid tissues examined in 5/6 pigs in the intracranial <6 months group, 6/7 intracranial >6 months group, 5/6 pigs in the oral <6 months group, and 4/6 oral >6 months group. Overall, the MLN was positive in 14/19 (74%) of samples examined, the RPLN in 8/18 (44%), and the tonsil in 10/25 (40%).
***> Conclusions: This study demonstrates that PrPSc accumulates in lymphoid tissues from pigs challenged intracranially or orally with the CWD agent, and can be detected as early as 4 months after challenge. CWD-infected pigs rarely develop clinical disease and if they do, they do so after a long incubation period. This raises the possibility that CWD-infected pigs could shed prions into their environment long before they develop clinical disease. Furthermore, lymphoid tissues from CWD-infected pigs could present a potential source of CWD infectivity in the animal and human food chains.
MONDAY, NOVEMBER 23, 2020
***> Chronic Wasting Disease CWD TSE Prion Cervid State by State and Global Update November 2020
MONDAY, NOVEMBER 30, 2020
***> REPORT OF THE MEETING OF THE OIE SCIENTIFIC COMMISSION FOR ANIMAL DISEASES Paris, 9–13 September 2019 BSE, TSE, PRION
see updated concerns with atypical BSE from feed and zoonosis...terry
Monday, November 30, 2020
Tunisia has become the second country after Algeria to detect a case of CPD within a year
TUESDAY, NOVEMBER 17, 2020
The European Union summary report on surveillance for the presence of transmissible spongiform encephalopathies (TSE) in 2019 First published 17 November 2020
WEDNESDAY, OCTOBER 28, 2020
EFSA Annual report of the Scientific Network on BSE-TSE 2020 Singeltary Submission
WEDNESDAY, OCTOBER 28, 2020
EFSA Scientific Opinion Potential BSE risk posed by the use of ruminant collagen and gelatine in feed for non‐ruminant farmed animals
WEDNESDAY, DECEMBER 2, 2020
EFSA Evaluation of public and animal health risks in case of a delayed post-mortem inspection in ungulates EFSA Panel on Biological Hazards (BIOHAZ) ADOPTED: 21 October 2020
i wonder if a 7 month delay on a suspect BSE case in Texas is too long, on a 48 hour turnaround, asking for a friend???
TUESDAY, DECEMBER 01, 2020
Sporadic Creutzfeldt Jakob Disease sCJD and Human TSE Prion Annual Report December 14, 2020
Terry S. Singeltary Sr.
Bacliff, Texas USA 77518
