Biodegradation of bovine spongiform encephalopathy prions in compost
Shanwei Xu1*, SujeemaAbeysekara2 , Sandor Dudas3 , Stefanie Czub3 , Antanas Staskevicius4 , Gordon Mitchell4 , Kingsley K.Amoako5 & Tim A. McAllister2
To reduce the transmission risk of bovine spongiform encephalopathy prions (PrPBSE), specified risk materials (SRM) that can harbour PrPBSE are prevented from entering the feed and food chains. As composting is one approach to disposing of SRM, we investigated the inactivation of PrPBSE in lab-scale composters over 28 days and in bin composters over 106–120 days. Lab-scale composting was conducted using 45 kg of feedlot manure with and without chicken feathers. Based on protein misfolding cyclic amplification (PMCA), after 28 days of composting, PrPBSE seeding activity was reduced by 3–4 log10 with feathers and 3 log10 without. Bin composters were constructed using~ 2200 kg feedlot manure and repeated in 2017 and 2018. PMCA results showed that seeding activity of PrPBSE was reduced by 1–2 log10 in the centre, but only by 1 log10 in the bottom of bin composters. Subsequent assessment by transgenic (Tgbov XV) mouse bioassay confirmed a similar reduction in PrPBSE infectivity. Enrichment for proteolytic microorganisms through the addition of feathers to compost could enhance PrPBSE degradation. In addition to temperature, other factors including varying concentrations of PrPBSE and the nature of proteolytic microbial populations may be responsible for differential degradation of PrPBSE during composting.
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Discussion
Improving on analytical sensitivity, amplification assays such as PMCA have been used to detect trace amounts of PrPTSE in saliva, tongue, nasal mucosa, palatine tonsils, lymph nodes, ileocecal and muscular tissues of TSE infected animals27–29. However, quantification of PrPTSE in compost presents a significant challenge, due to the highly hydrophobic nature of PrPTSE and their tendency to aggregate into plaque-like complexes that exhibit a high affinity for soil particles30–32. Among detergents, SDS has been used to release PrPTSE from soil, manure, wood, metal and plastic for subsequent detection by WB18,33,34. Therefore, incorporation of PMCA after SDS extraction of PrPTSE could provide an ultrasensitive method to quantify PrPTSE degradation in compost. Currently, PMCA has been successfully applied to examine the contamination and persistence of PrPTSE in soil, wheat grass, wood, plastic, cement, metals, stream and wastewater35–38. Our research team successfully employed PMCA with SDS extraction to quantify a declined PrPTSE seeding activity with 2 log10 reduction in PrP263K and 3 log10 reduction in PrPCWD after 28 days of lab-scale composting18. However, we are unaware of studies that have used PMCA to assess the potential degradation of PrPBSE in compost. In the current study, we established a PMCA assay that could detect manure-bound PrPBSE at a 3–4 log10 sensitivity afer two rounds of PMCA, enabling us to estimate the biodegradation of PrPBSE in both lab and bin composters.
Animal bioassays represent the most definitive test for assessing the inactivation of PrPTSE. Although bioassays are expensive and require a considerable period of time to generate results39, their ability to measure the infectivity of prions is required to comprehensively assess PrPTSE infectivity after composting and to substantiate PMCA results. Stainless steel has a high affinity for prions, which can result in the transmission of prion disease from contaminated surgical instruments40,41. Our titration results indicated that the bioassay remained sensitive to wire-bound dilutions up to 10–3 to 10–6, with an average clinical dpi of ~400 to 500 days, a detection limit similar to our PMCA protocol. Moreover, our bioassay showed a 1–2 log10 reduction of PrPBSE infectivity in bin composters with a significantly higher reduction in the centre than the bottom layer, a fnding that agrees with our PMCA observations. Similarly, Yoshioka et al.42 reported a similar level of PrPBSE inactivation afer heat treatment (140–180°C for 1–3 h) of yellow grease using both PMCA and Tgbov mouse bioassay. Tis suggests that PMCA may be reflective of the results obtained through bioassays, making an attractive alternative owing to its highly sensitive and comparatively rapid ability to detect PrPBSE.
Poultry feathers are predominantly composed of β-keratin (90% of DM), which similar to PrPTSE are structurally rich in β-sheets43,44. Keratinases10,12 have the capacity to degrade feather protein and have shown activity against PrPTSE. Therefore, enrichment for keratinolytic microorganisms in compost through inclusion of feathers may be a means of promoting PrPTSE degradation. In an earlier study by our group18, mixing feathers with cattle manure in lab-scale composters enhanced PrP263K degradation by 1 log10 afer 14 days and PrPCWD by 0.5–1 log10 after 28 days. In the present study, there was also evidence that the degradation of PrPBSE in lab-scale composters was enhanced by~0.5 log10 as indicated from the different PrPBSE seeding activity between compost extract samples with and without feathers after 28 days. Responses to the inclusion of feathers were more evident for PrPCWD and PrPBSE after the second composting cycle, suggesting that feathers may have selected for enriched prion degrading bacterial communities after a sufficient thermophilic period. Previous studies found that enrichment of a compost straw matrix with feathers not only effectively increased proteolytic activity in the early composting process but also promoted the growth of keratinolytic fungi during the latter stages of composting45,46. Currently, approximately 100,000 tonnes of feathers are produced from the Canadian poultry industry annually with most of them being landfilled or incinerated47. Feather composts have been recently considered to be used as agricultural fertilizers to promote crop production48,49 and share properties in common with slow release N fertilizers. Consequently, composting of SRM and feathers together might be an option for co-disposal of both agricultural waste materials generated from agriculture production with the potential co-benefit of maximizing PrPBSE degradation in compost.
Composting of SRM has economic advantages as disposal is estimated at $120–180 CAN per tonne, two to three times lower than disposal methods such as landfill, incineration, or alkaline hydrolysis50. Composting also could be more amendable to remote areas where transport of SRM to a centralized processing facility is infeasible. Currently, CFIA regulations require at least 5 log10 inactivation of PrPBSE to approve a method for SRM disposal. Our previous work demonstrated a 4.8 log10 reduction in PrP263K infectivity after 230 days of field-scale static composting18. Turning of compost piles breaks up aggregates, increases porosity, redistributes moisture and promotes the microbial decomposition of organic matter, increasing the duration and temperature achieved during composting16,18. In practice, thermophilic composting (≥55°C) is recommended to inactivate environmental pathogens51 and guidelines from CCME (Canadian Council of Ministers of the Environment) and USEPA (United States Environmental Protection Agency) suggest temperatures should exceed 55°C for at least 15 consecutive days in windrows that are turned three times. Consequently, we elected to mix the compost to generate three heating cycles to subject PrPBSE to a period of thermophilic exposure that was the same as what was achieved for PrP263K in our static field-scale composters. In 2017 and 2018, similar sources of feedlot manure resulted in comparable changes in temperature, moisture and pH during the composting process. Both PMCA and bioassay results suggest that PrPBSE was reduced by 1–2 log10 in the centre of compost piles in both years. In practice, compost piles consist of a heterogenous mixture of animal tissues, manure and a carbon source such as woodchips or straw. Even after blending, spatial variability in temperature profles15 and the composition of microbial communities52 are often observed. In the present study, temperatures broadly varied among core and edge locations at all three depths of bin composters. In 2017, we did observe a large variation (1 log10 and 2 log10) in the reduction of PrPBSE seeding activity between duplicate manure spheres at the centre of the same compost pile as measured by PMCA as well as in the reduction of PrPBSE infectivity between bin composters (i.e., 0.4 log10 and 1.3 log10) as assessed by the bioassay. Therefore, the variability in matrix composition, microbial populations and temperature profiles generated in compost piles would make it virtually impossible to verify uniform degradation of PrPBSE throughout a field scale compost pile. In addition, we did observe higher temperatures in the upper layers of bin composters as compared to the bottom. Tis temperature distribution likely reflects the upward movement of warm gases arising from microbial activity. Similar results were observed in our previous lab-scale composting study for degradation of SRM in compost20. As a result of this temperature variability, we examined the degradation of PrPBSE at the centre and bottom compost layers in 2018. Both the PMCA and bioassay confirmed that the degradation of PrPBSE was less at the bottom layer as compared to the centre layer after 120 days. Tis finding likely reflects the lower microbial activity at the bottom of compost piles as temperatures≥55°C occurred for only 39 days at this location as compared to 80 days at the central location.
Several factors could significantly affect the accurate quantification of PrPBSE biodegradation in compost. First, it is possible that PrPBSE may have migrated from the manure spheres into the surrounding compost matrix or may be cleaved of the stainless steel wires without being degraded. We attempted to examine this possibility using PMCA to test for the presence of PrPBSE in the compost matrix surrounding the nylon bags, but no signals were detected. Previous studies have shown that the mobility of prions is limited in environmental samples such as soil7,53, composted yard waste and municipal solid waste54. The mobility of PrPTSE in these environmental samples was not observed even after exposure to stringent chaotropic agents, nonionic detergents or extreme pH55,56. These observations suggest that during composting, the mobility of PrPBSE from manure spheres or their cleavage from stainless steel wires was unlikely. Moreover, Tgbov mice developed TSE disease prior to and after the composting process, suggesting PrPBSE remained bound and infectious throughout composting. Tus, the reduction in PrPBSE likely reflects enzymatic degradation.
With multiple cycles of PMCA, it is possible for false positives to be generated from non-infectious BH24,25. To check for false-positives, BSE-negative samples including Tgbov BH and extract samples from fresh manure spheres inoculated with non-infectious BH were subjected to multiple rounds of PMCA. False positive signals were only observed after the ffh round of PMCA. Consequently, we restricted PMCA to three rounds to ensure that declines in PrPBSE seeding capacity during composting were not confounded by the spontaneous generation of false positive PrPBSE signals. It is also possible that dynamic changes in manure properties during composting may have impacted the binding of PrPBSE with manure/compost particles that could impact the subsequent PrPBSE extraction or templating activity for PMCA detection. Ionic concentration and pH have been reported to influence PrPTSE adsorption and conformational changes in soil57,58. Moreover, humic acid-prion interactions have also been shown to impact the extraction and detection of PrPCWD in soils59,60. The disease-associated form of prions binding to soil particles or minerals were reported to either increase61 or decrease62 the prion infectivity and PMCA replication efficiency. Our results showed that compost-bound PrPBSE enhanced PMCA replication efficiency than manure-bound PrPBSE (i.e., 5 log10 vs 3 log10 limit of detection). Tis suggests that our established extraction protocols recovered more PrPBSE from compost than fresh manure, resulting in more PrPBSE seeds available for amplification. Another possible explanation could be made that changes in composition and structure of organic compounds such as humic acids during composting63 may modify the afnity of PrPBSE to aged manure and enhance the ability of compost-bound prions to covert PrPC to PrPBSE, resulting in a better propensity of compost-bound PrPBSE to be amplified in our assay. However, these fndings further support that 1–2 log10 reduction of PrPBSE in bin compost is not due to differential PrPBSE recovery or templating activity between manure and compost, but as a result of microbial activity during composting.
In the present study, lab-scale composters achieved≥55°C for 3 days after two cycles over 28 days. Tis contrasts with bin composters where≥55°C was achieved for 60–80 days after composting for 106–120 days. Our previous work demonstrated that PrP263K was degraded during lab-scale composting and that degradation was enhanced as result of prolonged exposure to microbial activity in field-scale composters18. However, these differences in microbial activity in the current two composting systems did not result in more extensive inactivation of PrPBSE in bin composters even though we ensured that it was exposed to thermophilic period similar to that of PrP263K in previous field-scale composters. We observed up to a 3–4 log10 reduction in PrPBSE seeding activity in lab-scale composters as opposed to only a 1–2 log10 reduction in bin composters. Several factors could have contributed to these differing observations. Due to the large volume of BSE brain materials needed for this project, we used two different sources of PrPBSE in two composting systems. Te lab-scale composting experiment used brain tissues from a cattle intra-cranially challenged with infectious BH from a Canadian BSE field case, while materials used for bin composting experiment were from a cattle orally challenged using the brain tissues from the intra-cranially challenged animal used in lab-scale composting experiment. Moreover, brain tissues used for lab-scale composting experiment were strongly positive for PrPBSE with limit of detection at 30 µg by WB (Supplementary Figs. S1a and S5a), but materials used to make the homogenate in the bin composting experiment were moderate to strongly PrPBSE positive which was detected at the limit of 300 µg (Figs. S1a and S7a). Tis suggests a higher concentration of PrPBSE in the brain tissues used in lab-scale than bin composting experiment, which is also reflected by the fact that one more PMCA round is required for PrPBSE amplification in bin composting experiment brain tissues to achieve a sensitivity similar to that obtain in the brain tissues used in lab-scale composting experiment. Therefore, varying concentrations of PrPBSE in the infectious BH or even possibly difference in PrPBSE stability related to inoculation route or host variability (i.e., PrP expression level, co-factor variation/expression, breed composition etc.) may have impacted PrPBSE degradation during composting. Giles et al.64 reported that cattle PrPBSE was 1,000-fold more resistant to inactivation by acidic SDS treatment than mouse adapted PrPBSE 301V. In addition, we used two different sources of feedlot manure in two composting experiments. The fresh manure used for lab-scale composting experiment significantly had lower moisture and pH but higher TC content than the one in the bin composting experiment. Therefore, proteolytic microbial populations developed in lab-scale composters may have differed from those established in bin composters, particularly in lab-scale composters that were enriched for keratinolytic bacteria via the addition of feathers. Based on these results, temperature can be used as a proxy for microbial activity in compost, but lacks merit as direct indicator of PrPBSE degradation. Therefore, further investigation is needed to characterize the biodegradation of PrPBSE with consideration for other complex factors such as differences in PrPBSE strains and the proteolytic microbial communities in compost.
In general, thermophilic composting (i.e., temperature≥55°C) has a hierarchy of essential factors that facilitate the biodegradation of waste materials and inactivation of pathogens. In this study, we successfully stimulated thermophilic temperature profiles that were similar to that observed during field-scale composting18. However, our composting model in biocontainment did not fully represent the field scale composting systems that could be used for the disposal of SRM or cattle carcasses. In containment,~2 tonnes of feedlot manure were used over 120 days of composting, resulting in a~1 to 2 log10 PrPBSE infectivity reduction. In contrast, in our field scale model18,~100 tonnes of feedlot manure with 16 cattle carcasses were composted over 230 days resulting in a 4.8 log10 reduction in PrP263K infectivity. As a logical extrapolation, a longer composting duration and greater volume of biomass in the field-scale composters would likely enhance PrPBSE degradation. As a 4.8 log10 reduction of PrP263K infectivity was observed in the field-scale composters18, it would be surprising if inactivation of PrPBSE was not further enhanced in field-scale composting. In addition, Belondrade et al.65,66 demonstrated that commercial chemicals fully efficient on sterilization of PrP263K were inefcient for the inactivation of variant PrPCJD, suggesting PrP263K might not be a suitable model to validate the prion resistance to inactivation. Consequently, further investigation of PrPBSE degradation in field-scale composting is needed.
Previous studies documented the more recalcitrant nature of PrPBSE than other TSE agents. After exposure to acidic SDS, PrPBSE was 10 and 10 million fold more resistant to inactivation than PrPCJD and hamster PrPSc, respectively, as assessed by infectivity titration in transgenic mice64. Langeveld et al.67 also reported PrPBSE to be more resistant to wet heat conditions at 115°C than PrP263K and PrPCWD as measured by transgenic mouse bioassay. Our PMCA results suggested that 28 days of lab-scale composting resulted in a reduction of PrPTSE seeding capacity with~2 log10 in PrP263K and~3 log10 in PrPCWD in a previous study18 and~3 log10 in PrPBSE in the current study. Different from chemical treatment of prion inactivation, compost is an exceedingly complex biological system, owning to changing temperatures and pH, and dynamic changes in microbial communities and the enzymes they produce during composting. Once PrPTSE enter the compost environment, a wide variety of physicochemical and microbiological processes can impact PrPTSE infectivity and seeding capacity. Tese uncontrolled factors might help to account for the variable inactivation observed in our PrPTSE composting studies. While this variability calls into question the utility of our composters for complete PrPTSE inactivation, it is encouraging that when our compost conditions were optimal, 28 days of composting effectively destructed PrPBSE replication capacity in vitro by 3 log10 (i.e., at least 99.9%). Currently, the Canadian government68 enacted a regulation on the limited use of composting for disposal of SRM under a temporary permit. It also requires a 5-year respite from cattle access to pasture or grazing land amended with SRM compost and from direct human consumption of annual crops produced from SRM compost amended feld69. A recent study from UK7 reported that the same amount of PrPBSE infectivity remained in both clay and sandy soil over a 5-year period. Our studies suggests that the maximum PrPBSE degradation (up to 3–4 log10) can achieve in the lab-scale composters with the addition of feathers. Therefore, composting of BSE infected-SRM prior to subsequent land application could be an effective approach to reduce the risk of high titer PrPBSE persisting in the environment.
Conclusions
In this study, we successfully quantified PrPBSE degradation using PMCA and bioassay in two-scale composting systems. Afer 28 days,~3 log10 reduction of PrPBSE seeding activity was observed in lab-scale composters. Addition of chicken feathers to the compost enhanced PrPBSE degradation, likely as a result of enrichment for keratinolytic bacteria. After 106–120 days, both BSE associated seeding activity and infectivity were reduced by 1–2 log10 in the centre, but only by 1 log10 at the bottom of bin composters. This suggests that placement of SRM in the centre of compost piles would be more amendable for the biodegradation of PrPBSE. Current CFIA policy on SRM destruction methods require at least 5 log10 reduction of PrPBSE to approve composting for disposal of BSE positive SRM. Our field-scale composting study18 demonstrated that 230 days of composting resulted in a 4.8 log10 inactivation in hamster PrP263K infectivity. However, PrP263K might be not a suitable surrogate model to validate the PrPBSE resistance in compost. The outcomes generated from this study did not meet this criteria, but do lay the foundational work needed to further optimize the degradation of PrPBSE in compost. Spatial variability in microbial activity within static compost piles makes it unlikely that the procedure will ever achieve the 5 log10 reduction in PrPBSE required for full regulatory approval as a disposal method of SRM.
Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
Received: 18 August 2022; Accepted: 12 December 2022 Published online: 23 December 2022
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Any haste in full regulatory approval of composting for disposal of specified risk materials SRMs, without complete inactivation of all TSE Prion Strains, will only help spread further the TSE Prion agent.
DO NOT PUT THE CART BEFORE THE HORSE AGAIN WITH TSE PRION DISEASE, which has been done time and time again with TSE PRION strains and science, and that, in part, is why we are where we are at with Transmissible Spongiform Encephalopathy TSE Prion Globally.
WITH CHRONIC WASTING DISEASE CWD TSE PRION in Cervid, CWD transmitting to PIGS, SHEEP, and CERVID, BY ORAL ROUTES, AND NO TELLING ABOUT THE NEW LIVESTOCK TSE PRION DISEASE IN A NEW LIVESTOCK SPECIES I.E. THE CAMEL PRION DISEASE, AND THE FACT THAT THE BSE FEED REGULATION (21 CFR 589.2000) HAS BEEN AND STILL IS A TOTAL FAILURE, ANY WEAKENING OF SRM RULES OR WITH LITTLE SCIENCE TO VALIDATE COMPOSTING AS A WAY TO RID OURSELVES OF TSE PRION SRMs, any move to validate said protocols IMO, is another TSE Prion disaster waiting to happen, another added risk factor for monetary gains... imo...terry
***> Moreover, according to Tronina and Bubel (2008), composting may not fully inactivate pathogenic microorganisms.
Inactivation of indicator microorganisms and biological hazards by standard and/or alternative processing methods in Category 2 and 3 animal by-products and derived products to be used as organic fertilisers and/or soil improvers
EFSA Panel on Biological Hazards (BIOHAZ), Konstantinos Koutsoumanis, Ana Allende, Declan Bolton, Sara Bover-Cid, Marianne Chemaly, Robert Davies … See all authors
First published: 02 December 2021 doi.org/10.2903/j.efsa.2021.6932
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3.2.6 Feathers and down
Article 10 of Regulation (EC) 1069/2009, point 3, defines ‘feather’ as Category 3 ABP originating from: ‘(b) animals that have been slaughtered in a slaughterhouse and considered fit for human consumption following the ante-mortem inspection or game killed for human consumption in accordance with Community legislation; (h) live animals that did not show any signs of diseases communicable through that product to human or animals; (n) dead animals that did not show any sign of disease communicable to humans or animals.’
The poultry industry has become one of the largest food industries in the world, producing large quantities of feather waste. Between 5% and 10% of the total weight of a chicken is made of feathers (Callegaro et al., 2019). More than 1 million metric tonnes of feathers are produced annually as a by-product at European poultry slaughterhouses (Goerner-Hu et al., 2020). Due to a large variety of chemical hazards and microbiota present on the feathers, including pathogens, they must be treated quickly. Poultry feathers are rich in keratin protein, which makes them a good source of nitrogen fertiliser (Joardar and Rahman, 2018).
Chicken feather waste can be:
Incinerated. This process is effective at inactivating biological hazards but requires a high energy consumption (Saidan et al., 2017) and produces large amounts of carbon dioxide.
Composted with manure. The composting process is slow and subject to the special requirements of veterinary inspection and requires a closed composting area with a sewage carry system, and periodic microbiological tests according to Commission Regulation (EU) No 142/2011. A problem for composting is odorous emission of hydrogen sulfide that persists in the air for a long period. Moreover, according to Tronina and Bubel (2008), composting may not fully inactivate pathogenic microorganisms.
Hydrolysed (Tesfaye et al., 2017a,b). Feather hydrolysis provides valuable amino acids, proteins and peptides in the mixture with acylglycerols and higher fatty acids. Chemical hydrolysis leads to destruction of the native structure of keratin and the feather waste becomes more water soluble. Acidic hydrolysis is highly efficient but causes loss of some amino acids. Alkaline hydrolysis is slower and can be incomplete, but the loss of amino acids is lower. The yield of the hydrolytic processes depends on pH, temperature and reaction time, and also on the type and concentration of acid or base used. As a drawback, commonly applied hydrolysis leads to the requirement for subsequent recycling of the process solutions, including neutralisation and elimination of undesirable salts (Solcova et al., 2021).
Treated in dimethyl sulfoxide or other solvents to get value added products from feather keratin, generated in excess from various livestock industries (Azmi et al., 2018).
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3.8.1 Ash derived from incineration, co-incineration and combustion A wide range of biological hazards may occur in the ABP raw materials before their incineration, co-incineration or combustion. However, bacteria, viruses and parasites are generally sensitive to heat and cannot survive normal burning temperatures. Prions are considered the most resistant biological hazards. Even the risk of TSE infectivity from ash would be extremely small if incineration is conducted at 850°C (SEAC, 2003). Indeed, the incineration at > 850°C is recognised in the EU as the standard method for disposing of waste.26 Therefore, ash derived from incineration, co-incineration or combustion is typically considered safe and may be disposed of in landfills.
Ash generated from incineration, co-incineration or combustion (carried out in accordance with Annex III of Commission Regulation (EU) No 142/2011) requires operating temperatures of 850°C for at least 2 seconds or 1,100°C for at least 0.2 s.
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Summary of the 78th SEAC meeting
on 24th June 2003
The Spongiform Encephalopathy Advisory Committee (SEAC) held its 78th meeting in London on 24 June 2003, when it discussed the following matters:
Risk Assessment on Ox Tongue and Associate Tonsil Tissue
At an earlier meeting SEAC considered a new finding of BSE infectivity in bovine tonsil and its possible association with ox tongue. SEAC recommended that a risk assessment be conducted and this was commissioned by the Food Standards Agency, and presented at the June 2003 meeting. The risk assessment considered the possible range of human exposure to BSE infectivity from the consumption of ox tongue. The Committee concluded that it was not possible to advise the FSA precisely on the magnitude of the risk due to the substantial scientific uncertainty inherent in the risk assessment. However, the Committee agreed that the scientific evidence indicated that the potential risk of infectivity from eating tongue was likely to be very small. The Committee identified further scientific work that would help to refine the risk estimates.
Review of the use of MMBM in fertiliser
The Department of the environment, food and rural affairs (Defra) asked SEAC to provide scientific advice on the animal health implications of proposed changes to UK fertiliser controls. SEAC agreed that the proposed use of ash from the incineration of meat and bone meal (MBM) derived from category 2 and category 3 material without restriction on land would not result in significant additional risk to animal health. SEAC confirmed its earlier advice that mammalian MBM should not be permitted in fertilisers likely to be spread on agricultural land or land where animals may graze.
VLA Survey – Scrapie Surveillance in Sheep
The Committee noted the preliminary results of a report from the Veterinary Laboratories Agency estimating the prevalence of scrapie in the national flock. The Committee also noted that a full report would be available in due course, containing all of the data from the study.
vCJD Update
The National CJD surveillance unit reported that 136 vCJD cases have been confirmed in the UK with 4 cases still alive. All vCJD cases tested to date are of the same genotype (Methionine homozygous at codon 129 of the PrP gene). All vCJD cases so far identified in 2003 have reported the onset of clinical signs in 2002. Therefore the total number of onsets in 2002 cannot yet be confirmed.
Report from the SEAC Epidemiology sub-group
The Chairman of this specialist sub-group reported to SEAC that there continues to be statistical evidence that the vCJD epidemic is no longer increasing at the rate seen previously and that the underlying incidence may have reached or be reaching a peak. However the possibility of susceptible genotypes other than methionine homozygotes and the theoretical possibility of other clinical manifestations of infection with the BSE agent other than vCJD means that prediction of the evolution of the epidemic is uncertain and continued surveillance is essential.
Expert Group on Strain Differentiation
SEAC received a report from the Chairman of an expert group of the EU Community TSE Reference Laboratory Committee, which met on 23 June 2003 to review progress on a trial to evaluate rapid TSE tests.
Quinquennial Review of SEAC
The Committee welcomed the recommendations outlined in the SEAC Quinquennial Review Report published in March 2003.
EU HEALTH, OPINION ON:
HYPOTHESES ON THE ORIGIN AND TRANSMISSION OF BSE ADOPTED BY THE SCIENTIFIC STEERING COMMITTEE AT ITS MEETING OF 29-30 NOVEMBER 2001
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III.2. HYPOTHESES FOR OTHER ‘THIRD WAYS’
Composted manure and stomach and intestinal contents
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III.2.1.3 Indirect transmission from cattle or other animal sources to the alimentary tract of cattle:
a) Risks from soil
The German, Federal Ministry for the Environment, Nature Conservation and Reactor Safety have produced a report of an International Expert Discussion on the Occurrence and Behaviour of BSE/TSE prions in soil, held on 8 December 2000 in Bonn (Report 2001). This report indicated the lack of knowledge about the contamination and degradation following pollution of soil and water and made useful recommendations for the correction of this deficiency but no conclusive final statement that had the support of all participants could be made. Nevertheless, they considered various routes for the pollution of soil and water including from organic fertilisers containing for example mammalian protein, industrial fertilisers from farms (faecal and urine sources), from composting of infected material after biogas production, sheep placentas, sewage sludge and dog and cat faeces. Deficits in particular knowledge were indicated in the role of soil nematodes but there was a consensus that prions might be bound in the superficial layers of soil and that degradation would be a slow process. This report noted that cattle could consume up to 1kg of soil per day suggesting a risk might be present should an effective oral dose of the BSE agent be present. Cattle would be less likely to consume leaves contaminated with dust from any distributed source of infection. In addition, the plants themselves would be devoid of risk because the roots cannot absorb protein molecules the size of PrP molecules. The role of dog and cat faeces was considered a negligible risk. It is also known that the scrapie agent is highly resistant to inactivation (Taylor 1996).
b) Experimental studies
Greig (1940), found it was very difficult from the analysis of field incidents of scrapie to determine whether or not scrapie could be transmitted from pasture upon which scrapie animals had grazed. He therefore undertook an experimental study in which 3 or 4 scrapie sheep and 20 sheep from flocks and regions with no history of scrapie. These animals were kept on separate fields without direct contact. About twice each week, the sheep in each pasture were exchanged, again without direct contact with each other. The ewes were mated and lambed. After three years, no cases of scrapie were seen in the contact sheep. They were moved to another farm that had never reported scrapie but between a further three months and a further two years and three months a total of 9 cases of scrapie developed and it was concluded that the origin of infection was the pasture that had been grazed by scrapie-affected sheep.
c) Experiences in Iceland
Indirect transmission of scrapie from a contaminated environment has been reported, notably from Iceland (Pálsson, 1979; Sigurdarson, 1991). This is plausible because there has been a high incidence of scrapie (rida) in some Icelandic flocks in fenced-off scrapie-affected regions, especially as there is close confinement of housed breeding sheep over the long winter period. In one recent occurrence in Iceland, scrapie returned to a flock following strict depopulation cleaning and disinfection after a period of seven years (S. Sigurdarson, personal communication). This suggests that the site might previously have been highly infected and was responsible for the new occurrence (Wilson, Anderson and Smith, 1950) or that an alternative source such as hay mites (see below) might be responsible. Scrapie infectivity could be transferred to the environment from infected placenta. Sheep placenta is a known source of scrapie infection and, if not consumed by the dam or unrelated sheep, could be taken by foxes or other carnivorous species across farm boundaries. Ravens in Iceland (Sigurdarson, 1991) and Black-backed gulls (Moon, 1978) have been suggested as vectors. However, ravens do not succumb to challenge with scrapie (S. Sigurdarson, Personal communication). Furthermore laboratory strains of high titre hamster scrapie can survive for up to three years in the soil, though at very much reduced titre, but still sufficient to produce disease by the i/c route in hamsters (Brown and Gajdusek, 1991). Transmission by the oral route following three years in soil has not been demonstrated.
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Faeces In cattle with natural or experimental BSE, the only plausible source of infection is from faeces (urine is discussed separately below), as other infected organs have no direct connection with the environment in the living animal. In this context there are theoretically three ways in which faeces might become infected and then theoretically transmit disease indirectly after composting (that is unlikely to completely inactivate TSE agents).
j) Composted manure and stomach and intestinal contents
Theoretical risks from spreading composted dung from captive wild ruminant species on farmland exist but should easily be detectable by epidemiological investigation. A greater risk might theoretically have occurred in the past (before the introduction of feed bans for non-ruminant animals) if stomach/intestinal contents or dung/droppings from abattoirs dealing with pigs and poultry fed mammalian protein was spread on to farmland grazed by cattle. The same could apply to composted dung or droppings from these species fed mammalian protein. This is a more realistic risk because it would be expected that the inclusion rate of such protein could be quite high (c. 15%) and infectivity if present in the raw material would mostly pass through the gut to enter the faeces. Whether or not an infectious oral dose of BSE for cattle could be consumed is more difficult to ascertain but it is not beyond the bounds of possibility if land was grazed quickly after distribution of these products.
SCIENTIFIC OPINION
Scientific Opinion on Composting on-farm of dead poultry1
EFSA Panel on Biological Hazards (BIOHAZ)2, 3
European Food Safety Authority (EFSA), Parma, Italy
2014 SCIENTIFIC REPORT OF EFSA Protocol for further laboratory investigations into the distribution of infectivity of Atypical BSE1
European Food Safety Authority2,3European Food Safety Authority (EFSA), Parma, Italy
DOPTED: 7 June 2018doi: 10.2903/j.efsa.2018.5314
Updated quantitative risk assessment (QRA) of the BSE risk posed by processed animal protein (PAP)
EFSA Panel on Biological Hazards (BIOHAZ), Antonia Ricci, Ana Allende, Declan Bolton, Marianne Chemaly, Robert Davies, Pablo Salvador Fernandez Escamez, Rosina Girones, Lieve Herman, Kostas Koutsoumanis, Roland Lindqvist, Birgit Nørrung, Lucy Robertson, Giuseppe Ru, Moez Sanaa, Panagiotis Skandamis, Emma Snary, Niko Speybroeck, Benno Ter Kuile, John Threlfall,Helene Wahlstr€om, Amie Adkin, Matthias Greiner, Daniela Marchis, Marta Prado, Teresa Da Silva Felicio, Angel Ortiz-Pelaez and Marion Simmons
Abstract
EFSA was requested: to assess the impact of a proposed quantitative real-time polymerase chain reaction (qPCR)‘technical zero’ on the limit of detection of official controls for constituents of ruminant origin in feed, to review and update the 2011 QRA, and to estimate the cattle bovine spongiform encephalopathy (BSE) risk posed by the contamination of feed with BSE-infected bovine-derived processed animal protein (PAP), should pig PAP be re-authorised in poultry feed and vice versa, using both light microscopy and ruminant qPCR methods, and action limits of 100, 150, 200, 250 and 300DNA copies. The current qPCR cannot discriminate between legitimately added bovine material and unauthorised contamination, or determine if any detected ruminant material is associated with BSE infectivity. The sensitivity of the surveillance for the detection of material of ruminant origin in feed is currently limited due to the heterogeneous distribution of the material, practicalities of sampling and test performance. A ‘technical zero’ will further reduce it. The updated model estimated a total BSE infectivity four times lower than that estimated in 2011, with less than one new case of BSE expected to arise each year. In the hypothetical scenario of a whole carcass of an infected cow entering the feed chain without any removal of specified risk material (SRM) or reduction of BSE infectivity via rendering, up to four new cases of BSE could be expected at the upper 95th percentile. A second model estimated that at least half of the feed containing material of ruminant origin will not be detected or removed from the feed chain, if an interpretation cut-off point of 100 DNA copies or more is applied. If the probability of a contaminated feed sample increased to 5%, with an interpretation cut-off point of300 DNA copies, there would be a fourfold increase in the proportion of all produced feed that is contaminated but not detected.©2018 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.
Keywords: BSE, cattle, PAP, risk, qPCR, technical zero
Requestor: European Commission Question number: EFSA-Q-2017-00705 Correspondence: biohaz@efsa.europa.eu
EFSA Journal 2018;16(7):5314www.efsa.europa.eu/efsajournal
SNIP...
4.3.7. Infectivity of Bovine Tissues
4.3.7.1. Estimation of the BSE oral infectious dose 50 in cattle
The Veterinary Laboratory Agency (VLA)27in the in the UK has carried out experiments to determine the BSE minimal oral infectious dose of BSE in cattle. In this titration experiment groups of 10 calves were each fed 300g, 100g, 10g and 1g of an homogenate made from the brain stems from clinically sick animals.
According to titration in RIII mice the used brain homogenate contained 103.5 mouse i.c./i.p.ID50 per g of tissue.
All animals inoculated with 300g and 100g came down with BSE, and 7 out of 10 in both the 10g and 1g trials. The incubation periods for both the 1g and 10g trials were comparable (between 44–71months).
As it was not possible to determine an ID50 dose from this experiment, an extension of this titration experiment was carried out with doses of 1g, 100mg, 10mg and 1mg (Wells et al., 2007). The results show 3 of 5 in the 1g trial group, 7 out of 15 animals in the 100mg group, 1 out of 15 in the10mg group, and 1 out of 15 in the 1mg group, positive for BSE. Incubation periods for the positive results in both the 1 and 10mg groups were similar to those for the 1 g trial, but two of the animals in the 100mg group had incubation periods in excess of 90 months.
In their study, Wells et al. (2007) report that the ID50 estimate from these experiments is equivalent to 0.20 g of the brain homogenate used (i.e. 5 ID50/g) with a 95% confidence interval of0.04–1.00g. This value also indicates that 1 cattle oral ID50 is approximately equivalent to 102.8mouse i.c./i.p ID50 in RIII mice.4.3.7.2.
Infectivity in the Brain and spinal cord
From titrations conducted in mice on brain from clinical or clinical suspect cases of BSE, a wide range of titres have been obtained: 102.4–105.2 mouse i.c.or i.c./i.p. ID50/g (Fraser et al., 1992;Taylor et al., 1994). These data were used to estimate the titre at clinical onset and its variability. From this, the mean titre of brain at clinical onset was given by 103.3 mouse i.c.or i.c./i.p. ID50/g with standard deviation of 100.58 (Arnold et al., 2009). The working group preparing the EFSA QRA report considered that with higher titres of BSE affected brain the range could extend to 300 ID50/g’(see section III.2 of EFSA, 2005) and decided to take a precautionary view and assuming that the infectivity titre in brain of a clinically BSE infected bovine follows the following distribution:
Log normal distribution with•
•Median (50th percentile): 5 Co ID50/gram
•Higher 99th percentile: 100 Co ID50/gram
For the present assessment this distribution was considered as a reasonable representation of the infectivity level in the CNS of a cattle affected with BSE.
4.3.7.3 Infectivity in the Dorsal root ganglia
In their 2009 paper Arnold et al. (2009) estimated the infectious titre in cervical and thoracic dorsal root ganglia from cattle orally inoculated with 100g brain material at different time points of their incubation. According to this study the titre in the DRG was lower than CNS, with the thoracic and cervical DRG having mean titres approximately 1 and 1.5 log10 mouse i.c./i.p. ID50/g lower than CNS respectively.
4.3.7.4. Infectivity in the Peripheral Nervous System There have been a number of studies reporting detection of infectivity using transgenic PrP bovine mice (Buschmann and Groschup, 2005; Espinosa et al., 2007) or PrPSc(Iwata et al., 2006) in some peripheral nervous system tissues.
27Now known as Animal and Plant Health Agency (APHA).
Updated QRA of BSE in PAPwww.efsa.europa.eu/efsajournal63EFSA Journal 2018;16(7):5314
According to the data reported by Buschmann and Groschup (2005) the infectivity could be detected in some but not all nerves samples from a BSE affected animal. In this study the infectivity level in the positive nerves could be estimated to be about 5-6 log10 folds lower than that in the brain from the same animal. These data are consistent with those reported by Espinosa et al., (2007) indifferent BSE infected animals using another bovine PrP transgenic mouse model. In this study the author report the detection of infectivity in the sciatic nerve from 30 and 33 months post cattle exposure, but its absence in animals killed at 20, 24 and 27 months post exposure (n=1 cattle per timepoint).
Iwata et al. (2006) reported the detection of PrPSc in some but not all nerves from 2 naturally BSE infected cases (preclinical stage of the disease). On the basis of PrPSc biochemical detection (WesternBlot) it was estimated that the infectivity in the femoral and lumbar nerves of an affected cattle was1,000 to 1,400 fold less than the PrPSc amount detected in the spinal cord.
4.3.7.5. Infectivity in non Nervous System tissues
A large range of tissues collected at various stages of the incubation were tested for the presence of BSE infectivity by mouse bioassay (conventional or bovine transgenic) (Arnold et al., 2009;Buschmann and Groschup, 2005; Espinosa et al., 2007). A more limited range of tissues was also tested by intracerebral inoculation into calves (Wells et al., 2005).
The only non-nervous tissues shown to harbour consistent infectivity in these experiments are the distal ileum and lingual tonsil.
In the distal ileum infectivity was evidenced as early as 6 months post oral exposure and seems to persist all along the incubation period. The infectious titres in the distal ileum were estimated to range between 100.06and 101.94 i.c./i.p. ID50 in RIII mice per gram depending on the age of the individual (Arnold et al., 2009) (i.e. between 1 and 3 log10 fold lower than in the mean level of infectivity found in the brain from BSE affected individuals).
In lingual tonsil, infectivity was detected
•In one out of 5 calves inoculated intracerebrally with a pool of tonsil collected in orally inoculated cattle killed 10 months post exposure (Wells et al., 2005). There were no other positive results for tonsil at subsequent time points of the study (18, 22, 26, 32 and 36 months post exposure).
•In cattle killed at 20 - 24 - 27–30–33 months (n = 1 animal per time point–no younger animal tested) post inoculation in transgenic mice expressing the bovine PrP gene (1/6 mice in each case) (Espinosa et al., 2007).
On the basis of these data, it was estimated (EFSA, 2008) that the infectivity in the tonsil tissue was less than 1 bovine i.c. ID50/g or 10-6.5 Co ID50/g.
Finally, detection of minute amounts of infectivity were reported (bioassay in transgenic bovine mice) in one striated muscle sample collected in a BSE affected cattle (Buschmann and Groschup,2005). The authors failed to detect infectivity in other muscle samples from the same animal. Using another transgenic bovine PrP mouse model other authors failed to detected infectivity in striated muscle samples (one sample per cattle) collected in cattle orally challenged with BSE (100g) and killed at 20 - 24 - 27–30–33 months (n=1 animal per time point) (Espinosa et al., 2007). These data remain difficult to interpret. In particular, it is unclear if the detected infectivity was associated to nervous ramifications present in the muscle sample or to striated muscular cells, as reported in other TSE models (Andreoletti et al., 2004; Thomzig et al., 2004).
With the current state of knowledge it cannot be considered that striated muscles cells are harbouring BSE infectivity in cattle.
4.3.7.6. Total infectivity amount in a BSE clinical case
The total infectivity in a clinical case of BSE is summarised in Table3. The weights of the various tissues are mainly taken from the LFRA and MLC report (LFRA and MLC, 1997) and the infectivity values are as discussed above, with the infectivity for whole brain taken to be 5 Co ID50/g. It can be seen that 90% of the infectivity is associated with central and peripheral nervous system tissues, with about 10% associated with the distal ileum.
4.3.7.7. Development of the infectivity in tissues through incubation period
SNIP...
DEFRA U.K. What is the risk of Chronic Wasting Disease CWD being introduced into Great Britain? A Qualitative Risk Assessment October 2012
snip...
In the USA, under the Food and Drug Administration's BSE Feed Regulation (21 CFR 589.2000) most material (exceptions include milk, tallow, and gelatin) from deer and elk is prohibited for use in feed for ruminant animals. With regards to feed for non-ruminant animals, under FDA law, CWD positive deer may not be used for any animal feed or feed ingredients. For elk and deer considered at high risk for CWD, the FDA recommends that these animals do not enter the animal feed system. However, this recommendation is guidance and not a requirement by law.
Animals considered at high risk for CWD include:
1) animals from areas declared to be endemic for CWD and/or to be CWD eradication zones and
2) deer and elk that at some time during the 60-month period prior to slaughter were in a captive herd that contained a CWD-positive animal.
Therefore, in the USA, materials from cervids other than CWD positive animals may be used in animal feed and feed ingredients for non-ruminants.
The amount of animal PAP that is of deer and/or elk origin imported from the USA to GB can not be determined, however, as it is not specified in TRACES. It may constitute a small percentage of the 8412 kilos of non-fish origin processed animal proteins that were imported from US into GB in 2011.
Overall, therefore, it is considered there is a __greater than negligible risk___ that (nonruminant) animal feed and pet food containing deer and/or elk protein is imported into GB.
There is uncertainty associated with this estimate given the lack of data on the amount of deer and/or elk protein possibly being imported in these products.
snip...
Very low oral exposure to prions of brain or saliva origin can transmit chronic wasting disease
Nathaniel D. Denkers ,Clare E. Hoover ,Kristen A. Davenport,Davin M. Henderson,Erin E. McNulty,Amy V. Nalls,Candace K. Mathiason,Edward A. Hoover
Published: August 20, 2020
We report that oral exposure to as little as 300 nanograms (ng) of CWD-positive brain or to saliva containing seeding activity equivalent to 300 ng of CWD-positive brain, were sufficient to transmit CWD disease. This was true whether the inoculum was administered as a single bolus or divided as three weekly 100 ng exposures. However, when the 300 ng total dose was apportioned as 10, 30 ng doses delivered over 12 weeks, no infection occurred. While low-dose exposures to prions of brain or saliva origin prolonged the time from inoculation to first detection of infection, once infection was established, we observed no differences in disease pathogenesis. These studies suggest that the CWD minimum infectious dose approximates 100 to 300 ng CWD-positive brain (or saliva equivalent), and that CWD infection appears to conform more with a threshold than a cumulative dose dynamic.
WE know now, and we knew decades ago, that 5.5 grams of suspect feed in TEXAS was enough to kill 100 cows.
look at the table and you'll see that as little as 1 mg (or 0.001 gm) caused 7% (1 of 14) of the cows to come down with BSE;
Risk of oral infection with bovine spongiform encephalopathy agent in primates
Corinne Ida Lasmézas, Emmanuel Comoy, Stephen Hawkins, Christian Herzog, Franck Mouthon, Timm Konold, Frédéric Auvré, Evelyne Correia, Nathalie Lescoutra-Etchegaray, Nicole Salès, Gerald Wells, Paul Brown, Jean-Philippe Deslys
Summary The uncertain extent of human exposure to bovine spongiform encephalopathy (BSE)--which can lead to variant Creutzfeldt-Jakob disease (vCJD)--is compounded by incomplete knowledge about the efficiency of oral infection and the magnitude of any bovine-to-human biological barrier to transmission. We therefore investigated oral transmission of BSE to non-human primates. We gave two macaques a 5 g oral dose of brain homogenate from a BSE-infected cow. One macaque developed vCJD-like neurological disease 60 months after exposure, whereas the other remained free of disease at 76 months. On the basis of these findings and data from other studies, we made a preliminary estimate of the food exposure risk for man, which provides additional assurance that existing public health measures can prevent transmission of BSE to man.
snip...
BSE bovine brain inoculum
100 g 10 g 5 g 1 g 100 mg 10 mg 1 mg 0·1 mg 0·01 mg
Primate (oral route)* 1/2 (50%)
Cattle (oral route)* 10/10 (100%) 7/9 (78%) 7/10 (70%) 3/15 (20%) 1/15 (7%) 1/15 (7%)
RIII mice (ic ip route)* 17/18 (94%) 15/17 (88%) 1/14 (7%)
PrPres biochemical detection
The comparison is made on the basis of calibration of the bovine inoculum used in our study with primates against a bovine brain inoculum with a similar PrPres concentration that was inoculated into mice and cattle.8 *Data are number of animals positive/number of animals surviving at the time of clinical onset of disease in the first positive animal (%). The accuracy of bioassays is generally judged to be about plus or minus 1 log. ic ip=intracerebral and intraperitoneal.
Table 1: Comparison of transmission rates in primates and cattle infected orally with similar BSE brain inocula
Published online January 27, 2005
It is clear that the designing scientists must
also have shared Mr Bradley’s surprise at the results because all the dose
levels right down to 1 gram triggered infection.
6. It also appears to me that Mr Bradley’s answer (that it would take less than say 100
grams) was probably given with the benefit of hindsight; particularly if one
considers that later in the same answer Mr Bradley expresses his surprise that it
could take as little of 1 gram of brain to cause BSE by the oral route within the
same species. This information did not become available until the "attack rate"
experiment had been completed in 1995/96. This was a titration experiment
designed to ascertain the infective dose. A range of dosages was used to ensure
that the actual result was within both a lower and an upper limit within the study
and the designing scientists would not have expected all the dose levels to trigger
infection. The dose ranges chosen by the most informed scientists at that time
ranged from 1 gram to three times one hundred grams. It is clear that the designing
scientists must have also shared Mr Bradley’s surprise at the results because all the
dose levels right down to 1 gram triggered infection.
***> cattle, pigs, sheep, cwd, tse, prion, oh my!
***> In contrast, cattle are highly susceptible to white-tailed deer CWD and mule deer CWD in experimental conditions but no natural CWD infections in cattle have been reported (Sigurdson, 2008; Hamir et al., 2006).
Sheep and cattle may be exposed to CWD via common grazing areas with affected deer but so far, appear to be poorly susceptible to mule deer CWD (Sigurdson, 2008). In contrast, cattle are highly susceptible to white-tailed deer CWD and mule deer CWD in experimental conditions but no natural CWD infections in cattle have been reported (Sigurdson, 2008; Hamir et al., 2006). It is not known how susceptible humans are to CWD but given that the prion can be present in muscle, it is likely that humans have been exposed to the agent via consumption of venison (Sigurdson, 2008). Initial experimental research suggests that human susceptibility to CWD is low and there may be a robust species barrier for CWD transmission to humans (Sigurdson, 2008), however the risk appetite for a public health threat may still find this level unacceptable.
i am thinking of that 10,000,000 POUNDS OF BLOOD LACED MEAT AND BONE MEAL IN COMMERCE WARNING LETTER back in 2007, see;
a review of banned mad cow feed in USA;
BANNED MAD COW FEED IN COMMERCE IN ALABAMA
Date: September 6, 2006 at 7:58 am PST PRODUCT
a) EVSRC Custom dairy feed, Recall # V-130-6;
b) Performance Chick Starter, Recall # V-131-6;
c) Performance Quail Grower, Recall # V-132-6;
d) Performance Pheasant Finisher, Recall # V-133-6.
CODE None RECALLING FIRM/MANUFACTURER Donaldson & Hasenbein/dba J&R Feed Service, Inc., Cullman, AL, by telephone on June 23, 2006 and by letter dated July 19, 2006. Firm initiated recall is complete.
REASON
Dairy and poultry feeds were possibly contaminated with ruminant based protein.
VOLUME OF PRODUCT IN COMMERCE 477.72 tons
DISTRIBUTION AL
______________________________
http://www.fda.gov/bbs/topics/enforce/2006/ENF00968.html
PRODUCT Bulk custom dairy pre-mixes,
Recall # V-120-6 CODE None RECALLING FIRM/MANUFACTURER Ware Milling Inc., Houston, MS, by telephone on June 23, 2006. Firm initiated recall is complete. REASON Possible contamination of dairy animal feeds with ruminant derived meat and bone meal.
VOLUME OF PRODUCT IN COMMERCE 350 tons
DISTRIBUTION AL and MS
______________________________
PRODUCT
a) Tucker Milling, LLC Tm 32% Sinking Fish Grower, #2680-Pellet, 50 lb. bags, Recall # V-121-6;
b) Tucker Milling, LLC #31120, Game Bird Breeder Pellet, 50 lb. bags, Recall # V-122-6;
c) Tucker Milling, LLC #31232 Game Bird Grower, 50 lb. bags, Recall # V-123-6;
d) Tucker Milling, LLC 31227-Crumble, Game Bird Starter, BMD Medicated, 50 lb bags, Recall # V-124-6;
e) Tucker Milling, LLC #31120, Game Bird Breeder, 50 lb bags, Recall # V-125-6;
f) Tucker Milling, LLC #30230, 30 % Turkey Starter, 50 lb bags, Recall # V-126-6;
g) Tucker Milling, LLC #30116, TM Broiler Finisher, 50 lb bags, Recall # V-127-6
CODE All products manufactured from 02/01/2005 until 06/20/2006 RECALLING FIRM/MANUFACTURER Recalling Firm: Tucker Milling LLC, Guntersville, AL, by telephone and visit on June 20, 2006, and by letter on June 23, 2006. Manufacturer: H. J. Baker and Brothers Inc., Stamford, CT. Firm initiated recall is ongoing.
REASON Poultry and fish feeds which were possibly contaminated with ruminant based protein were not labeled as "Do not feed to ruminants".
VOLUME OF PRODUCT IN COMMERCE 7,541-50 lb bags
DISTRIBUTION AL, GA, MS, and TN
END OF ENFORCEMENT REPORT FOR AUGUST 9, 2006
###
http://www.fda.gov/bbs/topics/ENFORCE/2006/ENF00964.html
Subject: MAD COW FEED RECALL AL AND FL VOLUME OF PRODUCT IN COMMERCE 125 TONS Products manufactured from 02/01/2005 until 06/06/2006
Date: August 6, 2006 at 6:16 pm PST PRODUCT
a) CO-OP 32% Sinking Catfish, Recall # V-100-6;
b) Performance Sheep Pell W/Decox/A/N, medicated, net wt. 50 lbs, Recall # V-101-6;
c) Pro 40% Swine Conc Meal -- 50 lb, Recall # V-102-6;
d) CO-OP 32% Sinking Catfish Food Medicated, Recall # V-103-6;
e) "Big Jim's" BBB Deer Ration, Big Buck Blend, Recall # V-104-6;
f) CO-OP 40% Hog Supplement Medicated Pelleted, Tylosin 100 grams/ton, 50 lb. bag, Recall # V-105-6;
g) Pig Starter Pell II, 18% W/MCDX Medicated 282020, Carbadox -- 0.0055%, Recall # V-106-6;
h) CO-OP STARTER-GROWER CRUMBLES, Complete Feed for Chickens from Hatch to 20 Weeks, Medicated, Bacitracin Methylene Disalicylate, 25 and 50 Lbs, Recall # V-107-6;
i) CO-OP LAYING PELLETS, Complete Feed for Laying Chickens, Recall # 108-6;
j) CO-OP LAYING CRUMBLES, Recall # V-109-6;
k) CO-OP QUAIL FLIGHT CONDITIONER MEDICATED, net wt 50 Lbs, Recall # V-110-6;
l) CO-OP QUAIL STARTER MEDICATED, Net Wt. 50 Lbs, Recall # V-111-6;
m) CO-OP QUAIL GROWER MEDICATED, 50 Lbs, Recall # V-112-6 CODE
Product manufactured from 02/01/2005 until 06/06/2006
RECALLING FIRM/MANUFACTURER Alabama Farmers Cooperative, Inc., Decatur, AL, by telephone, fax, email and visit on June 9, 2006. FDA initiated recall is complete.
REASON Animal and fish feeds which were possibly contaminated with ruminant based protein not labeled as "Do not feed to ruminants".
VOLUME OF PRODUCT IN COMMERCE 125 tons
DISTRIBUTION AL and FL
END OF ENFORCEMENT REPORT FOR AUGUST 2, 2006
###
http://www.fda.gov/bbs/topics/enforce/2006/ENF00963.html
MAD COW FEED RECALL USA EQUALS 10,878.06 TONS NATIONWIDE Sun Jul 16, 2006 09:22 71.248.128.67
RECALLS AND FIELD CORRECTIONS: VETERINARY MEDICINE -- CLASS II
______________________________
PRODUCT
a) PRO-LAK, bulk weight, Protein Concentrate for Lactating Dairy Animals, Recall # V-079-6;
b) ProAmino II, FOR PREFRESH AND LACTATING COWS, net weight 50lb (22.6 kg), Recall # V-080-6;
c) PRO-PAK, MARINE & ANIMAL PROTEIN CONCENTRATE FOR USE IN ANIMAL FEED, Recall # V-081-6;
d) Feather Meal, Recall # V-082-6 CODE
a) Bulk
b) None
c) Bulk
d) Bulk
RECALLING FIRM/MANUFACTURER H. J. Baker & Bro., Inc., Albertville, AL, by telephone on June 15, 2006 and by press release on June 16, 2006. Firm initiated recall is ongoing.
REASON
Possible contamination of animal feeds with ruminent derived meat and bone meal.
VOLUME OF PRODUCT IN COMMERCE 10,878.06 tons
DISTRIBUTION Nationwide
END OF ENFORCEMENT REPORT FOR July 12, 2006
###
http://www.fda.gov/bbs/topics/enforce/2006/ENF00960.html
10,000,000+ LBS. of PROHIBITED BANNED MAD COW FEED I.E. BLOOD LACED MBM IN COMMERCE USA 2007
Date: March 21, 2007 at 2:27 pm PST
RECALLS AND FIELD CORRECTIONS: VETERINARY MEDICINES -- CLASS II
___________________________________
PRODUCT
Bulk cattle feed made with recalled Darling's 85% Blood Meal, Flash Dried, Recall # V-024-2007
CODE
Cattle feed delivered between 01/12/2007 and 01/26/2007
RECALLING FIRM/MANUFACTURER
Pfeiffer, Arno, Inc, Greenbush, WI. by conversation on February 5, 2007.
Firm initiated recall is ongoing.
REASON
Blood meal used to make cattle feed was recalled because it was cross- contaminated with prohibited bovine meat and bone meal that had been manufactured on common equipment and labeling did not bear cautionary BSE statement.
VOLUME OF PRODUCT IN COMMERCE
42,090 lbs.
DISTRIBUTION
WI
___________________________________
PRODUCT
Custom dairy premix products: MNM ALL PURPOSE Pellet, HILLSIDE/CDL Prot- Buffer Meal, LEE, M.-CLOSE UP PX Pellet, HIGH DESERT/ GHC LACT Meal, TATARKA, M CUST PROT Meal, SUNRIDGE/CDL PROTEIN Blend, LOURENZO, K PVM DAIRY Meal, DOUBLE B DAIRY/GHC LAC Mineral, WEST PIONT/GHC CLOSEUP Mineral, WEST POINT/GHC LACT Meal, JENKS, J/COMPASS PROTEIN Meal, COPPINI - 8# SPECIAL DAIRY Mix, GULICK, L-LACT Meal (Bulk), TRIPLE J - PROTEIN/LACTATION, ROCK CREEK/GHC MILK Mineral, BETTENCOURT/GHC S.SIDE MK-MN, BETTENCOURT #1/GHC MILK MINR, V&C DAIRY/GHC LACT Meal, VEENSTRA, F/GHC LACT Meal, SMUTNY, A- BYPASS ML W/SMARTA, Recall # V-025-2007
CODE
The firm does not utilize a code - only shipping documentation with commodity and weights identified.
RECALLING FIRM/MANUFACTURER
Rangen, Inc, Buhl, ID, by letters on February 13 and 14, 2007. Firm initiated recall is complete.
REASON
Products manufactured from bulk feed containing blood meal that was cross contaminated with prohibited meat and bone meal and the labeling did not bear cautionary BSE statement.
VOLUME OF PRODUCT IN COMMERCE
9,997,976 lbs.
DISTRIBUTION
ID and NV
END OF ENFORCEMENT REPORT FOR MARCH 21, 2007
http://www.fda.gov/Safety/Recalls/EnforcementReports/2007/ucm120446.htm
RE-Inactivation of porcine endogenous retrovirus in pigs using CPISPR-Cas9
TERRY S. SINGELTARY SR. - retired
- Mr.
seems that the USA feed ban for ruminant protein is still a serious problem, so there seems to still be a risk factor for pigs and Transmissible Spongiform Encephalopathy TSE prion disease. now with the updated science showing that pigs are susceptible to the Chronic Wasting Disease TSE Prion ORALLY, and cwd running rampant in the USA, any use of porcine organs should be tested for the CWD TSE Prion...
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 item Kunkle, Robert item Kondru, Naveen item Manne, Sireesha item Smith, Jodi item Kanthasamy, Anumantha item West Greenlee, M 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.
CONFIDENTIAL
EXPERIMENTAL PORCINE SPONGIFORM ENCEPHALOPATHY
While this clearly is a cause for concern we should not jump to the conclusion that this means that pigs will necessarily be infected by bone and meat meal fed by the oral route as is the case with cattle. ...
we cannot rule out the possibility that unrecognised subclinical spongiform encephalopathy could be present in British pigs though there is no evidence for this: only with parenteral/implantable pharmaceuticals/devices is the theoretical risk to humans of sufficient concern to consider any action.
Our records show that while some use is made of porcine materials in medicinal products, the only products which would appear to be in a hypothetically ''higher risk'' area are the adrenocorticotrophic hormone for which the source material comes from outside the United Kingdom, namely America China Sweden France and Germany. The products are manufactured by Ferring and Armour. A further product, ''Zenoderm Corium implant'' manufactured by Ethicon, makes use of porcine skin - which is not considered to be a ''high risk'' tissue, but one of its uses is described in the data sheet as ''in dural replacement''. This product is sourced from the United Kingdom.....
snip...see much more here ;
OIE Bulletin
Camel prion disease: a possible emerging disease in dromedary camel populations?
The identification of a new prion disease in dromedary camels in Algeria and Tunisia, called camel prion disease (CPD), extends the spectrum of animal species naturally susceptible to prion diseases and opens up new research areas for investigation.
Camel prion disease was identified in 2018 in adult camels showing clinical signs at the ante mortem inspection at slaughterhouses in the region of Ouargla (Algeria), and in 2019 in the region of Tataouine (Tunisia). It adds to the group of existing animal prion diseases, including scrapie in sheep and goats, chronic wasting disease (CWD) in cervids and BSE (mainly in bovines). The detection of a new prion disease in the dromedary population requires attention and investigation needs to be carried out to assess the risks of this disease to animal and public health. As of today, very limited epidemiological information is available to assess the prevalence, geographical distribution and dynamic of the transmission of the disease.
Based on the clinical signs suggesting prion disease, CPD seems to have occurred in 3.1% of the dromedaries brought to the abattoir in Ouargla. Pathognomonic neurodegeneration and disease specific prion protein (PrPSc) were detected in brain tissue from three symptomatic animals (source:
In May 2019, the OIE received a report from Tunisia on a single case of a 12-year-old slaughtered dromedary camel showing neurological signs confirmed as CPD by the Istituto Superiore di Sanità (ISS) based in Italy.
©B. Babelhadj/University Kasdi Merbah, Algeria
2
Is camel prion disease transmissible in natural conditions?
The involvement of lymphoid tissue in prion replication, observed both in the Algeria and Tunisia cases, is suggestive of a peripheral pathogenesis, which is thought to be a prerequisite for prion shedding into the environment. As with other animal prion diseases, such as scrapie and CWD, in which lymphoid tissues are extensively involved and horizontal transmission occurs efficiently under natural conditions, the detection of prion proteins in lymph nodes is suggestive of the infectious nature of CPD and concurs to hypothesise the potential impact of CPD on animal health. No evidence is currently available with which to argue for the relevance of CPD for human health. However, no absolute species barrier exists in prion diseases and minimising the exposure of humans to prion-infected animal products is an essential aspect of public health protection. As for the relationship between CPD and other animal prion diseases, preliminary analyses suggest that CPD prions have a different molecular signature from scrapie and BSE.
Actions on the follow up of CPD
Since the first description of CPD, the OIE promoted discussions on the impact of this new disease through the OIE Scientific Commission for Animal Diseases (Scientific Commission). The Scientific Commission consulted two OIE ad hoc Groups, one on BSE risk status evaluation of Members and the other on camelids. It analysed the information available from the Algeria and Tunisia cases to evaluate if CPD should be considered an ‘emerging disease’ based on the criteria listed in the Terrestrial Animal Health Code1 .
The OIE Scientific Commission noted that limited surveillance data were available on the prevalence of CPD and that the evidence was not sufficient to measure, at that time, the impact of the disease on animal or public health. Therefore, it was concluded that, with the current knowledge, CPD did not currently meet the criteria to be considered an emerging disease. Nonetheless, it was emphasised that CPD should be considered as a new disease not to be overlooked and called for the collection of further scientific evidence through research and surveillance in the affected countries and in countries with dromedary camel populations to measure the impact of the disease. As new scientific evidence becomes available, the OIE Scientific Commission will reassess whether this disease should be considered as an emerging disease.
The worldwide camel population is ~35 million head (FAO, 2019), 88% of which is found in Africa. The camel farming system is evolving rapidly, and these animals represent vital sources of meat, milk and transportation for millions of people living in the most arid regions of the world. This makes it necessary to assess the risk for animal and human health and to develop evidence-based policies to control and limit the spread of the disease in animals, and to minimise human exposure. As a first step, the awareness of Veterinary Services about CPD and its diagnostic capacity needs to be improved in all countries where dromedaries are part of the domestic livestock.
At the regional level, CPD was first discussed in the 18th Joint Permanent Committee of the Mediterranean Animal Health Network (REMESA) held in Cairo, Egypt, in June 2019 where an expert 1 a new occurrence in an animal of a disease, infection or infestation, causing a significant impact on animal or public health resulting from a) a change of a known pathogenic agent or its spread to a new geographic area or species, or b) a previously unrecognised pathogenic agent or disease diagnosed for the first time www.oiebulletin.com
3
from ISS, Italy, shared the knowledge available on the new disease with the 15 REMESA Member Countries. The discussion highlighted the need to strengthen surveillance systems in order to collect epidemiological data to inform the risk assessments. The results of these risk assessments will support the implementation of evidence-based policies to manage the risks in both animals and humans.
CPD was recently discussed atthe 15thConference of the OIE Regional Commission for the Middle East in November. During this conference, the CAMENET (Camel Middle East Network) launched a wide ranging proposal for training, coordinated surveillance and research on CPD. In addition, the ERFAN (Enhancing Research for Africa Network), a platform aimed at enhancing scientific cooperation between Africa and Italy, during its 2nd ERFAN meeting for North Africa, presented a project on CPD with the objective of increasing CPD coordinated surveillance in North Africa.
The OIE, through its Reference Laboratories for prion diseases, and by involving the above scientific initiatives, is keeping a close watch on the evolution of the disease to gather scientific evidence and to allow a proper and more thorough assessment of the risk associated with this novel disease.
◼ December 2019
Monday, November 14, 2022
Prion Diseases in Dromedary Camels (CPD) 2022 Review
THE tse prion aka mad cow type disease is not your normal pathogen.
The TSE prion disease survives ashing to 600 degrees celsius, that’s around 1112 degrees farenheit.
you cannot cook the TSE prion disease out of meat.
you can take the ash and mix it with saline and inject that ash into a mouse, and the mouse will go down with TSE.
Prion Infected Meat-and-Bone Meal Is Still Infectious after Biodiesel Production as well.
the TSE prion agent also survives Simulated Wastewater Treatment Processes.
IN fact, you should also know that the TSE Prion agent will survive in the environment for years, if not decades.
you can bury it and it will not go away.
The TSE agent is capable of infected your water table i.e. Detection of protease-resistant cervid prion protein in water from a CWD-endemic area.
it’s not your ordinary pathogen you can just cook it out and be done with.
***> that’s what’s so worrisome about Iatrogenic mode of transmission, a simple autoclave will not kill this TSE prion agent.
1: J Neurol Neurosurg Psychiatry 1994 Jun;57(6):757-8
***> Transmission of Creutzfeldt-Jakob disease to a chimpanzee by electrodes contaminated during neurosurgery.
Gibbs CJ Jr, Asher DM, Kobrine A, Amyx HL, Sulima MP, Gajdusek DC.
Laboratory of Central Nervous System Studies, National Institute of
Neurological Disorders and Stroke, National Institutes of Health,
Bethesda, MD 20892.
Stereotactic multicontact electrodes used to probe the cerebral cortex of a middle aged woman with progressive dementia were previously implicated in the accidental transmission of Creutzfeldt-Jakob disease (CJD) to two younger patients. The diagnoses of CJD have been confirmed for all three cases. More than two years after their last use in humans, after three cleanings and repeated sterilisation in ethanol and formaldehyde vapour, the electrodes were implanted in the cortex of a chimpanzee. Eighteen months later the animal became ill with CJD. This finding serves to re-emphasise the potential danger posed by reuse of instruments contaminated with the agents of spongiform encephalopathies, even after scrupulous attempts to clean them.
PMID: 8006664 [PubMed - indexed for MEDLINE]
New studies on the heat resistance of hamster-adapted scrapie agent: Threshold survival after ashing at 600°C suggests an inorganic template of replication
Prion Infected Meat-and-Bone Meal Is Still Infectious after Biodiesel Production
MONDAY, APRIL 19, 2021
Evaluation of the application for new alternative biodiesel production process for rendered fat including Category 1 animal by-products (BDI-RepCat® process, AT) ???
Detection of protease-resistant cervid prion protein in water from a CWD-endemic area
A Quantitative Assessment of the Amount of Prion Diverted to Category 1 Materials and Wastewater During Processing
Rapid assessment of bovine spongiform encephalopathy prion inactivation by heat treatment in yellow grease produced in the industrial manufacturing process of meat and bone meals
THURSDAY, FEBRUARY 28, 2019
BSE infectivity survives burial for five years with only limited spread
5 or 6 years quarantine is NOT LONG ENOUGH FOR CWD TSE PRION !!!
QUARANTINE NEEDS TO BE 21 YEARS FOR CWD TSE PRION !
FRIDAY, APRIL 30, 2021
Should Property Evaluations Contain Scrapie, CWD, TSE PRION Environmental Contamination of the land?
***> Confidential!!!!
***> As early as 1992-3 there had been long studies conducted on small pastures containing scrapie infected sheep at the sheep research station associated with the Neuropathogenesis Unit in Edinburgh, Scotland. Whether these are documented...I don't know. But personal recounts both heard and recorded in a daily journal indicate that leaving the pastures free and replacing the topsoil completely at least 2 feet of thickness each year for SEVEN years....and then when very clean (proven scrapie free) sheep were placed on these small pastures.... the new sheep also broke out with scrapie and passed it to offspring. I am not sure that TSE contaminated ground could ever be free of the agent!! A very frightening revelation!!!
---end personal email---end...tss
and so it seems...
Scrapie Agent (Strain 263K) Can Transmit Disease via the Oral Route after Persistence in Soil over Years
Published: May 9, 2007
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Our results showed that 263K scrapie agent can persist in soil at least over 29 months. Strikingly, not only the contaminated soil itself retained high levels of infectivity, as evidenced by oral administration to Syrian hamsters, but also feeding of aqueous soil extracts was able to induce disease in the reporter animals. We could also demonstrate that PrPSc in soil, extracted after 21 months, provides a catalytically active seed in the protein misfolding cyclic amplification (PMCA) reaction. PMCA opens therefore a perspective for considerably improving the detectability of prions in soil samples from the field.
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Dr. Paul Brown Scrapie Soil Test BSE Inquiry Document
ENVIRONMENT FACTORS FOR THE TRANSMISSION OF CWD TSE PRP
Sensitive detection of chronic wasting disease prions recovered from environmentally relevant surfaces
Environment International
Available online 13 June 2022, 107347
Environment International
Sensitive detection of chronic wasting disease prions recovered from environmentally relevant surfaces
Qi Yuana Gag e Rowdenb Tiffany M.Wolfc Marc D.Schwabenlanderb Peter A.LarsenbShannon L.Bartelt-Huntd Jason C.Bartza
a Department of Medical Microbiology and Immunology, Creighton University, Omaha, Nebraska, 68178, United States of America
b Department of Veterinary and Biomedical Sciences, University of Minnesota, Saint Paul, MN, 55108, United States of America
c Department of Veterinary Population Medicine, University of Minnesota, Saint Paul, MN, 55108, United States of America
d Department of Civil and Environmental Engineering, Peter Kiewit Institute, University of Nebraska-Lincoln, Omaha, Nebraska, 68182, United States of America
Received 26 April 2022, Revised 8 June 2022, Accepted 9 June 2022, Available online 13 June 2022.
Get rights and content
Under a Creative Commons license Open access
Highlights • An innovative method for prion recovery from swabs was developed.
• Recovery of prions decreased as swab-drying time was increased.
• Recovery of CWD prions from stainless steel and glass was approximately 30%.
• RT-QuIC enhanced CWD prion detection by 4 orders of magnitude.
• Surface-recovered CWD prion was sufficient for efficient RT-QuIC detection.
Abstract
Chronic wasting disease (CWD) has been identified in 30 states in the United States, four provinces in Canada, and recently emerged in Scandinavia. The association of CWD prions with environmental materials such as soil, plants, and surfaces may enhance the persistence of CWD prion infectivity in the environment exacerbating disease transmission. Identifying and quantifying CWD prions in the environment is significant for prion monitoring and disease transmission control. A systematic method for CWD prion quantification from associated environmental materials, however, does not exist. In this study, we developed an innovative method for extracting prions from swabs and recovering CWD prions swabbed from different types of surfaces including glass, stainless steel, and wood. We found that samples dried on swabs were unfavorable for prion extraction, with the greatest prion recovery from wet swabs. Using this swabbing technique, the recovery of CWD prions dried to glass or stainless steel was approximately 30% in most cases, whereas that from wood was undetectable by conventional prion immunodetection techniques. Real-time quake-induced conversion (RT-QuIC) analysis of these same samples resulted in an increase of the detection limit of CWD prions from stainless steel by 4 orders of magnitude. More importantly, the RT-QuIC detection of CWD prions recovered from stainless steel surfaces using this method was similar to the original CWD prion load applied to the surface. This combined surface swabbing and RT-QuIC detection method provides an ultrasensitive means for prion detection across many settings and applications.
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5. Conclusions
Chronic wasting disease is spreading in North America and it is hypothesized that in CWD-endemic areas environmental persistence of CWD prions can exacerbate disease transmission. The development of a sensitive CWD prion detection method from environmentally relevant surfaces is significant for monitoring, risk assessment, and control of CWD. In this study, we developed a novel swab-extraction procedure for field deployable sampling of CWD prions from stainless steel, glass, and wood. We found that extended swab-drying was unfavorable for extraction, indicating that hydrated storage of swabs after sampling aided in prion recovery. Recoverable CWD prions from stainless steel and glass was approximately 30%, which was greater than from wood. RT-QuIC analysis of the swab extracts resulted in an increase of the detection limit of CWD prions from stainless steel by 4 orders of magnitude compared to conventional immunodetection techniques. More importantly, the RT-QuIC detection of CWD prions recovered from stainless steel surfaces using this developed method was similar to the original CWD prion load without surface contact. This method of prion sampling and recovery, in combination with ultrasensitive detection methods, allows for prion detection from contaminated environmental surfaces.
Research Paper
Cellular prion protein distribution in the vomeronasal organ, parotid, and scent glands of white-tailed deer and mule deer
Anthony Ness, Aradhana Jacob, Kelsey Saboraki, Alicia Otero, Danielle Gushue, Diana Martinez Moreno, Melanie de Peña, Xinli Tang, Judd Aiken, Susan Lingle & Debbie McKenzie ORCID Icon show less
Pages 40-57 | Received 03 Feb 2022, Accepted 13 May 2022, Published online: 29 May 2022
Download citation
ABSTRACT
Chronic wasting disease (CWD) is a contagious and fatal transmissible spongiform encephalopathy affecting species of the cervidae family. CWD has an expanding geographic range and complex, poorly understood transmission mechanics. CWD is disproportionately prevalent in wild male mule deer and male white-tailed deer. Sex and species influences on CWD prevalence have been hypothesized to be related to animal behaviours that involve deer facial and body exocrine glands. Understanding CWD transmission potential requires a foundational knowledge of the cellular prion protein (PrPC) in glands associated with cervid behaviours. In this study, we characterized the presence and distribution of PrPC in six integumentary and two non-integumentary tissues of hunter-harvested mule deer (Odocoileus hemionus) and white-tailed deer (O. virginianus). We report that white-tailed deer expressed significantly more PrPC than their mule deer in the parotid, metatarsal, and interdigital glands. Females expressed more PrPC than males in the forehead and preorbital glands. The distribution of PrPC within the integumentary exocrine glands of the face and legs were localized to glandular cells, hair follicles, epidermis, and immune cell infiltrates. All tissues examined expressed sufficient quantities of PrPC to serve as possible sites of prion initial infection, propagation, and shedding.
KEYWORDS: Prion chronic wasting diseasesex differences species differences disease prevalence cervid protein expression glands
Paper
Rapid recontamination of a farm building occurs after attempted prion removal
Kevin Christopher Gough BSc (Hons), PhD Claire Alison Baker BSc (Hons) Steve Hawkins MIBiol Hugh Simmons BVSc, MRCVS, MBA, MA Timm Konold DrMedVet, PhD, MRCVS … See all authors
First published: 19 January 2019 https://doi.org/10.1136/vr.105054
The data illustrates the difficulty in decontaminating farm buildings from scrapie, and demonstrates the likely contribution of farm dust to the recontamination of these environments to levels that are capable of causing disease.
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This study clearly demonstrates the difficulty in removing scrapie infectivity from the farm environment. Practical and effective prion decontamination methods are still urgently required for decontamination of scrapie infectivity from farms that have had cases of scrapie and this is particularly relevant for scrapiepositive goatherds, which currently have limited genetic resistance to scrapie within commercial breeds.24 This is very likely to have parallels with control efforts for CWD in cervids.
***>This is very likely to have parallels with control efforts for CWD in cervids.
***> Infectious agent of sheep scrapie may persist in the environment for at least 16 years
***> Nine of these recurrences occurred 14–21 years after culling, apparently as the result of environmental contamination, but outside entry could not always be absolutely excluded.
JOURNAL OF GENERAL VIROLOGY Volume 87, Issue 12
Infectious agent of sheep scrapie may persist in the environment for at least 16 years Free
Gudmundur Georgsson1, Sigurdur Sigurdarson2, Paul Brown3
Front. Vet. Sci., 14 September 2015 | https://doi.org/10.3389/fvets.2015.00032
Objects in contact with classical scrapie sheep act as a reservoir for scrapie transmission
imageTimm Konold1*, imageStephen A. C. Hawkins2, imageLisa C. Thurston3, imageBen C. Maddison4, imageKevin C. Gough5, imageAnthony Duarte1 and imageHugh A. Simmons1
The findings of this study highlight the role of field furniture used by scrapie-infected sheep to act as a reservoir for disease re-introduction although infectivity declines considerably if the field furniture has not been in contact with scrapie-infected sheep for several months. PMCA may not be as sensitive as VRQ/VRQ sheep to test for environmental contamination.
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Discussion
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In conclusion, the results in the current study indicate that removal of furniture that had been in contact with scrapie-infected animals should be recommended, particularly since cleaning and decontamination may not effectively remove scrapie infectivity (31), even though infectivity declines considerably if the pasture and the field furniture have not been in contact with scrapie-infected sheep for several months. As sPMCA failed to detect PrPSc in furniture that was subjected to weathering, even though exposure led to infection in sheep, this method may not always be reliable in predicting the risk of scrapie infection through environmental contamination.
***> 172. Establishment of PrPCWD extraction and detection methods in the farm soil
Kyung Je Park, Hoo Chang Park, In Soon Roh, Hyo Jin Kim, Hae-Eun Kang and Hyun Joo Sohn
Foreign Animal Disease Division, Animal and Plant Quarantine Agency, Gimcheon, Gyeongsangbuk-do, Korea
Conclusions: Our studies showed that PrPCWD persist in 0.001% CWD contaminated soil for at least 4 year and natural CWD-affected farm soil. When cervid reintroduced into CWD outbreak farm, the strict decontamination procedures of the infectious agent should be performed in the environment of CWD-affected cervid habitat.
Published: 06 September 2021
***> Chronic wasting disease: a cervid prion infection looming to spillover
Alicia Otero, Camilo Duque Velásquez, Judd Aiken & Debbie McKenzie
Veterinary Research volume 52, Article number: 115 (2021)
October 6th-12th, 126th Meeting 2022 Resolutions
RESOLUTION NUMBER: 30 Approved
SOURCE: COMMITTEE ON WILDLIFE
SUBJECT MATTER: Chronic Wasting Disease Carcass Disposal Dumpster Management and Biosecurity
BACKGROUND INFORMATION:
State and tribal wildlife agencies may identify collection points (dumpsters) within an identified chronic wasting disease (CWD) management zone for the disposal of hunter-harvested cervid carcasses to remove potentially infected carcasses off the landscape for disposal by an approved method (Gillin & Mawdsley, 2018, chap.14). However, depending on their placement and maintenance these dumpsters could potentially increase the risk of CWD transmission.
In several different states, photographic evidence has shown dumpsters in state identified CWD management zones overflowing with deer carcasses and limbs scattered on the land nearby. This could provide an opportunity for scavengers to potentially move infected carcass material to non-infected zones or increase contamination of the ground material around the dumpster’s location.
Federal guidance does not explicitly address uniform standards for collection locations for carcasses of free-ranging cervids; however, the United States Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Services Program Standards on CWD outlines procedures for carcass disposal, equipment sanitation, and decontamination of premises for captive cervid facilities.
RESOLUTION:
The United States Animal Health Association urges the Association of Fish and Wildlife Agencies (AFWA), Wildlife Health Committee to further refine the AFWA Technical Report on Best Management Practices for Prevention, Surveillance, and Management of Chronic Wasting Disease; Chapter 14, Carcass Disposal to address the placement and management of chronic wasting disease carcass disposal dumpsters or other carcass collection containers.
Reference:
1. Gillin, Colin M., and Mawdsley, Jonathan R. (eds.). 2018. AFWA Technical Report on Best Management Practices for Surveillance, Management and Control of Chronic Wasting Disease. Association of Fish and Wildlife Agencies, Washington, D. C. 111 pp.
PRION CONFERENCE 2022 ABSTRACTS CWD TSE PrP ZOONOSIS and ENVIRONMENTAL FACTORS
Chronic wasting disease detection in environmental and biological samples from a taxidermy site
Paulina Sotoa,b, J. Hunter Reedc, Mitch Lockwoodc, and Rodrigo Moralesa,b aDepartment of Neurology, McGovern Medical School, University of Texas Health Science Center at Houston, Texas, USA; bUniversidad Bernardo O’Higgins, Santiago, Chile; cTexas Parks and Wildlife Department, Texas, USA
Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy affecting captive and free-ranging cervids (e.g., mule deer, white-tailed deer, elk, reindeer, and moose). Nowadays, CWD is widely distributed in North America. It is suggested that CWD spreads due to direct animal contact or through exposure to contaminated environments previously inhabited by infected animals. CWD may also be spread through the movement of infected animals and carcasses. Taxidermy practices involve processing deer tissues (or whole animal carcasses). In many cases, the CWD status of processed animals is unknown. This can generate risks of disease spread and transmission. Taxidermy practices include different steps involving physical, chemical, and biological procedures. Without proper tissue handling or disposal practices, taxidermist facilities may become a focus of prion infectivity.
Aims: In this study, we evaluated the presence of infectious prions in a taxidermy facility believed to be exposed to CWD. Detection was performed using the Protein Misfolding Cyclic Amplification (PMCA) technique in biological and inert environmental samples. Methods: We collected biological and environmental samples (plants, soils, insects, excreta, and others) from a taxidermy facility, and we tested these samples using the PMCA technique. In addition, we swabbed different surfaces possibly exposed to CWD-infected animals. For the PMCA reaction, we directly used a swab piece or 10 µL of 20% w/v homogenized samples.
Results: The PMCA analysis demonstrated CWD seeding activity in some of the components of this facility, including insects involved in head processing, soils, and a trash dumpster.
Conclusions: Different areas of this property were used for various taxidermy procedures. We were able to detect the presence of prions in
i) soils that were in contact with the heads of dead animals,
ii) insects involved in the cleaning of skulls, and
iii) an empty dumpster where animal carcasses were previously placed.
This is the first report demonstrating that swabbing is a helpful method to screen for prion infectivity on surfaces potentially contaminated with CWD. These findings are relevant as this swabbing and amplification strategy may be used to evaluate the disease status of other free-ranging and captive settings where there is a concern for CWD transmissions, such as at feeders and water troughs with CWD-exposed properties. This approach could have substantial implications for free-ranging cervid surveillance as well as in epidemiological investigations of CWD.
PRION CONFERENCE 2022 ABSTRACTS CWD TSE PrP ZOONOSIS
Transmission of prion infectivity from CWD-infected macaque tissues to rodent models demonstrates the zoonotic potential of chronic wasting disease.
Samia Hannaouia, Ginny Chenga, Wiebke Wemheuerb, Walter J. Schulz-Schaefferb, Sabine Gilcha, and Hermann M. Schätzla aDepartment of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine & Hotchkiss Brain Institute; University of Calgary, Calgary, Canada; bInstitute of Neuropathology, Medical Faculty, Saarland University, Homburg/Saar, Germany
Aims: Chronic wasting disease (CWD) is a prion disease of cervids. Its rapid geographic expansion, shedding of infectivity and persistence in the environment for many years are of concern for humans. Here, we provide the first evidence by transmission experiments to different transgenic mouse models and bank voles that Cynomolgus macaques inoculated via different routes with CWD-positive cervid tissues harbor infectious prions that elicit clinical disease in rodents.
Material and Methods: We used tissue materials from macaques inoculated with CWD to inoculate transgenic mice overexpressing cervid PrPCfollowed by transmission into bank voles. We used RT-QuIC, immunoblot and PET blot analysis to assess brains, spinal cords, and tissues of the gastrointestinal tract (GIT) for the presence of prions.
Results: Our results show that of the macaque materials that induced clinical disease in transgenic mice,73% were from the CNS (46% spinal cord and 27% brain), and 27% were from the spleen, although attack rates were low around 20%. Clinical mice did not display PK-resistant PrPSc(PrPres) in immunoblot, but showed low-levels of prion seeding activity. Transmission into bank voles from clinical transgenic mice led to a 100% attack rate with typical PrPressignature in immunoblot, which was different from that of voles inoculated directly with CWD or scrapie prions. High-level prion seeding activity in brain and spinal cord and PrPresdeposition in the brain were present. Remarkably, we also found prion seeding activity in GIT tissues of inoculated voles. Second passage in bank voles led to a 100% attack rate in voles inoculated with brain, spinal cord and small intestine material from first round animals, with PrPresin immunoblot, prion seeding activity, and PrPresdeposition in the brain. Shortened survival times indicate adaptation in the new host. This also shows that prions detected in GIT tissues are infectious and transmissible. Transmission of brain material from sick voles back to cervidized mice revealed transmission in these mice with a 100% attack rate, and interestingly, with different biochemical signature and distribution in the brain.
Conclusions: Our findings demonstrate that macaques, considered the best model for the zoonotic potential of prions, were infected upon CWD challenge, including oral one. The disease manifested as atypical in macaques and transgenic mice, but with infectivity present at all times, as unveiled in the bank vole model with an unusual tissue tropism.
Funded by: The National Institutes of Health, USA, and the Alberta Prion Research Institute/Alberta Innovates Canada. Grant number: 1R01NS121016-01; 201,600,023
Acknowledgement: We thank Umberto Agrimi, Istituto Superiore di Sanità, Rome, Italy, and Michael Beekes, Robert-Koch Institute Berlin, Germany, for providing the bank vole model. We thank the University of Calgary animal facility staff and Dr. Stephanie Anderson for animal care.
Transmission of Cervid Prions to Humanized Mice Demonstrates the Zoonotic Potential of CWD
Samia Hannaouia, Irina Zemlyankinaa, Sheng Chun Changa, Maria Immaculata Arifina, Vincent Béringueb, Debbie McKenziec, Hermann M. Schatzla, and Sabine Gilcha
aDepartment of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine; Hotchkiss Brain Institute; University of Calgary, Calgary, Canada; bUniversité Paris-Saclay, INRAE, UVSQ, VIM, Jouy-en-Josas, France; cDepartment of Biological Sciences, Center for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Canada
Aims: Chronic wasting disease (CWD), a prion disease of cervids, spreads efficiently among wild and farmed animals. Potential transmission to humans of CWD is a growing concern due to its increasing prevalence. Here, we aimed to determine the zoonotic potential of CWD using a mouse model for human prion diseases.
Material and Methods: Transgenic mice overexpressing human PrPChomozygous for methionine at codon 129 (tg650) were inoculated intracerebrally with brain homogenates of white-tailed deer infected with Wisc-1/CWD1 or 116AG CWD strains. Mice were monitored for clinical signs and were euthanized at terminal disease. Brains were tested by RT-QuIC, western blot upon PK digestion, and immunohistochemistry; fecal homogenates were analyzed by RT-QuIC. Brain/spinal cord and fecal homogenates of CWD-inoculated tg650 mice were inoculated into tg650 mice or bank voles. Brain homogenates of bank voles inoculated with fecal homogenates of CWD-infected tg650 mice were used for second passage in bank voles.
Results: Here, we provide the strongest evidence supporting the zoonotic potential of CWD prions, and their possible phenotype in humans. Inoculation of mice expressing human PrPCwith deer CWD isolates (strains Wisc-1 and 116AG) resulted in atypical clinical manifestations in > 75% of the mice, with myoclonus as leading clinical sign. Most of tg650 brain homogenates were positive for seeding activity in RT-QuIC. Clinical disease and presentation was transmissible to tg650 mice and bank voles. Intriguingly, protease-resistant PrP in the brain of tg650 mice resembled that found in a familial human prion disease and was transmissible upon passage. Abnormal PrP aggregates upon infection with Wisc-1 were detectable in thalamus, hypothalamus, and midbrain/pons regions.
Unprecedented in human prion disease, feces of CWD-inoculated tg650 mice harbored prion seeding activity and infectious prions, as shown by inoculation of bank voles and tg650 with fecal homogenates.
Conclusions: This is the first evidence that CWD can infect humans and cause disease with a distinctive clinical presentation, signature, and tropism, which might be transmissible between humans while current diagnostic assays might fail to detect it. These findings have major implications for public health and CWD-management.
Funded by: We are grateful for financial support from the Natural Sciences and Engineering Research Council of Canada, the National Institutes of Health, Genome Canada, and the Alberta Prion Research Institute. SG is supported by the Canada Research Chairs program.
Acknowledgement: We thank Dr. Trent Bollinger, WCVM, University of Saskatchewan, Saskatoon, Canada, for providing brain tissue from the WTD-116AG isolate, Dr. Stéphane Haïk, ICM, Paris, France, for providing brain tissue from vCJD and sCJD cases, and Dr. Umberto Agrimi, Istituto Superiore di Sanità, Italy, for the bank vole model. We thank animal facility staff for animal care, Dr. Stephanie Anderson for veterinary oversight, and Yo-Ching Cheng for preparing recombinant PrP substrates. Thank you to Dr. Stephanie Booth and Jennifer Myskiw, Public Health Agency of Canada, Canada.
The chronic wasting disease agent from white-tailed deer is infectious to humanized mice after passage through raccoons
Eric Cassmanna, Xu Qib, Qingzhong Kongb, and Justin Greenleea
aNational Animal Disease Center, Agricultural Research Service, US Department of Agriculture, Ames, IA, USA bDepartments of Pathology, Neurology, National Center for Regenerative Medicine, and National Prion Disease Pathology Surveillance Center, Case Western Reserve University, Cleveland, Ohio, USA
Aims: Evaluate the zoonotic potential of the raccoon passaged chronic wasting disease (CWD) agent in humanized transgenic mice in comparison with the North American CWD agent from the original white-tailed deer host.
Material and Methods: Pooled brain material (GG96) from a CWD positive herd was used to oronasally inoculate two white-tailed deer with wild-type prion protein genotype and intracranially inoculate a raccoon. Brain homogenates (10% w/v) from the raccoon and the two white-tailed deer were used to intracranially inoculate separate groups of transgenic mice that express human prion protein with methionine (M) at codon 129 (Tg40h). Brains and spleens were collected from mice at experimental endpoints of clinical disease or approximately 700 days post-inoculation. Tissues were divided and homogenized or fixed in 10% buffered neutral formalin. Immunohistochemistry, enzyme immunoassay, and western blot were used to detect misfolded prion protein (PrPSc) in tissue.
Results: Humanized transgenic mice inoculated with the raccoon passaged CWD agent from white-tailed deer exhibited a 100% (12/12) attack rate with an average incubation period of 605 days. PrPScwas detected in brain tissue by enzyme immunoassay with an average optical density of 3.6/4.0 for positive brains. PrPScalso was detected in brain tissue by western blot and immunohistochemistry. No PrPScwas detected in the spleens of mice inoculated with the raccoon passaged CWD agent. Humanized mice inoculated with the CWD agent from white-tailed deer did not have detectable PrPScusing conventional immunoassay techniques.
Conclusions: The host range of the CWD agent from white-tailed deer was expanded in our experimental model after one passage through raccoons.
Funded by: This research was funded in its entirety by congressionally appropriated funds to the United States Department of Agriculture, Agricultural Research Service. The funders of the work did not influence study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Acknowledgement: We thank Quazetta Brown, Lexi Frese, Rylie Frese, Kevin Hassall, Leisa Mandell, and Trudy Tatum for providing excellent technical support to this project.
Stable and highly zoonotic cervid prion strain is possible
Manuel Camacho, Xu Qi, Liuting Qing, Sydney Smith, Jieji Hu, Wanyun Tao, Ignazio Cali, and Qingzhong Kong Department of Pathology, Case Western Reserve University, Cleveland, USA
Aims: Whether CWD prions can infect humans remains unclear despite the very substantial scale and long history of human exposure of CWD in some areas. Multiple in vitro conversion experiments and in vivo animal studies suggest that the CWD-to-human transmission barrier is not unbreakable. A major public health concern on CWD zoonosis is the emergence of highly zoonotic CWD strains. We aim to address the question of whether highly zoonotic CWD strains are possible.
Material and Methods: We inoculated a few sCJD brain samples into cervidized transgenic mice, which were intended as negative controls for bioassays of brain tissues from sCJD cases who had hunted or consumed vension from CWD-endemic states. Some of these mice became infected and their brain tissues were further examined by serial passages in humanized or cervidized mice.
Results: Passage of sCJDMM1 in transgenic mice expressing elk PrP (Tg12) resulted in a ‘cervidized’ CJD strain that we termed CJDElkPrP. We observed 100% transmission of CJDElkPrPin transgenic mice expressing human PrP (Tg40h). We passaged CJDElkPrPtwo more times in the Tg12 mice. We found that such second and third passage CJDElkPrPprions also led to 100% infection in the Tg40h mice. In contrast, we and others found zero or poor transmission of natural elk CWD isolates in humanized mice, despite that natural elk CWD isolates and CJDElkPrPshare the same elk PrP sequence.
Conclusions: Our data demonstrate that highly zoonotic cervid prion strains are not only possible but also can be stably maintained in cervids and that CWD zoonosis is prion strain-dependent.
Funded by: NIH
Grant number: R01NS052319, R01NS088604, R01NS109532
Acknowledgement: We want to thank the National Prion Disease Pathology Surveillance Center and Drs. Allen Jenny and Katherine O’Rourke for providing the sCJD samples and the CWD samples, respectively.
Adaptation of chronic wasting disease (CWD) prion strains in hosts with different PRNP genotypes
Camilo Duque Velasqueza,c, Elizabeth Triscotta,c, Chiye Kima,c, Diana Morenoa,c, Judd Aikenb,c, and Debbie McKenziea,c
aDepartment of Biological Science, University of Alberta, Edmonton, AB T6G 2G8, Canada; bDepartment of Agriculture, Food & Nutritional Science, University of Alberta, Edmonton, AB T6G 2G8, Canada; cCentre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, AB T6G 2M8, Canada
Aims: The contagious nature of CWD epizootics and the PrPCamino acid variation of cervids (and susceptible sympatric species) guarantee the expansion of prion conformational diversity and selective landscapes where new strains can arise. CWD strains can have novel transmission properties including altered host range that may increase zoonotic risk as circulating strains diversify and evolve. We are characterizing the host adaptability of characterized CWD strains as well as CWD isolates from different cervid species in various enzootic regions.
Material and Methods: Characterized CWD strains as well as a number of isolates from hunter-harvested deer were bioassayed in our rodent panel (transgenic mice expressing cervid alleles G96, S96 and H95-PrPC, elk PrPC, bovine PrPC, and both hamsters and non-transgenic laboratory mice). Strain characteristics were compared using computer based scoring of brain pathology (e.g. PrPCWDbrain distribution), western blot and protein misfolding cyclic amplification (PMCA).
Results: Transmission of various isolates resulted in the selection of strain mixtures in hosts expressing similar PrPC, particularly for polymorphic white-tailed deer and for Norwegian reindeer. As of the second passage, transmission of P153 moose prions from Norway has not resulted in emergence of strains with properties similar to any North American CWD strains in our taxonomic collection (Wisc-1, CWD2, H95+and 116AG).
Conclusions: Our data indicates polymorphic white-tailed deer can favor infection with more than one strain. Similar to transmission studies of Colorado CWD isolates from cervids expressing a single PrPCprimary structure, the isolate from Norway reindeer (V214) represents a strain mixture, suggesting intrinsic strain diversity in the Nordfjella epizootic. The diversity of CWD strains with distinct transmission characteristics represents a threat to wildlife, sympatric domestic animals and public health.
Funded by: Genome Canada and Genome Alberta (Alberta Prion Research Institute and Alberta Agriculture & Forestry); NSERC Grant number: #LSARP 10205; NSERC RGPIN-2017-05539
Acknowledgement: We would like to thank Margo Pybus (Alberta Environment and Parks) Trent Bollinger (University of Saskatchewan) for providing us with tissue samples from hunter-harvested deer and Sylvie Benestad for providing moose and reindeer samples.
Application of PMCA to understand CWD prion strains, species barrier and zoonotic potential
Sandra Pritzkowa, Damian Gorskia, Frank Ramireza, Fei Wanga, Glenn C. Tellingb, Justin J. Greenleec, Sylvie L. Benestadd, and Claudio Sotoa aDepartment of Neurology, University of Texas Medical School at Houston, Houston, Texas, USA; bDepartment of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA; cVirus and Prion Research Unit, United States Department of Agriculture, Ames, Iowa, USA; dNorwegian Veterinary Institute, OIE Reference Laboratory for CWD, Ås, Norway
Aims: Chronic wasting disease (CWD) is a prion disease affecting various species of cervids that continues to spread uncontrollably across North America and has recently been detected in Scandinavia (Norway, Sweden and Finland). The mechanisms responsible for the natural transmission of CWD are largely unknown. Furthermore, the risk of CWD transmission to other species, including humans, is also unknown and remains a dangerous enigma. In this study, we investigated the potential of CWD prions to infect several other animal species (sheep, cattle, pig, hamster, and mouse) including humans, by examining their capacity to convert the normal prion protein of distinct species in a PMCA reaction. Moreover, we also investigated whether the in vivo passage of CWD through intermediate species alters their capacity for zoonotic transmission, which may represent a major hazard to human health.
Material and Methods: For these studies, we used brain material from CWD-infected white-tailed deer (Odocoileus virginianus), elk (Cervus canadensis), and mule deer (Odocoileus hemionus) as species native to North America. We also used CWD-infected Moose (Alces alces), reindeer (Rangifer tarandus) and red deer (Cervus elaphus) as Norwegian cervids. We also used brains from cattle, sheep and pigs experimentally infected by CWD. To study interspecies-transmission and zoonotic potential, samples were tested via PMCA for the conversion of PrPCinto PrPScusing different combinations of inoculum and host species. Based on these analyses we estimated the spillover and zoonotic potential for different CWD isolates. We define and quantify spillover and zoonotic potential indices as the efficiency by which CWD prions sustain prion generation in vitro at the expense of normal prion proteins from various mammals and human, respectively.
Results: Our results show that prions from some cervid species, especially those found in Northern Europe, have a higher potential to transmit disease characteristics to other animals. Conversely, CWD-infected cervids originated in North America appear to have a greater potential to generate human PrPSc. We also found that in vivo transmission of CWD to cattle, but not to sheep or pigs substantially increases the ability of these prions to convert human PrPCby PMCA.
Conclusions: Our findings support the existence of different CWD prion strains with distinct spillover and zoonotic potentials. We also conclude that transmission of CWD to other animal species may increase the risk for CWD transmission to humans. Our studies may provide a tool to predict the array of animal species that a given CWD prion could affect and may contribute to understanding the risk of CWD for human health.
Funded by: National Institute of Health Grant number: P01 AI077774
Generation of human chronic wasting disease in transgenic mice
Zerui Wanga, Kefeng Qinb, Manuel V. Camachoa, Ignazio Cali a,c, Jue Yuana, Pingping Shena, Tricia Gillilanda, Syed Zahid Ali Shaha, Maria Gerasimenkoa, Michelle Tanga, Sarada Rajamanickama, Anika Yadatia, Lawrence B. Schonbergerd, Justin Greenleee, Qingzhong Konga,c, James A. Mastriannib, and Wen-Quan Zoua,c
aDepartment of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH, USA; bDepartment of Neurology and Center for Comprehensive Care and Research on Memory Disorders, the University of Chicago Pritzker School of Medicine, Chicago, USA; cNational Prion Disease Pathology Surveillance Center, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA; dDivision of High-Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, 1600 Clifton Rd, Atlanta, GA, USA; eVirus and Prion Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, 1920 Dayton Avenue, Ames, IA, USA
Aims: Chronic wasting disease (CWD) results from the accumulation of an infectious misfolded conformer (PrPSc) of cellular prion protein (PrPC) in the brains of deer and elk. It has been spreading rapidly throughout many regions of North America, exported inadvertently to South Korea, and more recently identified in Europe. Mad cow disease has caused variant Creutzfeldt-Jakob disease (vCJD) in humans and is currently the only known zoonotic prion disease. Whether CWD is transmissible to humans remains uncertain. The aims of our study were not only to confirm whether CWD prion isolates can convert human brain PrPCinto PrPScin vitro by serial protein misfolding cyclic amplification (sPMCA) but also to determine whether the sPMCA-induced CWD-derived human PrPScis infectious.
Material and Methods: Eight CWD prion isolates from 7 elks and 1 deer were used as the seeds while normal human brain homogenates containing either PrP-129 MM (n = 2) or PrP-129 VV (n = 1) were used as the substrates for sPMCA assay. A normal elk brain tissue sample was used as a negative control seed. Two lines of humanized transgenic (Tg) mice expressing either human PrP-129VV or −129 MM polymorphism were included for transmission studies to determine the infectivity of PMCA-amplified PrPSc. Wester blotting and immunohistochemistry and hematoxylin & eosin staining were used for determining PrPScand neuropathological changes of inoculated animals.
Results: We report here the generation of the first CWD-derived infectious human PrPScusing elk CWD PrPScto initiate conversion of human PrPCfrom normal human brain homogenates with PMCA in vitro. Western blotting with a human PrP selective antibody confirmed that the PMCA-generated protease-resistant PrPScwas derived from the human brain PrPCsubstrate. Two lines of humanized transgenic mice expressing human PrPCwith either Val or Met at the polymorphic codon 129 developed clinical prion disease following intracerebral inoculation with the PMCA-generated CWD-derived human PrPSc. Diseased mice exhibited distinct PrPScpatterns and neuropathological changes in the brain.
Conclusions: Our study, using PMCA and animal bioassays, provides the first evidence that CWD PrPSchas the potential to overcome the species barrier and directly convert human PrPCinto infectious PrPScthat can produce bona fide prion disease when inoculated into humanized transgenic mice.
Funded by: CJD Foundation and NIH
Mortality surveillance of persons potentially exposed to chronic wasting disease
R.A. Maddoxa, R.F. Klosb, L.R. Willb, S.N. Gibbons-Burgenerb, A. Mvilongoa, J.Y. Abramsa, B.S. Applebyc, L.B. Schonbergera, and E.D. Belaya aNational Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention (CDC), Atlanta, USA; bWisconsin Department of Health Services (WDHS), Division of Public Health, Madison, USA; cNational Prion Disease Pathology Surveillance Center (NPDPSC), Case Western Reserve University, Cleveland, USA
Aims: It is unknown whether chronic wasting disease (CWD), a prion disease of cervids, can infect people, but consumption of meat from infected animals would be the most likely route of transmission. Wisconsin Department of Health Services, Division of Public Health (WDHS) personnel maintain a database consisting of information collected from hunters who reported eating, or an intention to eat, venison from CWD-positive cervids. These data, collected since 2003, allow for the evaluation of causes of mortality in individuals potentially exposed to CWD.
Material and Methods: The WDHS database contains the name, date of birth, when available, year of CWD-positive deer harvest, and city and state of residence for each potentially exposed individual. The database also includes information on how the deer was processed (self-processed or by a commercial operator) and when applicable, names of others with whom the venison was shared. Duplicate entries (i.e., those who consumed venison from CWD-positive deer in multiple hunt years) are determined by first name, last name, and date of birth. All names in the database are cross-checked with reported cases of human prion disease in Wisconsin and cases in the National Prion Disease Pathology Surveillance Center (NPDPSC) diagnostic testing database. Persons with date of birth available are also cross-checked with prion disease decedents identified through restricted-use national multiple cause-of-death data via a data use agreement with the National Center for Health Statistics (NCHS).
Results: The database currently consists of 1561 records for hunt years 2003–2017 and 87 additional records for 2018–2019. Of these, 657 records have accompanying date of birth; 15 entries were removed as duplicates leaving 642 unique individuals. Of these individuals, 278 of 426 (66%) who ate venison from a CWD-positive deer and provided processing information reported self-processing. No matches were found among any persons in the database cross-checked with WDHS human prion disease surveillance data, NPDPSC data (February 2022 update), and NCHS data through 2020.
Conclusions: Because of the linkage of person and CWD-positive animal in the WDHS database, reviewing the cause of mortality in potentially exposed persons is possible. The number of individuals cross-checked so far is likely only a small percentage of those potentially exposed to CWD in Wisconsin, and many more years of vital status tracking are needed given an expected long incubation period should transmission to humans occur. Nevertheless, the findings of this ongoing review are thus far reassuring.
Prion disease incidence, United States, 2003–2020
R.A. Maddoxa, M.K. Persona, K. Kotobellib, A. Mvilongoa, B.S. Applebyb, L.B. Schonbergera, T.A. Hammetta, J.Y. Abramsa, and E.D. Belaya aNational Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention (CDC), Atlanta, USA; bNational Prion Disease Pathology Surveillance Center (NPDPSC), Case Western Reserve University, Cleveland, USA
Aims: Mortality data, in conjunction with neuropathological and genetic testing results, are used to estimate prion disease incidence in the United States.
Material and Methods: Prion disease decedents for 2003–2020 were identified from restricted-use U.S. national multiple cause-of-death data, via a data use agreement with the National Center for Health Statistics, and from the National Prion Disease Pathology Surveillance Center (NPDPSC) database. NPDPSC decedents with neuropathological or genetic test results positive for prion disease for whom no likely match was found in the NCHS multiple cause-of-death data were added as cases for incidence calculations, while those with negative neuropathology results but with cause-of-death data indicating prion disease were removed. Unmatched cases in the NPDPSC database lacking neuropathological testing but with a positive real-time quaking-induced conversion (RT-QuIC) test result were additionally assessed. Age-specific and age-adjusted average annual incidence rates were calculated from the combined data; the year 2000 as the standard population and the direct method were used for age-adjustment.
Results: A total of 7,921 decedents were identified as having prion disease during 2003–2020 for an age-adjusted average annual incidence of 1.2 per million population. The age-adjusted incidence between males and females (1.3 and 1.1 per million, respectively) differed significantly (p < 0.0001). The age-specific average annual incidence among those <55 and ≥55 years of age was 0.2 and 4.8 per million, respectively; incidence among those ≥65 was 6.1 per million. Eighteen cases were <30 years of age for an age-specific incidence of 8.0 per billion; only 6 of these very young cases were sporadic (3 sporadic CJD, 3 sporadic fatal insomnia), with the rest being familial (9), variant (2), or iatrogenic (1). The age-adjusted annual incidence for the most recent year of data, 2020, was 1.3 per million. However, assessment of RT-QuIC positive cases lacking neuropathology in the NPDPSC database suggested that approximately 20% more cases may have occurred in that year; the addition of a subset of these cases that had date of death information available (n = 44) increased the 2020 rate to 1.4 per million.
Conclusions: Mortality data supplemented with the results of neuropathological, CSF RT-QuIC, and genetic testing can be used to estimate prion disease incidence. However, the identification in the NPDPSC database of RT-QuIC-positive cases lacking date of death information suggests that this strategy may exclude a number of probable prion disease cases. Prion disease cases <30 years of age, especially those lacking a pathogenic mutation, continue to be very rare.
Shedding of Chronic Wasting Disease Prions in Multiple Excreta Throughout Disease Course in White-tailed Deer
Nathaniel D. Denkersa, Erin E. McNultya, Caitlyn N. Krafta, Amy V. Nallsa, Joseph A. Westricha, Wilfred Goldmannb, Candace K. Mathiasona, and Edward A. Hoovera
aPrion Research Center, College of Veterinary Medicine and Biological Sciences, Department of Microbiology, Immunology, and Pathology; Colorado State University, Fort Collins, CO, USA; bDivision of Infection and Immunity, The Roslin Institute and the Royal Dick School of Veterinary Studies, University of Edinburgh, Midlothian, UK
Aims: Chronic wasting disease (CWD) now infects cervids in South Korea, North America, and Scandinavia. CWD is unique in its efficient transmission and shedding of prions in body fluids throughout long course infections. Questions remain as to the magnitude of shedding and the route of prion acquisition. As CWD continues to expand, the need to better understand these facets of disease becomes more pertinent. The purpose of the studies described was to define the longitudinal shedding profile of CWD prions in urine, saliva, and feces throughout the course of infection in white-tailed deer.
Material and Methods: Twelve (12) white-tailed deer were inoculated with either 1 mg or 300ng of CWD. Urine, saliva, and feces were collected every 3-month post-inoculation (MPI) throughout the study duration. Cohorts were established based on PNRP genotype: codon 96 GG (n = 6) and alternate codons 96 GS (n = 5) & 103NT (n = 1). Urine and saliva were analyzed using iron-oxide magnetic extraction (IOME) and real-time quaking induced conversion (RT-QuIC)(IQ). Feces were subjected to IOME, followed by 4 rounds protein misfolding cyclic amplification (PMCA) with products analyzed by RT-QuIC (IPQ). To determine whether IPQ may be superior to IQ, a subset of urine and saliva were also tested by IPQ. Results were compared with clinical disease status.
Results: Within the 96 GG cohort, positive seeding activity was detected in feces from all deer (100%), in saliva from 5 of 6 (83%), and in urine from 4 of 6 (66%). Shedding in all excreta occurred at, or just after, the first positive tonsil biopsy result. In the 96 GS/103NT cohort, positive seeding activity could be detected in feces from 3 of 6 (50%) deer, saliva in 2 of 6 (33%), and urine in 1 of 6 (16%). Shedding in excreta was detected >5 months after the first tonsil positive result. Four of six 96 GG deer developed clinical signs of CWD, whereas only 2 of the 96 GS/103NT did. Shedding was more frequently detected in deer with clinical disease. The IPQ protocol did not significantly improve detection in saliva or urine samples, however, it significantly augmented detection in feces by eliminating non-specific background commonly experienced with IQ. Negative control samples remained negative in samples tested.
Conclusions: These studies demonstrate: (a) CWD prion excretion occurs throughout infection; (2) PRNP genotype (GG≫GS/NT) influences the excreta shedding; and (3) detection sensitivity in excreta can vary with different RT-QuIC protocols. These results provide a more complete perspective of prion shedding in deer during the course of CWD infection.
Funded by: National Institutes of Health (NIH)
Grant number: RO1-NS061902-09 R to EAH, PO1-AI077774 to EAH, and R01-AI112956-06 to CKM
Acknowledgement: We abundantly thank Sallie Dahmes at WASCO and David Osborn and Gino D’Angelo at the University of Georgia Warnell School of Forestry and Natural Resources for their long-standing support of this work through provision of the hand-raised, CWD-free, white-tailed deer used in these studies
Large-scale PMCA screening of retropharyngeal lymph nodes and in white-tailed deer and comparisons with ELISA and IHC: the Texas CWD study
Rebeca Benaventea, Paulina Sotoa, Mitch Lockwoodb, and Rodrigo Moralesa
aDepartment of Neurology, McGovern Medical School, University of Texas Health Science Center at Houston, Texas, USA; bTexas Park and Wildlife Department, Texas, USA
Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy that affects various species of cervids, and both free-ranging and captive animals. Until now, CWD has been detected in 3 continents: North America, Europe, and Asia. CWD prevalence in some states may reach 30% of total animals. In Texas, the first case of CWD was reported in a free-range mule deer in Hudspeth and now it has been detected in additional 14 counties. Currently, the gold standard techniques used for CWD screening and detection are ELISA and immunohistochemistry (IHC) of obex and retropharyngeal lymph nodes (RPLN). Unfortunately, these methods are known for having a low diagnostic sensitivity. Hence, many CWD-infected animals at pre-symptomatic stages may be misdiagnosed. Two promising in vitro prion amplification techniques, including the real-time quaking-induced conversion (RT-QuIC) and the protein misfolding cyclic amplification (PMCA) have been used to diagnose CWD and other prion diseases in several tissues and bodily fluids. Considering the low cost and speed of RT-QuIC, two recent studies have communicated the potential of this technique to diagnose CWD prions in RPLN samples. Unfortunately, the data presented in these articles suggest that identification of CWD positive samples is comparable to the currently used ELISA and IHC protocols. Similar studies using the PMCA technique have not been reported.
Aims: Compare the CWD diagnostic potential of PMCA with ELISA and IHC in RPLN samples from captive and free-range white-tailed deer. Material and Methods: In this study we analyzed 1,003 RPLN from both free-ranging and captive white-tailed deer collected in Texas. Samples were interrogated with the PMCA technique for their content of CWD prions. PMCA data was compared with the results obtained through currently approved techniques.
Results: Our results show a 15-fold increase in CWD detection in free-range deer compared with ELISA. Our results unveil the presence of prion infected animals in Texas counties with no previous history of CWD. In the case of captive deer, we detected a 16% more CWD positive animals when compared with IHC. Interestingly, some of these positive samples displayed differences in their electroforetic mobilities, suggesting the presence of different prion strains within the State of Texas.
Conclusions: PMCA sensitivity is significantly higher than the current gold standards techniques IHC and ELISA and would be a good tool for rapid CWD screening.
Funded by: USDA
Grant number: AP20VSSPRS00C143
ATYPRION project: assessing the zoonotic potential of interspecies transmission of CWD isolates to livestock (preliminary results).
Enric Vidala,b, Juan Carlos Espinosac, Samanta Gilera,b, Montserrat Ordóñeza,b, Guillermo Canteroa,b, Vincent Béringued, Justin J. Greenleee, and Juan Maria Torresc
aUnitat mixta d’Investigació IRTA-UAB en Sanitat Animal. Centre de Recerca en Sanitat Animal (CReSA). Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, Catalonia; bIRTA. Programa de Sanitat Animal. Centre de Recerca en Sanitat Animal (CReSA). Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, Catalonia; cCentro de Investigación en Sanidad Animal, CISA-INIA-CSIC, Valdeolmos, Madrid, Spain; dMolecular Virology and Immunology, French National Research Institute for Agriculture, Food and Environment (INRAE), Université Paris-Saclay, Jouy-en-Josas, France; eVirus and Prion Research Unit, National Animal Disease Center, ARS, United States Department of Agriculture, Ames, IA, USA
Aims: Since variant Creutzfeldt-Jackob disease was linked to the consumption of bovine spongiform encephalopathy prions, the study of the pathobiological features of animal prions, particularly their zoonotic potential, is of great concern to the scientific community and public health authorities. Furthermore, interspecies transmission of prions has been demonstrated as a putative evolutionary mechanism for prions, that can lead to the emergence of new features including the ability to infect humans. For instance, small ruminants’ atypical scrapie prions, when propagated in a bovine or porcine host, can shift to a classical BSE phenotype thus posing a potential risk in case of human exposure. So far, no hard evidence of zoonotic transmission of cervids’ chronic wasting disease (CWD) to humans has been published, however experimental transmission to bovine, ovine and caprine hosts has been achieved. Our goal is to investigate if, once passaged through these domestic species, CWD prions might become infectious to humans.
Material and Methods: Different CWD isolates experimentally adapted to cattle, sheep and goat (Hamir et al, 2005, 2006, 2007, Greenlee et al 2012) have been intracerebrally inoculated to transgenic mouse models expressing the human cellular prion protein either homozygous for methionine or valine at codon 129 (Tg340-Met129 and Tg362-Val129). Additionally, inocula obtained from experimental transmission of elk CWD to ovinized (Tg501) and bovinized (BoTg110) transgenic mice, as well as white-tailed deer CWD to BoTg110 mice, are currently being bioassayed in both human PrPCtransgenic models.
Results and conclusions: No evidence of transmission has been found on first passage for bovine adapted elk and mule deer CWD to none of the humanized models. The remaining bioassays are ongoing without showing clinical signs yet, as well as second passages for the negative 1stpassages.
Funded by: La Marató de TV3 foundation. Grant number: ATYPRION (201,821–30-31-32)
PRION CONFERENCE 2022 ABSTRACTS CWD TSE PrP ZOONOSIS and ENVIRONMENTAL FACTORS
Chronic wasting disease detection in environmental and biological samples from a taxidermy site
Paulina Sotoa,b, J. Hunter Reedc, Mitch Lockwoodc, and Rodrigo Moralesa,b aDepartment of Neurology, McGovern Medical School, University of Texas Health Science Center at Houston, Texas, USA; bUniversidad Bernardo O’Higgins, Santiago, Chile; cTexas Parks and Wildlife Department, Texas, USA
Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy affecting captive and free-ranging cervids (e.g., mule deer, white-tailed deer, elk, reindeer, and moose). Nowadays, CWD is widely distributed in North America. It is suggested that CWD spreads due to direct animal contact or through exposure to contaminated environments previously inhabited by infected animals. CWD may also be spread through the movement of infected animals and carcasses. Taxidermy practices involve processing deer tissues (or whole animal carcasses). In many cases, the CWD status of processed animals is unknown. This can generate risks of disease spread and transmission. Taxidermy practices include different steps involving physical, chemical, and biological procedures. Without proper tissue handling or disposal practices, taxidermist facilities may become a focus of prion infectivity. Aims: In this study, we evaluated the presence of infectious prions in a taxidermy facility believed to be exposed to CWD. Detection was performed using the Protein Misfolding Cyclic Amplification (PMCA) technique in biological and inert environmental samples. Methods: We collected biological and environmental samples (plants, soils, insects, excreta, and others) from a taxidermy facility, and we tested these samples using the PMCA technique. In addition, we swabbed different surfaces possibly exposed to CWD-infected animals. For the PMCA reaction, we directly used a swab piece or 10 µL of 20% w/v homogenized samples. Results: The PMCA analysis demonstrated CWD seeding activity in some of the components of this facility, including insects involved in head processing, soils, and a trash dumpster. Conclusions: Different areas of this property were used for various taxidermy procedures. We were able to detect the presence of prions in i) soils that were in contact with the heads of dead animals, ii) insects involved in the cleaning of skulls, and iii) an empty dumpster where animal carcasses were previously placed. This is the first report demonstrating that swabbing is a helpful method to screen for prion infectivity on surfaces potentially contaminated with CWD. These findings are relevant as this swabbing and amplification strategy may be used to evaluate the disease status of other free-ranging and captive settings where there is a concern for CWD transmissions, such as at feeders and water troughs with CWD-exposed properties. This approach could have substantial implications for free-ranging cervid surveillance as well as in epidemiological investigations of CWD.
Funded by: USDA Grant number: AP20VSSPRS00C143
Large-scale PMCA screening of retropharyngeal lymph nodes and in white-tailed deer and comparisons with ELISA and IHC: the Texas CWD study
Rebeca Benaventea, Paulina Sotoa, Mitch Lockwoodb, and Rodrigo Moralesa aDepartment of Neurology, McGovern Medical School, University of Texas Health Science Center at Houston, Texas, USA; bTexas Park and Wildlife Department, Texas, USA
Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy that affects various species of cervids, and both free-ranging and captive animals. Until now, CWD has been detected in 3 continents: North America, Europe, and Asia. CWD prevalence in some states may reach 30% of total animals. In Texas, the first case of CWD was reported in a free-range mule deer in Hudspeth and now it has been detected in additional 14 counties. Currently, the gold standard techniques used for CWD screening and detection are ELISA and immunohistochemistry (IHC) of obex and retropharyngeal lymph nodes (RPLN). Unfortunately, these methods are known for having a low diagnostic sensitivity. Hence, many CWD-infected animals at pre-symptomatic stages may be misdiagnosed. Two promising in vitro prion amplification techniques, including the real-time quaking-induced conversion (RT-QuIC) and the protein misfolding cyclic amplification (PMCA) have been used to diagnose CWD and other prion diseases in several tissues and bodily fluids. Considering the low cost and speed of RT-QuIC, two recent studies have communicated the potential of this technique to diagnose CWD prions in RPLN samples. Unfortunately, the data presented in these articles suggest that identification of CWD positive samples is comparable to the currently used ELISA and IHC protocols. Similar studies using the PMCA technique have not been reported. Aims: Compare the CWD diagnostic potential of PMCA with ELISA and IHC in RPLN samples from captive and free-range white-tailed deer. Material and Methods: In this study we analyzed 1,003 RPLN from both free-ranging and captive white-tailed deer collected in Texas. Samples were interrogated with the PMCA technique for their content of CWD prions. PMCA data was compared with the results obtained through currently approved techniques. Results: Our results show a 15-fold increase in CWD detection in free-range deer compared with ELISA. Our results unveil the presence of prion infected animals in Texas counties with no previous history of CWD. In the case of captive deer, we detected a 16% more CWD positive animals when compared with IHC. Interestingly, some of these positive samples displayed differences in their electroforetic mobilities, suggesting the presence of different prion strains within the State of Texas. Conclusions: PMCA sensitivity is significantly higher than the current gold standards techniques IHC and ELISA and would be a good tool for rapid CWD screening.
Funded by: USDA Grant number: AP20VSSPRS00C143
Protein misfolding cyclic amplification (PMCA) as an ultra-sensitive technique for the screening of CWD prions in different sample types
Francisca Bravo‐Risia,b, Paulina Sotoa,b, Rebeca Benaventea, Hunter Reedc, Mitch Lockwoodc, Tracy Nicholsd, and Rodrigo Moralesa,b aDepartment of Neurology, The University of Texas Health Science Center at Houston, Houston, TX, USA; bCentro Integrativo de Biologia y Quimica Aplicada (CIBQA), Universidad Bernardo O’Higgins, Santiago, Chile; cTexas Park and Wildlife Department, Texas, USA; dVeterinary Services Cervid Health Program, United States Department of Agriculture, Animal and Plant Health Inspection Service, Fort Collins, Colorado, USA
Chronic wasting disease (CWD) is a prion disease that affects farmed and free-ranging cervids. The infectious agent in CWD is a misfolded form of the prion protein (PrPSc) that promotes conformational changes in the host’s cellular prion protein (PrPC). Currently, definitive CWD status is confirmed in the brain and lymphoid tissues by immunohistochemistry. The limitation of this technique is its poor sensitivity. Protein misfolding cyclic amplification (PMCA) and real-time quaking-induced conversion (RT- QuIC) are ultra-sensitive techniques that overcome these issues. PMCA mimics the self- propagation of infectious prions in vitro through multiple incubation/sonication cycles, increasing the number of prion particles present in a given sample. The detection of proteinase K (PK) -resistant PrPScby PMCA has been performed in experimental and natural samples that might harbor subclinical levels of prions. These samples include several tissues, bodily fluids, excreta, and different manmade and natural materials, including mineral licks, soils, and plants. Aims: In this study, we highlight recent advances and contributions that our group has performed in the detection of CWD prions from samples collected in farmed and free-ranging cervids, as well as other specimens involving the environment that contains CWD-infected deer. Material and Methods: A set of diverse samples analyzed in this study were collected by USDA and TPWD personnel in breeding and taxidermy facilities, and deer breeding facilities. These included animal and environmental samples. Additional samples from free-ranging animals were provided by hunters. Results: The diverse range of samples successfully detected for CWD prion infection in this study include blood, semen, feces, obex, retropharyngeal lymph node, fetuses (neural and peripheral tissues) and gestational tissues, parasites, insects, plants, compost/soil mixtures, and swabs from trash containers. Importantly, these results helped to identify seeding-competent prions in places reported to be free of CWD. The levels of prion infectivity in most of these samples are currently being investigated. Conclusions: Our findings contribute to the understanding of the transmission dynamics and prevalence of CWD. In addition, our data have helped to identify CWD in areas previously considered to be free of CWD. We also demonstrate that PMCA is a powerful technique for the screening of biological and environmental samples. Overall, our research suggests that PMCA may be a useful tool to implement for the surveillance and management of CWD. Funded by: NIH/NIAID and USDA Grant number: 1R01AI132695 (NIH) and AP20VSSPRS00C143 (USDA)
Nasal bot: an emerging vector for natural chronic wasting disease transmission
Paulina Sotoa,b, Francisca Bravo-Risia,b, Carlos Kramma, Nelson Pereza, Rebeca Benaventea, J. Hunter Reedc, Mitch Lockwoodc, Tracy A. Nicholsd, and Rodrigo Moralesa,b aDepartment of Neurology, McGovern Medical School, University of Texas Health Science Center at Houston, Texas, USA; bUniversidad Bernardo O’Higgins, Santiago, Chile; cTexas Park and Wildlife Department, Texas, USA; dVeterinary Services Cervid Health Program, United States Department of Agriculture, Animal and Plant Health Inspection Service, Fort Collins, Colorado, USA
Chronic wasting disease (CWD) is a fatal neurodegenerative disease that affects farmed and free-ranging cervids populations. The spread of CWD in cervids is thought to occur through the direct contact between cervids or through the exposure of naïve animals to contaminated environments. Parasites are known vectors of multiple diseases in animals. However, the potential role of parasites in CWD transmission remains unclear. Aims: The main objective of this study was to determine if CWD prions could be detected in the larvae of deer nasal bot flies, a common deer parasite, taken from CWD-infected white-tailed deer (Odocoileus virginianus). Methods: Bot fly larvae were collected from the nasal cavity of naturally infected CWD- positive or CWD non-detect white-tailed deer. The CWD seeding activity of the larvae was interrogated by PMCA. Prion infectivity was also evaluated in cervidized transgenic mouse bioassay (intra-cerebral administration in Tg1536 mice). Mice inoculated with bot larvae homogenate were sacrificed when they showed established signs of prion disease, or at extended periods after treatment (600 days). All inoculated mouse brains were evaluated for protease resistant prions to confirm clinical or sub-clinical infection. Bot larvae from CWD non-detect deer were used as controls. To further mimic environmental transmission, bot larvae homogenates were mixed with soils and plants were grown on them. Both plants and soils were tested for prion seeding activity. Results: PMCA analysis demonstrated CWD seeding activity in nasal bot larvae from captive and free-ranging white-tailed deer. CWD-contaminated bots efficiently infected transgenic mice, with attack rates and incubation periods suggesting high infectivity titers. Further analyses of treated animals (biochemical characterization of protease resistant prions and immunohistochemistry) confirmed prion infection. Analyses on dissected parts of the bot larvae demonstrate that the infectivity is concentrated in the larvae cuticle (outer part). Nasal bot larvae extracts mixed with
soils showed seeding activity by PMCA. Interestingly, plants grown in soil contaminated with the nasal bot larvae extract were found to produce seeding activity by PMCA. Conclusion: In this study we described for the first time that deer nasal bot larvae from CWD-infected deer carry high CWD infectivity titers. We also demonstrate that CWD prions in these parasites can interact with other environmental components relevant for disease transmission. Considering this information, we propose that deer nasal bot larvae could act as vectors for CWD transmission in wild and farming settings. Funded by: NIH/NIAID and USDA/APHIS Grant number: R01AI132695 and AP20VSSPRS00C143 PRION 2022 ABSTRACTS, AND A BIG THANK YOU TO On behalf of the Prion2020/2022 Congress Organizing Committee and the NeuroPrion Association, we heartily invite you to join us for the International Conference Prion2020/2022 from 13.-16. September 2022 in Göttingen.
Prion 2022 Conference abstracts: pushing the boundaries
Shedding of Chronic Wasting Disease Prions in Multiple Excreta Throughout Disease Course in White-tailed Deer
Nathaniel D. Denkersa, Erin E. McNultya, Caitlyn N. Krafta, Amy V. Nallsa, Joseph A. Westricha, Wilfred Goldmannb, Candace K. Mathiasona, and Edward A. Hoovera
aPrion Research Center, College of Veterinary Medicine and Biological Sciences, Department of Microbiology, Immunology, and Pathology; Colorado State University, Fort Collins, CO, USA; bDivision of Infection and Immunity, The Roslin Institute and the Royal Dick School of Veterinary Studies, University of Edinburgh, Midlothian, UK
Aims: Chronic wasting disease (CWD) now infects cervids in South Korea, North America, and Scandinavia. CWD is unique in its efficient transmission and shedding of prions in body fluids throughout long course infections. Questions remain as to the magnitude of shedding and the route of prion acquisition. As CWD continues to expand, the need to better understand these facets of disease becomes more pertinent. The purpose of the studies described was to define the longitudinal shedding profile of CWD prions in urine, saliva, and feces throughout the course of infection in white-tailed deer.
Material and Methods: Twelve (12) white-tailed deer were inoculated with either 1 mg or 300ng of CWD. Urine, saliva, and feces were collected every 3-month post-inoculation (MPI) throughout the study duration. Cohorts were established based on PNRP genotype: codon 96 GG (n = 6) and alternate codons 96 GS (n = 5) & 103NT (n = 1). Urine and saliva were analyzed using iron-oxide magnetic extraction (IOME) and real-time quaking induced conversion (RT-QuIC)(IQ). Feces were subjected to IOME, followed by 4 rounds protein misfolding cyclic amplification (PMCA) with products analyzed by RT-QuIC (IPQ). To determine whether IPQ may be superior to IQ, a subset of urine and saliva were also tested by IPQ. Results were compared with clinical disease status.
Results: Within the 96 GG cohort, positive seeding activity was detected in feces from all deer (100%), in saliva from 5 of 6 (83%), and in urine from 4 of 6 (66%). Shedding in all excreta occurred at, or just after, the first positive tonsil biopsy result. In the 96 GS/103NT cohort, positive seeding activity could be detected in feces from 3 of 6 (50%) deer, saliva in 2 of 6 (33%), and urine in 1 of 6 (16%). Shedding in excreta was detected >5 months after the first tonsil positive result. Four of six 96 GG deer developed clinical signs of CWD, whereas only 2 of the 96 GS/103NT did. Shedding was more frequently detected in deer with clinical disease. The IPQ protocol did not significantly improve detection in saliva or urine samples, however, it significantly augmented detection in feces by eliminating non-specific background commonly experienced with IQ. Negative control samples remained negative in samples tested.
Conclusions: These studies demonstrate: (a) CWD prion excretion occurs throughout infection; (2) PRNP genotype (GG≫GS/NT) influences the excreta shedding; and (3) detection sensitivity in excreta can vary with different RT-QuIC protocols. These results provide a more complete perspective of prion shedding in deer during the course of CWD infection.
Funded by: National Institutes of Health (NIH)
Grant number: RO1-NS061902-09 R to EAH, PO1-AI077774 to EAH, and R01-AI112956-06 to CKM
Acknowledgement: We abundantly thank Sallie Dahmes at WASCO and David Osborn and Gino D’Angelo at the University of Georgia Warnell School of Forestry and Natural Resources for their long-standing support of this work through provision of the hand-raised, CWD-free, white-tailed deer used in these studies
Carrot plants as potential vectors for CWD transmission
Paulina Sotoa,b, Francisca Bravo-Risia,b, Claudio Sotoa, and Rodrigo Moralesa,b
aDepartment of Neurology, McGovern Medical School, University of Texas Health Science Center at Houston, Texas, USA; bUniversidad Bernardo O’Higgins, Santiago, Chile
Prion diseases are infectious neurodegenerative disorders afflicting humans and other mammals. These diseases are generated by the misfolding of the cellular prion protein into a disease-causing isoform. Chronic wasting disease (CWD) is a prevalent prion disease affecting cervids (captive and free-range). CWD is thought to be transmitted through direct animal contact or by indirect exposure to contaminated environments. Many studies have shown that infectious prions can enter the environment through saliva, feces, or urine from infected animals and decaying carcasses. However, we do not fully understand the specific contribution of each component to disease transmission events. Plants are logical environmental components to be evaluated since they grow in environments contaminated with CWD prions and are relevant for animal and human nutrition.
Aims: The main objective of this study is to study whether prions are transported to the roots and leaves of carrots, an edible plant commonly used in the human diet and as deer bait.
Methods: We have grown carrot plants in CWD-infected soils. After 90 days, we harvested the carrots and separated them from the leaves. The experiment was controlled by growing plants in soil samples treated with brain extracts from healthy animals. These materials were interrogated for their prion seeding activity using the Protein Misfolding Cyclic Amplification (PMCA) technique. Infectivity was evaluated in mouse bioassays (intracerebral injections in Tg1536 mice). The animals were sacrificed when they showed established signs of prion disease. Animals not displaying clinical signs were sacrificed at 600 days post-inoculation.
Results: The PMCA analysis demonstrated CWD seeding activity in soils contaminated with CWD prions, as well as in carrot plants (leaves and roots) grown on them. Bioassays demonstrated that both leaves and roots contained CWD prions in sufficient quantities to induce disease (92% attack rate). As expected, animals treated with prion-infected soils developed prion disease at shorter incubation periods (and complete attack rates) compared to plant components. Animals treated with soil and plant components exposed with CWD-free brain extracts did not display prion-associated clinical signs or evidence of sub-clinical prion infection.
Conclusions: We show that edible plant components can absorb prions from CWD contaminated soils and transport them to their aerial parts. Our results indicate that plants could participate as vectors of CWD transmission. Importantly, plants designated for human consumption represent a risk of introducing CWD prions into the human food chain.
Funded by: NIH
Grant number: R01AI132695
Prion Conference 2018 Abstracts
P190 Human prion disease mortality rates by occurrence of chronic wasting disease in freeranging cervids, United States
Abrams JY (1), Maddox RA (1), Schonberger LB (1), Person MK (1), Appleby BS (2), Belay ED (1)
(1) Centers for Disease Control and Prevention (CDC), National Center for Emerging and Zoonotic Infectious Diseases, Atlanta, GA, USA (2) Case Western Reserve University, National Prion Disease Pathology Surveillance Center (NPDPSC), Cleveland, OH, USA.
Background
Chronic wasting disease (CWD) is a prion disease of deer and elk that has been identified in freeranging cervids in 23 US states. While there is currently no epidemiological evidence for zoonotic transmission through the consumption of contaminated venison, studies suggest the CWD agent can cross the species barrier in experimental models designed to closely mimic humans. We compared rates of human prion disease in states with and without CWD to examine the possibility of undetermined zoonotic transmission.
Methods
Death records from the National Center for Health Statistics, case records from the National Prion Disease Pathology Surveillance Center, and additional state case reports were combined to create a database of human prion disease cases from 2003-2015. Identification of CWD in each state was determined through reports of positive CWD tests by state wildlife agencies. Age- and race-adjusted mortality rates for human prion disease, excluding cases with known etiology, were determined for four categories of states based on CWD occurrence: highly endemic (>16 counties with CWD identified in free-ranging cervids); moderately endemic (3-10 counties with CWD); low endemic (1-2 counties with CWD); and no CWD states. States were counted as having no CWD until the year CWD was first identified. Analyses stratified by age, sex, and time period were also conducted to focus on subgroups for which zoonotic transmission would be more likely to be detected: cases <55 years old, male sex, and the latter half of the study (2010-2015).
Results
Highly endemic states had a higher rate of prion disease mortality compared to non-CWD states (rate ratio [RR]: 1.12, 95% confidence interval [CI] = 1.01 - 1.23), as did low endemic states (RR: 1.15, 95% CI = 1.04 - 1.27). Moderately endemic states did not have an elevated mortality rate (RR: 1.05, 95% CI = 0.93 - 1.17). In age-stratified analyses, prion disease mortality rates among the <55 year old population were elevated for moderately endemic states (RR: 1.57, 95% CI = 1.10 – 2.24) while mortality rates were elevated among those ≥55 for highly endemic states (RR: 1.13, 95% CI = 1.02 - 1.26) and low endemic states (RR: 1.16, 95% CI = 1.04 - 1.29). In other stratified analyses, prion disease mortality rates for males were only elevated for low endemic states (RR: 1.27, 95% CI = 1.10 - 1.48), and none of the categories of CWD-endemic states had elevated mortality rates for the latter time period (2010-2015).
Conclusions
While higher prion disease mortality rates in certain categories of states with CWD in free-ranging cervids were noted, additional stratified analyses did not reveal markedly elevated rates for potentially sensitive subgroups that would be suggestive of zoonotic transmission. Unknown confounding factors or other biases may explain state-by-state differences in prion disease mortality.
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P172 Peripheral Neuropathy in Patients with Prion Disease
Wang H(1), Cohen M(1), Appleby BS(1,2)
(1) University Hospitals Cleveland Medical Center, Cleveland, Ohio (2) National Prion Disease Pathology Surveillance Center, Cleveland, Ohio.
Prion disease is a fatal progressive neurodegenerative disease due to deposition of an abnormal protease-resistant isoform of prion protein. Typical symptoms include rapidly progressive dementia, myoclonus, visual disturbance and hallucinations. Interestingly, in patients with prion disease, the abnormal protein canould also be found in the peripheral nervous system. Case reports of prion deposition in peripheral nerves have been reported. Peripheral nerve involvement is thought to be uncommon; however, little is known about the exact prevalence and features of peripheral neuropathy in patients with prion disease.
We reviewed autopsy-proven prion cases from the National Prion Disease Pathology Surveillance Center that were diagnosed between September 2016 to March 2017. We collected information regarding prion protein diagnosis, demographics, comorbidities, clinical symptoms, physical exam, neuropathology, molecular subtype, genetics lab, brain MRI, image and EMG reports. Our study included 104 patients. Thirteen (12.5%) patients had either subjective symptoms or objective signs of peripheral neuropathy. Among these 13 patients, 3 had other known potential etiologies of peripheral neuropathy such as vitamin B12 deficiency or prior chemotherapy. Among 10 patients that had no other clear etiology, 3 (30%) had familial CJD. The most common sCJD subtype was MV1-2 (30%), followed by MM1-2 (20%). The Majority of cases wasere male (60%). Half of them had exposure to wild game. The most common subjective symptoms were tingling and/or numbness of distal extremities. The most common objective finding was diminished vibratory sensation in the feet. Half of them had an EMG with the findings ranging from fasciculations to axonal polyneuropathy or demyelinating polyneuropathy.
Our study provides an overview of the pattern of peripheral neuropathy in patients with prion disease. Among patients with peripheral neuropathy symptoms or signs, majority has polyneuropathy. It is important to document the baseline frequency of peripheral neuropathy in prion diseases as these symptoms may become important when conducting surveillance for potential novel zoonotic prion diseases.
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P177 PrP plaques in methionine homozygous Creutzfeldt-Jakob disease patients as a potential marker of iatrogenic transmission
Abrams JY (1), Schonberger LB (1), Cali I (2), Cohen Y (2), Blevins JE (2), Maddox RA (1), Belay ED (1), Appleby BS (2), Cohen ML (2)
(1) Centers for Disease Control and Prevention (CDC), National Center for Emerging and Zoonotic Infectious Diseases, Atlanta, GA, USA (2) Case Western Reserve University, National Prion Disease Pathology Surveillance Center (NPDPSC), Cleveland, OH, USA.
Background
Sporadic Creutzfeldt-Jakob disease (CJD) is widely believed to originate from de novo spontaneous conversion of normal prion protein (PrP) to its pathogenic form, but concern remains that some reported sporadic CJD cases may actually be caused by disease transmission via iatrogenic processes. For cases with methionine homozygosity (CJD-MM) at codon 129 of the PRNP gene, recent research has pointed to plaque-like PrP deposition as a potential marker of iatrogenic transmission for a subset of cases. This phenotype is theorized to originate from specific iatrogenic source CJD types that comprise roughly a quarter of known CJD cases.
Methods
We reviewed scientific literature for studies which described PrP plaques among CJD patients with known epidemiological links to iatrogenic transmission (receipt of cadaveric human grown hormone or dura mater), as well as in cases of reported sporadic CJD. The presence and description of plaques, along with CJD classification type and other contextual factors, were used to summarize the current evidence regarding plaques as a potential marker of iatrogenic transmission. In addition, 523 cases of reported sporadic CJD cases in the US from January 2013 through September 2017 were assessed for presence of PrP plaques.
Results
We identified four studies describing 52 total cases of CJD-MM among either dura mater recipients or growth hormone recipients, of which 30 were identified as having PrP plaques. While sporadic cases were not generally described as having plaques, we did identify case reports which described plaques among sporadic MM2 cases as well as case reports of plaques exclusively in white matter among sporadic MM1 cases. Among the 523 reported sporadic CJD cases, 0 of 366 MM1 cases had plaques, 2 of 48 MM2 cases had kuru plaques, and 4 of 109 MM1+2 cases had either kuru plaques or both kuru and florid plaques. Medical chart review of the six reported sporadic CJD cases with plaques did not reveal clinical histories suggestive of potential iatrogenic transmission.
Conclusions
PrP plaques occur much more frequently for iatrogenic CJD-MM cases compared to sporadic CJDMM cases. Plaques may indicate iatrogenic transmission for CJD-MM cases without a type 2 Western blot fragment. The study results suggest the absence of significant misclassifications of iatrogenic CJD as sporadic. To our knowledge, this study is the first to describe grey matter kuru plaques in apparently sporadic CJD-MM patients with a type 2 Western blot fragment.
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P180 Clinico-pathological analysis of human prion diseases in a brain bank series
Ximelis T (1), Aldecoa I (1,2), Molina-Porcel L (1,3), Grau-Rivera O (4), Ferrer I (5), Nos C (6), Gelpi E (1,7), Sánchez-Valle R (1,4)
(1) Neurological Tissue Bank of the Biobanc-Hospital ClÃnic-IDIBAPS, Barcelona, Spain (2) Pathological Service of Hospital ClÃnic de Barcelona, Barcelona, Spain (3) EAIA Trastorns Cognitius, Centre Emili Mira, Parc de Salut Mar, Barcelona, Spain (4) Department of Neurology of Hospital ClÃnic de Barcelona, Barcelona, Spain (5) Institute of Neuropathology, Hospital Universitari de Bellvitge, Hospitalet de Llobregat, Barcelona (6) General subdirectorate of Surveillance and Response to Emergencies in Public Health, Department of Public Health in Catalonia, Barcelona, Spain (7) Institute of Neurology, Medical University of Vienna, Vienna, Austria.
Background and objective:
The Neurological Tissue Bank (NTB) of the Hospital Clínic-Institut d‘Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain is the reference center in Catalonia for the neuropathological study of prion diseases in the region since 2001. The aim of this study is to analyse the characteristics of the confirmed prion diseases registered at the NTB during the last 15 years.
Methods:
We reviewed retrospectively all neuropathologically confirmed cases registered during the period January 2001 to December 2016.
Results:
176 cases (54,3% female, mean age: 67,5 years and age range: 25-86 years) of neuropathological confirmed prion diseases have been studied at the NTB. 152 cases corresponded to sporadic Creutzfeldt-Jakob disease (sCJD), 10 to genetic CJD, 10 to Fatal Familial Insomnia, 2 to GerstmannSträussler-Scheinker disease, and 2 cases to variably protease-sensitive prionopathy (VPSPr). Within sCJD subtypes the MM1 subtype was the most frequent, followed by the VV2 histotype.
Clinical and neuropathological diagnoses agreed in 166 cases (94%). The clinical diagnosis was not accurate in 10 patients with definite prion disease: 1 had a clinical diagnosis of Fronto-temporal dementia (FTD), 1 Niemann-Pick‘s disease, 1 Lewy Body‘s Disease, 2 Alzheimer‘s disease, 1 Cortico-basal syndrome and 2 undetermined dementia. Among patients with VPSPr, 1 had a clinical diagnosis of Amyotrophic lateral sclerosis (ALS) and the other one with FTD.
Concomitant pathologies are frequent in older age groups, mainly AD neuropathological changes were observed in these subjects.
Discussion:
A wide spectrum of human prion diseases have been identified in the NTB being the relative frequencies and main characteristics like other published series. There is a high rate of agreement between clinical and neuropathological diagnoses with prion diseases. These findings show the importance that public health has given to prion diseases during the past 15 years. Continuous surveillance of human prion disease allows identification of new emerging phenotypes. Brain tissue samples from these donors are available to the scientific community. For more information please visit:
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P192 Prion amplification techniques for the rapid evaluation of surface decontamination procedures
Bruyere-Ostells L (1), Mayran C (1), Belondrade M (1), Boublik Y (2), Haïk S (3), Fournier-Wirth C (1), Nicot S (1), Bougard D (1)
(1) Pathogenesis and control of chronic infections, Etablissement Français du Sang, Inserm, Université de Montpellier, Montpellier, France. (2) Centre de Recherche en Biologie cellulaire de Montpellier, CNRS, Université de Montpellier, Montpellier, France. (3) Inserm U 1127, CNRS UMR 7225, Sorbonne Universités, UPMC Université Paris 06 UMR S 1127, Institut du Cerveau et de la Moelle épinière, ICM, Paris, France.
Aims:
Transmissible Spongiform Encephalopathies (TSE) or prion diseases are a group of incurable and always fatal neurodegenerative disorders including Creutzfeldt-Jakob diseases (CJD) in humans. These pathologies include sporadic (sCJD), genetic and acquired (variant CJD) forms. By the past, sCJD and vCJD were transmitted by different prion contaminated biological materials to patients resulting in more than 400 iatrogenic cases (iCJD). The atypical nature and the biochemical properties of the infectious agent, formed by abnormal prion protein or PrPTSE, make it particularly resistant to conventional decontamination procedures. In addition, PrPTSE is widely distributed throughout the organism before clinical onset in vCJD and can also be detected in some peripheral tissues in sporadic CJD. Risk of iatrogenic transmission of CJD by contaminated medical device remains thus a concern for healthcare facilities. Bioassay is the gold standard method to evaluate the efficacy of prion decontamination procedures but is time-consuming and expensive. Here, we propose to compare in vitro prion amplification techniques: Protein Misfolding Cyclic Amplification (PMCA) and Real-Time Quaking Induced Conversion (RT-QuIC) for the detection of residual prions on surface after decontamination.
Methods:
Stainless steel wires, by mimicking the surface of surgical instruments, were proposed as a carrier model of prions for inactivation studies. To determine the sensitivity of the two amplification techniques on wires (Surf-PMCA and Surf-QuIC), steel wires were therefore contaminated with serial dilutions of brain homogenates (BH) from a 263k infected hamster and from a patient with sCJD (MM1 subtype). We then compared the different standard decontamination procedures including partially and fully efficient treatments by detecting the residual seeding activity on 263K and sCJD contaminated wires. We completed our study by the evaluation of marketed reagents endorsed for prion decontamination.
Results:
The two amplification techniques can detect minute quantities of PrPTSE adsorbed onto a single wire. 8/8 wires contaminated with a 10-6 dilution of 263k BH and 1/6 with the 10-8 dilution are positive with Surf-PMCA. Similar performances were obtained with Surf-QuIC on 263K: 10/16 wires contaminated with 10-6 dilution and 1/8 wires contaminated with 10-8 dilution are positive. Regarding the human sCJD-MM1 prion, Surf-QuIC allows us to detect 16/16 wires contaminated with 10-6 dilutions and 14/16 with 10-7 . Results obtained after decontamination treatments are very similar between 263K and sCJD prions. Efficiency of marketed treatments to remove prions is lower than expected.
Conclusions:
Surf-PMCA and Surf-QuIC are very sensitive methods for the detection of prions on wires and could be applied to prion decontamination studies for rapid evaluation of new treatments. Sodium hypochlorite is the only product to efficiently remove seeding activity of both 263K and sCJD prions.
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WA2 Oral transmission of CWD into Cynomolgus macaques: signs of atypical disease, prion conversion and infectivity in macaques and bio-assayed transgenic mice
Schatzl HM (1, 2), Hannaoui S (1, 2), Cheng Y-C (1, 2), Gilch S (1, 2), Beekes M (3), SchulzSchaeffer W (4), Stahl-Hennig C (5) and Czub S (2, 6)
(1) University of Calgary, Calgary Prion Research Unit, Calgary, Canada (2) University of Calgary, Faculty of Veterinary Medicine, Calgary, Canada, (3) Robert Koch Institute, Berlin, Germany, (4) University of Homburg/Saar, Homburg, Germany, (5) German Primate Center, Goettingen, Germany, (6) Canadian Food Inspection Agency (CFIA), Lethbridge, Canada.
To date, BSE is the only example of interspecies transmission of an animal prion disease into humans. The potential zoonotic transmission of CWD is an alarming issue and was addressed by many groups using a variety of in vitro and in vivo experimental systems. Evidence from these studies indicated a substantial, if not absolute, species barrier, aligning with the absence of epidemiological evidence suggesting transmission into humans. Studies in non-human primates were not conclusive so far, with oral transmission into new-world monkeys and no transmission into old-world monkeys. Our consortium has challenged 18 Cynomolgus macaques with characterized CWD material, focusing on oral transmission with muscle tissue. Some macaques have orally received a total of 5 kg of muscle material over a period of 2 years. After 5-7 years of incubation time some animals showed clinical symptoms indicative of prion disease, and prion neuropathology and PrPSc deposition were found in spinal cord and brain of euthanized animals. PrPSc in immunoblot was weakly detected in some spinal cord materials and various tissues tested positive in RT-QuIC, including lymph node and spleen homogenates. To prove prion infectivity in the macaque tissues, we have intracerebrally inoculated 2 lines of transgenic mice, expressing either elk or human PrP. At least 3 TgElk mice, receiving tissues from 2 different macaques, showed clinical signs of a progressive prion disease and brains were positive in immunoblot and RT-QuIC. Tissues (brain, spinal cord and spleen) from these and preclinical mice are currently tested using various read-outs and by second passage in mice. Transgenic mice expressing human PrP were so far negative for clear clinical prion disease (some mice >300 days p.i.). In parallel, the same macaque materials are inoculated into bank voles. Taken together, there is strong evidence of transmissibility of CWD orally into macaques and from macaque tissues into transgenic mouse models, although with an incomplete attack rate. The clinical and pathological presentation in macaques was mostly atypical, with a strong emphasis on spinal cord pathology. Our ongoing studies will show whether the transmission of CWD into macaques and passage in transgenic mice represents a form of non-adaptive prion amplification, and whether macaque-adapted prions have the potential to infect mice expressing human PrP. The notion that CWD can be transmitted orally into both new-world and old-world non-human primates asks for a careful reevaluation of the zoonotic risk of CWD.
See also poster P103
***> The notion that CWD can be transmitted orally into both new-world and old-world non-human primates asks for a careful reevaluation of the zoonotic risk of CWD.
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WA16 Monitoring Potential CWD Transmission to Humans
Belay ED
Centers for Disease Control and Prevention (CDC), National Center for Emerging and Zoonotic Infectious Diseases, Atlanta, GA, USA.
The spread of chronic wasting disease (CWD) in animals has raised concerns about increasing human exposure to the CWD agent via hunting and venison consumption, potentially facilitating CWD transmission to humans. Several studies have explored this possibility, including limited epidemiologic studies, in vitro experiments, and laboratory studies using various types of animal models. Most human exposures to the CWD agent in the United States would be expected to occur in association with deer and elk hunting in CWD-endemic areas. The Centers for Disease Control and Prevention (CDC) collaborated with state health departments in Colorado, Wisconsin, and Wyoming to identify persons at risk of CWD exposure and to monitor their vital status over time. Databases were established of persons who hunted in Colorado and Wyoming and those who reported consumption of venison from deer that later tested positive in Wisconsin. Information from the databases is periodically cross-checked with mortality data to determine the vital status and causes of death for deceased persons. Long-term follow-up of these hunters is needed to assess their risk of development of a prion disease linked to CWD exposure.
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P166 Characterization of CJD strain profiles in venison consumers and non-consumers from Alberta and Saskatchewan
Stephanie Booth (1,2), Lise Lamoureux (1), Debra Sorensen (1), Jennifer L. Myskiw (1,2), Megan Klassen (1,2), Michael Coulthart (3), Valerie Sim (4)
(1) Zoonotic Diseases and Special Pathogens, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg (2) Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg (3) Canadian CJD Surveillance System, Public Health Agency of Canada, Ottawa (4) Division of Neurology, Department of Medicine Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton.
Chronic wasting disease (CWD) is spreading rapidly through wild cervid populations in the Canadian provinces of Alberta and Saskatchewan. While this has implications for tourism and hunting, there is also concern over possible zoonotic transmission to humans who eat venison from infected deer. Whilst there is no evidence of any human cases of CWD to date, the Canadian CJD Surveillance System (CJDSS) in Canada is staying vigilant. When variant CJD occurred following exposure to BSE, the unique biochemical fingerprint of the pathologic PrP enabled a causal link to be confirmed. However, we cannot be sure what phenotype human CWD prions would present with, or indeed, whether this would be distinct from that see in sporadic CJD. Therefore we are undertaking a systematic analysis of the molecular diversity of CJD cases of individuals who resided in Alberta and Saskatchewan at their time of death comparing venison consumers and non-consumers, using a variety of clinical, imaging, pathological and biochemical markers. Our initial objective is to develop novel biochemical methodologies that will extend the baseline glycoform and genetic polymorphism typing that is already completed by the CJDSS. Firstly, we are reviewing MRI, EEG and pathology information from over 40 cases of CJD to select clinically affected areas for further investigation. Biochemical analysis will include assessment of the levels of protease sensitive and resistant prion protein, glycoform typing using 2D gel electrophoresis, testing seeding capabilities and kinetics of aggregation by quaking-induced conversion, and determining prion oligomer size distributions with asymmetric flow field fractionation with in-line light scattering. Progress and preliminary data will be presented. Ultimately, we intend to further define the relationship between PrP structure and disease phenotype and establish a baseline for the identification of future atypical CJD cases that may arise as a result of exposure to CWD.
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Source Prion Conference 2018 Abstracts
Volume 24, Number 8—August 2018 Research Susceptibility of Human Prion Protein to Conversion by Chronic Wasting Disease Prions
Marcelo A. BarriaComments to Author , Adriana Libori, Gordon Mitchell, and Mark W. Head Author affiliations: National CJD Research and Surveillance Unit, University of Edinburgh, Edinburgh, Scotland, UK (M.A. Barria, A. Libori, M.W. Head); National and OIE Reference Laboratory for Scrapie and CWD, Canadian Food Inspection Agency, Ottawa, Ontario, Canada (G. Mitchell)
Abstract Chronic wasting disease (CWD) is a contagious and fatal neurodegenerative disease and a serious animal health issue for deer and elk in North America. The identification of the first cases of CWD among free-ranging reindeer and moose in Europe brings back into focus the unresolved issue of whether CWD can be zoonotic like bovine spongiform encephalopathy. We used a cell-free seeded protein misfolding assay to determine whether CWD prions from elk, white-tailed deer, and reindeer in North America can convert the human prion protein to the disease-associated form. We found that prions can convert, but the efficiency of conversion is affected by polymorphic variation in the cervid and human prion protein genes. In view of the similarity of reindeer, elk, and white-tailed deer in North America to reindeer, red deer, and roe deer, respectively, in Europe, a more comprehensive and thorough assessment of the zoonotic potential of CWD might be warranted.
snip...
Discussion Characterization of the transmission properties of CWD and evaluation of their zoonotic potential are important for public health purposes. Given that CWD affects several members of the family Cervidae, it seems reasonable to consider whether the zoonotic potential of CWD prions could be affected by factors such as CWD strain, cervid species, geographic location, and Prnp–PRNP polymorphic variation. We have previously used an in vitro conversion assay (PMCA) to investigate the susceptibility of the human PrP to conversion to its disease-associated form by several animal prion diseases, including CWD (15,16,22). The sensitivity of our molecular model for the detection of zoonotic conversion depends on the combination of 1) the action of proteinase K to degrade the abundant human PrPC that constitutes the substrate while only N terminally truncating any human PrPres produced and 2) the presence of the 3F4 epitope on human but not cervid PrP. In effect, this degree of sensitivity means that any human PrPres formed during the PMCA reaction can be detected down to the limit of Western blot sensitivity. In contrast, if other antibodies that detect both cervid and human PrP are used, such as 6H4, then newly formed human PrPres must be detected as a measurable increase in PrPres over the amount remaining in the reaction product from the cervid seed. Although best known for the efficient amplification of prions in research and diagnostic contexts, the variation of the PMCA method employed in our study is optimized for the definitive detection of zoonotic reaction products of inherently inefficient conversion reactions conducted across species barriers. By using this system, we previously made and reported the novel observation that elk CWD prions could convert human PrPC from human brain and could also convert recombinant human PrPC expressed in transgenic mice and eukaryotic cell cultures (15).
A previous publication suggested that mule deer PrPSc was unable to convert humanized transgenic substrate in PMCA assays (23) and required a further step of in vitro conditioning in deer substrate PMCA before it was able to cross the deer–human molecular barrier (24). However, prions from other species, such as elk (15) and reindeer affected by CWD, appear to be compatible with the human protein in a single round of amplification (as shown in our study). These observations suggest that different deer species affected by CWD could present differing degrees of the olecular compatibility with the normal form of human PrP.
The contribution of the polymorphism at codon 129 of the human PrP gene has been extensively studied and is recognized as a risk factor for Creutzfeldt-Jakob disease (4). In cervids, the equivalent codon corresponds to the position 132 encoding methionine or leucine. This polymorphism in the elk gene has been shown to play an important role in CWD susceptibility (25,26). We have investigated the effect of this cervid Prnp polymorphism on the conversion of the humanized transgenic substrate according to the variation in the equivalent PRNP codon 129 polymorphism. Interestingly, only the homologs methionine homozygous seed–substrate reactions could readily convert the human PrP, whereas the heterozygous elk PrPSc was unable to do so, even though comparable amounts of PrPres were used to seed the reaction. In addition, we observed only low levels of human PrPres formation in the reactions seeded with the homozygous methionine (132 MM) and the heterozygous (132 ML) seeds incubated with the other 2 human polymorphic substrates (129 MV and 129 VV). The presence of the amino acid leucine at position 132 of the elk Prnp gene has been attributed to a lower degree of prion conversion compared with methionine on the basis of experiments in mice made transgenic for these polymorphic variants (26). Considering the differences observed for the amplification of the homozygous human methionine substrate by the 2 polymorphic elk seeds (MM and ML), reappraisal of the susceptibility of human PrPC by the full range of cervid polymorphic variants affected by CWD would be warranted.
In light of the recent identification of the first cases of CWD in Europe in a free-ranging reindeer (R. tarandus) in Norway (2), we also decided to evaluate the in vitro conversion potential of CWD in 2 experimentally infected reindeer (18). Formation of human PrPres was readily detectable after a single round of PMCA, and in all 3 humanized polymorphic substrates (MM, MV, and VV). This finding suggests that CWD prions from reindeer could be more compatible with human PrPC generally and might therefore present a greater risk for zoonosis than, for example, CWD prions from white-tailed deer. A more comprehensive comparison of CWD in the affected species, coupled with the polymorphic variations in the human and deer PRNP–Prnp genes, in vivo and in vitro, will be required before firm conclusions can be drawn. Analysis of the Prnp sequence of the CWD reindeer in Norway was reported to be identical to the specimens used in our study (2). This finding raises the possibility of a direct comparison of zoonotic potential between CWD acquired in the wild and that produced in a controlled laboratory setting. (Table).
The prion hypothesis proposes that direct molecular interaction between PrPSc and PrPC is necessary for conversion and prion replication. Accordingly, polymorphic variants of the PrP of host and agent might play a role in determining compatibility and potential zoonotic risk. In this study, we have examined the capacity of the human PrPC to support in vitro conversion by elk, white-tailed deer, and reindeer CWD PrPSc. Our data confirm that elk CWD prions can convert the human PrPC, at least in vitro, and show that the homologous PRNP polymorphisms at codon 129 and 132 in humans and cervids affect conversion efficiency. Other species affected by CWD, particularly caribou or reindeer, also seem able to convert the human PrP. It will be important to determine whether other polymorphic variants found in other CWD-affected Cervidae or perhaps other factors (17) exert similar effects on the ability to convert human PrP and thus affect their zoonotic potential.
Dr. Barria is a research scientist working at the National CJD Research and Surveillance Unit, University of Edinburgh. His research has focused on understanding the molecular basis of a group of fatal neurologic disorders called prion diseases.
Acknowledgments We thank Aru Balachandran for originally providing cervid brain tissues, Abigail Diack and Jean Manson for providing mouse brain tissue, and James Ironside for his critical reading of the manuscript at an early stage.
This report is independent research commissioned and funded by the United Kingdom’s Department of Health Policy Research Programme and the Government of Scotland. The views expressed in this publication are those of the authors and not necessarily those of the Department of Health or the Government of Scotland.
Author contributions: The study was conceived and designed by M.A.B. and M.W.H. The experiments were conducted by M.A.B. and A.L. Chronic wasting disease brain specimens were provided by G.M. The manuscript was written by M.A.B. and M.W.H. All authors contributed to the editing and revision of the manuscript.
Prion 2017 Conference Abstracts
First evidence of intracranial and peroral transmission of Chronic Wasting Disease (CWD) into Cynomolgus macaques: a work in progress Stefanie Czub1, Walter Schulz-Schaeffer2, Christiane Stahl-Hennig3, Michael Beekes4, Hermann Schaetzl5 and Dirk Motzkus6 1
University of Calgary Faculty of Veterinary Medicine/Canadian Food Inspection Agency; 2Universitatsklinikum des Saarlandes und Medizinische Fakultat der Universitat des Saarlandes; 3 Deutsches Primaten Zentrum/Goettingen; 4 Robert-Koch-Institut Berlin; 5 University of Calgary Faculty of Veterinary Medicine; 6 presently: Boehringer Ingelheim Veterinary Research Center; previously: Deutsches Primaten Zentrum/Goettingen
This is a progress report of a project which started in 2009.
21 cynomolgus macaques were challenged with characterized CWD material from white-tailed deer (WTD) or elk by intracerebral (ic), oral, and skin exposure routes. Additional blood transfusion experiments are supposed to assess the CWD contamination risk of human blood product. Challenge materials originated from symptomatic cervids for ic, skin scarification and partially per oral routes (WTD brain). Challenge material for feeding of muscle derived from preclinical WTD and from preclinical macaques for blood transfusion experiments. We have confirmed that the CWD challenge material contained at least two different CWD agents (brain material) as well as CWD prions in muscle-associated nerves.
Here we present first data on a group of animals either challenged ic with steel wires or per orally and sacrificed with incubation times ranging from 4.5 to 6.9 years at postmortem. Three animals displayed signs of mild clinical disease, including anxiety, apathy, ataxia and/or tremor. In four animals wasting was observed, two of those had confirmed diabetes. All animals have variable signs of prion neuropathology in spinal cords and brains and by supersensitive IHC, reaction was detected in spinal cord segments of all animals. Protein misfolding cyclic amplification (PMCA), real-time quaking-induced conversion (RT-QuiC) and PET-blot assays to further substantiate these findings are on the way, as well as bioassays in bank voles and transgenic mice.
At present, a total of 10 animals are sacrificed and read-outs are ongoing. Preclinical incubation of the remaining macaques covers a range from 6.4 to 7.10 years. Based on the species barrier and an incubation time of > 5 years for BSE in macaques and about 10 years for scrapie in macaques, we expected an onset of clinical disease beyond 6 years post inoculation.
PRION 2017 DECIPHERING NEURODEGENERATIVE DISORDERS ABSTRACTS REFERENCE
8. Even though human TSE‐exposure risk through consumption of game from European cervids can be assumed to be minor, if at all existing, no final conclusion can be drawn due to the overall lack of scientific data. In particular the US data do not clearly exclude the possibility of human (sporadic or familial) TSE development due to consumption of venison. The Working Group thus recognizes a potential risk to consumers if a TSE would be present in European cervids. It might be prudent considering appropriate measures to reduce such a risk, e.g. excluding tissues such as CNS and lymphoid tissues from the human food chain, which would greatly reduce any potential risk for consumers. However, it is stressed that currently, no data regarding a risk of TSE infections from cervid products are available.
SATURDAY, FEBRUARY 23, 2019
Chronic Wasting Disease CWD TSE Prion and THE FEAST 2003 CDC an updated review of the science 2019
TUESDAY, NOVEMBER 04, 2014
Six-year follow-up of a point-source exposure to CWD contaminated venison in an Upstate New York community: risk behaviours and health outcomes 2005–2011
Authors, though, acknowledged the study was limited in geography and sample size and so it couldn't draw a conclusion about the risk to humans. They recommended more study. Dr. Ermias Belay was the report's principal author but he said New York and Oneida County officials are following the proper course by not launching a study. "There's really nothing to monitor presently. No one's sick," Belay said, noting the disease's incubation period in deer and elk is measured in years. "
Transmission Studies
Mule deer transmissions of CWD were by intracerebral inoculation and compared with natural cases {the following was written but with a single line marked through it ''first passage (by this route)}....TSS
resulted in a more rapidly progressive clinical disease with repeated episodes of synocopy ending in coma. One control animal became affected, it is believed through contamination of inoculum (?saline). Further CWD transmissions were carried out by Dick Marsh into ferret, mink and squirrel monkey. Transmission occurred in ALL of these species with the shortest incubation period in the ferret.
snip....
Prion Infectivity in Fat of Deer with Chronic Wasting Disease▿
Brent Race#, Kimberly Meade-White#, Richard Race and Bruce Chesebro* + Author Affiliations
In mice, prion infectivity was recently detected in fat. Since ruminant fat is consumed by humans and fed to animals, we determined infectivity titers in fat from two CWD-infected deer. Deer fat devoid of muscle contained low levels of CWD infectivity and might be a risk factor for prion infection of other species.
Prions in Skeletal Muscles of Deer with Chronic Wasting Disease
Here bioassays in transgenic mice expressing cervid prion protein revealed the presence of infectious prions in skeletal muscles of CWD-infected deer, demonstrating that humans consuming or handling meat from CWD-infected deer are at risk to prion exposure.
*** now, let’s see what the authors said about this casual link, personal communications years ago, and then the latest on the zoonotic potential from CWD to humans from the TOKYO PRION 2016 CONFERENCE.
see where it is stated NO STRONG evidence. so, does this mean there IS casual evidence ???? “Our conclusion stating that we found no strong evidence of CWD transmission to humans”
From: TSS
Subject: CWD aka MAD DEER/ELK TO HUMANS ???
Date: September 30, 2002 at 7:06 am PST
From: "Belay, Ermias"
To: Cc: "Race, Richard (NIH)" ; ; "Belay, Ermias"
Sent: Monday, September 30, 2002 9:22 AM
Subject: RE: TO CDC AND NIH - PUB MED- 3 MORE DEATHS - CWD - YOUNG HUNTERS
Dear Sir/Madam,
In the Archives of Neurology you quoted (the abstract of which was attached to your email), we did not say CWD in humans will present like variant CJD.. That assumption would be wrong. I encourage you to read the whole article and call me if you have questions or need more clarification (phone: 404-639-3091). Also, we do not claim that "no-one has ever been infected with prion disease from eating venison." Our conclusion stating that we found no strong evidence of CWD transmission to humans in the article you quoted or in any other forum is limited to the patients we investigated.
Ermias Belay, M.D. Centers for Disease Control and Prevention
-----Original Message-----
From: Sent: Sunday, September 29, 2002 10:15 AM
Subject: TO CDC AND NIH - PUB MED- 3 MORE DEATHS - CWD - YOUNG HUNTERS
Sunday, November 10, 2002 6:26 PM .......snip........end..............TSS
Thursday, April 03, 2008
A prion disease of cervids: Chronic wasting disease 2008 1: Vet Res. 2008 Apr 3;39(4):41 A prion disease of cervids: Chronic wasting disease Sigurdson CJ.
snip...
*** twenty-seven CJD patients who regularly consumed venison were reported to the Surveillance Center***,
snip... full text ;
> However, to date, no CWD infections have been reported in people.
sporadic, spontaneous CJD, 85%+ of all human TSE, did not just happen. never in scientific literature has this been proven.
if one looks up the word sporadic or spontaneous at pubmed, you will get a laundry list of disease that are classified in such a way;
sporadic = 54,983 hits https://www.ncbi.nlm.nih.gov/pubmed/?term=sporadic
spontaneous = 325,650 hits https://www.ncbi.nlm.nih.gov/pubmed/?term=spontaneous
key word here is 'reported'. science has shown that CWD in humans will look like sporadic CJD. SO, how can one assume that CWD has not already transmitted to humans? they can't, and it's as simple as that. from all recorded science to date, CWD has already transmitted to humans, and it's being misdiagnosed as sporadic CJD. ...terry
*** LOOKING FOR CWD IN HUMANS AS nvCJD or as an ATYPICAL CJD, LOOKING IN ALL THE WRONG PLACES $$$ ***
> However, to date, no CWD infections have been reported in people.
key word here is ‘reported’. science has shown that CWD in humans will look like sporadic CJD. SO, how can one assume that CWD has not already transmitted to humans? they can’t, and it’s as simple as that. from all recorded science to date, CWD has already transmitted to humans, and it’s being misdiagnosed as sporadic CJD. …terry
*** LOOKING FOR CWD IN HUMANS AS nvCJD or as an ATYPICAL CJD, LOOKING IN ALL THE WRONG PLACES $$$ ***
*** These results would seem to suggest that CWD does indeed have zoonotic potential, at least as judged by the compatibility of CWD prions and their human PrPC target. Furthermore, extrapolation from this simple in vitro assay suggests that if zoonotic CWD occurred, it would most likely effect those of the PRNP codon 129-MM genotype and that the PrPres type would be similar to that found in the most common subtype of sCJD (MM1).***
CWD TSE PRION AND ZOONOTIC, ZOONOSIS, POTENTIAL
Subject: Re: DEER SPONGIFORM ENCEPHALOPATHY SURVEY & HOUND STUDY
Date: Fri, 18 Oct 2002 23:12:22 +0100
From: Steve Dealler
Reply-To: Bovine Spongiform Encephalopathy Organization: Netscape Online member
To: BSE-L@ References:
Dear Terry,
An excellent piece of review as this literature is desperately difficult to get back from Government sites.
What happened with the deer was that an association between deer meat eating and sporadic CJD was found in about 1993. The evidence was not great but did not disappear after several years of asking CJD cases what they had eaten. I think that the work into deer disease largely stopped because it was not helpful to the UK industry...and no specific cases were reported. Well, if you dont look adequately like they are in USA currenly then you wont find any!
Steve Dealler ===============
''The association between venison eating and risk of CJD shows similar pattern, with regular venison eating associated with a 9 FOLD INCREASE IN RISK OF CJD (p = 0.04).''
CREUTZFELDT JAKOB DISEASE SURVEILLANCE IN THE UNITED KINGDOM THIRD ANNUAL REPORT AUGUST 1994
Consumption of venison and veal was much less widespread among both cases and controls. For both of these meats there was evidence of a trend with increasing frequency of consumption being associated with increasing risk of CJD. (not nvCJD, but sporadic CJD...tss) These associations were largely unchanged when attention was restricted to pairs with data obtained from relatives. ...
Table 9 presents the results of an analysis of these data.
There is STRONG evidence of an association between ‘’regular’’ veal eating and risk of CJD (p = .0.01).
Individuals reported to eat veal on average at least once a year appear to be at 13 TIMES THE RISK of individuals who have never eaten veal.
There is, however, a very wide confidence interval around this estimate. There is no strong evidence that eating veal less than once per year is associated with increased risk of CJD (p = 0.51).
The association between venison eating and risk of CJD shows similar pattern, with regular venison eating associated with a 9 FOLD INCREASE IN RISK OF CJD (p = 0.04).
There is some evidence that risk of CJD INCREASES WITH INCREASING FREQUENCY OF LAMB EATING (p = 0.02).
The evidence for such an association between beef eating and CJD is weaker (p = 0.14). When only controls for whom a relative was interviewed are included, this evidence becomes a little STRONGER (p = 0.08).
snip...
It was found that when veal was included in the model with another exposure, the association between veal and CJD remained statistically significant (p = < 0.05 for all exposures), while the other exposures ceased to be statistically significant (p = > 0.05).
snip...
In conclusion, an analysis of dietary histories revealed statistical associations between various meats/animal products and INCREASED RISK OF CJD. When some account was taken of possible confounding, the association between VEAL EATING AND RISK OF CJD EMERGED AS THE STRONGEST OF THESE ASSOCIATIONS STATISTICALLY. ...
snip...
In the study in the USA, a range of foodstuffs were associated with an increased risk of CJD, including liver consumption which was associated with an apparent SIX-FOLD INCREASE IN THE RISK OF CJD. By comparing the data from 3 studies in relation to this particular dietary factor, the risk of liver consumption became non-significant with an odds ratio of 1.2 (PERSONAL COMMUNICATION, PROFESSOR A. HOFMAN. ERASMUS UNIVERSITY, ROTTERDAM). (???...TSS)
snip...see full report ;
http://web.archive.org/web/20090506050043/http://www.bseinquiry.gov.uk/files/yb/1994/08/00004001.pdf
http://web.archive.org/web/20090506050007/http://www.bseinquiry.gov.uk/files/yb/1994/10/00003001.pdf
http://web.archive.org/web/20090506050244/http://www.bseinquiry.gov.uk/files/yb/1994/07/00001001.pdf
Stephen Dealler is a consultant medical microbiologist deal@airtime.co.uk
BSE Inquiry Steve Dealler
Management In Confidence
BSE: Private Submission of Bovine Brain Dealler
snip...see full text;
MONDAY, FEBRUARY 25, 2019
***> MAD DOGS AND ENGLISHMEN BSE, SCRAPIE, CWD, CJD, TSE PRION A REVIEW 2019
***> ''The association between venison eating and risk of CJD shows similar pattern, with regular venison eating associated with a 9 FOLD INCREASE IN RISK OF CJD (p = 0.04).''
***> In conclusion, sensory symptoms and loss of reflexes in Gerstmann-Sträussler-Scheinker syndrome can be explained by neuropathological changes in the spinal cord. We conclude that the sensory symptoms and loss of lower limb reflexes in Gerstmann-Sträussler-Scheinker syndrome is due to pathology in the caudal spinal cord. <***
***> The clinical and pathological presentation in macaques was mostly atypical, with a strong emphasis on spinal cord pathology.<***
***> The notion that CWD can be transmitted orally into both new-world and old-world non-human primates asks for a careful reevaluation of the zoonotic risk of CWD. <***
***> All animals have variable signs of prion neuropathology in spinal cords and brains and by supersensitive IHC, reaction was detected in spinal cord segments of all animals.<***
***> In particular the US data do not clearly exclude the possibility of human (sporadic or familial) TSE development due to consumption of venison. The Working Group thus recognizes a potential risk to consumers if a TSE would be present in European cervids.'' Scientific opinion on chronic wasting disease (II) <***
***Moreover, sporadic disease has never been observed in breeding colonies or primate research laboratories, most notably among hundreds of animals over several decades of study at the National Institutes of Health25, and in nearly twenty older animals continuously housed in our own facility.***
Even if the prevailing view is that sporadic CJD is due to the spontaneous formation of CJD prions, it remains possible that its apparent sporadic nature may, at least in part, result from our limited capacity to identify an environmental origin.
Notice of Request To Renew an Approved Information Collection: Specified Risk Materials DOCKET NUMBER Docket No. FSIS-2022-0027 Singeltary Submission
Singeltary further comments in attachment Specified Risk Materials DOCKET NUMBER Docket No. FSIS-2022-0027
Specified Risk Materials DOCKET NUMBER Docket No. FSIS-2022-0027 Singeltary Submission Attachment
https://downloads.regulations.gov/FSIS-2022-0027-0002/attachment_1.pdf
Monday, December 5, 2022
Notice of Request To Renew an Approved Information Collection: Specified Risk Materials DOCKET NUMBER Docket No. FSIS-2022-0027 Singeltary Submission
Tuesday, September 10, 2019
FSIS [Docket No. FSIS–2019–0021] Notice of Request To Renew an Approved Information Collection: Specified Risk Materials Singeltary Submission
Sunday, January 10, 2021APHIS Concurrence With OIE Risk Designation for Bovine Spongiform Encephalopathy [Docket No. APHIS-2018-0087] Singeltary Submission June 17, 2019APHIS Concurrence With OIE Risk Designation for Bovine Spongiform Encephalopathy [Docket No. APHIS-2018-0087] Singeltary SubmissionGreetings APHIS et al,I would kindly like to comment on APHIS Concurrence With OIE Risk Designation for Bovine Spongiform Encephalopathy [Docket No. APHIS-2018-0087], and my comments are as follows, with the latest peer review and transmission studies as references of evidence.THE OIE/USDA BSE Minimal Risk Region MRR is nothing more than free pass to import and export the Transmissible Spongiform Encephalopathy TSE Prion disease. December 2003, when the USDA et al lost it's supposedly 'GOLD CARD' ie BSE FREE STATUS (that was based on nothing more than not looking and not finding BSE), once the USA lost it's gold card BSE Free status, the USDA OIE et al worked hard and fast to change the BSE Geographical Risk Statuses i.e. the BSE GBR's, and replaced it with the BSE MRR policy, the legal tool to trade mad cow type disease TSE Prion Globally. The USA is doing just what the UK did, when they shipped mad cow disease around the world, except with the BSE MRR policy, it's now legal.Also, the whole concept of the BSE MRR policy is based on a false pretense, that atypical BSE is not transmissible, and that only typical c-BSE is transmissible via feed. This notion that atypical BSE TSE Prion is an old age cow disease that is not infectious is absolutely false, there is NO science to show this, and on the contrary, we now know that atypical BSE will transmit by ORAL ROUTES, but even much more concerning now, recent science has shown that Chronic Wasting Disease CWD TSE Prion in deer and elk which is rampant with no stopping is sight in the USA, and Scrapie TSE Prion in sheep and goat, will transmit to PIGS by oral routes, this is our worst nightmare, showing even more risk factors for the USA FDA PART 589 TSE PRION FEED ban.The FDA PART 589 TSE PRION FEED ban has failed terribly bad, and is still failing, since August 1997. there is tonnage and tonnage of banned potential mad cow feed that went into commerce, and still is, with one decade, 10 YEARS, post August 1997 FDA PART 589 TSE PRION FEED ban, 2007, with 10,000,000 POUNDS, with REASON, Products manufactured from bulk feed containing blood meal that was cross contaminated with prohibited meat and bone meal and the labeling did not bear cautionary BSE statement. you can see all these feed ban warning letters and tonnage of mad cow feed in commerce, year after year, that is not accessible on the internet anymore like it use to be, you can see history of the FDA failure August 1997 FDA PART 589 TSE PRION FEED ban here, but remember this, we have a new outbreak of TSE Prion disease in a new livestock species, the camel, and this too is very worrisome.WITH the OIE and the USDA et al weakening the global TSE prion surveillance, by not classifying the atypical Scrapie as TSE Prion disease, and the notion that they want to do the same thing with typical scrapie and atypical BSE, it's just not scientific.WE MUST abolish the BSE MRR policy, go back to the BSE GBR risk assessments by country, and enhance them to include all strains of TSE Prion disease in all species. With Chronic Wasting CWD TSE Prion disease spreading in Europe, now including, Norway, Finland, Sweden, also in Korea, Canada and the USA, and the TSE Prion in Camels, the fact the the USA is feeding potentially CWD, Scrapie, BSE, typical and atypical, to other animals, and shipping both this feed and or live animals or even grains around the globe, potentially exposed or infected with the TSE Prion. this APHIS Concurrence With OIE Risk Designation for Bovine Spongiform Encephalopathy [Docket No. APHIS-2018-0087], under it's present definition, does NOT show the true risk of the TSE Prion in any country. as i said, it's nothing more than a legal tool to trade the TSE Prion around the globe, nothing but ink on paper.AS long as the BSE MRR policy stays in effect, TSE Prion disease will continued to be bought and sold as food for both humans and animals around the globe, and the future ramifications from friendly fire there from, i.e. iatrogenic exposure and transmission there from from all of the above, should not be underestimated. ...
APHIS Indemnity Regulations [Docket No. APHIS-2021-0010] RIN 0579-AE65 Singeltary Comment SubmissionComment from Singeltary Sr., TerryPosted by the Animal and Plant Health Inspection Service on Sep 8, 2022Comments 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 RegulationsCommentsSubject: [Docket No. FSIS-2006-0011] FSIS Harvard Risk Assessment of Bovine Spongiform Encephalopathy (BSE) Page 1 of 98 8/3/2006Greetings 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 07182.024FULL 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.SATURDAY, AUGUST 16, 2008Qualitative Analysis of BSE Risk Factors in the United States February 13, 2000 at 3:37 pm PST (BSE red book)Tuesday, September 14, 2010Transmissible 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 diseaseDate: Mon, 02 May 2005 16:59:07 -0500From: "Terry S. Singeltary Sr."Greetings Honorable Paul Feeney, Keith Arnold, and William Busbyet al at OIG, ...............snip...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 xxxxDate: June 14, 2005 at 1:46 pm PSTIn Reply to:Re: Transcript Ag. Secretary Mike Johanns and Dr. John Clifford, Regarding further analysis of BSE Inconclusive Test Resultsposted 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 ***Suppressed peer review of Harvard study October 31, 2002October 31, 2002Review 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 UniversityFinal ReportHarvard Risk Assessment of Bovine Spongiform Encephalopathy (BSE) Update; Notice of Availability and Technical MeetingOwens, JulieFrom: Terry S. Singeltary Sr. [flounder9@verizon.net]Sent: Monday, July 24, 2006 1:09 PMTo: FSIS RegulationsCommentsSubject: [Docket No. FSIS-2006-0011] FSIS Harvard Risk Assessment of Bovine Spongiform Encephalopathy (BSE)Response to Public Comments on the Harvard Risk Assessment of Bovine Spongiform Encephalopathy Update,October 31, 2005INTRODUCTIONThe United States Department of Agriculture’s Food Safety and Inspection Service (FSIS) held a public meeting on July 25, 2006 in Washington, D.C. to present findings from the Harvard Risk Assessment of Bovine Spongiform Encephalopathy Update, October 31, 2005 (report and model located on the FSIS website: http://www.fsis.usda.gov/Science/Risk_Assessments/index.asp). 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, RCALF 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:03-025IFA03-025IFA-2Terry S. SingeltaryFrom: Terry S. Singeltary Sr. [flounder9@verizon.net]Sent: Thursday, September 08, 2005 6:17 PMTo: fsis.regulationscomments@fsis.usda.govSubject: [Docket No. 03-025IFA] FSIS Prohibition of the Use of Specified Risk Materials for Human Food and Requirements for the Disposition of Non-Ambulatory Disabled CattleONE final comment tonight, i just cannot take anymore. well, ill just let the facts speak for themselves, no need to even comment ;Section 2. Testing Protocols and Quality Assurance ControlsIn November 2004, USDA announced that its rapid screening test, Bio-Rad Enzyme Linked Immunosorbent Assay (ELISA), produced an inconclusive BSE test result as part of its enhanced BSE surveillance program. The ELISA rapid screening test performed at a BSE contract laboratory produced three high positive reactive results.40 As required,41 the contract laboratory forwarded the inconclusive sample to the APHIS National Veterinary Services Laboratories (NVSL) for confirmatory testing. NVSL repeated the ELISA testing and again produced three high positive reactive results.42 In accordance with its established protocol, NVSL ran its confirmatory test, an immunohistochemistry (IHC) test, which was interpreted as negative for BSE. In addition, NVSL performed a histological43 examination of the tissue and did not detect lesions44 consistent with BSE.Faced with conflicting results, NVSL scientists recommended additional testing to resolve the discrepancy but APHIS headquarters officials concluded no further testing was necessary because testing protocols were followed. In our discussions with APHIS officials, they justified their decision not to do additional testing because the IHC is internationally recognized as the "gold standard." Also, they believed that conducting additional tests would undermine confidence in USDA’s established testing protocols.full text 130 pages ;PDF]Freas, William TSS SUBMISSIONFile Format: PDF/Adobe Acrobat - 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://web.archive.org/web/20170301223601/https://www.fda.gov/OHRMS/DOCKETS/AC/01/slides/3681s2_09.pdfWEDNESDAY, NOVEMBER 30, 2022USDA Bovine Spongiform Encephalopathy BSE, Scrapie, CWD, Testing and Surveillance 2022 A Review of History
Control of Chronic Wasting Disease OMB Control Number: 0579-0189 APHIS-2021-0004 Singeltary Submission
Docket No. APHIS-2018-0011 Chronic Wasting Disease Herd Certification
Comment from Terry Singeltary Sr.
Posted by the Food and Drug Administration on May 17, 2016
Docket No. FDA-2003-D-0432 (formerly 03D-0186) Use of Material from Deer and Elk in Animal Feed Singeltary Submission
Greetings again FDA and Mr. Pritchett et al,
MY comments and source reference of sound science on this very important issue are as follows ;
Docket No. FDA-2003-D-0432 (formerly 03D-0186) Use of Material from Deer and Elk in Animal Feed Singeltary Submission
I kindly wish to once again submit to Docket No. FDA-2003-D-0432 (formerly 03D-0186) Use of Material from Deer and Elk in Animal Feed.
Thank you kindly for allowing me to comment again, ...and again...and again, on a topic so important, why it is 'NON-BINDING' is beyond me.
this should have been finalized and made 'BINDING' or MANDATORY OVER A DECADE AGO.
but here lay the problem, once made 'BINDING' or 'MANDATORY', it is still nothing but ink on paper.
we have had a mad cow feed ban in place since August 1997, and since then, literally 100s of millions of pounds BANNED MAD COW FEED has been sent out to commerce and fed out (see reference materials).
ENFORCEMENT OF SAID BINDING REGULATIONS HAS FAILED US TOO MANY TIMES.
so, in my opinion, any non-binding or voluntary regulations will not work, and to state further, 'BINDING' or MANDATORY regulations will not work unless enforced.
with that said, we know that Chronic Wasting Disease CWD TSE Prion easily transmits to other cervid through the oral route.
the old transmission studies of BSE TSE floored scientist once they figured out what they had, and please don't forget about those mink that were fed 95%+ dead stock downer cow, that all came down with TME. please see ;
It is clear that the designing scientists must also have shared Mr Bradleys surprise at the results because all the dose levels right down to 1 gram triggered infection.
it is clear that the designing scientists must have also shared Mr Bradleys surprise at the results because all the dose levels right down to 1 gram triggered infection.
Evidence That Transmissible Mink Encephalopathy Results from Feeding Infected Cattle
Over the next 8-10 weeks, approximately 40% of all the adult mink on the farm died from TME.
snip...
The rancher was a ''dead stock'' feeder using mostly (>95%) downer or dead dairy cattle...
To further complicate things, we now know that science has shown that plants and vegetables can uptake the TSE Prion, and that the Scrapie agent can still be infectious from soil 16 years later. a frightening thought with the CWD running rampant now in North America (please see source reference materials below).
IF we don't do this, we have failed, and the TSE Prion agent will continue to spread, as it is doing as we speak.
I strenuously once again urge the FDA and its industry constituents, to make it MANDATORY that all ruminant feed be banned to all ruminants, and this should include all cervids, as well as non-ruminants such as cats and dogs as well, as soon as possible for the following reasons...
31 Jan 2015 at 20:14 GMT
*** Ruminant feed ban for cervids in the United States? ***
31 Jan 2015 at 20:14 GMT
see Singeltary comment ;
Notice of Request To Renew an Approved Information Collection: Specified Risk Materials DOCKET NUMBER Docket No. FSIS-2022-0027 Singeltary Submission
Singeltary further comments in attachment;
Specified Risk Materials DOCKET NUMBER Docket No. FSIS-2022-0027 Singeltary Submission Attachment
https://downloads.regulations.gov/FSIS-2022-0027-0002/attachment_1.pdf
please see further ;
REFERENCE MATERIALS
snip...
Terry S. Singeltary Sr.
Sunday, March 20, 2016
Docket No. FDA-2003-D-0432 (formerly 03D-0186) Use of Material from Deer and Elk in Animal Feed Singeltary Submission
Singeltary previous submission to DOCKET-- 03D-0186 -- FDA Issues Draft Guidance on Use of Material From Deer and Elk in Animal Feed; Availability
DOCKET-- 03D-0186 -- FDA Issues Draft Guidance on Use of Material From Deer and Elk in Animal Feed; Availability Fri, 16 May 2003 11:47:37 0500 EMC 1 Terry S. Singeltary Sr. Vol #: 1
Date: Fri, 16 May 2003 11:47:37 0500 EMC 1 Terry S. Singeltary Sr. Vol #: 1
2003D-0186 Guidance for Industry: Use of Material From Deer and Elk In Animal Feed
EMC 1 Terry S. Singeltary Sr. Vol #: 1
PLEASE SOURCE REFERENCES UPLOADED FILES AT BOTTOM, SEE ATTACHMENT...
SEE SINGELTARY ATTACHMENT;
MONDAY, DECEMBER 12, 2022
Idiopathic Brainstem Neuronal Chromatolysis (IBNC) TSE Prion disease
TUESDAY, DECEMBER 13, 2022
Scrapie and CJD, Suspect Symptoms, Like Lambs To the Slaughter, a review 2022
THURSDAY, NOVEMBER 10, 2022
Annual Report of the Scientific Network on BSE‐TSE 2022
SUNDAY, OCTOBER 30, 2022 Why is USDA "only" testing 25,000 samples a year?
https://bovineprp.blogspot.com/2022/10/why-is-usda-only-testing-25000-samples.htmlSUNDAY, DECEMBER 11, 2022
SEAC SPONGIFORM ENCEPHALOPATHY ADVISORY COMMITTEE Minutes of the 99th meeting held on 14th December 2007 Singeltary Submission
FRIDAY, DECEMBER 02, 2022
Creutzfeldt Jacob Disease CJD TSE Prion December 2022 Annual Update
Terry S. Singeltary Sr.
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