Monday, July 28, 2014

Mitigating the Risk of Transmission and Environmental Contamination of Transmissible Spongiform Encephalopathies 2013 Annual Report

Research Project: Mitigating the Risk of Transmission and Environmental Contamination of Transmissible Spongiform Encephalopathies Location: Animal Diseases Research


2013 Annual Report


1a.Objectives (from AD-416): Objective 1: Determine whether goats are a transmission reservoir for ovine scrapie by developing and validating diagnostic methods for detecting goat scrapie. Determine the genetic predisposition and transmission route(s) of goat scrapie.


Subobjective 1.1: Improve eradication efforts by developing improved methods for antemortem scrapie diagnosis.


Subobjective 1.2: Determine if placenta and milk from goats are potential sources of scrapie to sheep.


Objective 2: Develop methods to mitigate infectivity of soil-associated prions by screening soil microbes for potential candidates for bioremediation.


1b.Approach (from AD-416): Scrapie is a complex and rare disorder affecting outbred farm animals held under a wide variety of husbandry conditions and exposed to an agent for which the transmissible and pathogenic events remain largely unknown (125). The work described in the research plan is an extension of the previous highly productive studies by this research group, addressing the need for implementation of federal regulations based on the best available science, often in the face of relatively small sample numbers in the natural host. The work includes development of specific management and diagnostic tools and is presented as an integrated series of research objectives. This approach was selected over a hypothesis based approach. After consulting Glass and Hall (46), the group determined that the work presented in the following plan was best represented by goal statements rather than hypotheses because the work increases the density of data necessary for progress (103) and for support of current and proposed federal regulations. This project addresses only scrapie, the TSE of sheep and goats. Chronic wasting disease (CWD) is the TSE of North America cervids (deer and elk). No live animal work with CWD is included in this project plan since CWD is not endemic in Washington State, the disease appears to be highly communicable, the modes of transmission are unknown, and we do not have suitable biocontainment facilities to conduct CWD studies in large animals.


3.Progress Report: Progress was made on characterizing caprine scrapie in sheep and goats through continued monitoring and data collection from natural and experimental cases of sheep-origin or goat-origin scrapie. Tissues were banked from postmortem examinations on animals developing terminal disease for use in an origin/strain discrimination study. A study demonstrating the infectious nature of the goat placenta to goats and sheep through oral exposure at birth contributed to a better understanding of typical modes of transmission in mixed sheep and goat operations. We completed second year sampling and analyses to determine the impact of lactation cycle, local inflammation and small ruminant lentivirus co-infection on prions in milk, and made progress adapting a seeded-conversion assay for detecting misfolded prion proteins in milk. Continued collection and analysis of rectal tissue samples from sheep and goats of various PrP genotypes will contribute towards improved diagnostic testing of goats, and the results should be useful for optimization of biopsy sampling and processing procedures for use with standard diagnostic assays. Progress on development of blood testing includes using bioassay in sheep and transgenic mice to determine which blood fractions harbor scrapie prions. Certain blood fractions could be detected by bioassay even when derived from the smaller blood sample volumes routinely collected under field conditions. Accumulation profiles of disease-associated prion protein were compared between hemal nodes and lymph nodes of sheep and goats to demonstrate the distribution in the circulatory system. As an alternative to bioassay we have begun developing additional transgenic cell lines that express species-specific prion proteins and are permissive to animal-derived as well as culture-adapted scrapie strains. Other work included determination that only one strain is evident in the widely studied Stetsonville isolate of transmissible mink encephalopathy and that TME in mink is a relevant model of extrinsic prion infections of man. The potential for genetic control of chronic wasting disease in North American cervids was reviewed and the diagnostic accuracy of rectal biopsy for detecting chronic wasting disease in captive North American white tailed deer was determined. We began development of methods to mitigate prion contamination of soil using a primary cell line of sheep microglia. Further testing of this cell line found it to be robustly permissive to geographically divergent authentic sources of scrapie prions. In addition, these cells have been adapted successfully to a high throughput format. By developing and testing immortalized ovine and caprine microglial cell lines of relevant prion protein genotypes and working toward expressing and purifying recombinant ovine and caprine prion protein that can be utilized in a cell-free misfolding assay we have contributed to the contingency plan to reduce the use of Tg mice for bioassay. These advances create the real potential to replace the need for bioassay in sorption and mitigation studies in soil with more timely and cost efficient in vitro methods.


4.Accomplishments 1. Detection of scrapie prions in routine blood samples. The presence of infectious scrapie prions in the blood of some sheep indicates the possibility of developing a blood-based diagnostic test. However, initial studies utilized blood volumes much larger than those routinely collected for diagnostic work. ARS researchers at the Animal Disease Research Unit in Pullman, Washington, therefore determined if infectious prions could be detected in volumes of blood typically collected under field conditions. The results demonstrate that the presence of disease could be determined from these smaller blood samples after isolating and testing the specific cells known to harbor scrapie prions. The ability to utilize blood sample volumes routinely collected under field conditions is an important step toward developing a sensitive blood-based test for scrapie disease.


2. A live-animal test to monitor chronic wasting disease in free-ranging elk herds. Keeping track of chronic wasting disease – a fatal prion disease affecting the brain of deer and elk – is important to managing North America herds. In some areas, the presence of disease in free-ranging herds can be monitored by testing tissues collected from hunter-harvested animals, but such surveillance is generally not possible in herds managed within national parks. Thus a live-animal test was needed. In this project, ARS researchers at the Animal Disease Research Unit in Pullman, Washington, teamed up with colleagues in the National Park Service and at Colorado State University to determine if biopsy collection of small amounts of tissue from the rectal mucosa could be used to fill this surveillance gap in free-ranging elk. Despite a limited ability to detect disease during the early stage of infection in individual elk, the live-animal test did prove useful in estimating the minimum prevalence of disease in the herd. Thus, the live animal testing procedure should be a useful tool for researchers and managers of free-ranging elk herds.


3. The placenta of goats with scrapie is infectious to other goats and sheep. Scrapie disease has long been known to affect both sheep and goats but very little is known about the disease specifically in goats. Control of disease depends on accurate knowledge of the disease process including identifying the major sources for transmission of disease. In infected sheep, for example, the placenta shed at lambing is known to be both a major transmission source to other sheep and goats as well as a source of prions contaminating the environment. Though this information about the disease in sheep plays a key role in conducting disease investigations, the role of the placenta shed from goats with scrapie is not known. In this project, ARS researchers at the Animal Disease Research Unit in Pullman, Washington, determined that, even though the pathology associated with scrapie disease in the placenta of goats is significantly less than that observed in sheep, the goat’s placenta was an infectious source of scrapie prions to goats and sheep exposed by a natural route. Thus, like for sheep, the placenta and postpartum period of goats must be considered transmission risks for other susceptible small ruminants and environmental contamination.


4. Pathologic prion protein accumulates in a unique blood-filtering organ of small ruminants. The discovery that the pathologic form of the prion protein can be detected in in the blood of scrapie infected small ruminants suggests development of a blood-based test for scrapie may be possible. But little more is known about the pathologic prion protein in the blood except that it is present very limited amounts. To learn more its structure and circulation in the body, ARS researchers at the Animal Disease Research Unit in Pullman, Washington, studied its accumulation in small blood-filtering organs known as hemal nodes, an anatomic feature unique to ruminants. The results demonstrate that the pathologic prion protein in the hemal nodes of sheep and goats is of similar structure and accumulates at a similar rate and in similar immune cells as that accumulating in lymph nodes. These results support the conclusion that the pathologic prion protein circulating in the blood of sheep and goats may be a suitable target for development of a blood-based diagnostic test.


Review Publications Monello, R., Powers, J.G., Hobbs, N.T., Spraker, T.R., O'Rourke, K., Wild, M.A. 2013. Efficacy of antemortem rectal biopsies to diagnose and estimate prevalence of chronic wasting disease in free-ranging elk (Cervus elaphus nelsoni). Journal of Wildlife Diseases. DOI: 10.7589/WD.2011-12-362.


 Dassanayake, R.P., Truscott, T.C., Özyigit, Ö.M., Zhuang, D., Schneider, D.A., O'Rourke, K.I. 2013. Accumulation of PrP-Sc in hemal nodes of naturally and experimentally scrapie-infected sheep. BioMed Central (BMC) Veterinary Research. doi:10.1186/1746-6148-9-82.


Schneider, D.A., Harrington, R., Zhuang, D., Yan, H., Truscott, T.C., Dassanayake, R., Orourke, K.I. 2012. Disease-associated prion protein in neural and lymphoid tissues of mink (Mustela vison) inoculated with transmissible mink encephalopathy. Journal of Comparative Pathology. 147:508-521.


Thomsen, B.V., Schneider, D.A., O'Rourke, K., Gidlewski, T., Mclane, J., Allen, R.W., Mcisaac, A.A., Mitchell, G.B., Keane, D.P., Spraker, T., Balachandran, A. 2012. Diagnostic accuracy of rectal mucosa biopsy testing for chronic wasting disease within white-tailed deer (Odocoileus virginianus) herds in North America:Effects of age,sex,polymorphism at PRNP codon 96,and disease progression. J Vet Diagn Invest. 24(5):878-87.


Stanton, J.B., Schneider, D.A., Dinkel, K.D., Balmer, B.F., Baszler, T.V., Mathison, B.A., Boykin, D.W. 2012. Discovery of a novel, monocationic, small-molecule inhibitor of scrapie prion accumulation in cultured sheep microglia and rov cells PLoS one. Antimicrobial Agents and Chemotherapy. PloSone 7(11):e51173.



Research Project: Transmission, Differentiation, and Pathobiology of Transmissible Spongiform Encephalopathies 2013 Annual Report


1a.Objectives (from AD-416): 1. Investigate the pathobiology of atypical transmissible spongiform encephalopathies (TSEs) in natural hosts. A. Investigate the pathobiology of atypical scrapie. B. Investigate the pathobiology of atypical bovine spongiform encephalopathy (BSE). 2. Investigate the horizontal transmission of TSEs. A. Assess the horizontal transmission of sheep scrapie in the absence of lambing. B. Determine routes of transmission in chronic wasting disease (CWD) infected premises. C. Assess oral transmission of CWD in reindeer. 3. Investigate determinants of CWD persistence. A. Determine CWD host range using natural routes of transmission. B. Investigate the pathobiology of CWD.


1b.Approach (from AD-416): The studies will focus on three animal transmissible spongiform encephalopathy (TSE) agents found in the United States: bovine spongiform encephalopathy (BSE); scrapie of sheep and goats; and chronic wasting disease (CWD) of deer, elk, and moose. The research will address sites of accumulation, routes of infection, environmental persistence, and ante mortem diagnostics with an emphasis on controlled conditions and natural routes of infection. Techniques used will include clinical exams, histopathology, immunohistochemistry and biochemical analysis of proteins. The enhanced knowledge gained from this work will help mitigate the potential for unrecognized epidemic expansions of these diseases in populations of animals that could either directly or indirectly affect food animals.


3.Progress Report: Research efforts directed toward meeting objective 1 of our project plan, Investigate the pathobiology of atypical TSEs in natural hosts, include work in previous years starting with the inoculation of animals for studies designed to address the pathobiology of atypical scrapie, atypical bovine spongiform encephalopathy (BSE), as well as a genetic version of BSE. Animals inoculated with atypical scrapie have not yet developed disease. Atypical BSE animals have developed disease and evaluation of the samples is currently underway. Animals inoculated with a genetic version of BSE have developed disease and the manuscript has been published (2012). In addition, we have investigated the possibility that atypical scrapie was present earlier than previously detected in the national flock by analyzing archived field isolates using methods that were unavailable at the time of original diagnosis. Sample quality was sufficiently degraded that modern methods were not suitable for evaluation. In research pertaining to objective 2, Investigate the horizontal transmission of TSEs, we have initiated a study to determine if cohousing non-lambing scrapie inoculated sheep is sufficient to transmit scrapie to neonatal lambs. At this time, scrapie inoculated sheep are being co-housed with pregnant ewes.


4.Accomplishments 1. Partial effectiveness of a common cleaning product to disinfect prions. Diseases such as mad cow disease, scrapie, and chronic wasting disease of animals and Creutzfeldt-Jakob disease in humans are collectively known as transmissible spongiform encephalopathies. They are caused by an infectious protein, rather than a virus or bacterium, which is notoriously difficult to inactivate. Decontaminating surfaces or instruments is essential to prevent potential spread of these diseases. ARS researchers in Ames, IA tested whether sodium percarbonate, a chemical similar to hydrogen peroxide and contained in such products as OxiClean, in combination with a detergent (sodium dodecyl sulfate) could effectively disinfect surfaces contaminated with prions. The treatment rendered the prions sensitive to degradation with protein-destroying enzymes, but only limited reduction in infectivity was observed. Thus, this treatment alone is not sufficient to inactivate prions, but could be used in combination with other treatments to decontaminate prion contaminated areas.


2. Determined the likelihood of transmission of chronic wasting disease (CWD) to cattle. CWD is a prion disease, similar to mad cow disease in cattle, which naturally occurs in cervid animals, such as deer and elk, in the United States. Prion diseases are caused by an infectious protein, rather than a virus or bacterium. The prion that causes CWD is contagious through animal contact and through the environment, so cattle could be exposed to the agent of CWD through contact with infected farmed or free-ranging cervids, or exposure to contaminated premises. The purpose of this study by ARS researchers in Ames, IA, was to determine whether cattle could become infected with CWD if it was inoculated directly into the brain, a much more rigorous route of infection. Only 2 of the 14 cattle inoculated this way developed disease. Additional studies are required to assess the potential for cattle to develop CWD through a more natural route of exposure, but a low rate of transmission following inoculation into the brain suggests a low risk of transmission through contact with infected animals or a contaminated environment. However, if this ever occurred it was also determined that currently used diagnostic techniques would detect and differentiate CWD from other prion diseases in cattle, such as mad cow disease.


3. Produced a genetically unique calf for studies of inherited bovine spongiform encephalopathy (BSE). Diseases such as BSE in cattle, more commonly known as mad cow disease, and Creutzfeldt-Jakob disease (CJD) in humans are caused by an infectious protein called a prion, rather than a virus or bacterium. A usually non-harmful form of this prion protein is normally produced in animals and humans, however, animals and humans born with a certain genetic form of this protein are more susceptible to developing an inherited form of these diseases. Proteins are made up of subunits called amino acids, and these amino acids are encoded by the DNA that makes up genes. In humans, when the amino acid lysine is encoded by the DNA in a certain position of the prion gene, instead of the commonly occurring amino acid glutamate, a genetic form of CJD can occur. Recently, occurrence of this same amino acid in a cow was associated with a case of BSE. Before this case of BSE, the lysine for glutamate amino acid substitution had not been identified in cattle. Researchers at ARS, National Animal Disease Center, Ames, Iowa, in collaboration with Iowa State University were able to produce a calf with this lysine for glutamate amino acid substitution. This animal and its progeny will be used to determine if a genetic form of BSE occurs and for further study of BSE in cattle. It is important to distinguish whether cases of BSE that arise in the United States are due to an infectious or genetic cause.


Review Publications Greenlee, J.J., Nicholson, E.M., Smith, J.D., Kunkle, R.A., Hamir, A.N. 2012. Susceptibility of cattle to the agent of chronic wasting disease from elk after intracranial inoculation. Journal of Veterinary Diagnostic Investigation. 24(6):1087-1093.


 Lennon, C.W., Ross, W., Martin-Tumasz, S., Toulokhonov, I., Vrentas, C.E., Rutherford, S.T., Lee, J.H., Butcher, S.E., Gourse, R.L. 2012. Direct interactions between the coiled-coil tip of DksA and the trigger loop of RNA polymerase mediate transcriptional regulation. Genes and Development. 26(23):2634-2646.


Di Bari, M.A., Nonno, R., Castilla, J., D'Agostino, C., Pirisinu, L., Riccardi, G., Conte, M., Richt, J., Kunkle, R., Langeveld, J., Vaccari, G., Agrimi, U. 2013. Chronic wasting disease in bank voles: characterisation of the shortest incubation time model for prion diseases. PLoS Pathogens. 9(3):e1003219.


Smith, J.D., Nicholson, E.M., Foster, G.H., Greenlee, J.J. 2013. Exposure of RML scrapie agent to a sodium percarbonate-based product and sodium dodecyl sulfate renders PrPSc protease sensitive but does not eliminate infectivity. BioMed Central (BMC) Veterinary Research. 9:8.


Bose, S., Schonenbrucher, H., Richt, J.A., Casey, T., Rasmussen, M.A., Kehrli, Jr., M.E., Petrich, J.W. 2013. Fluorescence spectroscopy of the retina from scrapie-infected mice. Photochemistry and Photobiology. 89(4):864-868. Available:


Comoy, E.E., Mikol, J., Ruchoux, M., Durand, V., Luccantoni-Freire, S., Dehen, C., Correia, E., Casalone, C., Richt, J.A., Greenlee, J.J., Torres, J.M., Brown, P., Deslys, J. 2013. Evaluation of the zoonotic potential of transmissible mink encephalopathy. Pathogens. 2:(3)520-532.


Franz, M., Eiden, M., Balkema-Buschmann, A., Greenlee, J., Schatzl, H., Fast, C., Richt, J., Hildebrandt, J.P., Groschup, M.H. 2012. Detection of PrP(Sc) in peripheral tissues of clinically affected cattle after oral challenge with bovine spongiform encephalopathy. Journal of General Virology. 93(Pt12):2740-2748.


Smith, J.D., Nicholson, E.M., Greenlee, J.J. 2013. Evaluation of a combinatorial approach to prion inactivation using an oxidizing agent, SDS, and proteinase K. BioMed Central (BMC) Veterinary Research. 9:151.


Vrentas, C.E., Greenlee, J.J., Baron, T., Caramelli, M., Czub, S., Nicholson, E.M. 2013. Stability properties of PrPSc from cattle with experimental transmissible spongiform encephalopathies: use of a rapid whole homogenate, protease-free assay. BMC Veterinary Research. 9:167.





1a.Objectives (from AD-416): We will develop highly sensitive diagnostic tests to detect transmissible spongiform encephalopathy (TSE) in livestock and wildlife animal species prior to the onset of clinical disease. We will also develop tests to confirm the presence or absence of TSE disease agents in ingredients of animal origin and decontaminated environments.


1b.Approach (from AD-416): The threat of BSE continues to affect export economics for US meat. Meanwhile scrapie continues to influence sheep profits and herd biosecurity, and CWD is spreading throughout North America. Thus U.S. animal industry stakeholders have identified detection of the TSE infectious agent (prions) as a priority biosecurity research issue essential for prevention of TSE diseases. We will build on our previous successes using mass spectrometry (MS) for high-sensitivity and specificity in detection of PrPsc as a marker for TSE infectivity in blood using a hamster scrapie model. We will also develop a novel PrP-null mouse strain and related myeloma cell culture system for production of monoclonal antibodies (MAb), which may be specific for PrPsc. We will then choose MS or MAb and validate our novel diagnostic for preclinical diagnosis of scrapie in sheep blood. Whereas MS and MAb methods rely on dissolved samples, contamination of agricultural products and environmental surfaces is associated with solid samples. So we will produce a cell culture based assay for TSE infectivity that is surface-adsorbed. After using the relatively convenient hamster model for early development, we will validate this technology for detection of scrapie in sheep brain on meat-and-bone meal and stainless steel. All work with infectious material will take place within our APHIS-approved BL2 biocontainment facilities labs at the Western Regional Research Center (WRRC), while mass spectrometry will be performed on non-infectious material under BL1 containment. Replacing 5325-32000-007-00D (3/19/2008).


3.Progress Report: This is the final report for this project which terminated in April of 2013 and was replaced with 5325-32000-009-00D, "Innumodiagnostics to Detect Prions and Other Important Animal Pathogens". Substantial results were realized over the project period. New analytical techniques and materials were developed for detection of infectious prions with improved results compared to conventional methods. A prion gene knockout mouse was developed and immunized with purified infectious prions resulting in the generation of sensitive anti-prion monoclonal antibodies. The impact of this research was the production of distinct anti-prion monoclonal antibodies that bind novel prion epitopes for use in the development of commercial immunoassays. The binding properties of these monoclonal antibodies were extensively characterized and their utility in a variety of immunoassay formats were demonstrated. A patent has been filed for this technology and three of these antibodies have been transferred to academic and government partners including the National Veterinary Services Laboratory (NVSL). License negotiations are currently ongoing with a commercial partner. The slow accumulation of prions and limited concentration in target tissues confounds detection. Sample preparative methods were defined that significantly enrich prions in tissue extract using isolation with lipid raft. The resulting increase in prion sample concentration results in improved immunoassay detection. The impact of this research is a method to detect prions from infected asymptomatic animal tissue. A patent has been filed and papers published on this methodology. Enzyme-linked immunosorbent assays (ELSIA) are frequently used for the detection of prions from target tissues. A chaotropic agent was effectively used in a modified immunohistochemical assay and an ELISA format to increase the sensitivity of prion detection. This technical modification resulted in a in a seminal paper with a broad impact on antigen detection by immunoassay. Infectious prion strains reflect alternate abnormal tertiary protein structure that stably propagate and produce distinct disease pathology. Analytical mass spectrometry has been used to elucidate structural differences in prion protein strains and quantitate the concentration of prion in a given sample. The impact of this research has been to further the understanding of prion strain differences and identify potential anti-prion binding epitopes for selective modification. This knowledge can be used to build immunoassays capable of discriminating prion strains. The overall impact of these accomplishments is improved prion immunoassay technologies for use in ongoing prion research and surveillance efforts with commercial application.


4.Accomplishments 1. Improved anti-prion monoclonal antibodies for prion detection. Using purified prions from infected animals, ARS scientist in Albany, California, have generated anti-prion DRM monoclonal antibodies from immunized prion knockout mice. This approach effectively overcame the poor immune response to prion immunization in normal mice and our research efforts defined an improved method to yield a purified infectious prion immunogen used to generate high-titer antibody response in a new mouse model. A novel differential screening methods based on preferential binding to infectious prion antigen was used in the identification and selection of hybridoma cell lines producing anti-prion monoclonal antibodies. The properties of these antibodies have been extensively characterized, their binding epitopes defined and their utility in a number of immunoassay formats validated. These antibodies have been transferred to both academic and national laboratories for use in prion immunoassays. A patent has been filed (US Patent # 13/157,216) and a manuscript has been published describing these anti-prion monoclonal antibodies. Negotiations with a major international biotechnology company are in progress to issue a license for commercialization of this technology.


2. Novel peptide sequences for specific binding to infectious prions. Using a proteomic peptide array consisting of peptides corresponding to functional domains of endogenous proteins involved in protein-protein interaction ARS scientists in Albany, California, have identified specific amino acid sequences that bind only the infectious form of prion proteins. Using these peptides as part of a modified immunoassay for the selective capture of infectious prion protein from animal tissues, ARS scientists have demonstrated the ability to discriminate prion infected from uninfected samples using conventional anti-prion antibodies for detection. This approach allows for the direct capture and detection of infectious prions without the interference of the normal endogenous prion protein that confounds traditional assay techniques. The identification of several prion specific binding peptides represents important components of new peptide-based detection assays and important targets to facilitate research toward understanding the mechanism of prion conformational conversion. A patent has been issued (US Patent #12/571,275; Aug 2013) for this technology.


3. Novel methods to isolate prions in non-diagnostic tissues to improve detection of scrapie and chronic wasting disease (CWD). ARS scientists in Albany, California, along with an international consortium of scientists have developed an efficient means of isolating and detecting prions in non-diagnostic portions of brain tissue from sheep naturally infected with scrapie and elk infected with CWD. Conventional methods rely on accumulation of infectious prions in a brainstem tissue called the obex for diagnosis of disease. Other areas of the brain tend to have less prion and consequently have been excluded from sampling and analysis. The small size and lipid rich aspect of the obex has limited the application of prion detection assays. ARS scientists have devised new methods to enhance prion detection from brain regions that have been previously ignored for use in confirmatory diagnostic testing. These results have been published in the scientific literature and provide alternate techniques to augment current prion testing protocols.


Review Publications Stanker, L.H., Scotcher, M.C., Lin, A.V., Mcgarvey, J.A., Prusiner, S., Hnasko, R.M. 2012. Novel epitopes identified by Anti-PrP monoclonal antibodies produced following immunization of Prnp0/0 Balb/cJ mice with purified scrapie prions. Hybridoma. 31(5):312-324. doi:10.1089/hyb.2012.0022.


 Vasquez-Fernandez, E., Alonso, J., Pastrana, M.A., Ramos, A., Stitz, L., Vidal, E., Dynin, I.A., Petsch, B., Silva, C.J., Requena, J.R. 2012. Structural organization of mammalian prions as probed by limited proteolysis. PLoS One. 7(11):e50111. doi:10.1371/journal.pone.0050111.


Silva, C.J., Dynin, I.A., Erickson-Beltran, M.L., Requena, J.R., Balachandran, A., Onisko, B.C., Hui, C., Carter, J.M. 2013. Using mass spectrometry to detect prions and oxidized prions in scrapie-infected sheep and CWD-infected elk. Biochemistry. 52:2139-2147. doi:10.1021/bi3016795.







item Greenlee, Justin


Submitted to: American Veterinary Medical Association Abstract Publication Type: Abstract Only Publication Acceptance Date: May 1, 2013 Publication Date: July 19, 2013 Citation: Greenlee, J.J. 2013. Atypical BSE: role of the E211K prion polymorphism. 2013 American Veterinary Medical Association (AVMA) Annual Convention, July 19-23, 2013, Chicago, Illinois. Paper No. 13920. p. 45.


Technical Abstract: Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases that naturally affect several species including human beings. These chronic diseases are associated with the accumulation of a protease-resistant, disease-associated isoform of the prion protein (PrPSc) in the central nervous system and other tissues, depending on the species affected. In humans, TSEs can be acquired through exposure to infectious material, inherited as germline polymorphisms in the prion gene (prnp), or occur spontaneously. Bovine spongiform encephalopathy (BSE) or mad cow disease cases can be subclassified into at least 3 distinct disease forms with the predominant form known as classical BSE and the others collectively referred to as atypical BSE. Atypical BSE is further subdivided into H-type and L-type cases that are distinct from classical BSE and from each other on the basis of their unique molecular profiles. Both atypical BSE subtypes are believed to have arisen spontaneously and today researchers are investigating atypical BSE as a possible origin of classical BSE that was a feed-borne epidemic primarily affecting cattle in Europe where BSE-contaminated animal protein sources derived from central nervous system tissues were previously fed to ruminants. Several hypotheses have been proposed to explain atypical BSE cases. At the forefront of this discussion is the possibility that both H-type and L-type BSE may be spontaneous diseases in cattle. Support for atypical BSE occurring spontaneously is drawn from parallels to sporadic prion disease in humans, specifically, occurrence in older hosts and a comparable low incidence rate. Furthermore, atypical BSE occurs as isolated, sporadic cases in contrast to the clustering of cases observed for feedborne classical BSE. Recognition of a spontaneous prion disease in cattle, coupled with evidence indicating atypical BSE can convert to classical BSE upon serial passage in mice, has broad implications for our understanding of the origins of the classical form of the disease. In 2006 a critical discovery was made when one case of H-type BSE was associated with a heritable mutation in the prion protein gene referred to as E211K. This case was diagnosed in the U.S. and led to the identification of a new prion protein (prnp) allele, K211, that is associated with H-type BSE and is heritable. This presentation will present data demonstrating a rapid onset of disease in an animal with the E211K mutation following experimental inoculation with H-type BSE from the original E211K H-type BSE case. Interestingly, disease associated prion protein was widespread in neural tissues this animal, and antemortem retinal thinning and functional deficits of the visual system were observed prior to the onset of clinical disease. The existence of genetic forms of BSE offers new explanations for the potential origins of BSE.



Research Project: TRANSMISSION, DIFFERENTIATION, AND PATHOBIOLOGY OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES Title: Evaluation of the zoonotic potential of transmissible mink encephalopathy




item Comoy, Emmanuel - item Mikol, Jacqueline - item Ruchoux, Marie-Madeleine - item Durand, Valerie - item Luccantoni-Freire, Sophie - item Dehen, Capucine - item Correia, Evelyne - item Casalone, Cristina - item Richt, Juergen item Greenlee, Justin item Torres, Juan Maria - item Brown, Paul - item Deslys, Jean-Philippe -


Submitted to: Pathogens Publication Type: Peer Reviewed Journal Publication Acceptance Date: July 30, 2013 Publication Date: July 30, 2013 Citation: Comoy, E.E., Mikol, J., Ruchoux, M., Durand, V., Luccantoni-Freire, S., Dehen, C., Correia, E., Casalone, C., Richt, J.A., Greenlee, J.J., Torres, J.M., Brown, P., Deslys, J. 2013. Evaluation of the zoonotic potential of transmissible mink encephalopathy. Pathogens. 2:(3)520-532.


Interpretive Summary: Cases of bovine spongiform encephalopathy (BSE) or mad cow disease can be subclassified into at least 3 distinct disease forms with the predominate form known as classical BSE and the others collectively referred to as atypical BSE. Atypical BSE can be further subdivided into H-type and L-type cases that are distinct from classical BSE and from each other. Both of the atypical BSE subtypes are believed to occur spontaneously, whereas classical BSE is spread through feeding contaminated meat and bone meal to cattle. Transmissible mink encephalopathy (TME) is another prion disease that transmits to cattle and show similarities to L-type BSE when subjected to laboratory testing. The purpose of this study was to use non-human primates (cynomologous macaque) and transgenic mice expressing the human prion protein to determine if TME could represent a potential risk to human health. TME from two sources (cattle and raccoons) was able to infect non-human primates and transgenic mice after exposure by the intracranial route. This result suggest that humans may be able to replicate TME prions after an exposure that allows infectious material access to brain tissue. At this time, it is unknown whether non-human primates or transgenic mice would be susceptible to TME prions after oral exposure. The results obtained in these animal models were similar to those obtained for L-type BSE. Although rare, the existence of TME and that it transmits to cattle, non-human primates, and transgenic mice suggest that feed bans preventing the feeding of mammalian tissues to cattle should stay in place and that regular prion surveillance during the slaughter should remain in place. Parties with interest in the cattle and beef industries and regulatory officials responsible for safe feeding practices of cattle will be interested in this work. Technical Abstract: Successful transmission of Transmissible Mink Encephalopathy (TME) to cattle supports the bovine hypothesis to the still controversial origin of TME outbreaks. Human and primate susceptibility to classical Bovine Spongiform Encephalopathy (c-BSE) and the transmissibility of L-type BSE to macaques assume a low cattle-to-primate species barrier: we therefore evaluated the zoonotic potential of cattle-adapted TME. In less than two years, this strain induced in cynomolgus macaques a neurological disease similar to L-BSE and distinct from c-BSE. TME derived from another donor species (raccoon) induced a similar disease with shorter incubation periods. L-BSE and cattle-adapted TME were also transmissible to transgenic mice expressing human PrP. Interestingly, secondary transmissions to transgenic mice expressing bovine PrP showed the maintenance of prion strain features for the three tested bovine prion strains (cattle TME, c-BSE and L-BSE) regardless of intermediate host. Thus, TME is the third animal prion strain transmissible to both macaques and humanized transgenic mice, suggesting zoonotic potentials that should be considered in the risk analysis of animal prion diseases for human health. Moreover, the similarities between TME and L-BSE are highly suggestive of a link between those strains, and of the presence of L-BSE decades prior to its identification in USA and Europe.



TME in mink was documented in the early 1960s. it was first thought that the TME out break was from scrapie infected sheep, until a investigation was done on feed practices at these mink facilities, and it was later found that the mink had been fed 95%+ dead stock downer cows. and later, the Late Richard Marsh tried to warn the feds of the pending mad cow debacle. they refused to listen. ...


some interesting reading on pages 26 to 33





TME originates from feeding mink, scrapie infected materials...



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.




The rancher was a ''dead stock'' feeder using mostly (>95%) downer or dead dairy cattle...





another nice bit of history on cwd can be found here ;



I know scarpie was first documented around 1947 here in the USA, how long it was here before that, I don’t know, some say over 250 years.



Saturday, June 25, 2011


Transmissibility of BSE-L and Cattle-Adapted TME Prion Strain to Cynomolgus Macaque


"BSE-L in North America may have existed for decades"



Sunday, December 15, 2013





Thursday, July 24, 2014


Protocol for further laboratory investigations into the distribution of infectivity of Atypical BSE SCIENTIFIC REPORT OF EFSA



Discussion: The C, L and H type BSE cases in Canada exhibit molecular characteristics similar to those described for classical and atypical BSE cases from Europe and Japan. *** This supports the theory that the importation of BSE contaminated feedstuff is the source of C-type BSE in Canada. *** It also suggests a similar cause or source for atypical BSE in these countries. ***


see page 176 of 201 pages...tss



*** Singeltary reply ; Molecular, Biochemical and Genetic Characteristics of BSE in Canada Singeltary reply ;



Saturday, August 14, 2010


BSE Case Associated with Prion Protein Gene Mutation (g-h-BSEalabama) and VPSPr PRIONPATHY


(see mad cow feed in COMMERCE IN ALABAMA...TSS)









Conclusions. To our knowledge, this is the first established experimental model of CWD in TgSB3985. We found evidence for co-existence or divergence of two CWD strains adapted to Tga20 mice and their replication in TgSB3985 mice. Finally, we observed phenotypic differences between cervid-derived CWD and CWD/Tg20 strains upon propagation in TgSB3985 mice. Further studies are underway to characterize these strains.


We conclude that TSE infectivity is likely to survive burial for long time periods with minimal loss of infectivity and limited movement from the original burial site. However PMCA results have shown that there is the potential for rainwater to elute TSE related material from soil which could lead to the contamination of a wider area. These experiments reinforce the importance of risk assessment when disposing of TSE risk materials.


The results show that even highly diluted PrPSc can bind efficiently to polypropylene, stainless steel, glass, wood and stone and propagate the conversion of normal prion protein. For in vivo experiments, hamsters were ic injected with implants incubated in 1% 263K-infected brain homogenate. Hamsters, inoculated with 263K-contaminated implants of all groups, developed typical signs of prion disease, whereas control animals inoculated with non-contaminated materials did not.


Our data establish that meadow voles are permissive to CWD via peripheral exposure route, suggesting they could serve as an environmental reservoir for CWD. Additionally, our data are consistent with the hypothesis that at least two strains of CWD circulate in naturally-infected cervid populations and provide evidence that meadow voles are a useful tool for CWD strain typing.


Conclusion. CWD prions are shed in saliva and urine of infected deer as early as 3 months post infection and throughout the subsequent >1.5 year course of infection. In current work we are examining the relationship of prionemia to excretion and the impact of excreted prion binding to surfaces and particulates in the environment.


Conclusion. CWD prions (as inferred by prion seeding activity by RT-QuIC) are shed in urine of infected deer as early as 6 months post inoculation and throughout the subsequent disease course. Further studies are in progress refining the real-time urinary prion assay sensitivity and we are examining more closely the excretion time frame, magnitude, and sample variables in relationship to inoculation route and prionemia in naturally and experimentally CWD-infected cervids.


Conclusions. Our results suggested that the odds of infection for CWD is likely controlled by areas that congregate deer thus increasing direct transmission (deer-to-deer interactions) or indirect transmission (deer-to-environment) by sharing or depositing infectious prion proteins in these preferred habitats. Epidemiology of CWD in the eastern U.S. is likely controlled by separate factors than found in the Midwestern and endemic areas for CWD and can assist in performing more efficient surveillance efforts for the region.


Conclusions. During the pre-symptomatic stage of CWD infection and throughout the course of disease deer may be shedding multiple LD50 doses per day in their saliva. CWD prion shedding through saliva and excreta may account for the unprecedented spread of this prion disease in nature.


see full text and more ;


Monday, June 23, 2014





Thursday, July 03, 2014


*** How Chronic Wasting Disease is affecting deer population and what’s the risk to humans and pets?



Tuesday, July 01, 2014





***P.170: Potential detection of oral transmission of H type atypical BSE in cattle using in vitro conversion


Sandor Dudas, John G Gray, Renee Clark, and Stefanie Czub Canadian Food Inspection Agency; Lethbridge, AB Canada


Keywords: Atypical BSE, oral transmission, RT-QuIC


The detection of bovine spongiform encephalopathy (BSE) has had a significant negative impact on the cattle industry worldwide. In response, governments took actions to prevent transmission and additional threats to animal health and food safety. While these measures seem to be effective for controlling classical BSE, the more recently discovered atypical BSE has presented a new challenge. To generate data for risk assessment and control measures, we have challenged cattle orally with atypical BSE to determine transmissibility and mis-folded prion (PrPSc) tissue distribution. Upon presentation of clinical symptoms, animals were euthanized and tested for characteristic histopathological changes as well as PrPSc deposition.


The H-type challenged animal displayed vacuolation exclusively in rostral brain areas but the L-type challenged animal showed no evidence thereof. To our surprise, neither of the animals euthanized, which were displaying clinical signs indicative of BSE, showed conclusive mis-folded prion accumulation in the brain or gut using standard molecular or immunohistochemical assays. To confirm presence or absence of prion infectivity, we employed an optimized real-time quaking induced conversion (RT-QuIC) assay developed at the Rocky Mountain Laboratory, Hamilton, USA.


Detection of PrPSc was unsuccessful for brain samples tests from the orally inoculated L type animal using the RT-QuIC. It is possible that these negative results were related to the tissue sampling locations or that type specific optimization is needed to detect PrPSc in this animal. We were however able to consistently detect the presence of mis-folded prions in the brain of the H-type inoculated animal. Considering the negative and inconclusive results with other PrPSc detection methods, positive results using the optimized RT-QuIC suggests the method is extremely sensitive for H-type BSE detection. This may be evidence of the first successful oral transmission of H type atypical BSE in cattle and additional investigation of samples from these animals are ongoing.


P.169: PrPSc distribution in brain areas of a natural German H-type BSE case


Anne Balkema-Buschmann, Grit Priemer, Markus Keller, and Martin H Groschup Friedrich Loeffler Institut, Institute for Novel and Emerging Infectious Diseases; Greifswald, Insel Riems, Germany


Keywords: BSE H-type, brain, muscle


Ten years after the initial description of atypical BSE cases of the H-type and L-type, the distribution of PrPSc in different brain areas and peripheral tissues of natural cases of these BSE forms is still not fully understood. Intracerebral challenge experiments have been performed with both atypical BSE forms in cattle, and the distribution of the abnormal prion protein and infectivity has been analysed in a variety of tissues, confirming the general restriction to the central nervous system as it was already generally acknowledged for classical BSE, but showing a slightly earlier and stronger involvement of the peripheral nervous system and the skeletal muscle. Prion 105


However, data from cattle orally challenged with atypical BSE, which might mimic the natural situation, are not yet available. Unfortunately, for most natural cases of atypical BSE, only the obex region is available for further analysis. The PrPSc distribution in the brains of natural L-type BSE cases in Italy has been described in some detail, but comparably few such data are yet available for natural H-type cases. Here we describe the analysis of different brain areas and muscle samples of a natural H-type BSE case diagnosed in Germany in 2014, and compare these data with those obtained from the respective samples collected from cattle challenged intracerebrally with H-type BSE.


P.159: Transgenic mice overexpressing rabbit prion protein are susceptible to BSE, BASE and scrapie prion strains but resistant to CWD and atypical scrapie


Natalia Fernández-Borges,1 Enric Vidal,2 Belén Pintado,4 Hasier Eraña,1 Montserrat Ordóñez,3 Mercedes Márquez,5 Francesca Chianini,6 Dolors Fondevila,5 Manuel A Sánchez-Martín,7 Olivier Andréoletti,8 Mark P Dagleish,6 Martí Pumarola,5 and Joaquín Castilla1,3 1CIC bioGUNE; Parque tecnológico de Bizkaia; Derio; Bizkaia, Spain; 2Centre de Recerca en Sanitat Animal (CReSA); UAB-IR TA, Campus de la Universitat Autònoma de Barcelona; Bellaterra; Barcelona, Catalonia, Spain; 3IKERBASQUE; Basque Foundation for Science; Bilbao, Bizkaia, Spain; 4Centro Nacional de Biotecnología (CNB), Campus de Cantoblanco; Cantoblanco; Madrid, Spain; 5Department of Animal Medicine and Surgery; Veterinary faculty; Universitat Autònoma de Barcelona (UAB); Bellaterra (Cerdanyola del Vallès); Barcelona, Catalonia, Spain; 6Moredun Research Institute; Bush Loan, Penicuik, Scotland, UK; 7Unidad de Generación de OMGs. S.E.A. Department of Medicine; University of Salamanca; Salamanca, Spain; 8Ecole Nationale du Veterinaire; Service de Pathologie du Bétail; Toulouse, France


Interspecies transmission of prions is a well established phenomenon, both experimentally and in field conditions. Upon passage through new hosts prion strains have proven their capacity to change their properties. It is, in fact, a source of strain diversity which needs to be considered when assessing the potential risks associated with consumption of prion contaminated protein sources.


Rabbits were considered for decades a prion resistant species until proven recently otherwise. To determine the extent of rabbit susceptibility to prions and to assess their effects on the passage of different prion strains through this species, a transgenic mouse model overexpressing rabbit PrPC was developed (TgRab). Intracerebral challenges with prion strains originating from a variety of species including field isolates (SSBP1 scrapie, Nor98-like scrapie, BSE, BASE and CWD), experimental murine strains (ME7 and RML), experimentally obtained strains (sheepBSE) and strains obtained by in vitro crossing of the species barrier using saPMCA (BSE-RabPrPres, SSBP1-RabPrPres and CWD-RabPrPres) have been performed.


Interestingly, on first passage, TgRab were susceptible to the majority of prions tested with the exception of SSBP1 scrapie, CWD and Nor98 scrapie. Furthermore TgRab were capable of propagating strain-specific features such as differences in incubation periods, brain lesion and PrPd deposition profiles and PK resistant western blotting band patterns. Our results confirm previous studies shattering the myth that rabbits are resistant to prion infection and this should be taken into account when choosing protein sources to feed rabbits.


P.168: Evolution of the biological properties of L-BSE after passage in sheep with susceptible and resistant PrP genotypes


Michele A Di Bari, Umberto Agrimi, Claudia D’Agostino, Geraldina Riccardi, Stefano Marcon, Elena Esposito, Paolo Frassanito, Flavio Torriani, Shimon Simson, and Romolo Nonno Istituto Superiore di Sanità (ISS) Department of Veterinary Public Health and Food Safety; Rome, Italy


Background. Cattle L-BSE was efficiently transmitted to sheep with susceptible (QQ171) and resistant (QR171) PrP genotypes. 1 Notably, the PrPSc signature of L-BSE was preserved in QQ171 sheep but not in QR171 sheep.2 Notwithstanding, bioassay in transgenic mice expressing bovine or ovine (ARQ) PrPC showed that L-BSE strain was preserved in both, QQ171 and QR171 sheep-passaged L-BSE.3


Here we studied the biological properties of sheep-passaged L-BSE by bioassay in bank voles and transgenic mice expressing the ovine VRQ PrP (tg338), both characterized by a comparatively low susceptibility to cattle L-BSE.


Material and Methods. Voles and tg338 mice were intracerebrally inoculated with cattle L-BSE and sheep-passaged (QQ171 and QR171) L-BSE isolates. Survival time, lesion profiles, Pet-blot and WB analysis were used for strain typing. Results. Cattle L-BSE transmitted quite inefficiently to tg338 mice, with survival time >400 days post-infection (d.p.i.), while sheep-passaged inocula were much more efficient and all gave terminal disease by ~140 d.p.i. However, after sub-passage all inocula converged to a survival time of ~145 d.p.i.. and showed overlapping pathological phenotypes.


In voles, cattle L-BSE transmitted with very long survival times (~800 d.p.i.) and was accompanied by an upward shift of the PrPSc type. Again, all sheep-passaged L-BSE isolates transmitted much more efficiently, with similar survival times of ~360 d.p.i.. Upon second passage, three different strains were isolated in vole, characterized by distinct pathological phenotypes. This divergence is epitomized by the different survival times of vole-adapted L-BSE strains, which were ~400 d.p.i. for cattle L-BSE, ~130 d.p.i. for QQ171-passaged L-BSE and ~225 d.p.i. for QR171-passaged L-BSE.


Conclusions. These findings, along with previously published data,3 show that the original L-BSE strain was recovered after passage in sheep when bioassay was performed in animal models expressing bovine or ovine PrPC. In contrast, strain changes were observed in both, QQ171- and QR171-passaged L-BSE by bioassay in vole, a species with divergent PrP sequence compared to ruminants. Importantly, QQ171- and QR171-passaged L-BSE were characterised by different PrPSc types and, accordingly, showed different biological properties when transmitted to voles, but not when transmitted to other animal models.


Overall, our work support the hypothesis that prion isolates are likely composed of multiple prion components, emphasizes the role of host PrP polymorphisms on strain selection and mutation, and highlights the risk for new potentially zoonotic strains that could emerge from prion evolution in animal reservoirs.


P.172: BSE exposure risk from bovine intestine and mesentery


Fulvio Barizzone,1 Herbert Budka,2 Christine Fast,3 John N Griffin,4 Giuseppe Ru,5 Pietro Stella1 and Olivier Andréoletti6 1European Food Safety Authority; Parma, Italy; 2Institute of Neuropathology; University Hospital Zurich; Zurich, Switzerland; 3Friedrich-Loeffler-Institut; Institute of Novel and Emerging Infectious Diseases; Isle of Riems, Germany; 4Department of Agriculture, Food and the Marine; Backweston, Celbridge, Co. Kildare, Ireland; 5Istituto Zooprofilattico Sperimentale del Piemonte; Liguria e Valle d’Aosta; Biostatistics Epidemiology and Analysis of Risk (BEAR) unit; Turin, Italy; 6UMR Interactions Hôtes Agents Pathogènes; Ecole Nationale Vétérinaire INR A; ENVT; Toulouse, France


Keywords: Bovine Spongiform Encephalopathy (BSE), cattle, intestine, mesentery, specified risk material (SRM), quantitative risk assessment (QRA)


Bovine intestines and mesenteries in the European Union (EU) are considered among the tissues potentially containing the highest level of BSE infectivity and have to be removed from the food and feed chain. A quantitative assessment of the BSE infectious load potentially entering the food and feed chain yearly in the European Union (EU) was developed. The evolution of the BSE infectious titre and of the weight of the structures accumulating infectivity was considered. The number of BSE infected cattle entering undetected in the food and feed chain yearly was estimated. A model (TSEi) was developed to estimates the evolution of the BSE infectious load in animals and the total yearly infectious load that could enter the food and feed chain. In a BSE infected bovine, the distribution of infectivity in intestines and mesentery varies with the age. Up to 36 months of age the infectivity is mainly associated (on average more than 90%) with the last 4 metres of small intestine and the caecum, over 36 and under 60 months of age, there is an inter-individual variability, from 60 months of age the infectivity is mainly associated (on average more than 90%) with the mesenteric nerves and the celiac and mesenteric ganglion complex. The total amount of infectivity peaks, about 15 BoID50, in animals younger than 18 months, it declines to 8-9 BoID50 (24–48 months of age) and it drops to 0.7 BoID50 in animals older than 60 months. The ileocaecal plate is the most infectious part of the intestine and it can be used to estimate the potential maximum level of exposure for an individual consumer.


In the EU, between 2007 and 2012, the yearly amount of BSE infectivity associated with intestine and mesentery from animals entering the food and feed chain was reduced by a factor of 10 (from about 23,000 to about 2,000 BoID50).


However, the maximum level of exposure to the BSE agent from intestine remained stable (on average about 1.5-1.6 BoID50 per meter).


In case of re-emergence of BSE in the EU there would be an increase of the potential maximum level of exposure to BSE from intestine. According to the TSEi model the removal of the last four metres of the small intestine and of the caecum from the food and feed chain would result in a major reduction of the BSE exposure risk associated with intestine and mesentery in cattle.


P.131: Transmission of sheep-bovine spongiform encephalopathy in pigs


Carlos Hedman,1 Belén Marín,1 Fabian Corbière,3 Hicham Filali,1 Francisco Vázquez, José Luis Pitarch,1 William Jirón,1 Rodrigo S Hernandez,1 Bernardino Moreno,1 Martí Pumarola,2 Olivier Andréoletti,3 Juan José Badiola,1 and Rosa Bolea1 1University of Zaragoza; Zaragoza, Spain; 2University of Barcelona; Barcelona, Spain; 3Institut National de la Recherche (INR A); Toulouse, France


Introduction. The transmissible spongiform encephalopathies (TSE) don´t occur in swine in natural conditions. However, the bovine spongiform encephalopathy (BSE) agent, inoculated by 3 simultaneous routes in pigs, is able to reproduce a neurological disease in these animals. On the other hand, the BSE agent after passage in sheep under experimental conditions (sheep- BSE) exhibits altered pathobiologic properties. This new agent is able to cross the cattle-pig transmission barrier more efficiently than BSE. The potential propagation of TSE in animals from the human food chain, including pigs, needs to be assessed regarding the risk for human infection by animals other than TSE-infected ruminants. The aim of this work was to determine the susceptibility of pigs to the Sheep-BSE agent and describe the pathological findings and PrPSc deposition in different tissues.


Material and Methods. Seven minipigs were challenged intracerebrally with sheep-BSE agent. Clinical observation and postmortem histopathology, immunohistochemistry (antibody 2G11) and Western blotting were performed on central nervous system (CNS), peripheral nervous system (PNS) and other tissues.


Results. One pig was culled in an early incubation stage, and remaining six were culled at the presence of clinical sings. Pigs developed a clinical disease with locomotor disorders in an average time of 23 months post inoculation, showing clinical findings in most of them earlier than those described in the BSE in pigs experimental infection. TSE wasn´t confirmed in the preclinical pig. In clinical pigs, the entire cerebral cortex showed severe neuropil vacuolation, extensive and severe vacuolar changes affecting the thalamus, hippocampus and cerebellum. PrPSc was found in CNS of all clinical pigs (6/6). Intracellular (intraneuronal and intraglial) and neuropil-associated PrPSc deposition was consistently observed in the brainstem, thalamus, and deeper layers of the cerebral cortex. Also, PrPSc was observed in PNS, mainly in the myenteric plexus and also in nerves belonging to the skeleton muscle. Moreover, the glycosylation profile showed a 3 band pattern with a predominant monoglycosylated band in positive pig samples.


This features concern on the potential risk of utilization of meat and bound meal of small ruminants in feeding pigs.


P.177: Elements modulating the prion species barrier and its passage consequences


Juan-Carlos Espinosa,1 Patricia Aguilar-Calvo,1 Ana Villa-Diaz,1 Olivier Andréoletti,2 and Juan María Torres1 1Centro de Investigación en Sanidad Animal (CISA-INI A); Valdeolmos, Madrid, Spain; 2UMR INR A-ENVT 1225; Interactions Hôte Agent Pathogène; École Nationale Vétérinaire de Toulouse; Toulouse, France


The phenotypic features of Transmissible Spongiform Encephalopathy (TSE) strains may be modified during passage across a species barrier. In this study we investigated the biochemical and biological characteristics of Bovine Spongiform Encephalopathy (BSE) infectious agent after transmission in both natural host species (cattle, sheep, pigs, and mice) and in transgenic mice overexpressing the corresponding cellular prion protein (PrPC) in comparison with other non-BSE related prions from the same species. After these passages, most characteristics of the BSE agent remained unchanged. BSE-derived agents only showed slight modifications in the biochemical properties of the accumulated PrPSc, which were demonstrated to be reversible upon re-inoculation into transgenic mice expressing bovine-PrPC. Transmission experiments in transgenic mice expressing bovine, porcine or human-PrP revealed that all BSE-derived agents were transmitted with no or a weak transmission barrier. In contrast, a high species barrier was observed for the non-BSE related prions that harboured an identical PrP amino acid sequence such as sheep-scrapie, mouse RML or human sCJD isolates, supporting the theory that the prion transmission barrier is modulated by strain properties (presumably conformation-dependent) rather than by PrP amino acid sequence differences between host and donor.


As identical results were observed with prions propagated either in natural hosts or in transgenic mouse models, we postulate that the species barrier and its passage consequences are uniquely governed by the host PrPC sequence and not influenced by the PrPC expression level or genetic factors other than the PrPC amino acid sequence. All these findings unequivocally demonstrate that the species barrier and its passage consequences are uniquely driven by the PrPC sequence, and not by other host genetic factors, demonstrating the validity of transgenic PrP animals as models for studies of the species barrier.


The results presented herein reinforce the idea that the BSE agent is highly promiscuous, infecting other species, maintaining its properties in the new species, and even increasing its capabilities to jump to other species including humans. These data are essential for the development of an accurate risk assessment for BSE.




Monday, June 23, 2014





Transmissible Spongiform Encephalopathy TSE Prion Disease North America 2014


Transmissible Spongiform Encephalopathy TSE Prion Disease have now been discovered in a wide verity of species across North America. typical C-BSE, atypical L-type BASE BSE, atypical H-type BSE, atypical H-G BSE, of the bovine, typical and atypical Scrapie strains, in sheep and goats, with atypical Nor-98 Scrapie spreading coast to coast in about 5 years. Chronic Wasting Disease CWD in cervid is slowly spreading without any stopping it in Canada and the USA and now has mutated into many different strains. Transmissible Mink Encephalopathy TME outbreaks. These Transmissible Spongiform Encephalopathy TSE Prion Disease have been silently mutating and spreading in different species in North America for decades.


The USDA, FDA, et al have assured us of a robust Triple BSE TSE prion Firewall, of which we now know without a doubt, that it was nothing but ink on paper. Since the 1997 mad cow feed ban in the USA, literally tons and tons of banned mad cow feed has been put out into commerce, never to return, as late as December of 2013, serious, serious breaches in the FDA mad cow feed ban have been documented. The 2004 enhanced BSE surveillance program was so flawed, that one of the top TSE prion Scientist for the CDC, Dr. Paul Brown stated ; Brown, who is preparing a scientific paper based on the latest two mad cow cases to estimate the maximum number of infected cows that occurred in the United States, said he has "absolutely no confidence in USDA tests before one year ago" because of the agency's reluctance to retest the Texas cow that initially tested positive. see ;


The BSE surveillance and testing have also been proven to be flawed, and the GAO and OIG have both raised serious question as to just how flawed it has been (see GAO and OIG reports). North America has more documented TSE prion disease, in different documented species (excluding the Zoo BSE animals in the EU), then any other place on the Globe. This does not include the very likelihood that TSE prion disease in the domestic feline and canine have been exposed to high doses of the TSE prion disease vid pet food. To date, it’s still legal to include deer from cwd zone into pet food or deer food. Specified Risk Material i.e. SRM bans still being breach, as recently as just last month.


nvCJD or what they now call vCJD, another case documented in Texas last month, with very little information being released to the public on about this case? with still the same line of thought from federal officials, ‘it can’t happen here’, so another vCJD blamed on travel of a foreign animal disease from another country, while ignoring all the BSE TSE Prion risk factors we have here in the USA and Canada, and the time that this victim and others, do spend in the USA, and exposed to these risk factors, apparently do not count in any way with regard to risk factor. a flawed process of risk assessment.


sporadic CJD, along with new TSE prion disease in humans, of which the young are dying, of which long duration of illness from onset of symptoms to death have been documented, only to have a new name added to the pot of prion disease i.e. sporadic GSS, sporadic FFI, and or VPSPR. I only ponder how a familial type disease could be sporadic with no genetic link to any family member? when the USA is the only documented Country in the world to have documented two different cases of atypical H-type BSE, with one case being called atypical H-G BSE with the G meaning Genetic, with new science now showing that indeed atypical H-type BSE is very possible transmitted to cattle via oral transmission (Prion2014). sporadic CJD and VPSPR have been rising in Canada, USA, and the UK, with the same old excuse, better surveillance. You can only use that excuse for so many years, for so many decades, until one must conclude that CJD TSE prion cases are rising. a 48% incease in CJD in Canada is not just a blip or a reason of better surveillance, it is a mathematical rise in numbers. More and more we are seeing more humans exposed in various circumstance in the Hospital, Medical, Surgical arenas to the TSE Prion disease, and at the same time in North America, more and more humans are becoming exposed to the TSE prion disease via consumption of the TSE prion via deer and elk, cattle, sheep and goats, and for those that are exposed via or consumption, go on to further expose many others via the iatrogenic modes of transmission of the TSE prion disease i.e. friendly fire. I pondered this mode of transmission via the victims of sporadic FFI, sporadic GSS, could this be a iatrogenic event from someone sub-clinical with sFFI or sGSS ? what if?


Two decades have passed since Dr. Ironside first confirmed his first ten nvCJD victims in 1995. Ten years later, 2005, we had Dr. Gambetti and his first ten i.e. VPSPR in younger victims. now we know that indeed VPSPR is transmissible. yet all these TSE prion disease and victims in the USA and Canada are being pawned off as a spontaneous event, yet science has shown, the spontaneous theory has never been proven in any natural case of TSE prion disease, and scientist have warned, that they have now linked some sporadic CJD cases to atypical BSE, to atypical Scrapie, and to CWD, yet we don’t here about this in the public domain. We must make all human and animal TSE prion disease reportable in every age group, in ever state and internationally, we must have a serious re-evaluation and testing of the USA cattle herds, and we must ban interstate movement of all cervids. Any voluntary effort to do any of this will fail. Folks, we have let the industry run science far too long with regards to the TSE prion disease. While the industry and their lobbyist continues to funnel junk science to our decision policy makers, Rome burns. ...end




[all scientific peer review studies and other scientific information I have put into blogs, to shorten reference data. I DO NOT advertise or make money from this, this information is for education use...lost my mom to the hvCJD, and just made a promise, never forget, and never let them forget. ...TSS]




Sunday, June 29, 2014


*** Transmissible Spongiform Encephalopathy TSE Prion Disease North America 2014



Saturday, June 14, 2014


Rep. Rosa DeLauro (D-CT) Calls for Briefing on Beef Recalled for Mad Cow Potential Rep. Rosa DeLauro (D-CT)



Thursday, June 12, 2014


Missouri Firm Recalls Ribeye and Carcass Products That May Contain Specified Risk Materials 4,012 pounds of fresh beef products because the dorsal root ganglia may not have been completely removed



Monday, June 02, 2014


Confirmed Human BSE aka mad cow Variant CJD vCJD or nvCJD Case in Texas



Monday, June 9, 2014


TEXAS MAD COW COVER UP (human BSE) AGAIN IN TEXAS, Mr. President Sir, we need your help please



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





(Adopted by the International Committee of the OIE on 23 May 2006)


11. Information published by the OIE is derived from appropriate declarations made by the official Veterinary Services of Member Countries. The OIE is not responsible for inaccurate publication of country disease status based on inaccurate information or changes in epidemiological status or other significant events that were not promptly reported to the Central Bureau,



Wednesday, February 12, 2014


USDA/APHIS NOTICE: Final Rule Regarding Imports and BSE Effective March 4, 2014



Sunday, July 06, 2014


Dietary Risk Factors for Sporadic Creutzfeldt-Jakob Disease: A Confirmatory Case-Control Study


Conclusions—The a priori hypotheses were supported.


*Consumption of various meat products may be one method of transmission of the infectious agent for sCJD.





No comments:

Post a Comment