Duration: 10/01/2005 to 09/30/2009

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

Transmissible spongiform encephalopathies represent a family of emerging, potentially zoonotic diseases affecting US Agriculture and Wildlife management. While most of these disorders are species-specific, they have the potential to cause massive economic losses due to general concern over the potential consequences of a zoonotic spread. This was clearly demonstrated when Bovine Spongiform Encephalopathy was found to cause a novel disease in humans. Although at least one of these diseases (Scrapie) has been recognized for more than two centuries, it has only recently been demonstrated that the infectious agent is a new, little understood variant of a normal cellular protein known as a prion. Most of these prion diseases appear to be species specific and the general nature of the disease pathogenesis appears to be highly conserved across species. While the United States has in the past had incidences of prion diseases affecting sheep, elk, deer, and mink, the zoonotic bovine form (BSE) was only recently identified within the North American cattle herd. Due to the devastating effect of BSE on the European beef and cattle industries, a strong network of prion researchers currently exists within the European Community. Until recently most research in the United States was confined to TSEs affecting individual species, with studies conducted by individual investigators focused on specifically defined problems. These individual research projects focused primarily on molecular, cellular, physiologic or epidemiologic problems, with limited integration. It is the broad, long-term goal of this proposal to form a co-operative of basic and applied researchers focused on animal prion diseases that will rapidly and markedly expand the collaboration and focus of US-based TSE research. The goal of this proposal is to develop and maintain that activity and provide immediate support in the form of new research towards understanding, controlling, and responding to prion diseases such as BSE.

Related, Current and Previous Work

This committee follows from the original creation of the rapid response committee established in February of 2004. Currently, researchers within the multistate committee are pursuing individual research projects focused on investigating molecular, cellular, immunological, neurological, diagnostic, and epidemiological aspects of prion diseases. A search of the CRIS database using keywords prion, scrapie, Chronic Wasting Disease, or Bovine Spongiform Encephalopathy yields 118 hits. After exclusion of the multistate Committee NC505 itself, as well as several meeting grants, the majority of the remaining projects are carried out by PIs represented on the existing NC505 and proposed NC_Temp1882. While many of the individual projects form a component of the efforts of the proposed multistate committee, there is no duplication of individual research efforts. Instead, NC505 has served as the catalyst to bring these interests together to pool research and technological resources for greater efficiency in US research efforts. At its initial meeting, members shared their interests with the committee, and a number of initial collaborations were established. At that time, despite the diversity of the group, it was decided that a principal focus of the research collaborations should be a greater understanding of Chronic Wasting Disease (CWD), given its apparently unique transmission and specificity to North America. A mandate of this initial meeting was to work towards establishment of shared research resources, including the support of a research core for CWD. While BSE is of major public interest, current guidelines severely limit study of this agent, and therefore CWD and Scrapie are used primarily as models for TSEs. In response to a request from the Experiment Station Committee on Organization and Policy, the committee is now preparing a white paper on TSEs in the US. At the second meeting of NC505, called to coincide with an International meeting on Animal Prion Diseases in the Americas where NC505 was well represented, it was decided that the current committee should be extended beyond its initial 2-year mandate in order to continue this valuable work.

Objectives

1. To compare and contrast the native pathogenesis of TSEs, in order to better understand the progression and potential transmission of disease at the individual level.
   
2. To extend existing and develop novel diagnostic tools in live and postmortem animals which can provide better epidemiological information on individual TSEs.
   
3. To investigate both the role of environmental contamination in population-level transmission of TSEs, as well as novel means to inactivate prions in the environment.
   
4. To develop an epidemiological model of individual TSEs which can be used to develop evidence and population -based animal management plans for TSE control.
   
5. To investigate the nature and effect of prion strains on the pathogenesis, progression, and transmissibility of TSEs in both individual and population based approaches.
   
6. Use pathogenomics to determine the role of genetic material in the pathogenesis of prion diseases, and identify surrogate genetic markers for the diagnosis of prion diseases.
   

Methods

Objective 1: To compare and contrast the native pathogenesis of TSEs, in order to better understand the progression and potential transmission of disease at the individual level. While prion diseases may be perceived by the public to be relatively recent, occurrence of the disease in sheep has been reported as early as the 18th century (Detwiler and Baylis, 2003; Lasmezas, 2003). For centuries, Scrapie was characterized as a "Slow Viral Disease", caused by an extremely inefficient virus. Despite the obvious appearance of symptoms in domestic sheep flocks, there is no evidence that scrapie can be transmitted directly to humans (Belay et al., 2004). Nonetheless, public concern remains about potential cross-species transmission of this and other prion diseases that may result in new outbreaks in the human population. Occurrence of a transmissible human form of prion disease was first reported in New Guinea as Kuru resulting from ritualistic cannibalism of brain tissue within an isolated tribe (Lasmezas 2003). Similar diseases were later identified in other species, including farmed mink and cervids, and eventually as BSE in 1986 (Prince et al., 2003). Although many TSEs may be transmissible to other species via intracerebral inoculation, BSE appears to be unique in it's ability to cause infection of a wide variety of species, including humans, through oral transmission (Hill et al., 1997). Public reactions to this disease have economic implications which far outweigh it's potential human health risk, likely due to the nature of infection (food-borne) and principal target organ (the central nervous system). It is important to note that most TSE's, once successfully passaged through a given species, will infect that species more readily in subsequent trials. CWD is particularly intriguing, given that it is the only known infectious prion disorder that appears to affect animals in the wild and that the precise mode of transmission remains unclear (Williams and Miller, 2003; Belay et al., 2004; Miller and Williams, 2004). Furthermore, while most other diseases are rapidly becoming of worldwide concern, CWD appears to be of primary concern to North America. Although no reliable link has been found between CWD and disease in humans, worry remains about the potential for human infection similar to that observed during the BSE concern in Europe. It is therefore crucial to develop adequate laboratory models of naturally occurring prion diseases that can be used by multiple investigators to study this disease family. Although the natural route of prion infection is believed to be oral, the precise means whereby the infectious agent spreads remains unclear. Experimental data have clearly indicated that both neuronal and lymphoid organs are involved in natural pathogenesis. The greatest limitation to existing research lies in the lack of availability of native research models and tissues for use by the prion research community. As described above, the NADC has been particularly co-operative at providing collaborative access to naturally infected tissues and animals to outside researchers, whereas additional work has been directed towards establishing rodent models of prion diseases. A number of investigators within the Multistate Research committee will continue established collaborations to investigate the natural pathogenesis of disease. The NADC and Colorado State University will pursue collaborations with Montana to investigate the tongue as a natural route of prion infection in CWD. This will involve sharing existing research resources and experimental material harvested from research animals housed in Colorado. South Dakota will collaborate with the NADC and Colorado State to extend ongoing investigations of the role(s) of Follicular Dendritic Cells and B cell subsets in the initial pathogenesis of Prion diseases through sharing of both research expertise and research resources. Minnesota, Montana, and South Dakota will collaborate to establish new animal models of CWD through existing and new grant applications. The NADC and Creighton will continue to collaborate in the investigation of the molecular mechanisms and pathogenesis of individual prion strains in disease using both hamster and ovine models. Success in these collaborations will be demonstrated through collaborative publications, grant applications, and translational research. Objective 2: To extend existing and develop novel pre- and post-harvest diagnostic tools which can provide better epidemiological information on individual TSEs. Although the causative agent of Scrapie and other TSE's appears to be due to altered self- protein, relatively little is known regarding the pathogenesis of the disease. Furthermore, diagnosis has previously been limited to complex histological techniques relying on examination of brain tissue-clearly unsuitable for identification and diagnosis of living animals (O'Rourke et al., 2000). As a result of data demonstrating that leukocytes also express the normal form of prion protein (PrPc) and are capable of hosting the PrPSc, a new Scrapie test has been developed involving examination of lymphoid tissue in the third eyelid for the presence of pathogenic prion protein (O'Rourke et al., 2000). While effective, this test still relies on a rather complex biopsy of living tissue and may not be directly applicable to other species. Optimal tests, providing potential early disease detection, would involve sampling and identification of infective agent from blood. To date, efforts to identify PrPSc in blood plasma and unseparated peripheral blood lymphocytes have been largely unsuccessful. Nonetheless, B cells, Follicular Dendritic Cells (FDCs), and Dendritic Cells (DCs) have all been shown to be associated with PrPSc in vivo, resulting in IHC and ELISA-based tests of lymphoid organs in prion-suspect animals (McBride et al., 1992; Cashman et al., 1990; Mabbott et al., 1997; Burthem et al., 2001). A number of blood-based tests for prion infection are currently in development. This Multistate committee will continue to perform baseline research on novel diagnostics, as well as evaluate existing and novel methods as they become available. South Dakota will continue to provide expertise on existing and novel commercial methodologies, as well as collaborate with the NADC on novel means to propogate prions in vitro for diagnosis. Minnesota will collaborate with South Dakota, Colorado, and Chronix Biomedical in assessment of the utility of RNA aptamers and other nucleic markers as diagnostic tools for prions. South Dakota will share research material and expertise to further these research goals. The NADC and Canadian Food Inspection Agency will continue to co-ordinate overall efforts on diagnostics, including further analysis of strain-typing techniques and other immune-based therapies. Colorado and Wisconsin will coordinate with USDA:APHIS to obtain samples from the existing TSE surveillance programs for the validation of the new developed tests. Success in these collaborations will be demonstrated through collaborative publications, grant applications, and translational research. Objective 3: To investigate both the role of environmental contamination in population-level transmission of TSEs, as well as novel means to inactivate prions in the environment. A major challenge to both prion science and public concern over prions has been the relative stability of the agents in the environment. Chronic Wasting Disease in particular has been shown to remain in the environment for extended periods of time, providing apparent future reservoirs of infection. While Objective 1 will focus on disease transmission at the individual animal level, including minimum infectious doses, current methods to inactivate prions are not practical for large-scale disinfection of pastureland or other grazing areas thought to propagate CWD infection. Briefly, current disinfection procedures involve either extended treatment with 40% household bleach or 2N NaOH, or alternatively lengthy autoclaving or incineration procedures. Experimental reports have recently suggested that biological alternatives (ie chicken keratinase) may exist to aid in prion inactivation, although these methods are also limited in their utility. For this reason, collaborators will be recruited to the existing committee to focus on novel technologies to inactivate prions in both the laboratory and the environment, including chemical, physical, and microbiological means to inactivate prions. Colorado State University will continue their research in determining the natural deactivation of the prion using the commercial decomposition of dairy carcasses. As research develops, further expansion to involve other members of the multistate committees is expected during validation processes. Objective 4: To define an epidemiological model of individual TSEs which can be used to develop evidence and population-based animal management plans for prion disease control. The first reported documentation of scrapie dates back to the early 18th century. As such, a large amount of baseline data has been obtained regarding disease occurrence, and animal (eg. age, gender, genotype), environment (eg. soil contamination) and management (eg. husbandry practices) factors. A critical issue in controlling prion diseases in animal populations is the long incubation period and diagnostic insensitivity. By the time a clinical case of disease is observed (domestic species) or detected (wild species), a significant proportion of the population may have become infected and disease control is a challenge. Studies of the transmission of scrapie in sheep flocks have demonstrated disease expression is regulated both by susceptibility genotype and level of exposure to infectious material. While placenta contains high levels of infectious prion protein, horizontal transmission between rams, wethers, or other adult animals does not appear to occur. Efforts to detect infectivity in feces, urine, or other bodily secretions from scrapie-infected animals have been unsuccessful. Based on this information, current sheep management practices focus on controlling potential exposure to infectious materials during lambing, and eradication based on susceptibility genotypes and selective breeding of resistant genotypes. Based on preliminary data, it appears that Bovine Spongiform Encephalopathy is not efficiently transmitted horizontally, although susceptibility genotypes and potential infectivity of placental material remain unclear. In contrast, Chronic Wasting Disease appears to be significantly more infective, and infective levels of environmental contamination appear to be long-lasting. For a number of reasons, significantly less information is available regarding the epidemiology and transmission of CWD in deer and elk populations, due to both a lack of basic data regarding modes of infection and transmission, as well as a general lack of information on behavioural interactions in deer communities. Furthermore, while cross-species transmission has not been identified, the potential for infection of domestic animal species remains a concern. In order to address these concerns, basic disease researchers within the multistate research committee will team with wildlife experts to determine the most likely modes of transmission of CWD between susceptible animals, as well as the means whereby the disease is maintained. Colorado, Michigan, Texas. Wisconsin will form a basis for this collaboration, as they provide access to laboratory animals and material for research. In addition, the Cervid Research and Recovery Center will provide important expertise, and the involvement of the North American Deer Farmers Association will provide an important link to the management of domesticated deer herds. This research effort will be unique, in that participation and input from a complementary multistate committee will be encouraged as a crossover interest to further expand the expertise of the committee (NC1005). Quantitative simulation epidemiological models will be constructed using parameters obtained from the above experimental and observational data. A spread model for highly contagious infectious animal diseases was developed and currently under evaluation. This model will be used as a base-line structure to assess the spread of TSEs within captive populations. The model then can be utilized to assess the various management strategies in reducing the impact of the disease. Colorado State University will maintain the lead in pursing this model construction in collaboration with other research team members, and the University of Wisconsin will collaborate in development of epidemiological models of CWD in free-ranging white-tailed deer populations. Objective 5: To investigate the nature and effect of prion strains on the pathogenesis, progression, and transmissibility of TSEs. The most useful animal model for strain typing of TSE isolates, which was developed by researchers in Edinburgh, Scotland, involves the inoculation of at least 20 mice from each of 3 inbred lines (RIII, VM, and C57Bl6) plus 1 F1 cross (C57Bl6 x VM), followed by a comparison of incubation periods and lesion profile (extent of spongiform change in defined anatomic regions of the brain). This method has been used for many years in Europe to characterize Scrapie strains and, more recently, to demonstrate a strain signature of BSE that is unique and that is maintained after cross-species transmission into other mammals and human beings. A western blot technique can used to examine and compare PrPSc from TSE isolates. The studies will determine if there are molecular characteristics that can be used to differentiate these agents from each other. In addition, comparing different isolates of the same agent may present an opportunity to select isolates with unique biochemical signatures that should be further characterized biologically. The analysis will include (1) migration distance of the three major PrPSc polypeptides, especially the unglycosylated polypeptide, and (2) glycoform profile (relative proportion of unglycosylated, mono- and di-glycosylated polypeptides). In addition, two other approaches will also be applied: differential antibody analysis using monoclonal antibodies to the N- and C- terminus and internal regions of PrPSc and measurement of relative proteinase K resistance. The validity of conclusions from these studies will be highly dependent on the repeatability of results, as confirmed through multiple repetitions and application of statistical evaluations. These studies will be conducted through shared research collaborations of both expertise and reagents by the NADC and Montana State University. Objective 6: Use pathogenomics to determine the role of genetic material in the pathogenesis of prion diseases, and identify surrogate genetic markers for the diagnosis of prion diseases. Genetic analysis has played a significant role in the study of prion diseases. Detection of mutations in the prion-coding region of the host genome has provided critical information about the risk of the host in developing clinical disease. The advent of technologies for large-scale comparison of genomes, transcriptomes and proteomes is revolutionizing molecular medicine. Analysis of gene expression creates the possibility to define a disease based on the coordinated action of a group of genes. An understanding of the molecular events that follow infection with a variety of strains or species-specific prions will help define the progression of this insidious condition and thus aid in the evolution of new ante mortem diagnostic markers as well as therapeutic modalities. In addition, these new strategies may be employed to more efficiently study host-pathogen interactions and disease progression. DNA microarrays exploit primary sequence data to measure expression at the transcript levels and detect sequence polymorphisms for thousands of genes simultaneously. By using microarrays that are representative of host species, one can explore host response at the gene expression level and provide a molecular description of the events that follow infection. Specific host genotypes may also have typical gene expression signatures unique for each genetically and phenotypically defined group(s) of hosts, thus providing a novel tool for diagnosis, prognosis, and clinical management of prion disease. Studies at the University of Minnesota (Dr. Skinner) and the National Microbiology Laboratory (NML), Division of Host Genetics and Prion Diseases, 1015 Arlington Street, Winnipeg R3E 3R2 Canada (Dr. Booth) have now established the utility of DNA microarray technology in studying neuronal responses to prions (Booth et al., 2004). . Collaborative studies between National Animal Disease Center, University of Minnesota, and NML (Canada) will be designed in the future to establish methods to study strain-specific variations in neuronal and microglial responses as well as in determining gene expression signatures that may aid in ante mortem diagnostics. The studies will also be aimed at defining a repertoire of surrogate markers for prion diagnostics. Recent studies suggest that non-coding, repetitive genetic elements may also be associated with risk of developing prion disease. An important goal is to gain understanding of the significance of both single function and repetitive genetic material to prion disease pathogenesis and to determine whether these gene elements can serve as surrogate markers for disease diagnosis. Collaborations between The Ohio State University, University of Minnesota (Dr. Sreevatsan), and Chronix Biomedical Inc., will be established to define families of circulating nucleic acids, definition of their binding specificities to prions in different conformations, their utility in ante mortem diagnosis, and their function in prion misfolding. A combinatorial library-based iterative technology will be applied to define novel ligands capable of differentiating prion conformations, which define a molecular signature of a strain. Proteomics provides an additional portal to TSE pathophysiology and new high-throughput techniques share may of the strengths of RNA profiling with additional advantages particularly applicable to TSEs; namely the ability to profile proteins and peptides without prior sequence knowledge, important when working with species such as elk and deer for which large scale genome projects do not exist. Quantitative profiling of proteins and peptides can also be done in readily accessible biological samples, (blood, cerebrospinal fluid and urine) which is vital in order to obtain the sample base necessary for the identification and validation of disease specific biomarkers. The facility at the NML in Canada is acquiring a SELDI mass spectrophotometer capable of such high throughput studies in 2005. South Dakota will collaborate with the NLM to develop proteomic markers of prion infection using samples derived from animal models.

Measurement of Progress and Results

Outputs

• Define the molecular and anatomical basis of prion replication during TSE pathogenesis. (Objective 1)
• Determination of specified risk material for both diagnosis and control of disease transmission. (Objectives 1, 2)
• Define the sensitivity of existing and novel diagnostic techniques for both research and disease diagnosis in the field. (Objective 2)
• Provide improved means for prion deactivation in both the field and the laboratory setting. (Objective 3)
• Define factors important in the control or elimination of TSEs in domestic and wild animal species. (Objective 4)
• Map the importance of prion strains in prion pathogenicity and host range. (Objective 5)
• Identify novel molecular techniques which can be used in both prion research and diagnostics. (Objective 6)

Outcomes or Projected Impacts

• A greater understanding of the relative risk and transmission of prion diverse prion disorders.
• Definition of recommendations for the control or eradication of prion diseases.
• Production of a white paper for ESCOP defining current understanding of prion disorders.
• Organization of an International Research Symposium in the 4th year of the proposal to disseminate information obtained as a result of the multistate committee.
• Composition of specific recommendations on biosafety protocols for prion research.
• Expansion of current TSE research expertise in the US through greater collaboration and student education.
• Creation of specific foci of research resources and expertise which can be exploited by the prion research community.

Milestones

(1):Publication of collaborative research papers in internationally recognized journals.

(1):mposition of a white paper on TSE diseases in the US.

(1):Definition of Biosafety Guidelines for prion disease research and animal husbandry in the US.

(4):ganization of an International Research Symposium in the 4th year of the proposal.

Projected Participation

View Appendix E: Participation

Outreach Plan

The results of collaborative efforts will be published in peer-reviewed journals and communicated to the public through lay channels. Organization of a scientific conference in the 3rd year of the proposal will further enhance dissemination to stakeholders and the public. Finally, preparation of a white paper on TSEs as well as Biosafety Guidelines for TSE studies as a result of the above research will provide direct, measurable output of the committees success.

Organization/Governance

The committee members will annually elect a Chair, Vice-Chair, and Secretary to serve as organizational leads. Each position may be renewed up to a maximum of 2 consecutive terms at the discretion of the committee. The Chair will send and receive correspondence on behalf of the committee, as well as serving as official representative of the committee in formal circumstances. These duties will be shared by the Vice-Chair when the Chair is unable to fulfill these obligations, as well as serving as the administrative organizer for the committee. The secretary will record all minutes of formal meetings and is ultimately responsible for reporting minutes to the Administrative Advisor. Meetings will be held annually.

Literature Cited

Belay, E.D., Maddox, R.A., Williams, E.S., Miller, M>W., Gambetti, P., Schonberger, L.B. 2004. Chronic wasting disease and potential transmission to humans. Emerging Infectious Diseases 10(6):977-984.

Booth, S.A., C. Bowman, R. Baumgartner, G.C. Sorensen, C. Robertson, M.B. Coulthart, C.S. Phillipson, R.L. Somorjai. Molecular Classification of Scrapie Strains in Infected Mice Using Gene Expression Profiling. Biochemical and Biophysical Research Communications 325, 1339-1345.

Booth, S.A., Christopher Bowman, Richard Baumgartner, G. Sorensen, C. Robertson, M. Coulthart, C. Phillipson and Rajmund L. Somorjai. Identification of central nervous system genes involved in the host response to the scrapie agent during clinical and pre-clinical infection. J. Gen. Virol. 85, 3459-3471 (2004)).

Burthem, J., Urban, B., Pain, A., Roberts, D.J. 2001. The normal cellular prion protein is strongly expressed by myeloid dendritic cells. Blood 98:3733-3738.

Cashman, N.R., Loertscher, R., Nalbantoglu, j., Shaw, I., Kascsak, R.J., Bolton, D.C., Bendheim, P.E. 1990. Cellular isoform of the scrapie agent protein participates in lymphocote activation. Cell 61:185-192.

Detwiler, L.A., Baylis, M. 2003. The epidemiology of scrapie. Rev. Sci. Tech. Off. Int. Epiz. 22(1):121-144
Hill, A.F., Desbruslais, M., Joiner, S., Sidle, K.C.L., Gowland, I., Collinge, J. 1997. The same prion strain causes vCJD and BSE. Nature 389:448-450.

Lasmezas, C.I. 2003. The transmissible spongiform encephalopathies. Rev. Sci. Tech. Off. Int. Epiz. 22(1):23-36
Mabbott, N.A., Mackay, F., Minns, F., Bruce, M.E. 2000. Tempoorary inactivation of follicular dendritic cells delays neuroinvasion of scrapie. Nature Medicine 6: 719-720.
McBride, P.A., Eikelenboom, P., Kraal, G., Fraser, H., Bruce, M.E. 1992. PrP protein is associated with follicular dendritic cells of spleens and lymph nodes in uninfected and scrapie-infected mice. J. Pathol. 168:413-418.

Miller, M.W., Williams, E.S. 2004. Chronic wasting disease of cervids. Curr. Topics in Microbiology and Immunology. 284:193-214.

O'Rourke, K.I., Baszler, T.V., Besser, T.E., Miller, J.M., Cutlip, R.C., Wells, G.A., Ryder, S.J., Parish, S.M., Hamir, A.N., Cockett, N.E., Jenny, A., Knowles, D.P. 2000.

Preclinical diagnosis of scrapie by immunohistochemistry of third eyelid lymphoid tissue. J. Clin. Microbiol. 38(9):3254-3259.

Prince, M.J., Bailey, J.A., Barrowman, P.R., Bishop, K.J., Campbell, G.R., Wood, J.M. 2003. Bovine Spongiform encephalopathy. Rev. Sci. Tech. Off. Int. Epiz. 22(1):37-60

Williams, E.S., Miller, M.W. 2003. Transmissible spongiform encephalopathies in non-domestic animals: origin, transmission, and risk factors. Rev. Sci. Tech. Off. Int. Epiz. 22(1):145-156.

Attachments

Land Grant Participating States/Institutions

CO, IA, MI, MN, SD, WI

Non Land Grant Participating States/Institutions

Chronix biomedical, Creighton University, Johns Hopkins University, LSU Agricultural Center, National Microbiology Laboratory, Winnipeg Manitoba R3E 3R2, USDA ARS