some might be interested in this.......tss
Subject: RESEARCH PROJECT: TRANSMISSION, DIFFERENTIATION, AND PATHOBIOLOGY OF TSE
Date: January 13, 2007 at 2:25 pm PST
Research Project: Transmission, Differentiation, and Pathobiology of Transmissible Spongiform Encephalopathies
Location: Virus and Prion Diseases of Livestock
2006 Annual Report
1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter?
This project is aligned to National Program (NP) 103 - Animal Health. This project deals with Transmissible Spongiform Encephalopathies (TSE), which are fatal degenerative diseases of the central nervous system that can affect several animal species, including humans. The causal agent is believed to be a cellular protein, the prion protein (PrP) that has assumed an unnatural form. Because the altered protein is resistant to protease degradation, it accumulates in nervous tissue and the resulting dysfunction ultimately leads to death. The specific TSEs being investigated in this project are scrapie in sheep, transmissible mink encephalopathy (TME), bovine spongiform encephalopathy (BSE) in cattle, and chronic wasting disease (CWD) in deer and elk. The major concern about these diseases is that BSE has been shown to cross the species barrier to cause a unique TSE in human beings. Although it has not been demonstrated that scrapie, TME, or CWD present any risk to human health, the BSE experience has raised many questions about the potential hazard these TSEs present for transmission to other animal species, especially domesticated livestock and wildlife. The current research is focused on direct experimental challenge of the species barrier effect by animal inoculation. These results are then compared to results obtained with a variety of laboratory procedures to determine if they can be used as predictive models for future risk assessments. These studies also provide information about the clinical and pathological disease characteristics that can be expected if a TSE crosses the species barrier, thus enabling animal health specialists to recognize such situations should they occur. Additional transmission studies focus on (i) determining the modes of transmission and disease development in scrapie of sheep and CWD of cervids, (ii) on the evaluation of changes in the retina of sheep infected with the scrapie agent. The latter studies might enable us to design appropriate intervention or ante mortem diagnostic tools that might enable us to devise strategies to control the spread of these diseases.
Because of the risk for BSE transmission to human beings, the presence of scrapie, TME, CWD, and BSE in the United States presents a potential liability to the U.S. livestock and hunting industries, because the safety of animals and animal products intended for domestic consumption and international trade may be questioned. Efforts are being made by both federal and state regulatory agencies to eradicate scrapie and CWD and to determine the prevalence of BSE. The effectiveness of these programs will depend heavily on having accurate information about the nature of these diseases, not only in the original hosts, but also in other species that may be affected.
The impact of this research project on U.S. agriculture, especially the cattle industry, is significant. The discovery of one BSE positive animal in December 2003 in Washington State resulted in significant losses to the U.S. beef industry. More than 50 countries (including major markets such as Japan and South Korea) banned import of U.S. cattle and beef products within days of the December 2003 announcement. Estimated losses arising from these bans during 2004 range from $3.2 billion to $4.7 billion. Many of these bans are still in effect in 2006 and with the discovery of a third case of BSE (second indigenous BSE case in the U.S.) in March 2006, significant losses from export bans of U.S. beef will most likely be also experienced also in 2006. In addition, the regulations introduced in 2004 led to changes in the beef industry with a net economic cost of approximately $200 million in 2004 only.
2.List by year the currently approved milestones (indicators of research progress)
Animal experiments:
Because of the long-term nature of most TSE experiments in animals, especially those involving cross-species transmission, most of the studies outlined in this project will not be completed within 30 months. Therefore, the following time line primarily presents expectations for when experiments will be initiated.
2003 Cattle inoculated with white-tailed deer CWD Fallow and white-tailed deer inoculated with CWD Cattle inoculated with TME Sheep inoculated with AV136QR171 and Idaho scrapie isolates
2004 Cattle inoculated with elk CWD Raccoons inoculated with TME, scrapie, and CWD isolates Mice inoculated for strain typing of 10 TSE isolates Swine inoculated with scrapie and CWD Reindeer inoculated with CWD White-tailed deer inoculated with scrapie
2005 Study to assess scrapie and CWD amplification in market age swine completed Mice inoculated for strain typing of 15 TSE isolates
2006 Initiate mouse bioassay of various TSE isolates to determine if this strain typing procedure is useful for identification of unique TSE strains in the U.S. Initiate cattle inoculation with both U.S. BSE isolates in order to amplify BSE material for subsequent pathogenesis studies. Inoculation of reindeer with the CWD agents derived from white-tailed deer, mule deer and elk. Initiate oral pathogenesis studies of raccoons with TME.
Laboratory studies: 2003 Validation of method for genotyping from paraffin sections. Methods developed for biochemical strain typing studies.
2004 Genotyping of archived scrapie tissues from the National Veterinary Services Laboratories (NVSL)/APHIS/VS/USDA. Evaluation of biochemical methods for strain typing of scrapie isolates. Development of mass spectrometry methods for characterization of protein expression in normal sheep brain.
2005 Evaluation of biochemical methods for differentiation of TSE agents after cross-species transmission. Comparison of protein expression (mass spectrometry) in scrapie brain to that in normal sheep brain.
2006 Determination of the feasibility of PrP**Sc detection from formalin fixed tissues. Evaluate methods of biochemical strain typing of TSE isolates.
4a.List the single most significant research accomplishment during FY 2006.
Identification and characterization of third U.S. BSE case: Studies were conducted which confirmed the BSE diagnosis of an inconclusive bovine brain sample. The PrP**res profile from the third BSE case diagnosed in the United States showed different molecular properties when compared to the PrP**res pattern described for the 2003 U.S. isolate, however had similar molecular properties as described for the second U.S. BSE case. This finding and our findings from the two previous cases of BSE support the presence of only atypical BSE in the U.S. indigenous cattle population.
This work is of critical relevance to the National Program Action Plan, Animal Health (103), program component pathogen detection, because it had an important impact on the confirmation of an inconclusive BSE diagnosis (based on a rapid test) and the incidence of BSE in the U.S. cattle population.
4b.List other significant research accomplishment(s), if any.
Intracerebral transmission of CWD from white-tailed deer to cattle: Tissues were collected from cattle inoculated intracerebrally with CWD from white-tailed deer (WTD) at the termination of a 4-year study. The WTD CWD attack rate (7/8) in cattle was higher and the incubation time shorter than that previously seen in cattle challenged intracerebrally with mule deer CWD (3/8). The absence of lesions of spongiform encephalopathy, the prion accumulation as detected by immunohistochemistry and the Western blot profiles were similar to those observed in cattle inoculated with mule deer and elk derived CWD.
Intracerebral transmission of mule deer CWD into sheep: Tissues were collected from sheep at the termination of the 6-year study to assess transmission of mule deer CWD to sheep by intracerebral inoculation. These tissues were analyzed by histopathology, immunohistochemistry and Western blot. Two of eight inoculated sheep had spongiform encephalopathy with prion accumulation detected by immunohistochemistry and Western blot. Results of analyses indicated a prion disease in sheep indistinguishable from sheep scrapie.
Intracerebral transmission of CWD from elk, mule-deer, and white-tailed deer into white-tailed deer: Tissues were collected from white-tailed deer inoculated intracerebrally with mule deer, elk, and white-tailed deer CWD at the termination of a 3-year study. The clinical course, lesions of spongiform encephalopathy, prion accumulation detected by immunohistochemistry and Western blot profiles did not differ between treatment groups. The results suggest that the host-species source of CWD does not affect the outcome of CWD presentation in white-tailed deer after intracerebral inoculation.
Transmission of scrapie and CWD to swine: Tissues from swine inoculated with scrapie and CWD by intracerebral and oral routes were collected at 6 months post-inoculation and were analyzed by histopathology, immunohistochemistry and Western blot. There were no ante mortem clinical signs suggestive of neurologic disease, no histologic lesions suggestive of spongiform encephalopathy, and no indication of prion accumulation by immunohistochemistry and Western blot. Monitoring of a replicate of inoculated littermates will continue until the termination of the study in approximately 5 years.
Retinal Pathology in sheep with scrapie: A study was completed which demonstrated that the retina responds to the accumulation to abnormal prion protein in a way that may affect visual system function. It could be shown that specific cell populations of the retina were affected in scrapie-infected sheep.
Relevance to ARS National Program Action Plan, Animal Health (103): Above described research accomplishments have a critical impact on pathogen detection, epidemiology of disease, host/pathogen interactions, genetic resistance to disease and disease control strategies. In particular, these studies also provide information about the clinical and pathological disease characteristics that can be expected if a TSE crosses the species barrier, thus enabling animal health specialists to recognize such situations should they occur. Transmission studies focusing on the mode of TSE transmission and disease development and on the evaluation of changes in the retina of animals infected with TSEs might enable us to design appropriate intervention or ante mortem diagnostic tools that might enable us to devise strategies to control the spread of these fatal diseases.
4c.List significant activities that support special target populations.
None.
5.Describe the major accomplishments to date and their predicted or actual impact.
This project was initiated January 28, 2002, as a result of the FY 2002 Appropriations Bill passed by Congress and signed by the President for research on Emerging & Exotic Diseases of Pests. The major objectives are to assess transmissibility of the TSEs that affect livestock and wildlife species, to develop methods for differentiation of TSE strains, and to determine the pathobiology of these diseases in the natural host and after cross-species transmission. Such studies are of utmost importance to the ARS National Program Action Plan, Animal Health (103), which includes pathogen detection, epidemiology of disease, host/pathogen interactions, genetic resistance to disease, and disease control strategies. Experiments are in progress to determine the transmissibility of different CWDs into white-tailed deer, cattle, sheep and swine. Based on results of our studies, it may be concluded that under natural conditions cattle exposed to mule deer CWD would require a rather large dose of inoculum, and an extremely long incubation time to develop a TSE-associated disease. In contrast, intracerebral inoculation of cattle with brain material from white-tailed deer results in a higher incidence rate (7/8 animals, 88%) compared to mule deer CWD (3/8 animals, 38%). In addition, intracerebral inoculation of cattle with brain material from mink with TME or cattle-passaged TME resulted in incubation times, lesions of spongiform encephalopathy and PrP**res distribution which resemble those found in cattle infected with BSE. Transmission of scrapie and CWD to swine revealed that CWD- or scrapie-infected market-aged swine (6-month-old) showed no ante mortem clinical signs, no histologic lesions suggestive of spongiform encephalopathy, and no indication of prion accumulation by immunohistochemistry and Western blot. We were able to compare the first U.S. BSE isolate (2003) with other typical BSE isolates (Canadian, European) and found them to be indistinguishable. In contrast, the molecular phenotype of the second and third U.S. BSE isolate (11/2004, diagnosed 06/2005; 03/2006) was different from the 2003 U.S. BSE isolate as determined by Western blot analysis. Completed work has determined that sheep scrapie can be transmitted to elk and that the resulting disease is indistinguishable from CWD in that species. When elk CWD was transmitted orally to elk of different genotypes, one genotype (LL) seems to be less susceptible to CWD. Biopsy of the gut-associated lymphoid tissue (GALT) present at the rectal-anal junction revealed that 3 of the 4 remaining LL elk contained PrP-Sc positive cells. White-tailed deer have been inoculated with CWD from elk, mule deer and white-tailed deer in order to determine if strain differences in the CWD agent exist that depend on the host of origin. Upon completion (6 years post infection) of a study with sheep intracerebrally inoculated with mule deer CWD revealed that two of eight inoculated sheep had spongiform encephalopathy with prion accumulation detected by immunohistochemistry and Western blot. The prion disease observed in sheep infected with mule deer CWD was indistinguishable from scrapie. These cross-species transmission experiments will provide information and tissues that can be used to evaluate the effectiveness of current diagnostic protocols for the TSEs. An important contribution of this project has been the demonstration of the utility of raccoons as an animal model to distinguish scrapie, CWD and TME based on attack rate and time to clinical disease. Raccoons inoculated with TME develop disease within 6 months time and as such represent one of the fastest available, non-transgenic models of TSE disease. We developed a method to extract DNA from formalin-fixed paraffin-embedded brainstem tissue to determine prion gene polymorphisms in scrapie-affected sheep. This method has been successfully transferred to APHIS and might have important implications for future scrapie surveillance efforts.
6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products?
Two Cooperative Research and Development Agreements (CRADAs) were established with industry.
7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below).
Invited lecture presented at the NAEBA meeting on "Chronic Wasting Disease Research at the NADC".
Invited lecture presented at the German TSE Platform Meeting titled "Natural and Experimental Prion Diseases of Cattle".
Invited lecture presented at Interagency Working Group (IWG) on Prion Science on "TSE Research at the National Animal Disease Center".
Invited lecture presented at OIE meeting "Prion Diseases of Domestic Livestock" titled "Identification and Characterization of U.S. BSE Cases".
Invited lecture presented at the Third International Rushmore Conference on "Natural and Experimental Prion Diseases of Cattle".
Invited lecture presented at the AAVLD Meeting titled "Identification and Characterization of U.S. BSE cases".
Invited to participate in a workshop held in Saskatoon, Canada, on February 8, 2006, to establish research priorities for PrioNet Canada and the Alberta Prion Research Institute (APRI) with respect to chronic wasting disease (CWD). Presented a seminar at the workshop titled "Transmissible Spongiform Encephalopathy (TSE) studies at the National Animal Disease Center NADC, Agricultural Research Service."
Review Publications
Hamir, A.N., Gidlewski, T., Spraker, T.R., Miller, J.M., Creekmore, L., Crocheck, M., Cline, T., Orourke, K.I. 2006. Preliminary observations of genetic susceptibility of elk (Cervus elaphus nelsoni) to chronic wasting disease by experimental oral inoculation. Journal of Veterinary Diagnostic Investigation. 18(1):110-114.
Hamir, A.N., Kunkle, R.A., Miller, J.M., Bartz, J.C., Richt, J.A. 2006. First and second cattle passage of transmissible mink encephalopathy (TME) by intracerebral inoculation. Veterinary Pathology. 43(2):118-126.
Hamir, A.N., Kunkle, R.A., Miller, J.M., Greenlee, J.J., Richt, J.A. 2006. Experimental second passage of chronic wasting disease (CWD(mule deer)) agent to cattle. Journal of Comparative Pathology. 134(1):63-69.
Hamir, A.N., Kunkle, R.A., Miller, J.M., Hall, S.M. 2006. Abnormal prion protein in ectopic lymphoid tissue in a kidney of an asymptomatic white-tailed deer experimentally inoculated with the agent of chronic wasting disease. Veterinary Pathology. 43(3):367-369.
Hamir, A.N., Kunkle, R.A., Miller, J.M., Richt, J.A. 2005. Second passage of sheep scrapie and transmissible mink encephalopathy (TME) agents in raccoons (Procyon lotor). Veterinary Pathology. 42(6):844-851.
Bessen, R.A., Dejoia, C., Dlakic, W., Sorg, R., O¿Connell, K., Tucker, T., Kunkle, R.A., Hamir, A.N., Richt, J.A. 2005. Prion infection of mucosal tissue [abstract]. TSE Forum, Prion 2005: Between Fundamentals and Society's Needs. p. 31.
Greenlee, J.J., Kunkle, R.A., Hamir, A.N. 2005. Experimental intracerebral and oral inoculation of scrapie to swine: preliminary report [abstract]. Proceedings of the American Association of Veterinary Laboratory Diagnosticians 48th Annual Conference. p. 38.
Hall, M.S., Richt, J.A., Davis, A.J., Levings, R.L. 2005. Where we've been and where we're going with BSE testing in the United States [abstract]. 48th Annual Meeting of the American Association of Veterinary Laboratory Diagnosticians. p. 20.
Hall, S.M., Richt, J., Davis, A., Kluge, J., Simmons, M., Stack, M., Spencer, Y. 2006. Identification and characterization of U.S. BSE cases [abstract]. Prion Diseases of Domestic Livestock. p. 25.
Hamir, A.N., Cutlip, R.C., Miller, J.M., Kunkle, R.A., Greenlee, J.J., Richt, J.A. 2005. Experimental transmission of transmissible spongiform encephalopathies (TSE) at the National Animal Disease Center, Ames, Iowa: an update [abstract]. American Association of Veterinary Laboratory Diagnosticians. p. 169.
Hamir, A.N., Cutlip, R.C., Miller, J.M., Kunkle, R.A., Greenlee, J.J., Richt, J.A. 2006. Cross-species TSE transmission studies at NADC [abstract]. 4th International Veterinary Vaccines and Diagnostics Conference. p. 89. Paper No. PO52.
Hamir, A.N., Kunkle, R.A., Miller, J.M., Bartz, J.C., Richt, J.A. 2005. Experimental transmission of transmissible mink encephalopathy (TME) to cattle by intracerebral inoculation [abstract]. American College of Veterinary Pathologists Meeting. 42:706.
Hamir, A.N., Kunkle, R.A., Miller, J.M., Greenlee, J.J., Richt, J.A. 2006. Experimental transmission of transmissible mink encephalopathy (TME) to cattle by intracerebral inoculation [abstract]. 4th International Veterinary Vaccines and Diagnostics Conference. p. 89. Paper No. PO53.
Nicholson, E.M., Richt, J.A. 2006. Transmissible spongiform encephalopathies: biology and disease. Encyclopedia of Animal Science [online]. 1(1). Available:
http://www.dekker.com/sdek/abstract~db= ... a713617944.
Nicholson, E.M., Richt, J.A. 2003. Transmissible spongiform encephalopathies: detection & diagnosis. Encyclopedia of Animal Science [online]. 1(1). http://www.dekker.com/sdek/abstract~db= ... a713617959.
Richt, J.A., Hamir, A.N., Kunkle, R.A., Nicholson, E.M., Greenlee, J.J., Miller, J.M., Cutlip, R., Davis, A.J., Hall, S.M. 2005. Natural and experimental prion diseases of cattle [abstract]. Third International Rushmore Conference on Enteric Diseases. Paper No. 14.
Richt, J. 2005. TSE research at the National Animal Disease Center. In: Proceedings of the Interagency Working Group (IWG) on Prion Science, August 8, 2005, Beltsville, Maryland. 2005 CDROM.
http://www.ars.usda.gov/research/projec ... 08&fy=2006
Research Project: Transmission, Differentiation, and Pathobiology of Transmissible Spongiform Encephalopathies
Location: Virus and Prion Diseases of Livestock
Title: Experimental Second Passage of Chronic Wasting Disease (Cwd(mule Deer)) Agent to Cattle
Authors
Hamir, Amirali
Kunkle, Robert
Miller, Janice - ARS RETIRED
Greenlee, Justin
Richt, Juergen
Submitted to: Journal of Comparative Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 25, 2005
Publication Date: January 1, 2006
Citation: Hamir, A.N., Kunkle, R.A., Miller, J.M., Greenlee, J.J., Richt, J.A. 2006. Experimental second passage of chronic wasting disease (CWD(mule deer)) agent to cattle. Journal of Comparative Pathology. 134(1):63-69.
Interpretive Summary: To compare the findings of experimental first and second passage of chronic wasting disease (CWD) in cattle, 6 calves were inoculated into the brain with CWD-mule deer agent previously (first) passaged in cattle. Two other uninoculated calves served as controls. Beginning 10-12 months post inoculation (PI), all inoculates lost appetite and weight. Five animals subsequently developed clinical signs of central nervous system (CNS) abnormality. By 16.5 months PI, all cattle had been euthanized because of poor prognosis. None of the animals showed microscopic lesions of spongiform encephalopathy (SE) but the CWD agent was detected in their CNS tissues by 2 laboratory techniques (IHC and WB). These findings demonstrate that inoculated cattle amplify CWD agent but also develop clinical CNS signs without manifestation of microscopic lesions of SE. This situation has also been shown to occur following inoculation of cattle with another TSE agent, namely, sheep scrapie. The current study confirms previous work that indicates that the diagnostic tests currently used for confirmation of bovine spongiform encephalopathy (BSE) in the U.S. would detect CWD in cattle, should it occur naturally. Furthermore, it raises the possibility of distinguishing CWD from BSE in cattle due to the absence of microscopic lesions and a unique multifocal distribution of PrPres, as demonstrated by IHC, which in this study, appears to be more sensitive than the WB.
Technical Abstract: To compare clinicopathological findings of first and second passage of chronic wasting disease (CWD) in cattle, a group of calves (n=6) were intracerebrally inoculated with CWD-mule deer agent previously (first) passaged in cattle. Two other uninoculated calves served as controls. Beginning 10-12 months post inoculation (PI), all inoculates lost appetite and lost weight. Five animals subsequently developed clinical signs of central nervous system (CNS) abnormality. By 16.5 months PI, all cattle had been euthanized because of poor prognosis. None of the animals showed microscopic lesions of spongiform encephalopathy (SE) but PrPres was detected in their CNS tissues by immunohistochemistry (IHC) and Western blot (WB) techniques. These findings demonstrate that intracerebrally inoculated cattle not only amplify CWD PrPres but also develop clinical CNS signs without manifestation of morphologic lesions of SE. This situation has also been shown to occur following inoculation of cattle with another TSE agent, scrapie. The current study confirms previous work that indicates the diagnostic techniques currently used for confirmation of bovine spongiform encephalopathy (BSE) in the U.S. would detect CWD in cattle, should it occur naturally. Furthermore, it raises the possibility of distinguishing CWD from BSE in cattle due to the absence of neuropathologic lesions and a unique multifocal distribution of PrPres, as demonstrated by IHC, which in this study, appears to be more sensitive than the WB.
http://www.ars.usda.gov/research/public ... 115=178318
Research Project: Characterization of Strains of Chronic Wasting Disease in North American Cervids
Location: Virus and Prion Diseases of Livestock
2006 Annual Report
4d.Progress report.
This report serves to document research conducted under a specific cooperative agreement between ARS and the Institute for Animal Health (IAH), Neuropathogenesis Unit (NPU), Edinburgh, UK. Additional details of research can be found in the report for the parent project 3625-32000-073-00D, Bovine Spongiform Encephalopathy and other Transmissible Spongiform Encephalopathies (TSEs). The aim of the cooperative research project conducted by ARS and the IAH in Edinburgh is to characterize strains of chronic wasting disease (CWD). Five brain samples of CWD received from the NADC were injected: two from white tailed deer, two from North American Elk; and one from a mule deer. The samples were injected into panels of inbred mouse strains (RIII, C57BL, VM and C57BLxVM). The first elk sample was injected in September 2003; the mule deer sample was injected in November 2003. Both experiments are still ongoing since some animals in both experimental groups are still alive >1000 days post inoculation. The two white tailed deer samples and a second elk sample were injected in February 2005. These transmission experiments are still ongoing and the mice continue to be maintained and clinically monitored.
http://www.ars.usda.gov/research/projec ... ue&fy=2006
Title: Experimental Transmission of Chronic Wasting Disease Agent to Cattle by Intracerebral Route
Authors
Hamir, Amirali
Kunkle, Robert
Cutlip, Randall - ARS RETIRED
Miller, Janice - ARS RETIRED
O`rourke, Katherine
Williams, Elizabeth - UNIVERSITY OF WYOMING
Miller, Michael - COLORADO DIV WILDLIFE
Stack, Mick - VET SERVICES AGENCY, UK
Chaplin, Melanie - VET SERVICES AGENCY, UK
Richt, Juergen
Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 3, 2005
Publication Date: May 1, 2005
Citation: Hamir, A.N., Kunkle, R.A., Cutlip, R.C., Miller, J.M., Orourke, K.I., Williams, E.S., Miller, M.W., Stack, M.J., Chaplin, M.J., Richt, J. 2005. Experimental transmission of chronic wasting disease agent to cattle by intracerebral route. Journal of Veterinary Diagnostic Investigation. 17:276-281.
Interpretive Summary: This communication reports final observations on experimental transmission of chronic wasting disease (CWD) from mule deer to cattle. Thirteen calves were inoculated into the brain with brain suspension from mule deer naturally affected with CWD. Three other calves were kept as uninoculated controls. The experiment was terminated 6 years post inoculation (PI). During that time, abnormal prion protein was demonstrated in the brain and spinal cord of 5 cattle by laboratory tests. However, consistent clinical signs and microscopic changes were not seen in any of these cattle. Age related changes were seen in both inoculated and control cattle. Findings of this study show that only 38% of the inoculated cattle were positive for CWD agent. Although inoculation directly into the brain is an unnatural route of exposure, and is the most severe challenge possible, this experiment shows that CWD transmission in cattle could have long incubation periods (up to 5 years). This finding suggests that oral exposure of cattle to CWD agent, a more natural potential route of exposure, would require not only a much larger dose of inoculum, but also, may not result in amplification of CWD agent within brain and spinal cord tissues during the normal lifespan of cattle. It is possible that a second bovine passage of material (cattle brain infected with CWD) from this study may result in a larger incidence of affected cattle with a shortened incubation time, and may produce different clinical and pathological findings. Such a study is now in progress. Also, experimental inoculations of cattle with CWD isolates from white-tailed deer and elk are needed to compare findings with the present study and these studies will be initiated in the near future. Impact: Results of this study show that although cattle inoculated directly into the brain with CWD succumb to the disease, the attack rate was rather small (38%) with this unnatural route of transmission. It is speculated that the oral route of infection may not result in replication of the agent during normal lifespan of cattle.
Technical Abstract: This communication reports final observations on experimental transmission of chronic wasting disease (CWD) from mule deer to cattle by the intracerebral route. Thirteen calves were inoculated intracerebrally with brain suspension from mule deer naturally affected with CWD. Three other calves were kept as uninoculated controls. The experiment was terminated 6 years post inoculation (PI). During that time, abnormal prion protein (PrPres) was demonstrated in the central nervous system (CNS) of 5 cattle by both immunohistochemistry (IHC) and Western blot (WB). However, microscopic lesions suggestive of spongiform encephalopathy in the brains of these PrPres positive animals were subtle in 3 cases and absent in 2 cases. The 3 uninoculated control cattle and 8 other inoculated animals euthanized during this time did not have PrPres in their CNS. Degenerative changes indicative of neuroaxonal dystrophy (NAD) were seen in dorsal medulla oblongata and appeared to be related to advancing age in both inoculated and control cattle. Analysis of the gene encoding bovine PRNP revealed homozygosity for alleles encoding 6 octapeptide repeats, serine (S) at codon 46 and S at codon 146 in all samples. Findings of this study show that although PrPres amplification occurred following direct inoculation into the brain, none of the affected animals had classic histopathologic lesions of spongiform encephalopathy. Furthermore, only 38% of the inoculated cattle demonstrated amplification of PrPres. Although intracerebral inoculation is an unnatural route of exposure, and is the most severe challenge possible, this experiment shows that CWD transmission in cattle could have long incubation periods (up to 5 years). This finding suggests that oral exposure of cattle to CWD agent, a more natural potential route of exposure, would require not only a much larger dose of inoculum, but also, may not result in amplification of PrPres within CNS tissues during the normal lifespan of cattle.
http://www.ars.usda.gov/research/public ... 115=166311
TSS
Subject: RESEARCH PROJECT: TRANSMISSION, DIFFERENTIATION, AND PATHOBIOLOGY OF TSE
Date: January 13, 2007 at 2:25 pm PST
Research Project: Transmission, Differentiation, and Pathobiology of Transmissible Spongiform Encephalopathies
Location: Virus and Prion Diseases of Livestock
2006 Annual Report
1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter?
This project is aligned to National Program (NP) 103 - Animal Health. This project deals with Transmissible Spongiform Encephalopathies (TSE), which are fatal degenerative diseases of the central nervous system that can affect several animal species, including humans. The causal agent is believed to be a cellular protein, the prion protein (PrP) that has assumed an unnatural form. Because the altered protein is resistant to protease degradation, it accumulates in nervous tissue and the resulting dysfunction ultimately leads to death. The specific TSEs being investigated in this project are scrapie in sheep, transmissible mink encephalopathy (TME), bovine spongiform encephalopathy (BSE) in cattle, and chronic wasting disease (CWD) in deer and elk. The major concern about these diseases is that BSE has been shown to cross the species barrier to cause a unique TSE in human beings. Although it has not been demonstrated that scrapie, TME, or CWD present any risk to human health, the BSE experience has raised many questions about the potential hazard these TSEs present for transmission to other animal species, especially domesticated livestock and wildlife. The current research is focused on direct experimental challenge of the species barrier effect by animal inoculation. These results are then compared to results obtained with a variety of laboratory procedures to determine if they can be used as predictive models for future risk assessments. These studies also provide information about the clinical and pathological disease characteristics that can be expected if a TSE crosses the species barrier, thus enabling animal health specialists to recognize such situations should they occur. Additional transmission studies focus on (i) determining the modes of transmission and disease development in scrapie of sheep and CWD of cervids, (ii) on the evaluation of changes in the retina of sheep infected with the scrapie agent. The latter studies might enable us to design appropriate intervention or ante mortem diagnostic tools that might enable us to devise strategies to control the spread of these diseases.
Because of the risk for BSE transmission to human beings, the presence of scrapie, TME, CWD, and BSE in the United States presents a potential liability to the U.S. livestock and hunting industries, because the safety of animals and animal products intended for domestic consumption and international trade may be questioned. Efforts are being made by both federal and state regulatory agencies to eradicate scrapie and CWD and to determine the prevalence of BSE. The effectiveness of these programs will depend heavily on having accurate information about the nature of these diseases, not only in the original hosts, but also in other species that may be affected.
The impact of this research project on U.S. agriculture, especially the cattle industry, is significant. The discovery of one BSE positive animal in December 2003 in Washington State resulted in significant losses to the U.S. beef industry. More than 50 countries (including major markets such as Japan and South Korea) banned import of U.S. cattle and beef products within days of the December 2003 announcement. Estimated losses arising from these bans during 2004 range from $3.2 billion to $4.7 billion. Many of these bans are still in effect in 2006 and with the discovery of a third case of BSE (second indigenous BSE case in the U.S.) in March 2006, significant losses from export bans of U.S. beef will most likely be also experienced also in 2006. In addition, the regulations introduced in 2004 led to changes in the beef industry with a net economic cost of approximately $200 million in 2004 only.
2.List by year the currently approved milestones (indicators of research progress)
Animal experiments:
Because of the long-term nature of most TSE experiments in animals, especially those involving cross-species transmission, most of the studies outlined in this project will not be completed within 30 months. Therefore, the following time line primarily presents expectations for when experiments will be initiated.
2003 Cattle inoculated with white-tailed deer CWD Fallow and white-tailed deer inoculated with CWD Cattle inoculated with TME Sheep inoculated with AV136QR171 and Idaho scrapie isolates
2004 Cattle inoculated with elk CWD Raccoons inoculated with TME, scrapie, and CWD isolates Mice inoculated for strain typing of 10 TSE isolates Swine inoculated with scrapie and CWD Reindeer inoculated with CWD White-tailed deer inoculated with scrapie
2005 Study to assess scrapie and CWD amplification in market age swine completed Mice inoculated for strain typing of 15 TSE isolates
2006 Initiate mouse bioassay of various TSE isolates to determine if this strain typing procedure is useful for identification of unique TSE strains in the U.S. Initiate cattle inoculation with both U.S. BSE isolates in order to amplify BSE material for subsequent pathogenesis studies. Inoculation of reindeer with the CWD agents derived from white-tailed deer, mule deer and elk. Initiate oral pathogenesis studies of raccoons with TME.
Laboratory studies: 2003 Validation of method for genotyping from paraffin sections. Methods developed for biochemical strain typing studies.
2004 Genotyping of archived scrapie tissues from the National Veterinary Services Laboratories (NVSL)/APHIS/VS/USDA. Evaluation of biochemical methods for strain typing of scrapie isolates. Development of mass spectrometry methods for characterization of protein expression in normal sheep brain.
2005 Evaluation of biochemical methods for differentiation of TSE agents after cross-species transmission. Comparison of protein expression (mass spectrometry) in scrapie brain to that in normal sheep brain.
2006 Determination of the feasibility of PrP**Sc detection from formalin fixed tissues. Evaluate methods of biochemical strain typing of TSE isolates.
4a.List the single most significant research accomplishment during FY 2006.
Identification and characterization of third U.S. BSE case: Studies were conducted which confirmed the BSE diagnosis of an inconclusive bovine brain sample. The PrP**res profile from the third BSE case diagnosed in the United States showed different molecular properties when compared to the PrP**res pattern described for the 2003 U.S. isolate, however had similar molecular properties as described for the second U.S. BSE case. This finding and our findings from the two previous cases of BSE support the presence of only atypical BSE in the U.S. indigenous cattle population.
This work is of critical relevance to the National Program Action Plan, Animal Health (103), program component pathogen detection, because it had an important impact on the confirmation of an inconclusive BSE diagnosis (based on a rapid test) and the incidence of BSE in the U.S. cattle population.
4b.List other significant research accomplishment(s), if any.
Intracerebral transmission of CWD from white-tailed deer to cattle: Tissues were collected from cattle inoculated intracerebrally with CWD from white-tailed deer (WTD) at the termination of a 4-year study. The WTD CWD attack rate (7/8) in cattle was higher and the incubation time shorter than that previously seen in cattle challenged intracerebrally with mule deer CWD (3/8). The absence of lesions of spongiform encephalopathy, the prion accumulation as detected by immunohistochemistry and the Western blot profiles were similar to those observed in cattle inoculated with mule deer and elk derived CWD.
Intracerebral transmission of mule deer CWD into sheep: Tissues were collected from sheep at the termination of the 6-year study to assess transmission of mule deer CWD to sheep by intracerebral inoculation. These tissues were analyzed by histopathology, immunohistochemistry and Western blot. Two of eight inoculated sheep had spongiform encephalopathy with prion accumulation detected by immunohistochemistry and Western blot. Results of analyses indicated a prion disease in sheep indistinguishable from sheep scrapie.
Intracerebral transmission of CWD from elk, mule-deer, and white-tailed deer into white-tailed deer: Tissues were collected from white-tailed deer inoculated intracerebrally with mule deer, elk, and white-tailed deer CWD at the termination of a 3-year study. The clinical course, lesions of spongiform encephalopathy, prion accumulation detected by immunohistochemistry and Western blot profiles did not differ between treatment groups. The results suggest that the host-species source of CWD does not affect the outcome of CWD presentation in white-tailed deer after intracerebral inoculation.
Transmission of scrapie and CWD to swine: Tissues from swine inoculated with scrapie and CWD by intracerebral and oral routes were collected at 6 months post-inoculation and were analyzed by histopathology, immunohistochemistry and Western blot. There were no ante mortem clinical signs suggestive of neurologic disease, no histologic lesions suggestive of spongiform encephalopathy, and no indication of prion accumulation by immunohistochemistry and Western blot. Monitoring of a replicate of inoculated littermates will continue until the termination of the study in approximately 5 years.
Retinal Pathology in sheep with scrapie: A study was completed which demonstrated that the retina responds to the accumulation to abnormal prion protein in a way that may affect visual system function. It could be shown that specific cell populations of the retina were affected in scrapie-infected sheep.
Relevance to ARS National Program Action Plan, Animal Health (103): Above described research accomplishments have a critical impact on pathogen detection, epidemiology of disease, host/pathogen interactions, genetic resistance to disease and disease control strategies. In particular, these studies also provide information about the clinical and pathological disease characteristics that can be expected if a TSE crosses the species barrier, thus enabling animal health specialists to recognize such situations should they occur. Transmission studies focusing on the mode of TSE transmission and disease development and on the evaluation of changes in the retina of animals infected with TSEs might enable us to design appropriate intervention or ante mortem diagnostic tools that might enable us to devise strategies to control the spread of these fatal diseases.
4c.List significant activities that support special target populations.
None.
5.Describe the major accomplishments to date and their predicted or actual impact.
This project was initiated January 28, 2002, as a result of the FY 2002 Appropriations Bill passed by Congress and signed by the President for research on Emerging & Exotic Diseases of Pests. The major objectives are to assess transmissibility of the TSEs that affect livestock and wildlife species, to develop methods for differentiation of TSE strains, and to determine the pathobiology of these diseases in the natural host and after cross-species transmission. Such studies are of utmost importance to the ARS National Program Action Plan, Animal Health (103), which includes pathogen detection, epidemiology of disease, host/pathogen interactions, genetic resistance to disease, and disease control strategies. Experiments are in progress to determine the transmissibility of different CWDs into white-tailed deer, cattle, sheep and swine. Based on results of our studies, it may be concluded that under natural conditions cattle exposed to mule deer CWD would require a rather large dose of inoculum, and an extremely long incubation time to develop a TSE-associated disease. In contrast, intracerebral inoculation of cattle with brain material from white-tailed deer results in a higher incidence rate (7/8 animals, 88%) compared to mule deer CWD (3/8 animals, 38%). In addition, intracerebral inoculation of cattle with brain material from mink with TME or cattle-passaged TME resulted in incubation times, lesions of spongiform encephalopathy and PrP**res distribution which resemble those found in cattle infected with BSE. Transmission of scrapie and CWD to swine revealed that CWD- or scrapie-infected market-aged swine (6-month-old) showed no ante mortem clinical signs, no histologic lesions suggestive of spongiform encephalopathy, and no indication of prion accumulation by immunohistochemistry and Western blot. We were able to compare the first U.S. BSE isolate (2003) with other typical BSE isolates (Canadian, European) and found them to be indistinguishable. In contrast, the molecular phenotype of the second and third U.S. BSE isolate (11/2004, diagnosed 06/2005; 03/2006) was different from the 2003 U.S. BSE isolate as determined by Western blot analysis. Completed work has determined that sheep scrapie can be transmitted to elk and that the resulting disease is indistinguishable from CWD in that species. When elk CWD was transmitted orally to elk of different genotypes, one genotype (LL) seems to be less susceptible to CWD. Biopsy of the gut-associated lymphoid tissue (GALT) present at the rectal-anal junction revealed that 3 of the 4 remaining LL elk contained PrP-Sc positive cells. White-tailed deer have been inoculated with CWD from elk, mule deer and white-tailed deer in order to determine if strain differences in the CWD agent exist that depend on the host of origin. Upon completion (6 years post infection) of a study with sheep intracerebrally inoculated with mule deer CWD revealed that two of eight inoculated sheep had spongiform encephalopathy with prion accumulation detected by immunohistochemistry and Western blot. The prion disease observed in sheep infected with mule deer CWD was indistinguishable from scrapie. These cross-species transmission experiments will provide information and tissues that can be used to evaluate the effectiveness of current diagnostic protocols for the TSEs. An important contribution of this project has been the demonstration of the utility of raccoons as an animal model to distinguish scrapie, CWD and TME based on attack rate and time to clinical disease. Raccoons inoculated with TME develop disease within 6 months time and as such represent one of the fastest available, non-transgenic models of TSE disease. We developed a method to extract DNA from formalin-fixed paraffin-embedded brainstem tissue to determine prion gene polymorphisms in scrapie-affected sheep. This method has been successfully transferred to APHIS and might have important implications for future scrapie surveillance efforts.
6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products?
Two Cooperative Research and Development Agreements (CRADAs) were established with industry.
7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below).
Invited lecture presented at the NAEBA meeting on "Chronic Wasting Disease Research at the NADC".
Invited lecture presented at the German TSE Platform Meeting titled "Natural and Experimental Prion Diseases of Cattle".
Invited lecture presented at Interagency Working Group (IWG) on Prion Science on "TSE Research at the National Animal Disease Center".
Invited lecture presented at OIE meeting "Prion Diseases of Domestic Livestock" titled "Identification and Characterization of U.S. BSE Cases".
Invited lecture presented at the Third International Rushmore Conference on "Natural and Experimental Prion Diseases of Cattle".
Invited lecture presented at the AAVLD Meeting titled "Identification and Characterization of U.S. BSE cases".
Invited to participate in a workshop held in Saskatoon, Canada, on February 8, 2006, to establish research priorities for PrioNet Canada and the Alberta Prion Research Institute (APRI) with respect to chronic wasting disease (CWD). Presented a seminar at the workshop titled "Transmissible Spongiform Encephalopathy (TSE) studies at the National Animal Disease Center NADC, Agricultural Research Service."
Review Publications
Hamir, A.N., Gidlewski, T., Spraker, T.R., Miller, J.M., Creekmore, L., Crocheck, M., Cline, T., Orourke, K.I. 2006. Preliminary observations of genetic susceptibility of elk (Cervus elaphus nelsoni) to chronic wasting disease by experimental oral inoculation. Journal of Veterinary Diagnostic Investigation. 18(1):110-114.
Hamir, A.N., Kunkle, R.A., Miller, J.M., Bartz, J.C., Richt, J.A. 2006. First and second cattle passage of transmissible mink encephalopathy (TME) by intracerebral inoculation. Veterinary Pathology. 43(2):118-126.
Hamir, A.N., Kunkle, R.A., Miller, J.M., Greenlee, J.J., Richt, J.A. 2006. Experimental second passage of chronic wasting disease (CWD(mule deer)) agent to cattle. Journal of Comparative Pathology. 134(1):63-69.
Hamir, A.N., Kunkle, R.A., Miller, J.M., Hall, S.M. 2006. Abnormal prion protein in ectopic lymphoid tissue in a kidney of an asymptomatic white-tailed deer experimentally inoculated with the agent of chronic wasting disease. Veterinary Pathology. 43(3):367-369.
Hamir, A.N., Kunkle, R.A., Miller, J.M., Richt, J.A. 2005. Second passage of sheep scrapie and transmissible mink encephalopathy (TME) agents in raccoons (Procyon lotor). Veterinary Pathology. 42(6):844-851.
Bessen, R.A., Dejoia, C., Dlakic, W., Sorg, R., O¿Connell, K., Tucker, T., Kunkle, R.A., Hamir, A.N., Richt, J.A. 2005. Prion infection of mucosal tissue [abstract]. TSE Forum, Prion 2005: Between Fundamentals and Society's Needs. p. 31.
Greenlee, J.J., Kunkle, R.A., Hamir, A.N. 2005. Experimental intracerebral and oral inoculation of scrapie to swine: preliminary report [abstract]. Proceedings of the American Association of Veterinary Laboratory Diagnosticians 48th Annual Conference. p. 38.
Hall, M.S., Richt, J.A., Davis, A.J., Levings, R.L. 2005. Where we've been and where we're going with BSE testing in the United States [abstract]. 48th Annual Meeting of the American Association of Veterinary Laboratory Diagnosticians. p. 20.
Hall, S.M., Richt, J., Davis, A., Kluge, J., Simmons, M., Stack, M., Spencer, Y. 2006. Identification and characterization of U.S. BSE cases [abstract]. Prion Diseases of Domestic Livestock. p. 25.
Hamir, A.N., Cutlip, R.C., Miller, J.M., Kunkle, R.A., Greenlee, J.J., Richt, J.A. 2005. Experimental transmission of transmissible spongiform encephalopathies (TSE) at the National Animal Disease Center, Ames, Iowa: an update [abstract]. American Association of Veterinary Laboratory Diagnosticians. p. 169.
Hamir, A.N., Cutlip, R.C., Miller, J.M., Kunkle, R.A., Greenlee, J.J., Richt, J.A. 2006. Cross-species TSE transmission studies at NADC [abstract]. 4th International Veterinary Vaccines and Diagnostics Conference. p. 89. Paper No. PO52.
Hamir, A.N., Kunkle, R.A., Miller, J.M., Bartz, J.C., Richt, J.A. 2005. Experimental transmission of transmissible mink encephalopathy (TME) to cattle by intracerebral inoculation [abstract]. American College of Veterinary Pathologists Meeting. 42:706.
Hamir, A.N., Kunkle, R.A., Miller, J.M., Greenlee, J.J., Richt, J.A. 2006. Experimental transmission of transmissible mink encephalopathy (TME) to cattle by intracerebral inoculation [abstract]. 4th International Veterinary Vaccines and Diagnostics Conference. p. 89. Paper No. PO53.
Nicholson, E.M., Richt, J.A. 2006. Transmissible spongiform encephalopathies: biology and disease. Encyclopedia of Animal Science [online]. 1(1). Available:
http://www.dekker.com/sdek/abstract~db= ... a713617944.
Nicholson, E.M., Richt, J.A. 2003. Transmissible spongiform encephalopathies: detection & diagnosis. Encyclopedia of Animal Science [online]. 1(1). http://www.dekker.com/sdek/abstract~db= ... a713617959.
Richt, J.A., Hamir, A.N., Kunkle, R.A., Nicholson, E.M., Greenlee, J.J., Miller, J.M., Cutlip, R., Davis, A.J., Hall, S.M. 2005. Natural and experimental prion diseases of cattle [abstract]. Third International Rushmore Conference on Enteric Diseases. Paper No. 14.
Richt, J. 2005. TSE research at the National Animal Disease Center. In: Proceedings of the Interagency Working Group (IWG) on Prion Science, August 8, 2005, Beltsville, Maryland. 2005 CDROM.
http://www.ars.usda.gov/research/projec ... 08&fy=2006
Research Project: Transmission, Differentiation, and Pathobiology of Transmissible Spongiform Encephalopathies
Location: Virus and Prion Diseases of Livestock
Title: Experimental Second Passage of Chronic Wasting Disease (Cwd(mule Deer)) Agent to Cattle
Authors
Hamir, Amirali
Kunkle, Robert
Miller, Janice - ARS RETIRED
Greenlee, Justin
Richt, Juergen
Submitted to: Journal of Comparative Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 25, 2005
Publication Date: January 1, 2006
Citation: Hamir, A.N., Kunkle, R.A., Miller, J.M., Greenlee, J.J., Richt, J.A. 2006. Experimental second passage of chronic wasting disease (CWD(mule deer)) agent to cattle. Journal of Comparative Pathology. 134(1):63-69.
Interpretive Summary: To compare the findings of experimental first and second passage of chronic wasting disease (CWD) in cattle, 6 calves were inoculated into the brain with CWD-mule deer agent previously (first) passaged in cattle. Two other uninoculated calves served as controls. Beginning 10-12 months post inoculation (PI), all inoculates lost appetite and weight. Five animals subsequently developed clinical signs of central nervous system (CNS) abnormality. By 16.5 months PI, all cattle had been euthanized because of poor prognosis. None of the animals showed microscopic lesions of spongiform encephalopathy (SE) but the CWD agent was detected in their CNS tissues by 2 laboratory techniques (IHC and WB). These findings demonstrate that inoculated cattle amplify CWD agent but also develop clinical CNS signs without manifestation of microscopic lesions of SE. This situation has also been shown to occur following inoculation of cattle with another TSE agent, namely, sheep scrapie. The current study confirms previous work that indicates that the diagnostic tests currently used for confirmation of bovine spongiform encephalopathy (BSE) in the U.S. would detect CWD in cattle, should it occur naturally. Furthermore, it raises the possibility of distinguishing CWD from BSE in cattle due to the absence of microscopic lesions and a unique multifocal distribution of PrPres, as demonstrated by IHC, which in this study, appears to be more sensitive than the WB.
Technical Abstract: To compare clinicopathological findings of first and second passage of chronic wasting disease (CWD) in cattle, a group of calves (n=6) were intracerebrally inoculated with CWD-mule deer agent previously (first) passaged in cattle. Two other uninoculated calves served as controls. Beginning 10-12 months post inoculation (PI), all inoculates lost appetite and lost weight. Five animals subsequently developed clinical signs of central nervous system (CNS) abnormality. By 16.5 months PI, all cattle had been euthanized because of poor prognosis. None of the animals showed microscopic lesions of spongiform encephalopathy (SE) but PrPres was detected in their CNS tissues by immunohistochemistry (IHC) and Western blot (WB) techniques. These findings demonstrate that intracerebrally inoculated cattle not only amplify CWD PrPres but also develop clinical CNS signs without manifestation of morphologic lesions of SE. This situation has also been shown to occur following inoculation of cattle with another TSE agent, scrapie. The current study confirms previous work that indicates the diagnostic techniques currently used for confirmation of bovine spongiform encephalopathy (BSE) in the U.S. would detect CWD in cattle, should it occur naturally. Furthermore, it raises the possibility of distinguishing CWD from BSE in cattle due to the absence of neuropathologic lesions and a unique multifocal distribution of PrPres, as demonstrated by IHC, which in this study, appears to be more sensitive than the WB.
http://www.ars.usda.gov/research/public ... 115=178318
Research Project: Characterization of Strains of Chronic Wasting Disease in North American Cervids
Location: Virus and Prion Diseases of Livestock
2006 Annual Report
4d.Progress report.
This report serves to document research conducted under a specific cooperative agreement between ARS and the Institute for Animal Health (IAH), Neuropathogenesis Unit (NPU), Edinburgh, UK. Additional details of research can be found in the report for the parent project 3625-32000-073-00D, Bovine Spongiform Encephalopathy and other Transmissible Spongiform Encephalopathies (TSEs). The aim of the cooperative research project conducted by ARS and the IAH in Edinburgh is to characterize strains of chronic wasting disease (CWD). Five brain samples of CWD received from the NADC were injected: two from white tailed deer, two from North American Elk; and one from a mule deer. The samples were injected into panels of inbred mouse strains (RIII, C57BL, VM and C57BLxVM). The first elk sample was injected in September 2003; the mule deer sample was injected in November 2003. Both experiments are still ongoing since some animals in both experimental groups are still alive >1000 days post inoculation. The two white tailed deer samples and a second elk sample were injected in February 2005. These transmission experiments are still ongoing and the mice continue to be maintained and clinically monitored.
http://www.ars.usda.gov/research/projec ... ue&fy=2006
Title: Experimental Transmission of Chronic Wasting Disease Agent to Cattle by Intracerebral Route
Authors
Hamir, Amirali
Kunkle, Robert
Cutlip, Randall - ARS RETIRED
Miller, Janice - ARS RETIRED
O`rourke, Katherine
Williams, Elizabeth - UNIVERSITY OF WYOMING
Miller, Michael - COLORADO DIV WILDLIFE
Stack, Mick - VET SERVICES AGENCY, UK
Chaplin, Melanie - VET SERVICES AGENCY, UK
Richt, Juergen
Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 3, 2005
Publication Date: May 1, 2005
Citation: Hamir, A.N., Kunkle, R.A., Cutlip, R.C., Miller, J.M., Orourke, K.I., Williams, E.S., Miller, M.W., Stack, M.J., Chaplin, M.J., Richt, J. 2005. Experimental transmission of chronic wasting disease agent to cattle by intracerebral route. Journal of Veterinary Diagnostic Investigation. 17:276-281.
Interpretive Summary: This communication reports final observations on experimental transmission of chronic wasting disease (CWD) from mule deer to cattle. Thirteen calves were inoculated into the brain with brain suspension from mule deer naturally affected with CWD. Three other calves were kept as uninoculated controls. The experiment was terminated 6 years post inoculation (PI). During that time, abnormal prion protein was demonstrated in the brain and spinal cord of 5 cattle by laboratory tests. However, consistent clinical signs and microscopic changes were not seen in any of these cattle. Age related changes were seen in both inoculated and control cattle. Findings of this study show that only 38% of the inoculated cattle were positive for CWD agent. Although inoculation directly into the brain is an unnatural route of exposure, and is the most severe challenge possible, this experiment shows that CWD transmission in cattle could have long incubation periods (up to 5 years). This finding suggests that oral exposure of cattle to CWD agent, a more natural potential route of exposure, would require not only a much larger dose of inoculum, but also, may not result in amplification of CWD agent within brain and spinal cord tissues during the normal lifespan of cattle. It is possible that a second bovine passage of material (cattle brain infected with CWD) from this study may result in a larger incidence of affected cattle with a shortened incubation time, and may produce different clinical and pathological findings. Such a study is now in progress. Also, experimental inoculations of cattle with CWD isolates from white-tailed deer and elk are needed to compare findings with the present study and these studies will be initiated in the near future. Impact: Results of this study show that although cattle inoculated directly into the brain with CWD succumb to the disease, the attack rate was rather small (38%) with this unnatural route of transmission. It is speculated that the oral route of infection may not result in replication of the agent during normal lifespan of cattle.
Technical Abstract: This communication reports final observations on experimental transmission of chronic wasting disease (CWD) from mule deer to cattle by the intracerebral route. Thirteen calves were inoculated intracerebrally with brain suspension from mule deer naturally affected with CWD. Three other calves were kept as uninoculated controls. The experiment was terminated 6 years post inoculation (PI). During that time, abnormal prion protein (PrPres) was demonstrated in the central nervous system (CNS) of 5 cattle by both immunohistochemistry (IHC) and Western blot (WB). However, microscopic lesions suggestive of spongiform encephalopathy in the brains of these PrPres positive animals were subtle in 3 cases and absent in 2 cases. The 3 uninoculated control cattle and 8 other inoculated animals euthanized during this time did not have PrPres in their CNS. Degenerative changes indicative of neuroaxonal dystrophy (NAD) were seen in dorsal medulla oblongata and appeared to be related to advancing age in both inoculated and control cattle. Analysis of the gene encoding bovine PRNP revealed homozygosity for alleles encoding 6 octapeptide repeats, serine (S) at codon 46 and S at codon 146 in all samples. Findings of this study show that although PrPres amplification occurred following direct inoculation into the brain, none of the affected animals had classic histopathologic lesions of spongiform encephalopathy. Furthermore, only 38% of the inoculated cattle demonstrated amplification of PrPres. Although intracerebral inoculation is an unnatural route of exposure, and is the most severe challenge possible, this experiment shows that CWD transmission in cattle could have long incubation periods (up to 5 years). This finding suggests that oral exposure of cattle to CWD agent, a more natural potential route of exposure, would require not only a much larger dose of inoculum, but also, may not result in amplification of PrPres within CNS tissues during the normal lifespan of cattle.
http://www.ars.usda.gov/research/public ... 115=166311
TSS
