terry
12-05-2006, 10:27 AM
Sent: Monday, December 04, 2006 10:23 PM
Subject: Transmission, Differentiation, and Pathobiology of Transmissible Spongiform Encephalopathies 2005 Annual Report
Research Project: Transmission, Differentiation, and Pathobiology of Transmissible Spongiform Encephalopathies
Location: Virus and Prion Diseases of Livestock
2005 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? What does it matter?
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 in the natural host will focus on determining the modes of transmission and disease development in scrapie and CWD so that appropriate intervention strategies can be devised that will 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 2005 and with the discovery of a second, indigenous case of BSE in June 2005, significant losses from export bans of U.S. beef will most likely be also experienced in 2005. 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.
2.List the milestones (indicators of progress) from your Project Plan.
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.
FY 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.
FY 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.
FY 2005 Study to assess scrapie and CWD amplification in market age swine completed. Mice inoculated for strain typing of 15 TSE isolates.
Laboratory studies:
FY 2003 Validation of method for genotyping from paraffin sections. Methods developed for biochemical strain typing studies.
FY 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.
FY 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.
3a.List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why.
Study to assess scrapie and CWD amplification in market age swine completed.
Milestone Fully Met Milestone Substantially Met
Mice inoculated for strain typing of 15 TSE isolates.
Milestone Not Met
Evaluation of biochemical methods for differentiation of TSE agents after cross-species transmission.
Milestone Substantially Met
Comparison of protein expression (mass spectrometry) in scrapie brain to that in normal sheep brain.
Milestone Not Met
3b.List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone?
FY 2006 Mouse bioassay with various TSE isolates to determine if this strain typing procedure is useful for identification of unique TSE strains in the U.S.
Cattle inoculation with both U.S. BSE isolates in order to amplify BSE material for subsequent pathogenesis studies.
Inoculation of reindeer with CWD agent.
FY 2007 and FY 2008 Because the incubation period for TSE transmission is typically 2 years or longer, it is anticipated that the major activity during these years will involve termination of many animal inoculation experiments that began in FY03. The work will include necropsies and tissue analysis to confirm TSE transmissions, followed by laboratory analysis using strain characterization methods developed and evaluated in FY 04 and FY05.
4a.What was the single most significant accomplishment this past year?
Transmissible Mink Encephalopathy (TME) transmission to cattle: Intracerebral inoculation of cattle with brain material from mink with TME and TME from cattle (i.e. from 1st cattle passage) resulted in lesions of spongiform encephalopathy and distribution of the abnormal form of the prion protein, PrPres (as determined by immunohistochemistry and Western blot) in the central nervous system (CNS) which resembles those found in cattle infected with Bovine Spongiform Encephalopathy (BSE). This indicates that TME is able to induce a neurological disease and pathological lesions in cattle which are similar to BSE. Comparison of cattle- passaged TME with both U.S. BSE cases by Western Blot analysis revealed differences in molecular weight. This would allow us to differentiate between cattle infected with BSE and TME. This is a significant finding that might enable us to distinguish BSE in cattle from the other animal TSEs should they ever appear in cattle.
4b.List other significant accomplishments, if any.
Intracerebral transmission of Chronic Wasting Disease (CWD) to white-tailed deer: Under free-ranging conditions, CWD has been observed in elk, mule deer and white-tailed deer. Since there is no information to indicate that CWD from these 3 cervid species is the same (or if these are 3 different strains of cervid CWDs), we inoculated CWD from these 3 sources to 3 groups of white-tailed deer fawns. White-tailed deer were susceptible to all three sources of CWD with similar incubation times. This finding is important in that it indicates that there are no differences in clinical susceptibility of white-tailed deer to CWD from different cervid sources.
Oral transmission of CWD to elk: To compare genetic susceptibility of elk to CWD, eight 8-month-old elk calves of 3 different genotypes (MM, LM and LL) for the prion protein were orally dosed with CWD infected brain material from elk. Elk with the MM and LM genotype developed CWD within 23 and 40 months, respectively, whereas LL elk are still alive and clinically normal at 4 years post inoculation. The finding that elk with the LL genotype have a reduced susceptibility to oral infection with the chronic wasting disease agent is important to elk farmers. This study will continue for additional 2 years.
Scrapie transmission to swine: Swine were inoculated with scrapie-affected sheep brain homogenate via the oral and intracerebral route and necropsied at 6 months post inoculation. No evidence of prion disease was found in these market aged swine. Monitoring of scrapie-inoculated littermates will continue until the termination of the study in approximately 5 years. This work is important for pork producers.
Retinal pathology in sheep with scrapie: A study of retinal pathology in scrapie-affected sheep has been completed. It was demonstrated that cells of the retina in scrapie-affected sheep with prion accumulation in the retina express proteins that indicate retinal degeneration, whereas cells of the normal sheep retina do not express these proteins. The implication of this report is that scrapie-affected sheep may have deficits of the visual system which are detectable by various methods. Laboratory verification of second U.S. BSE case: Studies were conducted which confirmed the BSE diagnosis of an inconclusive bovine brain sample. The PrPres profile from the second BSE case diagnosed in the United States showed different molecular properties when compared to the PrPres pattern described for the 2003 U.S. isolate. A germline mutation in the bovine PrP gene was not evident. This work benefited the APHIS-National Veterinary Services Laboratories.
B cells in sheep scrapie: In scrapie-infected sheep, an over-representation of the B-1 subset of B cells was detected in the peripheral blood. In addition, a significant reduction in the expression of the normal prion protein, PrPc, on B cells, was found. This correlated with the progression of scrapie in these animals. The implication of these findings is that scrapie-affected sheep may have detectable changes in the peripheral blood that may lead to a live animal test.
4c.List any significant activities that support special target populations.
None.
CONTINUED
Subject: Transmission, Differentiation, and Pathobiology of Transmissible Spongiform Encephalopathies 2005 Annual Report
Research Project: Transmission, Differentiation, and Pathobiology of Transmissible Spongiform Encephalopathies
Location: Virus and Prion Diseases of Livestock
2005 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? What does it matter?
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 in the natural host will focus on determining the modes of transmission and disease development in scrapie and CWD so that appropriate intervention strategies can be devised that will 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 2005 and with the discovery of a second, indigenous case of BSE in June 2005, significant losses from export bans of U.S. beef will most likely be also experienced in 2005. 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.
2.List the milestones (indicators of progress) from your Project Plan.
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.
FY 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.
FY 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.
FY 2005 Study to assess scrapie and CWD amplification in market age swine completed. Mice inoculated for strain typing of 15 TSE isolates.
Laboratory studies:
FY 2003 Validation of method for genotyping from paraffin sections. Methods developed for biochemical strain typing studies.
FY 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.
FY 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.
3a.List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why.
Study to assess scrapie and CWD amplification in market age swine completed.
Milestone Fully Met Milestone Substantially Met
Mice inoculated for strain typing of 15 TSE isolates.
Milestone Not Met
Evaluation of biochemical methods for differentiation of TSE agents after cross-species transmission.
Milestone Substantially Met
Comparison of protein expression (mass spectrometry) in scrapie brain to that in normal sheep brain.
Milestone Not Met
3b.List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone?
FY 2006 Mouse bioassay with various TSE isolates to determine if this strain typing procedure is useful for identification of unique TSE strains in the U.S.
Cattle inoculation with both U.S. BSE isolates in order to amplify BSE material for subsequent pathogenesis studies.
Inoculation of reindeer with CWD agent.
FY 2007 and FY 2008 Because the incubation period for TSE transmission is typically 2 years or longer, it is anticipated that the major activity during these years will involve termination of many animal inoculation experiments that began in FY03. The work will include necropsies and tissue analysis to confirm TSE transmissions, followed by laboratory analysis using strain characterization methods developed and evaluated in FY 04 and FY05.
4a.What was the single most significant accomplishment this past year?
Transmissible Mink Encephalopathy (TME) transmission to cattle: Intracerebral inoculation of cattle with brain material from mink with TME and TME from cattle (i.e. from 1st cattle passage) resulted in lesions of spongiform encephalopathy and distribution of the abnormal form of the prion protein, PrPres (as determined by immunohistochemistry and Western blot) in the central nervous system (CNS) which resembles those found in cattle infected with Bovine Spongiform Encephalopathy (BSE). This indicates that TME is able to induce a neurological disease and pathological lesions in cattle which are similar to BSE. Comparison of cattle- passaged TME with both U.S. BSE cases by Western Blot analysis revealed differences in molecular weight. This would allow us to differentiate between cattle infected with BSE and TME. This is a significant finding that might enable us to distinguish BSE in cattle from the other animal TSEs should they ever appear in cattle.
4b.List other significant accomplishments, if any.
Intracerebral transmission of Chronic Wasting Disease (CWD) to white-tailed deer: Under free-ranging conditions, CWD has been observed in elk, mule deer and white-tailed deer. Since there is no information to indicate that CWD from these 3 cervid species is the same (or if these are 3 different strains of cervid CWDs), we inoculated CWD from these 3 sources to 3 groups of white-tailed deer fawns. White-tailed deer were susceptible to all three sources of CWD with similar incubation times. This finding is important in that it indicates that there are no differences in clinical susceptibility of white-tailed deer to CWD from different cervid sources.
Oral transmission of CWD to elk: To compare genetic susceptibility of elk to CWD, eight 8-month-old elk calves of 3 different genotypes (MM, LM and LL) for the prion protein were orally dosed with CWD infected brain material from elk. Elk with the MM and LM genotype developed CWD within 23 and 40 months, respectively, whereas LL elk are still alive and clinically normal at 4 years post inoculation. The finding that elk with the LL genotype have a reduced susceptibility to oral infection with the chronic wasting disease agent is important to elk farmers. This study will continue for additional 2 years.
Scrapie transmission to swine: Swine were inoculated with scrapie-affected sheep brain homogenate via the oral and intracerebral route and necropsied at 6 months post inoculation. No evidence of prion disease was found in these market aged swine. Monitoring of scrapie-inoculated littermates will continue until the termination of the study in approximately 5 years. This work is important for pork producers.
Retinal pathology in sheep with scrapie: A study of retinal pathology in scrapie-affected sheep has been completed. It was demonstrated that cells of the retina in scrapie-affected sheep with prion accumulation in the retina express proteins that indicate retinal degeneration, whereas cells of the normal sheep retina do not express these proteins. The implication of this report is that scrapie-affected sheep may have deficits of the visual system which are detectable by various methods. Laboratory verification of second U.S. BSE case: Studies were conducted which confirmed the BSE diagnosis of an inconclusive bovine brain sample. The PrPres profile from the second BSE case diagnosed in the United States showed different molecular properties when compared to the PrPres pattern described for the 2003 U.S. isolate. A germline mutation in the bovine PrP gene was not evident. This work benefited the APHIS-National Veterinary Services Laboratories.
B cells in sheep scrapie: In scrapie-infected sheep, an over-representation of the B-1 subset of B cells was detected in the peripheral blood. In addition, a significant reduction in the expression of the normal prion protein, PrPc, on B cells, was found. This correlated with the progression of scrapie in these animals. The implication of these findings is that scrapie-affected sheep may have detectable changes in the peripheral blood that may lead to a live animal test.
4c.List any significant activities that support special target populations.
None.
CONTINUED