Thursday, September 24, 2009

Validation of Use of Rectoanal Mucosa-Associated Lymphoid Tissue for Immunohistochemical Diagnosis of Chronic Wasting Disease in White-Tailed Deer

Journal of Clinical Microbiology, May 2009, p. 1412-1417, Vol. 47, No. 5 0095-1137/09/$08.00+0 doi:10.1128/JCM.02209-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Validation of Use of Rectoanal Mucosa-Associated Lymphoid Tissue for Immunohistochemical Diagnosis of Chronic Wasting Disease in White-Tailed Deer (Odocoileus virginianus)

Delwyn Keane,1* Daniel Barr,1 Rebecca Osborn,2 Julie Langenberg,2 Katherine O'Rourke,3 David Schneider,3 and Philip Bochsler1 University of Wisconsin, Wisconsin Veterinary Diagnostic Laboratory, Madison, Wisconsin,1 Wisconsin Department of Natural Resources, Madison, Wisconsin,2 U.S. Department of Agriculture, Agricultural Research Service, Animal Disease Research Unit, 3003 ADBF, Pullman, Washington3

Received 18 November 2008/ Returned for modification 3 January 2009/ Accepted 20 February 2009

The examination of rectoanal mucosa-associated lymphoid tissue (RAMALT) biopsy specimens for the diagnosis of transmissible spongiform encephalopathies has been described in sheep, elk, and small numbers of mule and white-tailed deer. Previous sample numbers have been too small to validate examination of this type of tissue as a viable antemortem diagnostic test. In this study, we examined RAMALT collected postmortem from 76 white-tailed deer removed from a farm in Wisconsin known to be affected by chronic wasting disease (CWD) and from 210 free-ranging white-tailed deer harvested from an area in Wisconsin where the overall prevalence of CWD among the deer was approximately 4 to 6%. The results of immunohistochemical (IHC) staining of the RAMALT sections were compared to the results of IHC staining of sections from the brain stem at the convergence of the dorsal motor nucleus of the vagus nerve, sections of the medial retropharyngeal lymph nodes (RLNs), and sections of tonsil (sections of tonsil only from captive animals were tested). The sensitivities of the IHC staining test with RAMALT sections were 81% for the captive animals and 91% for the free-ranging animals. False-negative results were usually associated with early infection, indicated by a low intensity of immunostaining in the obex and/or a polymorphism at PRNP codon 96. While the RLN remains the tissue of choice for use for the diagnosis of CWD in white-tailed deer, the results of the present study further support the use of RAMALTs collected antemortem as an adjunct to testing of tonsil biopsy specimens and surveillance by necropsy for the screening of farmed deer which have been put at risk through environmental exposure or exposure to deer with CWD.


* Corresponding author. Mailing address: University of Wisconsin, Wisconsin Veterinary Diagnostic Laboratory, 445 Easterday Lane, Madison, WI 53706. Phone: (608) 262-5432. Fax: (847) 574-8085. E-mail:

Published ahead of print on 4 March 2009.


Journal of Clinical Microbiology, May 2009, p. 1412-1417, Vol. 47, No. 5 0095-1137/09/$08.00+0 doi:10.1128/JCM.02209-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved.


Based on the results of our study, the IHC detection of 1 PrPCWD in RAMALT should be considered as a useful tool for the preclinical diagnosis of CWD infection in white tailed deer. It had been previously shown that in white tailed deer, accumulation of PrPCWD 3 occurs in RLN or tonsil prior to accumulation in the obex (11) and the presence of PrPCWD 4 in RLN and tonsil is a reliable marker for the antemortem and preclinical postmortem diagnosis of CWD (26, 29, 30) . Obtaining lymphatic tissues of the head and neck of live animals is not an easy task; tonsil biopsy procedures require general anesthesia, the tonsil is difficult to visualize, biopsy collection requires experienced personnel and the samples tend to be small (4 or 6 mm). As a tool for screening free ranging or captive populations, this technique is not as efficient or as economical as rectal biopsy. The latter procedure can be performed without general anesthesia, visualization of the rectal mucosa is much easier than that of tonsil, and larger samples can be obtained without the need for specialized equipment. However, the diagnosis of CWD using RAMALT, as with tonsillar tissues, is dependent on obtaining adequate samples.



Research Project: Transmissible Spongiform Encephalopathies: the Role of Genetics, Strain Variation, and Environmental Contamination in Disease Control Location: Animal Diseases Research

Title: Validation of Use of Rectoanal Mucosa-Associated Lymphoid Tissue for Immunohistochemical Diagnosis of Chronic Wasting Disease in White-Tailed Deer (Odocoileus virginianus)


Keane, D - UNIV OF WISCONSIN Barr, D - UNIV OF WISCONSIN Osborn, R - WISC DEPT OF NAT RESOURCE Langenberg, J - WISC DEPT OF NAT RESOURCE Orourke, Katherine Schneider, David Bochsler, P - UNIV OF WISCONSIN

Submitted to: Journal of Veterinary Diagnostic Investigation Publication Type: Peer Reviewed Journal Publication Acceptance Date: February 20, 2009 Publication Date: May 1, 2009 Publisher's URL: Reprint URL: Citation: Keane, D., Barr, D., Osborn, R., Langenberg, J., Orourke, K.I., Schneider, D.A., Bochsler, P. 2009. Validation of Use of Rectoanal Mucosa-Associated Lymphoid Tissue for Immunohistochemical Diagnosis of Chronic Wasting Disease in White-Tailed Deer (Odocoileus virginianus). Journal of Veterinary Diagnostic Investigation. 47(5):1412-1417.

Interpretive Summary: The prion diseases are a group of fatal brain disorders of sheep, goats, cattle, deer and elk. An abnormally folded protein accumulates in some lymphoid tissues of sheep early in disease. Biopsy sampling of lymphoid tissue, including tissue in the rectum, is a suitable live animal test in sheep. Adaptation of that test for use in deer exposed to the cervid prion disease Chronic Wasting Disease has been proposed. In this paper, the investigators compared the results of testing rectal tissue with test results on brain and the lymphoid tissues currently used for early diagnosis of the disease. Deer from a captive farm with a high prevalence of disease and wild deer with a low prevalence of disease were included in the study. Nearly eighty percent of the deer with abnormal prions in lymphoid tissue or brain had detectable abnormal prion proteins in the rectal lymphoid tissues. Although lymphoid tissues of the head remain the tissue of choice for early diagnosis of the disease in deer, the use of rectal lymphoid tissue is a suitable adjunct, particularly for live-screening farmed deer at risk for chronic wasting disease. Technical Abstract: The transmissible spongiform encephalopathies are a family of fatal neurodegenerative diseases characterized by accumulation of abnormal prion proteins in the brain. The abnormal prion protein is the major constituent of the infectious agent and is a reliable marker for disease. The occurrence of a zoonotic prion disease in cattle has resulted in efforts to eradicate or control all prion diseases in domestic livestock, including scrapie of sheep and chronic wasting disease CWD of deer and elk. Antemortem testing of sheep, deer and elk is based on the finding that abnormal prion proteins accumulate in some lymphoid tissues months or years before being detectable in brain. Biopsy of tonsil is a suitable test for live deer but requires general anesthesia. Biopsy sampling of the recto-anal mucosal associated lymphoid tissue (RAMALT) has been suggested as an alternative site for antemortem testing in sheep. In this study, postmortem sampling of RAMALT tissue from deer was performed to estimate the diagnostic sensitivity and specificity of the test. Samples were assayed by monoclonal antibody based immunohistochemistry and the results of RAMALT testing were compared with testing of brain, tonsil and retropharyngeal lymph node, the currently preferred tissue for early diagnosis. Sensitivity of the test was 80% in a sample of 76 white tailed deer from a captive facility and 77% in a sample of 210 free ranging white tailed deer. While the retropharyngeal lymph node remains the tissue of choice for early diagnostic testing, RAMALT biopsy may provide a suitable adjunct, particularly for antemortem testing of herds of farmed deer with potential exposure to the disease.

Monday, August 24, 2009 Third International CWD Symposium July 22-24, 2009 – Park City, Utah ABSTRACTS

Antemortem detection of PrPCWD in preclinical, ranch-raised Rocky Mountain elk (Cervus elaphus nelsoni) by biopsy of the rectal mucosa T

erry R. Spraker, Kurt C. VerCauteren, Thomas Gidlewski, David A. Schneider, Randy Munger, Aru Balachandran, Katherine I. O’Rourke1


Antemortem biopsy of the rectal mucosa was evaluated as a method for the preclinical diagnosis of chronic wasting disease (CWD) in a herd of ranch-raised Rocky Mountain elk (Cervus elaphus nelsoni) quarantined because of exposure to CWD. Biopsy samples were obtained from 41 elk during the winter of 2005–2006 and from 26 elk from that herd still alive and available for testing during the winter of 2006–2007. Samples were examined for PrPCWD, the protein marker for CWD infection, by immunohistochemistry. PrPCWD was detected in follicles of the rectoanal mucosa-associated lymphoid tissue in biopsy samples from 1 elk with clinical signs of chronic wasting disease and 5 clinically normal elk. The diagnosis was confirmed in all 6 animals by postmortem analysis of brain and peripheral lymph nodes. PrPCWD was also observed in the submucosal plexus and myenteric plexus of the enteric nervous system, and in close association with nonmyelinated mucosal and submucosal nerve fibers. In antemortem rectal biopsy samples from positive animals, immunostaining was consistently observed in approximately 60% of the mucosa-associated lymphoid tissue follicles if 10 or more total follicles per biopsy were present for evaluation. Most antemortem biopsy samples obtained from elk younger than 6.5 years contained at least 10 follicles per rectal mucosal biopsy. These findings support the analysis of antemortem biopsy of the rectal mucosa samples as part of an integrated strategy to manage chronic wasting disease in Rocky Mountain elk. Key words: Antemortem diagnosis; chronic wasting disease; elk; transmissible spongiform encephalopathy. Introduction Chronic wasting disease (CWD), a transmissible spongiform encephalopathy, has been reported in captive and free-ranging mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus), Rocky Mountain elk (Cervus elaphus nelsoni), and moose (Alces alces shirasi).3,22,26,27 Chronic wasting disease has been a devastating disease in the captive elk industry. An estimated 12,000–14,000 captive elk have been killed in the last 8 years in attempts to control CWD. Several thousand free-ranging mule deer, white-tailed deer, and elk also have been killed through liberalized hunting and targeted sharpshooting in attempts to reduce the disease prevalence in the wild. Since captive cervids cannot be ruled out as a source of CWD infection for free-ranging cervids, eradication of CWD in captive herds is likely to be critical in controlling the spread of this disease. An accurate antemortem diagnostic test to identify preclinical CWD-infected elk is essential to the success of management strategies aiming to control and eradicate CWD. An abnormal isoform (PrPCWD) of the host prion protein is a reliable marker for CWD in white-tailed deer,22 mule deer,23 and Rocky Mountain elk.20 PrPCWD and the corresponding abnormal prion protein PrPSc in sheep accumulate in lymphoid tissues during the prolonged preclinical stage of disease, providing the basis for antemortem testing in these species.21,25,28 Preclinical tests for scrapie in domestic sheep include biopsy of lymphoid tissues from the palatine tonsil,24 third eyelid,14 and rectal mucosa.5,6 Similar preclinical tests have been described for mule deer using biopsy of the palatine tonsil and rectal mucosa.25,28 An immunohistochemical study21 of rectoanal mucosa–associated lymphoid tissue (RAMALT) collected postmortem from elk suggested that antemortem diagnosis may be possible in cases with preclinical disease. The present study assessed the feasibility of obtaining suitable antemortem rectal biopsies of RAMALT from a mixed-age herd of Rocky Mountain elk with known exposure to CWD. From the Colorado State University Diagnostic Laboratory, College of Veterinary Medicine, Colorado State University, Fort Collins, CO (Spraker); the National Wildlife Research Center, Wildlife Services (VerCauteren) and Veterinary Services (Gidlewski, Munger), U.S. Department of Agriculture, Animal and Plant Health Inspection Service, Fort Collins, CO; Agricultural Research Services, U.S. Department of Agriculture, Pullman, WA (Schneider, O’Rourke); and the OIE and National Reference Laboratory for Scrapie and CWD, Canadian Food Inspection Agency, Nepean, ON, Canada (Balachandran). 1 Corresponding Author: Katherine O’Rourke, USDA, ARS ADRU, 3003 ADBF, Pullman, WA 99164. katherine.o’rourke@ J Vet Diagn Invest 21:15–24 (2009) 15


SESSION II: session leader: Edward Hoover

11:50-12:10 P7. Detection of CWD by RAMALT biopsy in two white-tailed deer farms. Aru Balachandran

12:10-12:30 P8. Infectious prions in pre-clinical deer and transmission of CWD solely by environmental exposure. Candace Mathiason

12:30-12:50 P9. Detection of low level CWD infection in deer after oral exposure to urine and feces. Nicholas Haley

12:50-13:10 P10. CWD transmission via aerosol and oral lesions. Nathanial Denkers

13:10-13:30 P11. Environmental prion contamination: Prion protein adsorption in a soil matrix. Jason Bartz

13:30-13:50 P12. Trafficking of CWD prions via the enteric autonomic nervous system. Davis Seelig

13:50-14:10 P13. NeuroPrion cervid group update and the state of play in relation to the European CWD survey. Mick Stack

Workshop 1 : New developments in TSEs of domestic and wild animals (22 September 2009). Organized by EU funded projects NeuroPrion and goatBSE.

Download Workshop 1 Agenda here

It is a pleasure to again announce a workshop on natural TSEs in animals. The occurrence of TSEs in the field and farms carries potential risks of the agents in the environment and food. The ease of CWD spread and the shedding of CWD prions among cervids as well as the examples of prion infections in goat herds, and the transmission of scrapie through milk all argue for better containment and eventual eradication of these diseases.

As a follow-up to previous workshops, participants of NeuroPrion, BSEgoat and CWD research TSE projects again are organizing a workshop to bring researchers together to discuss these veterinary and public health issues. Presentations will be actively sought by the organizers and further selections will be made from interesting abstracts sent to the Prion2009 conference.

Therefore, we invite you to join this meeting and participate in the discussions. Lectures will be 20 minutes duration including the possibility for questions. Further details of the programme will be announced before August 24.

¦Mick Stack, Veterinary Laboratories Agencies, Addlestone, Surrey, United Kingdom ¦Jan Langeveld, Central Veterinary Institute of WageningenUR, Lelystad, The Netherlands ¦Edward Hoover, Colorado State University, Fort Collins, USA

J Vet Diagn Invest 20:698-703 (2008)

Chronic wasting disease in a Wisconsin white-tailed deer farm

Delwyn P. Keane,' Daniel J. Barr, Philip N. Bochsler, S. Mark Hall, Thomas Gidlewski.Katherine I. O'Rourke, Terry R. Spraker, Michael D. Samuel

APPENDIX D SAES-422 Format for Multistate Research Activity Accomplishments Report Note: This report is submitted each year of an activity’s duration and is due 60 calendar days following the annual meeting. The SAES-422 is submitted electronically by AAs into NIMSS. Annual Reports for MRF projects are available to CRIS and CSREES through NIMSS. Project/Activity Number: NC1024 Project/Activity Title: Domestic Surveillance, Diagnosis, and Therapy of Transmissible Spongiform Encephalopathies Period Covered: June 2007-June 2008 Annual Meeting Date(s): June 2008


In a USDA validation study, the IDEXX RAMALT rectal biopsy assay showed good results, with the provision that the difficulty lies in accurate and repeatable biopsy of sufficient lymphoid follicles for analysis. Lastly, a small study demonstrated no evidence for fence line transmission of scrapie between uninfected and infected animals, indicating a general lack of casual horizontal transmission. At the conclusion of the meeting, the group determined that maintenance of the Idaho flock is a high priority for providing access to affected tissues for continued study.


Tuesday, January 13, 2009

Antemortem detection of PrPCWD in preclinical, ranch-raised Rocky Mountain elk (Cervus elaphus nelsoni) by biopsy of the rectal mucosa Full Scientific Reports

Sunday, September 07, 2008

CWD LIVE TEST, and the political aspects or fallout of live testing for BSE in cattle in the USA

P.O. BOX 8805 SAINT JOSEPH, MO 64508 TEL: (816) 671-1144 FAX: (816) 671-1201 John Ascuaga’s Nugget Hotel Reno, Nevada

Copyright 2008 United States Animal Health Association Library of Congress Catalogue Control Number 2008903861 Meghan Richey and Rapid Solutions Group Kansas City, Missouri

Report of the comite CANADIAN FOOD INSPECTION AGENCY SCRAPIE ERADICATION PROGRAM UPDATE Penny Greenwood Canadian Food Inspection Agency

Report of the Comite on Scrapie

Chair: Jim R. Logan, Cheyenne, WY Vice Chair: Charles Palmer, Redding, CA


Katherine O’Rourke, Agricultural Research Services (ARS), USDA, presented an ARS research update. The report from ARS, Animal Disease Research Unit (ADRU) Pullman is summarized as follows: ADRU reported on their research on the minor scrapie forms, in particular Nor98 in sheep and classical scrapie in goats. Nor98 affects sheep of all genotypes; the etiology and transmissibility of Nor98 is unknown. Experimental infection of sheep highly resistant to classical scrapie (RR171) with a brain homogenate from an RR171 sheep with Nor98 is underway; blood, peripheral lymphoid tissues, and placenta will 641 be monitored to determine whether an infectious agent is present outside the central nervous system. Sheep with the 141FL genotype appear to be especially predisposed to Nor98. ADRU would like to acquire aged 141FL sheep from flocks without classical scrapie but that work will depend on clarification of the regulatory status of Nor98 sheep. Experimental and natural scrapie in goats is being addressed through assay of blood, placenta, and peripheral nodes to gather data on incubation time, optimal age for diagnosis, and role of prion genotype. The 2 goat genotypes reported to be associated with low susceptibility in European studies are of particular interest. The scrapie-free goat herd maintained at Washington State University will be diversified to include dairy and meat goats of those genotypes to produce kids for experimental studies. ADRU will request live goats exposed to sheep or goat scrapie for DNA analysis, live animal testing, and incubation time determination. In addition, ADRU will request tissues from goats collected in regulatory and slaughter surveillance and DNA from goats sampled in the upcoming goat NAHMS study. Requests for DNA from healthy herds will be made to the various dairy and meat goat industry groups. Linda Detwiler, presented information on new scrapie research, titled Scrapie: An Update on the Science. Information regarding scrapie has increased significantly over the years. While additional knowledge is always helpful, many of the new findings have actually increased the number of questions about the disease. It is well...


Dr. Steven Sundlof, Center for Veterinary Medicine (CVM), Food and Drug Administration (FDA), addressed the group on a number of topics. First was bovine spongiform encephalopathy (BSE). The CVM is committed to publishing a final rule on BSE but is still wading through some 850 comments received on the rule. Many comments accused FDA of underestimating the economic and environmental impacts of the rule, including the major issue of carcass disposal and disposal of additional banned materials. There is concern especially if rendering disappears as an option for disposal and other disposal options have not been identified. The final rule will include provision for time to come into compliance prior to the implementation of the rule and for the development of alternative disposal methods. ...


Dr. Heidi Schleicher, NAHLN, provided more information regarding the NAHLN, and some of its initiatives with improving its information technology resources. Schleicher also provided an update on surveillance activities, such as with BSE...


Unfortunately, there have been recent setbacks in market integration due to bovine spongiform encephalopathy (BSE). It is anticipated that this hurdle will be overcome and markets will continue to be further integrated, amplifying the need for improved surveillance for animal diseases and real time diagnostics for that surveillance.


Wednesday, July 1, 2009

Nor98 scrapie identified in the United States J Vet Diagn Invest 21:454-463 (2009)

SINCE THE TOPIC OF BSE/TSE (out of sight, out of mind) APPEARED TO BE OF NO CONCERN, EXCEPT FOR (i might have missed it),


2009 UPDATE ON TEXAS AND ALABAMA MAD COWS FOUND IN 2004 (finally documented after an act of Congress in 2005) AND 2006

Wednesday, September 23, 2009

Scientific Opinion on BSE Risk in Bovine Intestines Question number: EFSA-Q-2009-00226


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