Source: UNIVERSITY OF WYOMING submitted to
SUSCEPTIBILITY OF DOMESTIC CATTLE TO CHRONIC WASTING DISEASE BY ORAL INOCULATION AND NATURAL EXPOSURE: FINAL PHASE, 10-YEAR STUDY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0211708
Grant No.
(N/A)
Project No.
WYO-419-07
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Aug 1, 2007
Project End Date
Sep 30, 2008
Grant Year
(N/A)
Project Director
Jewell, J. E.
Recipient Organization
UNIVERSITY OF WYOMING
1000 E UNIVERSITY AVE DEPARTMENT 3434
LARAMIE,WY 82071-2000
Performing Department
VETERINARY SCIENCES
Non Technical Summary
CWD is a transmissible spongiform encephalopathy that infects wild and farmed deer and elk. It is related to the prion diseases scrapie of sheep and BSE of cattle. It has been shown that some cattle can develop a BSE-like disease 2 to 5 years afterwards when this CWD brain material is injected intracerebrally, but it is not known if a more natural route of exposure to the infectious agent can transmit the disease to cattle. Since cattle can encounter CWD-infected deer and/or elk, or the long-lasting infectious prion shed by them, on shared grazing range, a realistic assessment of the possibility they could become infected is needed. The BSE epidemic in the UK in the 1980's and subsequent transmission of the BSE prion to humans through infected beef products created an awareness of the possible economic and public health impacts of a similar occurrence in the U.S. This research seeks to determine if domestic cattle that have been kept in physical contact with chronic wasting disease-infected deer and elk or with contaminated premises for ten years also became infected. This research project will analyze brain and other tissues from the CWD-exposed cattle, none of which have died or developed clinical signs of disease, to determine if transmission has occurred. A separate project will examine cattle were given a large oral dose of macerated brain tissues from CWD-infected mule deer ten years ago. Whether CWD in cattle would become infectious for humans is not known, but it is a risk factor that would need to be evaluated.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113310104013%
3113310108010%
3113310116033%
3113399104012%
3113399116032%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3310 - Beef cattle, live animal; 3399 - Beef cattle, general/other;

Field Of Science
1080 - Genetics; 1160 - Pathology; 1040 - Molecular biology;
Goals / Objectives
The research objectives are to determine if domestic cattle can be infected with chronic wasting disease (CWD) after long-term exposure to infected animals and contaminated premises, as well as oral inoculation. CWD is a fatal transmissible spongiform encephalopathy (TSE) that infects free-ranging and farmed deer and elk in Wyoming and Colorado, as well as elsewhere in North America. It is specific to cervids but is related to scrapie of sheep and goats, and to BSE, which infects cattle; more importantly, BSE is the only animal prion disease that is known to be transmitted to humans. A collaborative study has shown that intracerebral inoculations of cattle with CWD will produce a TSE in some animals, but it is not known if natural routes will produce similar results. CWD is transmitted experimentally and probably naturally among deer and elk by animal-to-animal contact as well as via contaminated premises. Feeding brain or lymph node tissue from infected cervids to other deer and elk induces the disease with high efficiency. Based on that it is thought that entry of infectious prions from exposure to CWD-infected animals or environments is also via ingestion of PrPd in saliva or shed from the gastrointestinal tract via feces and persisting in soil or on browse plants. BSE, on the other hand, was not passed from animal to animal but depended on ingestion of feed-borne infectious material, and experimentally was transmitted to other cows by ingestion of brain macerate from BSE-infected cattle. In conjunction with a concurrent study of cattle that were given large oral doses of pooled brain material from CWD-infected mule deer and observed for ten years, in this portion of the project we will analyze tissues from cattle held since July 1997 at Colorado Division of Wildlife (11 animals) and Wyoming Game and Fish Dept (10 animals) research facilities in contact with CWD-infected deer and elk, to determine if, after ten years of continuous direct or environmental exposure, they have become infected with a TSE. These conditions simulate the possible exposure routes that cattle in North America might encounter if they are raised or grazed in areas where free-ranging or captive deer and elk are infected with CWD. We do not know if the presence of CWD in cattle would be a significant public health issue, but it would be a risk factor that would need to be evaluated. Because the potential implications for loss of market and public health concerns are major, CWD could be agriculturally very important if it were naturally transmissible to domestic livestock, specifically cattle, but we need to establish if this is or is not the case.
Project Methods
Twenty-one cattle that have been kept in physical contact with CWD-infected deer and elk or in contaminated premises for ten years will be sequentially euthanized for necropsy and collection of tissue samples. Approximately 20-30 histological sections will be examined primarily from nervous and lymphoid system tissues for histopathology and for PrP deposition by immunohistochemistry using conditions previously published for CWD, scrapie, and BSE. Approximately 12 specific areas of brain will be examined. Based on results published to date for PrPd deposition in central nervous system of cattle orally dosed with BSE and in CNS of cattle intracerebrally inoculated with CWD, the most relevant tissue samples to analyze for deposition of PrP in the CNS, peripheral nervous system, and lymphoreticular system are brainstem at the level of the obex and the lymphoid and enteric nervous system cells of the distal ileum. In addition, an assortment of other tissues in which disease-associated prions are commonly found in cervids with CWD will be sampled, such as palatine tonsil, retropharyngeal, submandibular and mesenteric lymph nodes, cervical, thoracic and lumbar spinal cord sections, retina, and adrenal gland. Replicate tissue samples will be frozen; Western blots on samples of brainstem and tonsil or lymph node will be performed, with additional frozen tissues held in reserve for tests depending on IHC results. Genomic DNA will be extracted from spleen tissue from each animal and cellular prion protein coding sequences will be amplified by polymerase chain reaction (PCR) using previously published conditions and PCR primers. Products will be purified and submitted for DNA sequencing. An important aspect of this project is the availability for analysis of animals that have survived without loss to accidents or other diseases for the full ten year period. To date also, none of the principals have shown clinical signs of neurologic disease. We will ultimately determine infection of cattle with CWD to be positive or negative based on the detection of disease-associated prions in brainstem sections by immunohistochemistry and Western immunoblot. Based on observations of age-related degenerative changes including neuronal vacuoles in normal cattle brains, we anticipate such changes will be observed in most or all of our ten-year-old animals. The untreated control animals will be important for distinguishing non-specific lesions, but primary identification of disease associated prions will be based on immunological detection methods.

Progress 08/01/07 to 09/30/08

Outputs
OUTPUTS: During the duration of this funding, all 34 cattle that formed the four experimental groups described in the project narrative were assessed for neurological status and transported to the Wyoming State Veterinary Laboratory from Colorado (11), or from Wyoming Game and Fish Dept. facilities at Sybille (10), or were on site in the WSVL isolation building facilities (10), or in the case of untreated controls, held in outdoor pastures on the grounds of the WSVL. During this period, all animals were euthanized, necropsied, and tissues were collected for formalin fixation and frozen archives. Approximately 20 to 30 fixed sections per animal, primarily from nervous and lymphoid system tissues, were processed, embedded in paraffin, sectioned, and stained. Tissues were representative samples of all major thoracic and abdominal organs, brain, lymphoid tissues, spinal cord, eyes, and representative skeletal muscles. Protein extracts from frozen samples of the brain of each animal were analyzed by Western immunoblot for the presence of protease-resistant prion protein. Genomic DNA was extracted from frozen spleen samples of each animal and used as template for PCR amplification of bovine PrP; DNA sequence was determined from amplified reactions and tabulated. Dissemination to date includes presentations at scientific meetings: 1) presentation at Wyoming Veterinary Medical Assoc. meeting, May 2008, Laramie, WY. Presenter: Jewell. 2)Oral presentation at USDA-CSREES NRI Awardees' Workshop, December 2008, Chicago, IL. Presenter: Jewell. 3) Poster abstract submitted and accepted for presentation at Third International CWD Symposium, July 2009, Park City, UT. Presenter: M. Hille (University of Wyoming graduate student). 4) Oral presentation on project and pathology results accepted for Third International CWD Symposium, July 2009, Park City, UT. Presenter: O'Toole. PARTICIPANTS: Dr. Donal O'Toole conducted or supervised necropsy of the animals, collection and handling of tissues for analysis; evaluated the slides for histopathology and PrP deposition, supervised histological treatments of materials, and prepared pathology reports on the cases. Provides expertise on all pathology aspects of the study and collaborates as co-author in preparation of the manuscript. Presents findings at scientific meeting. Dr. Jean Jewell organized, coordinated and facilitated the study, genotype and Western blot analyses, provided molecular biology expertise as needed, prepared reports and collaborates as co-author on preparation of final manuscript. Presents findings at scientific meetings. Dr. Terry Kreeger oversaw disposition of ten natural exposure cattle maintained for ten years at WGFD research facilities until their transfer to WSVL; collaborates as co-author on preparation of final manuscript. Dr. Michael Miller oversaw disposition of eleven natural exposure cattle maintained for ten years at Colorado Division of Wildlife facilities until their transfer to WSVL; collaborates as co-author on preparation of final manuscript. Dr. Jon Ayers provided release-time diagnostic veterinary pathologist services for a portion of Dr. O'Toole's participation in this study. Matthew Hille: laboratory technical support in Western blot analysis of brain tissues from the study animals. Daniel Siltman: undergraduate laboratory technical support in DNA sequence analysis of cattle PrP genes. University of Wyoming Veterinary Sciences Department and Wyoming State Veterinary Laboratory provided livestock isolation housing facilities and animal care and management personnel for the entire 11 years of the overall study. During the period of this project funding, WSVL also supplied transport of animals (10) from Wyoming Game and Fish Dept facilities at Sybille, WY to the WSVL at Laramie, WY and temporary holding facilities, euthanasia and necropsy operations and facilities for all 34 animals, personnel with expertise in tissue collection, handling and processing, carcass disposal by medical incineration of all 34 carcasses, tissue blocking, and histology services including immunohistochemical staining of fixed tissue sections. Colorado Division of Wildlife supplied funds for the original purchase of the study animals in 1997, livestock housing facilities and animal care and management personnel including veterinary services from 1997 to 2007, and transport of animals from La Porte, CO to Laramie, WY. Wyoming Game and Fish Dept. provided livestock housing facilities and animal care and management personnel including veterinary services from 1997 to 2007. Dr. Amir N. Hamir, Veterinary Medical Officer, National Animal Disease Center provided the paraffin-embedded block of CWD-infected bovine brain tissue used for a positive control in immunohistochemistry. The project provided training for veterinary school students during externships at the WSVL by their participation in the cattle necropsies; it also provided undergraduate molecular biology research opportunities to two students participating in the protein and DNA sequence analysis portions of the project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This study investigated the risk of domestic cattle developing a transmissible spongiform encephalopathy (TSE) after oral inoculation followed by a long incubation period, or by long-term natural exposure to cervids infected with chronic wasting disease (CWD). Previously,ten cattle were given large oral doses of pooled brain material from CWD-infected mule deer in late August 1997 and housed in isolation at the Wyoming State Veterinary Laboratory until September 2007. Two additional groups of cattle were penned outdoors with CWD-infected deer and elk or in CWD-contaminated premises at Colorado Division of Wildlife (n=11) and Wyoming Game and Fish Department (n=10) research facilities during the same ten-year period. These conditions simulated exposure routes that cattle in North America might encounter if they are raised or grazed in areas where free-ranging or captive deer and elk are infected with CWD. During the current project, all exposed and three untreated control cattle were killed, and select tissues were collected at necropsy. Samples from each animal were analyzed for the diagnostic hallmarks of TSEs by histological examination, immunohistochemistry and Western blot. DNA sequences were determined for the cellular prion protein gene in each animal. No proteinase-K resistant prion protein or anti-PrP immunoreactive IHC signals were detected in any tissues of exposed or control animals. None of these results, taken individually or together, support a diagnosis of TSE in cattle inoculated orally with a high dose of infectious CWD material or continually exposed by cohabitation with infected deer or elk, or transmission from contaminated premises despite an incubation period of up to 10 or 11 years.

Publications

  • No publications reported this period