Abstract

Chronic wasting disease (CWD), a debilitating and fatal neurodegenerative disease, was first described in mule deer (Odocoileus hemionus hemionus) as a spongiform encephalopathy in 1980.⁶ Chronic wasting disease became a reportable disease in Canada in 2000. In 2003, a retrospective study of cervid deaths at the Toronto Zoo (TZ) was undertaken to follow up on the histopathologic diagnosis of spongiform encephalopathy that had been recorded in one mule deer which had died in 1978. The objectives of the study were to:

• Determine the most likely source of introduction of CWD into the TZ cervid collection in the 1970s.
• Assess the likelihood of spread within the TZ, and to other zoos and collections through the sale or donation of animals.
• Assess the current CWD status of the TZ cervid collection.²

Through archived zoo records, including inventory records, keepers’ daily reports, animal health records, and pathology records, it was possible to develop an understanding of the distribution of cervids in space and time in the TZ from 1973 to the present. A list of the mule deer (and of other cervid species that may have had direct or indirect contact with them, through environment, facilities, or equipment) was developed, and the CWD status of these animals was investigated. Archived, formalin-fixed, paraffin-embedded blocks including lymphoid tissue and/or brain were available from many of the cervids, at least 1 y of age that had died at the TZ. These samples were retrieved from storage in the Pathobiology Department, University of Guelph, and sent to the Canadian Food Inspection Agency’s National Reference Laboratory in Nepean, Ontario, for evaluation by immunohistochemical staining (IHC).

Tissue sections from paraffin blocks were cut at 5 μm, mounted on positively charged glass slides, and immunostained with monoclonal antibody F99/97.6.1, which had been used in another study.⁵ The method used for the detection of prion protein (PrP) was that described by O’Rourke.⁴ For one mule deer, only H&E-stained slides were available for evaluation, and PrP immunostaining was possible after the slides had been placed in preheated xylene (60°C) and then on a slide warming table (40°C) until the cover glasses could be slid off. In this case, the same methodology was also applied to known positive and negative slides that had previously been stained with H&E, for comparison.²

Cervids were considered to be “suspect” if they were >12 months of age and had displayed one or more of the following signs: excess salivation, unusual behavior, neurologic signs, polydipsia/polyuria, weight loss, retention of winter hair coat, or pneumonia.¹ Of 12 CWD-suspect mule deer, tissues appropriate for IHC were available from nine animals, seven of which were found to be positive for CWD. The earliest case of CWD was identified in a Colorado mule deer that died 21 months after arrival at the TZ. Another suspect Colorado animal, an emaciated female, had been killed by a male 10 months after arrival; unfortunately no tissues were available for IHC evaluation.

Based on clinical observation, a number of other cervids in the Canadian Domain area of the TZ were considered as suspects, and of these, appropriate tissues were available from 13 of 14 suspect black-tailed deer (Odocoileus hemionus columbianus), eight of 18 white-tailed deer (Odocoileus virginianus), one of one reindeer (Rangifer tarandus tarandus), five of six caribou (Rangifer tarandus caribou), and one of one moose (Alces alces americana). All of these suspect samples, from cervids other than mule deer, were negative for CWD.

Appropriate tissues available from all non-suspect cervids >12 months old were also evaluated by IHC with negative results, except for one black-tailed deer. A group of black-tails had been moved into the mule deer exhibit 3 days after the last mule deer was moved out. Four months later, one of these animals died acutely and showed no clinical signs consistent with CWD. Its postmortem record indicated acute hemorrhagic nephritis, consistent with clostridial septicemia or toxemia, as the final diagnosis. In repeated tests, IHC was positive on brain tissue from this animal. This is an extremely unusual finding, since the black-tail had no previous history of exposure to mule deer at the TZ. It is now known that infectious prions can be found in the tonsils of mule deer as soon as 3 months after exposure,³ and perhaps it is possible to find prions in the brain within 4 months after exposure in black-tails. Significantly, a total of 31 black-tails were tested, and all others were negative.

This study determined that mule deer from a Colorado zoo were the likely source of CWD, and that lateral transmission of CWD to offspring and to animals from another source had occurred within the mule deer herd. A black-tailed deer that was exposed to the contaminated mule deer exhibit, but never had direct contact with the mule deer, was also infected. The last CWD-positive death, the black-tailed deer, occurred in 1981. No mule deer had been moved from the zoo site. Three black-tailed deer were sold in 1988, 7 y after the last CWD diagnosis; their whereabouts could not be traced, but it is unlikely that they were infected based on the history of the herd and the time lapse since the last CWD death. Since 2001, all cervids >12 months of age that die at the TZ have routinely undergone testing for CWD.

Acknowledgments

We thank the staff at the Toronto Zoo for their help and cooperation, particularly Drs. Bill Rapley and Graham Crawshaw. Special thanks to Mrs. Carol-Lee Ernst of the Department of Pathobiology, University of Guelph, who retrieved postmortem records and tissue blocks for testing. The authors also thank Dr. Terry Spraker of Colorado State University for his helpful comments on IHC diagnosis, and Dr. Katherine O’Rourke of ARS, USDA, Pullman, Washington, for providing antibodies. Finally, thanks to Bruce Ullett who performed histological preparations and IHC assays.

This paper is presented with permission of the Canadian Veterinary Journal (Can Vet J. 2006;47:1185–1193).²

Literature Cited

1.  Canadian Food Inspection Agency. 2002. Disease Control Manual. Section 24—Chronic wasting disease—Part 1, Eradication. Ottawa; July 10, 2002.

2.  Dubé C, Mehren KG, Barker IK, Peart BL, Balachandran A. Retrospective investigation of chronic wasting disease of cervids at the Toronto Zoo, 1973–2003. Can Vet J. 2006;47:1185–1193.

3.  JAVMA News. Study: chronic wasting disease spread through blood, saliva. J Am Vet Med Assoc. 2006;229:1710.

4.  O’Rourke KI, Baszler TV, Miller JM. Monoclonal antibody F89/160.1.5 defines a conserved epitope on the ruminant prion protein. J Clin Microbiol. 1998;36:1750–1755.

5.  Spraker TR, O’Rourke KI, Balachandran A. Validation of monoclonal antibody F99/97.6.1 for immunohistochemical staining of brain and tonsil in mule deer (Odocoileus hemionus) with chronic wasting disease. J Vet Diagn Invest. 2002;14:3–7.

6.  Willams ES, Young S. Chronic wasting disease of captive mule deer: a spongiform encephalopathy. J Wild Dis. 1980;16:89–98.