Journal of Wildlife Diseases, 47(3), 2011, pp # Wildlife Disease Association 2011

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1 Journal of Wildlife Diseases, 47(3), 2011, pp # Wildlife Disease Association 2011 Failure of Fallow Deer (Dama dama) to Develop Chronic Wasting Disease When Exposed to a Contaminated Environment and Infected Mule Deer (Odocoileus hemionus) Jack C. Rhyan, 1,6 Michael W. Miller, 2 Terry R. Spraker, 3 Matt McCollum, 1 Pauline Nol, 1 Lisa L. Wolfe, 2 Tracy R. Davis, 2 Lynn Creekmore, 4 and Katherine I. O Rourke 5 1 National Wildlife Research Center, US Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Services, Fort Collins, Colorado 80521, USA; 2 Colorado Division of Wildlife, Wildlife Research Center, 317 West Prospect Road, Fort Collins, Colorado , USA; 3 Veterinary Diagnostic Laboratory, Colorado State University, Fort Collins, Colorado 80523, USA; 4 Natural Resources Research Center, US Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Services, Fort Collins, Colorado 80526, USA; 5 US Department of Agriculture, Agricultural Research Service, 337 Bustad Hall, Washington State University, Pullman, Washington 99163, USA; 6 Corresponding author ( jack.c.rhyan@aphis.usda.gov) ABSTRACT: We monitored a herd of fallow deer (Dama dama) for evidence of prion infection for 7 yr by periodic postmortem examination of animals from the herd. The fallow deer were exposed to the chronic wasting disease (CWD) agent from mule deer by living in a paddock considered contaminated with infectivity from its history of housing CWD infected deer and, after the first year of the study, by comingling with infected mule deer (Odocoileus hemionus). At least 8 of 12 mule deer serving as sentinels for prion transmission and 25 additional mule deer serving as sources of infectivity developed clinical CWD or were otherwise confirmed to be infected with CWD via lymphoid tissue immunohistochemistry (IHC). In contrast, none of the 41 exposed fallow deer showed clinical signs suggestive of CWD, IHC staining of disease-associated prion in lymphoid or brain tissues, or evidence of spongiform degeneration in sections of brain stem at the level of the obex when sampled 18 mo to 7 yr after entering the mule deer paddock. The absence of clinical disease and negative IHC results in fallow deer housed in the same contaminated paddock for up to 7 yr and almost continuously exposed to CWDinfected mule deer for up to 6 yr suggests a species barrier or other form of resistance preventing fallow deer infection by the CWD agent or delaying progression of the disease in this species. Key words: Chronic wasting disease (CWD), Dama dama, fallow deer, prion, spongiform encephalopathy. Chronic wasting disease (CWD) is caused by a prion strain (or strains) that infects several species of cervids including mule deer (Odocoileus hemionus), whitetailed deer (Odocoileus virginianus), elk (Cervus elaphus nelsoni), red deer (Cervus elaphus elaphus), moose (Alces alces), and, in preliminary studies, muntjac deer (Muntiacus reevesi; Williams and Young, 1980, 1982; Spraker et al., 1997; Kreeger et al., 2006; Martin et al., 2009; A. Young, pers. comm.). The CWD agent can be transmitted from infected animals and from environments contaminated with the excrement or carcasses of infected animals (Miller and Williams, 2003; Miller et al., 2004; Tamgüney et al., 2009). Intracerebral inoculation with brain suspension from CWD-infected whitetailed deer and elk resulted in progressive clinical disease in four of 13 fallow deer (Dama dama) between 24 and 37 mo postinoculation (mpi; Hamir et al., 2008). The four affected fallow deer, all of which had received disease-associated prion protein (PrP d ) from white-tailed deer (WTD), and another clinically normal individual killed 26 mpi had small amounts of the abnormal PrP d in central nervous system (CNS) tissues but lacked spongiform degeneration in the neuropil and PrP d in lymphoid tissues (Hamir et al., 2008). At 4 yr postinoculation, the remaining inoculated fallow deer (one had received PrP d from WTD and 4 PrP d from elk) were alive and apparently healthy; however, between 51 and 60 mo all five became sick and were euthanized. On postmortem examination, all deer had spongiform encephalopathy and evidence of PrP d in CNS tissues (Hamir et al., 2011). We monitored a group of fallow deer and their offspring for evidence of prion infection 739

2 740 JOURNAL OF WILDLIFE DISEASES, VOL. 47, NO. 3, JULY 2011 for up to 7 yr of exposure to CWD through living in a paddock contaminated by previous CWD studies and inhabited by infected mule deer. Our study was conducted at the Colorado Division of Wildlife s (CDOW) Foothills Wildlife Research Facility (FWRF; Fort Collins, Colorado) between October 2000 and November 2007 under study protocols approved by the CDOW Animal Care and Use Committee (file ). In October 2000, 22 female and 3 male, 6-mo-old fallow deer were purchased from a private herd, in a state in which CWD had not been diagnosed, and transported to the FWRF (Fig. 1). The private herd conducted routine slaughter surveillance for CWD and had never had an animal positive. We placed fallow deer in a,2.0 ha paddock ( Paddock C in Fig. 2A of Miller and Wild, 2004) that was considered contaminated with the CWD agent because it had recently housed CWD-infected mule deer, contained fecal and urine residues from previous CWD studies (Miller and Williams, 2003; Miller and Wild, 2004; Miller et al., 2006), and was adjacent to paddocks in which a CWD-mule deer study was ongoing. Between June 11 and 25 August 2002, 18 fallow deer fawns (12 males, 6 females) were born to the original study animals in the contaminated paddock (Fig. 1); two died as neonates. The three original male fallow deer were subsequently vasectomized, and males born in the paddock were euthanized and necropsied in the first 2 yr of life to preclude birth of additional fawns. During the study, we added mule deer to the paddock holding the fallow deer (Table 1). Twelve of these mule deer were #151 days old and were not known to be exposed to CWD (Fig. 1). Nine of the 12 had been born to dams in the FWRF, and three were raised as orphans by individuals elsewhere in Colorado. These deer served as sentinels for CWD transmission in the paddock and are called sentinel mule deer hereafter. Another 27 mule FIGURE 1. Temporal relationships between the addition and removal of fallow deer (Dama dama; n541) and sentinel (n512) and infection-source (n525) mule deer (Odocoileus hemionus) in a study of fallow deer susceptibility to chronic wasting disease. None of the fallow deer showed evidence of prion infection when removed at time points mo after the start of the study. Infections were confirmed in eight of the sentinel mule deer (solid circles); two sentinels appeared to be uninfected at postmortem examination (open circles), and the infection status of two others (gray centers) were not determined. For infection source mule deer, the gray bars represent the number of potentially infected deer days (number of deer 3 number of days) of exposure for each month where at least one infection-source deer was present in the paddock. deer placed in the paddock were $352 days old and had been exposed to the CWD agent during other studies prior to their placement in the fallow deer study paddock; 16 of these were positive on tonsil biopsy immunohistochemistry (IHC; Wolfe et al., 2002) at the time they were added to the fallow deer paddock. In all, 25 of these animals, hereafter called infection-source mule deer, were shown to be infected and served as additional sources of infection. Two of the 27 exposed deer were placed in the paddock 2.5 and 15.5 mo prior to the end of the study. These two were negative at the end of the study and are not included in the infection-source mule deer. Syntopic fallow deer and mule deer used the same automatic water source, shelters, pasture space, and feed bunks but rarely socialized

3 SHORT COMMUNICATIONS 741 TABLE 1. Numbers of sentinel and infection-source mule deer (Odocoileus hemionus) and fallow deer (Dama dama) added to and removed from a prion-contaminated paddock during a study of fallow deer susceptibility to chronic wasting disease (CWD). Values in parentheses are the numbers of deer developing clinical signs of or testing positive for CWD. Study year a Total Mule deer added Sentinel Infection source Mule deer removed Sentinel 2 (0) 1 (0) 3 (2) 4 (4) 0 2 (2) 0 12 b (8) Infection source 0 1 (1) 4 (4) 13 (13) 4 (4) 1 (1) 2 (2) 25 (25) Fallow deer added Original animals birth cohort 0 16 c Fallow deer removed Original animals 0 5 (0) 6 (0) 8 (0) (0) 25 (0) 2002 birth cohort 0 0 b (0) 8 (0) 4 (0) (0) 16 (0) a Years are measured from November to October (e.g., November 2000 to October 2001), except that included part of October 2000 and included part of November b Two of the 12 developed clinical illness but laboratory testing was not done; two were euthanized at 7 mo of age and were PrP d negative in lymphoid tissue. c Two neonatal deaths not included in count. or made direct contact between species while grazing. All deer were observed daily for clinical signs of CWD, and mule deer were routinely euthanized after clinical signs of CWD were observed. Mule deer were necropsied, and their retropharygeal lymph nodes collected and tested by an enzyme-linked immunosorbent assay (Hibler et al., 2003) and IHC (Spraker et al., 2002), except in 10 cases that had previously tested positive by tonsil biopsy IHC. In all, 8 of 12 sentinel mule deer and all 25 infection-source mule deer were confirmed as infected with CWD via lymphoid tissue IHC over the 7-yr study (Table 1). Of the four sentinel mule deer that were not confirmed as infected, two were euthanized at 7 mo of age due to chronic diarrhea and were negative for PrP d in lymphoid tissue, and two were euthanized due to illness but laboratory specimens were lost prior to testing for prion infection (Fig. 1). Of the eight sentinel mule deer with laboratory evidence of CWD and the 25 infectionsource mule deer, three died of immobilization complications, two of CWD, and the rest were euthanized following development of clinical signs consistent with CWD. In contrast to mule deer, none of the 41 exposed fallow deer showed clinical signs suggestive of CWD. Fallow deer were euthanized and necropsied at various times during the study (Table 1). Tissues collected for IHC staining for PrP d included brain (obex at the level of the dorsal motor nucleus of the vagus), palatine tonsil, and medial retropharyngeal lymph node. In selected animals, pharyngeal tonsil (n514), ileum (n519), jejunum (n517), mesenteric lymph node (n513), and rectum (n58) also were examined by IHC for PrP d. IHC staining of PrP d was not detected in any of the tissue sections from fallow deer. No evidence of spongiform degeneration was

4 742 JOURNAL OF WILDLIFE DISEASES, VOL. 47, NO. 3, JULY 2011 found in any of the brain sections from fallow deer. We extracted DNA from liver specimens from 23 fallow deer, and the open reading frame of the PRNP gene was amplified and sequenced as described by Spraker et al. (2004). Sequences were identical to that reported in GenBank accession AY except for a single synonymous change (aac/aat, encoding asparagine) at codon 138. Fourteen of these fallow deer were heterozygous for the polymorphism, six were homozygous for the aat variant, and three were homozygous for the aac variant. Susceptibility to some prion diseases is associated with single amino acid substitutions in the prion protein. Further, the species barrier, which limits the transmission of prion strains between species (Pattison and Jones, 1968; Prusiner, 1998), also may be linked to single amino acid mismatches between the infected donor animal and the naive recipient. In this experiment, the prion protein amino acid differences between the putative infected donor (mule deer) and the naive recipient (fallow deer) are limited to two residues. The mule deer PRNP gene encodes serine at codon 138 (138S), and the fallow deer prion gene encodes asparagine (138N). The mule deer gene encodes glutamine at codon 226 (226Q), and the fallow deer prion gene encodes glutamic acid at that site (226E). The substitution at codon 226 had no effect on susceptibility to experimental oral challenge of red deer with CWD-infected elk brain (Balachandran et al., 2009), but the effect of this mismatch between mule deer and fallow deer is unknown. The extent of variability in the PRNP gene of fallow deer in the USA is not known. The fallow deer in this study had a single PRNP genotype; no other alleles of the PRNP gene in fallow deer have been reported in the small numbers examined (this publication; Gen- Bank accessions EF165089, EF139175, AY639094, AY286007; K. I. O Rourke, unpubl. data on farmed US fallow deer). If other alleles of the gene are prevalent in farmed US fallow deer, additional in vitro or in vivo studies on their effect on susceptibility are warranted. Eight of 10 sentinel mule deer maintained in the fallow deer paddock for $530 days including two individuals born in the paddock were positive for CWD, indicating that transmission of the agent was occurring throughout most if not all of the study period (Fig. 1). The absence of clinical disease and negative IHC results in fallow deer housed in a contaminated paddock for up to 7 yr with 6 yr of essentially continuous exposure to infection source and infected sentinel mule deer (Fig. 1) suggests that a species barrier or other form of resistance exists that either prevented fallow deer from becoming infected by the CWD agent or delayed progression of the disease such that infections could not be demonstrated. These findings are consistent with the absence of reported field cases of CWD in fallow deer, and with results of a recent intracerebral inoculation study using CWD agent from WTD and elk (Hamir et al., 2008). Those authors suggested that it may not be possible to transmit CWD to fallow deer by more natural routes and that a relatively strong species barrier against CWD infection may exist for fallow deer. The eventual development of spongiform encephalopathy with PrP d amplification in the deer 51 to 60 mpi is indicative of a delayed disease progression following intracerebral inoculation as compared with other species. Suffolk sheep developed spongioform change and clinical illness and demonstrated PrP d amplification as early as 36 mo postintracerebral inoculation with PrP d of mule deer origin (Hamir et al., 2006). Whether fallow deer are completely resistant to developing CWD following natural exposure or experience greatly delayed disease progression is unknown and may warrant further study. Our study was funded by the US Department of Agriculture (USDA), Ani-

5 SHORT COMMUNICATIONS 743 mal and Plant Health Inspection Service, Veterinary Services, and the Colorado Division of Wildlife, with some funding also provided by the USDA, Agriculture Research Service. We thank Tom Gidlewski, Karl Held, and Roger Thompson for assistance with necropsies. Tom Gidlewski provided helpful comments on an earlier draft of our manuscript. LITERATURE CITED BALACHANDRAN, A., N. P. HARRINGTON, J. ALGIRE, A. SOUTYRINE, T. R. SPRAKER, M. JEFFREY, L. GONZALEZ, AND K. I. O ROURKE Experimental oral transmission of chronic wasting disease to red deer (Cervus elaphus elaphus): Early detection and late stage distribution of protease-resistant prion protein. Canadian Veterinary Journal 51: HAMIR, A. N., J. J. GREENLEE,E.M.NICHOLSON,R.A. KUNKLE, J. A. RICHT, J. M. MILLER, AND S. M. HALL Experimental transmission of chronic wasting disease (CWD) from elk and white-tailed deer to fallow deer by intracerebral route: Final report. Canadian Journal of Veterinary Research 75: , R. A. KUNKLE,R.C.CUTLIP,J.M.MILLER,E. S. WILLIAMS, AND J. A. RICHT Transmission of chronic wasting disease of mule deer to Suffolk sheep following intracerebral inoculation. Journal of Veterinary Diagnostic Investigation 18: ,, E. M. NICHOLSON, J.M.MILLER, S. M. HALL, H. SCHOENENBRUECHER, B. W. BRU- NELLE, AND J. A. RICHT Preliminary observations on the experimental transmission of chronic wasting disease (CWD) from elk and white-tailed deer to fallow deer. Journal of Comparative Pathology 138: HIBLER, C. P., K. L. WILSON, T. R. SPRAKER, M. W. MILLER, R.R.ZINK, L.L.DEBUSE, E.ANDERSEN, D. SCHWEITZER, J. A. KENNEDY, L. A. BAETEN, J. F. SMELTZER, M. D. SALMAN, AND B. E. POWERS Field validation and assessment of an enzyme-linked immunosorbent assay for detecting chronic wasting disease in mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus), and Rocky Mountain elk (Cervus elaphus nelsoni). Journal of Veterinary Diagnostic Investigation 15: KREEGER, T. J., D. L. MONTGOMERY, J. E. JEWELL, W. SCHULZ, AND E. S. WILLIAMS Oral transmission of chronic wasting disease in captive Shira s moose. Journal of Wildlife Diseases 42: MARTIN, S., M. JEFFREY, L. GONZALEZ, S. SISO, H. W. REID, P.STEELE, M.P.DAGLEISH, M.J.STACK, M. J. CHAPLIN, AND A. BALACHANDRAN Immunohistochemical and biochemical characteristics of BSE and CWD in experimentally infected European red deer (Cervus elaphus elaphus). BMC Veterinary Research 5: 26. doi: / MILLER, M. W., N. T. HOBBS, AND S. J. TAVENER Dynamics of prion disease transmission in mule deer. Ecological Applications 16: , AND M. A. WILD Epidemiology of chronic wasting disease in captive white-tailed and mule deer. Journal of Wildlife Diseases 40: , AND E. S. WILLIAMS Horizontal prion transmission in mule deer. Nature 425: ,, N. T. HOBBS, AND L. L. WOLFE Environmental sources of prion transmission in mule deer. Emerging Infectious Diseases 10: PATTISON, I. H., AND K. M. JONES Modification of a strain of mouse-adapted scrapie by passage through rats. Research in Veterinary Science 9: PRUSINER, S. B Prions. Proceedings of the National Academy of Sciences USA 95: SPRAKER, T. R., A. BALACHANDRAN, D.ZHUANG, AND K. I. O ROURKE Variable patterns of distribution of PrP CWD in the obex and cranial lymphoid tissues of Rocky Mountain elk (Cervus elaphus nelsoni) with subclinical chronic wasting disease. Veterinary Record 155: , M. W. MILLER, E.S.WILLIAMS, D.M.GETZY, W. J. ADRIAN, G. G. SCHOONVELD, R. A. SPOWART, K. I. O ROURKE, J. M. MILLER, AND P. A. MERZ Spongiform encephalopathy in free-ranging mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus), and Rocky Mountain elk (Cervus elaphus nelsoni) in northcentral Colorado. Journal of Wildlife Diseases 33: 1 6., K. I. O ROURKE, A. BALACHANDRAN, R. R. ZINK, B. A. CUMMINGS, M. W. MILLER, AND B. E. POWERS Validation of monoclonal antibody F99/ for immunohistochemical staining of brain and tonsil in mule deer (Odocoileus hemionus) with chronic wasting disease. Journal of Veterinary Diagnostic Investigation 14: 3 7. TAMGÜNEY, G., M. W. MILLER, L. L. WOLFE, T. M. SIROCHMAN, D. V. GLIDDEN, C. PALMER, A. LEMUS, S. J. DEARMOND, AND S. B. PRUSINER Asymptomatic deer excrete infectious prions in faeces. Nature 461: WILLIAMS, E. S., AND S. YOUNG Chronic wasting disease of captive mule deer: A spongiform encephalopathy. Journal of Wildlife Diseases 16: , AND Spongiform encephalopathy of Rocky Mountain elk. Journal of Wildlife Diseases 18:

6 744 JOURNAL OF WILDLIFE DISEASES, VOL. 47, NO. 3, JULY 2011 WOLFE, L. L., M. M. CONNER, T.H.BAKER, V.J. DREITZ, K.P.BURNHAM, E.S.WILLIAMS, N.T. HOBBS, AND M. W. MILLER Evaluation of antemortem sampling to estimate chronic wasting disease prevalence in free-ranging mule deer. Journal of Wildlife Management 66: Submitted for publication 25 November Accepted 1 March 2011.