visual observation. In addition, deer were observed and body condition assessed visually at least monthly. In addition to collecting survival and spatial data, we used marked deer to conduct annual, ground-based mark-resight inventories [25,26] to estimate herd size and composition, as well as annual productivity. For these inventories, established routes were walked or driven daily during late November–early December until a target observation quota of $1,000 deer (including all repeated observations) was reached; this quota was based on the estimated sample needed to achieve a 10% coefficient of variation on the population estimate. Observers recorded the total number of $1 year old male deer, $1 year old female deer, and ,1 year old deer in each group encountered, as well as the unique collar identification number for each marked deer observed; the approximate geographic location of the group also was estimated based on a hand-held GPS unit. To assess long-term trends in deer abundance, we also reviewed and assembled original field data from unpublished mark-resight inventories and systematic counts conducted by the City of Boulder in the Table Mesa area in 1985–2001; although daily count data for routes corresponding to our study area were available for 1987–2001, we were only able to gather all of the information needed for a reliable mark-resight estimate of deer abundance for the data from 1988. We also examined the trend in mean of daily counts for each year as a proxy for trends in deer abundance because count data were available and consistently recorded for almost every year since 1987. Endpoints Regardless of infection status, we allowed marked deer to survive until their natural demise without intervention except as described. Once deer were enrolled in the study, they were not euthanized unless they became severely debilitated as a result of prion infection or other health problems. Field assistants blinded to infection status of individual deer observed all marked deer about once a month to assess health. Signs of prion disease [4,5,10] were subjectively scored (0 = not shown, 1 = subtle, 2 = obvious) for behavioral changes, loss of body condition, stumbling or lack of coordination, inability to rise or retreat from a threat, and salivation, regurgitation, or excess drinking, and a total score calculated (maximum clinical score = 14). Deer with clinical scores $10 were regarded as being in ‘‘end-stage disease’’ (prion or other) and euthanized. References 1. Prusiner SB (1998) Prions. Proc. Natl. Acad. Sci. USA 95: 13363–13383. 2. Hornlimann B, Riesner D, Kretzschmar H, eds (2006) Prions in Humans and Animals. Walter de Gruyter. 714 p. 3. Detwiler LA, Baylis M (2003) The epidemiology of scrapie. Rev sci tech Off int Epiz 22: 121–143. 4. Williams ES, Young S (1980) Chronic wasting disease of captive mule deer: a spongiform encephalopathy. J Wildl Dis 16: 89–98. 5. Williams ES (2005) Chronic wasting disease. Vet Path 42: 530–549. 6. Miller MW, Williams ES, McCarty CW, Spraker TR, Kreeger TJ, et al. (2000) Epizootiology of chronic wasting disease in free-ranging cervids in Colorado and Wyoming. J Wildl Dis 36: 676–690. 7. Gross JE, Miller MW (2001) Chronic wasting disease in mule deer: disease dynamics and control. J Wildl Manage 65: 205–215. 8. Miller MW, Hobbs NT, Tavener SJ (2006) Dynamics of prion disease transmission in mule deer. Ecol Appl 16: 2208–2214. 9. Miller MW, Williams ES (2003) Horizontal prion transmission in mule deer. Nature 425: 35–36. 10. Fox KA, Jewell JE, Williams ES, Miller MW (2006) Patterns of PrPCWD accumulation during the course of chronic wasting disease infection in orally inoculated mule deer (Odocoileus hemionus). J Gen Virol 87: 3451–3461. 11. Packer C, Holt RD, Hudson PJ, Lafferty KD, Dobson AP (2003) Keeping the herds healthy and alert: implications of predator control for infectious disease. Ecol Lett 6: 797–802. Lions&Prions&Deer Because this study was conducted partially in an urban area, deer severely injured during the course of the study (e.g., from collisions with vehicles) were euthanized on a case-by-case basis consistent with established operating policies and agency practices (e.g., darting and lethal injection, gunshot); in these cases, the cause of death was recorded as ‘‘trauma–euthanized’’, and these deer were not censored from survival calculations. Analysis We tabulated test-positive and -negative results annually and used them to estimate prevalence and incidence of prion infection. We compared survival of prion-infected and uninfected mule deer using matched case-control conditional logistic regression [27] in SAS 9.1; model selection was based on Akaike’s Information Criterion (AIC) [28]. Individual covariates included infection status, sex, and age. We also used PrP genotype and capture location in grouping deer, but did not use these covariates in our analyses; instead, we paired pairs of male and female deer by capture location and genotype and then used these groups of four individuals (n = 12 groups in 2005–06, 16 groups in 2006–07) as the sample unit for our analyses to account for other potential influences on survival. We estimated relative risks of specific causes of mortality (predation, vehicle collisions) for prion-infected mule deer as compared to uninfected individuals. For all comparisons, we used DAIC.2.0 [28] or a = 0.05 in ascribing significance to results of analyses. Acknowledgments We thank A. Curtis, J. Dulberger, C. Dunning, B. Espe, W. Grabow, W. Keeley, J. Koszalka, B. Lenth, M. McKibben, C. Nunes, M. Sirochman, H. Southwick, P. Taylor, M. Wright and others for field assistance, T. Spraker and T. Cornish for diagnostic assistance, W. Johnson, M. Schuette, J. Sherry, M. Valenta, and C. Wanner for database and GIS support, and C. Anderson, C. Bishop, and M. Baylis for valuable comments on our draft manuscript. Author Contributions Conceived and designed the experiments: MWM HMS LLW SLH CHS PML. Performed the experiments: MWM HMS LLW FGQ. Analyzed the data: MWM PML. Wrote the paper: MWM HMS LLW FGQ SLH CHS PML. 12. Ostfeld RS, Holt RD (2004) Are predators good for your health? Evaluating evidence for top-down regulation of zoonotic disease reservoirs. Front Ecol Environ 2: 13–20. 13. Wolfe LL, Miller MW, Williams ES (2004) Feasibility of ‘‘test-and-cull’’ for managing chronic wasting disease in urban mule deer. Wildl Soc Bull 32: 500–505. 14. Keesing F, Holt RD, Ostfeld RS (2006) Effects of species diversity on disease risk. Ecol Lett 9: 485–498. 15. Duffy MA (2007) Selective predation, parasitism, and trophic cascades in a bluegill-Daphnia-parasite system. Oecologia 153: 453–460. 16. Holt RD, Roy M (2007) Predation can increase the prevalence of infectious disease. Am Nat 169: 690–699. 17. Miller MW, Conner MM (2005) Epidemiology of chronic wasting disease in free-ranging mule deer: spatial, temporal, and demographic influences on observed prevalence patterns. J Wildl Dis 41: 275–290. 18. Unsworth JW, Pac DF, White GC, Bartmann RM (1999) Mule deer survival in Colorado, Idaho, and Montana. J Wildl Manage 63: 315–326. 19. Hudson PJ, Dobson AP, Newborn D (1992) Do parasites make prey vulnerable to predation? Red grouse and parasites. J Anim Ecol 61: 681–692. 20. Johnson PTJ, Stanton DE, Preu ER, Forshay KJ, Carpenter SR (2006) Dining on disease: how interactions between parasite infection and environmental conditions affect host predation risk. Ecology 87: 1973–1980. 21. Fryxell JM, Mosser A, Sinclair ARE, Packer C (2007) Group formation stabilizes predator–prey dynamics. Nature 449: 1041–1043. PLoS ONE | www.plosone.org 6 December 2008 | Volume 3 | Issue 12 | e4019