Lions&Prions&Deer Figure 2. Causes of death in prion-infected and uninfected mule deer at Table Mesa. Mortality was higher among prion-infected deer; only about half of the infected deer survived annually (September–August) in both years (2005–06 and 2006–07). doi:10.1371/journal.pone.0004019.g002 Study design We compared annual (September–August) survival of prion- infected and apparently uninfected adult mule deer using a cohort design. We captured, tonsil biopsied [22], and permanently marked 131 different deer over a 2-year period. In 2005, 80 adult deer were captured and tested. In 2006, we captured and tested 79 adult deer, including 44 individuals that were test-negative in 2005; we did not recapture tonsil biopsy-positive deer. (In 2007, we also recaptured and tested 37 surviving individuals that were test-negative in 2006 to determine their final infection status.) Although we tried to avoid capturing subadult (,2-year-old) deer, we inadvertently captured 16 individuals that were ,2 years old at the time of capture; these subadults were not used for survival comparisons because infection rates typically are low in this age class [6,17]. For each year, each tonsil biopsy positive deer was matched by sex, age, PrP genotype [23], and capture location (as a proxy for home range) with a biopsy negative deer; pairings for 2005–06 and 2006–07 were independent. In all, we monitored, located, and observed 57 pairs of prion infected–uninfected deer (25 pairs in 2005–06, 32 in 2006–07), and investigated causes of death. Regardless of infection status, we allowed deer to survive until their natural demise without intervention (except where individ- uals were judged to be ‘‘terminal’’ and euthanized for humane reasons, as detailed below). Field procedures Our field procedures were reviewed and approved by the Colorado Division of Wildlife Animal Care and Use Committee (CDOW ACUC; file 08-2005) and generally followed those developed elsewhere [13,22]. All deer were captured and handled during September–February using previously-approved methods (CDOW ACUC files 12-1999, 07-2001, 07-2002, 05-2003). We used tonsil immunohistochemistry (IHC), a sensitive and specific test for prion infection in mule deer that detects a high proportion of the infected subpopulation [10,22], to classify deer as infected or uninfected. We regarded deer with abnormal prion accumulation in $1 lymphoid follicle as infected with prion disease; individuals with no evidence of prion accumulation were regarded as uninfected at the time of sampling. We anesthetized all deer prior to sampling. Biopsies were performed by veterinarians, biologists, or technicians with previous training and experience in these procedures; methods were as described elsewhere [13,22]. We estimated ages of deer by dental replacement and wear patterns. All sampled deer received a single antibiotic dose (florfenicol; 40 mg/kg, subcutaneously). In conjunction with biopsies, we collected up to 50 ml of blood via jugular or cephalic venipuncture. We marked all sampled deer with individually-identifiable plastic ear tags and with collars equipped with mortality-sensing VHF radio transmitters prior to recovery and release. Residual sedation or anesthesia was antagonized to speed recovery. Marked deer were tracked twice weekly via telemetry to monitor survival and movements. Deer whose telemetry devices transmitted a mortality signal were tracked down immediately. Sites where study deer died were marked using a global positioning system (GPS) unit, examined, and photographed. The probable PLoS ONE | www.plosone.org 4 December 2008 | Volume 3 | Issue 12 | e4019