Margaret A. Wild

PRP GENOTYPES OF CAPTIVE AND FREE-RANGING ROCKY MOUNTAIN ELK (CERVUS ELAPHUS NELSONI) WITH CHRONIC WASTING DISEASE

The PrP gene encodes the putative causative agent of the transmissible spongiform encephalopathies (TSEs), a heterogeneous group of fatal, neurodegenerative disorders including human Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, ovine scrapie and chronic wasting disease (CWD) of North American deer and elk. Polymorphisms in the PrP gene are associated with variations in relative susceptibility, pathological lesion patterns, incubation times and clinical course of TSEs of humans, mice and sheep. Sequence analysis of the PrP gene from Rocky Mountain elk showed only one amino acid change (Met to Leu at cervid codon 132). Homozygosity for Met at the corresponding polymorphic site (Met to Val) in humans (human codon 129) predisposes exposed individuals to some forms of Creutzfeldt-Jakob disease. In this study, Rocky Mountain elk homozygous for PrP codon 1 32 Met were over-represented in both free-ranging and farm-raised CWD-affected elk when compared to unaffected control groups.

EPIDEMIOLOGY OF CHRONIC WASTING DISEASE IN CAPTIVE ROCKY MOUNTAIN ELK

Between June 1986 and May 1997, chronic wasting disease (CWD) was the only natural cause of adult mortality among captive Rocky Mountain elk (Cervus elaphus nelsoni) held at a wildlife research facility near Fort Collins, Colorado (USA). Of 23 elk that remained in this herd >15 mo, four (17%) developed CWD. All affected elk were unrelated females from the founding cohort, captured as neonates and raised in 1986. The index case was diagnosed in 1989; time intervals between subsequent cases ranged from 13 to 32 mo. Initial age at onset of clinical signs ranged from about 2.9 to 8.1 yr; duration of clinical disease ranged from 5 to 12 mo (mean = 7.5 mo) prior to death. Intraspecific lateral transmission of CWD seemed the most plausible explanation for the epidemic pattern observed; neither periparturient nor maternal transmission appeared necessary to sustain this outbreak. Early detection and elimination of incubating or clinical individuals may have aided in reducing exposure or infection rates as compared to a previous outbreak in the same facility. Transmission routes and rates, pathogenesis, antemortem diagnostic tools, and the potential role of reservoirs or environmental contamination in perpetuating CWD epidemics warrant further investigation.

Comparison of Histological Lesions and Immunohistochemical Staining of Proteinase-resistant Prion Protein in a Naturally Occurring Spongiform Encephalopathy of Free-ranging Mule Deer (Odocoileus hemionus) with Those of Chronic Wasting Disease of Captive Mule Deer

In this investigation, the nature and distribution of histologic lesions and immunohistochemical staining (IHC) of a proteinase-resistant prion protein were compared in free-ranging mule deer (Odocoileus hemionus) dying of a naturally occurring spongiform encephalopathy (SE) and captive mule deer dying of chronic wasting disease (CWD). Sixteen free-ranging deer with SE, 12 free-ranging deer without SE, and 10 captive deer with CWD were examined at necropsy. Tissue sections were stained with hematoxylin and eosin, and duplicate sections were stained with a monoclonal antibody (F89/160.1.5). Histological lesions in the freeranging deer with SE and captive deer with CWD were found throughout the brain and spinal cord but were especially prominent in the myelencephalon, diencephalon, and rhinencephalon. The lesions were characterized by spongiform degeneration of gray matter neuropil, intracytoplasmic vacuolation and degeneration of neurons, and astrocytosis. IHC was found throughout the brain and retina of deer with SE and CWD. Positive IHC was found in lymphoid tissue of deer with SE and CWD. Histologic lesions and IHC were not found in multiple sections of integument, digestive, respiratory, cardiovascular, endocrine, musculoskeletal, and urogenital systems of deer with SE or CWD. Comparison of histologic lesions and IHC in tissues of free-ranging deer with those of captive deer provides strong evidence that these two diseases are indistinguishable morphologically.

Preclinical diagnosis of chronic wasting disease in captive mule deer (Odocoileus hemionus) and white-tailed deer (Odocoileus virginianus) using tonsillar biopsy.

The usefulness of tonsillar biopsy on live deer for preclinical diagnosis of the transmissible spongiform encephalopathy chronic wasting disease (CWD) was evaluated. Disease was tracked in a CWD-endemic herd using serial tonsillar biopsies collected at 6 to 9 month intervals from 34 captive mule deer (Odocoileus hemionus) and five white-tailed deer (O. virginianus). Tonsillar biopsies were examined for accumulation of PrP(CWD), the protein marker for infection, using immunohistochemical (IHC) staining. 26/34 (76%) mule deer and 4/5 (80%) white-tailed deer had PrP(CWD) accumulation in tonsillar biopsies; CWD was subsequently confirmed by post-mortem examination in all 30 of these tonsillar-positive deer. Six mule deer with IHC-negative tonsillar biopsies had positive brain and tonsillar IHC staining upon death 12 to 40 months following the last biopsy. PrP(CWD) accumulation in tonsillar biopsy was observed 2 to 20 months before CWD-related death and up to 14 months before onset of clinical signs of CWD. Tonsillar biopsies from 3-month-old mule deer (n=6) were IHC negative, but PrP(CWD) accumulation was detected in tonsillar biopsies from 7/10 mule deer by 19 months of age. Tonsillar biopsy evaluated with IHC staining is a useful technique for the preclinical diagnosis of CWD in live mule deer and white-tailed deer when intensive management approaches are possible.

DETECTION OF PrP CWD IN FECES FROM NATURALLY EXPOSED ROCKY MOUNTAIN ELK (CERVUS ELAPHUS NELSONI) USING PROTEIN MISFOLDING CYCLIC AMPLIFICATION

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy affecting captive and free-ranging cervids. Currently, tests for CWD in live animals involve relatively invasive procedures to collect lymphoid tissue biopsies and examine them for CWD-associated, protease-resistant cervid prion protein (PrPCWD) detected by immunohistochemistry (IHC). We adapted an ultrasensitive prion detection system, protein misfolding cyclic amplification (PMCA), to detect PrPCWD in Rocky Mountain elk (Cervus elaphus nelsoni) feces. Our PMCA reproducibly detected a 1.2×107 dilution of PrPCWD (a 10% infected brain homogenate diluted 1.2×106-fold into 10% fecal homogenates), equivalent to approximately 100 pg of PrPCWD/g of feces. We developed a semiquantitative scoring system based on the first PMCA round at which PrPCWD was detected and fit a nonlinear regression curve to our serial dilutions to correlate PMCA scores with known PrPCWD concentrations. We used this PMCA scoring system to detect PrPCWD and estimate its concentration in feces from free-ranging elk from Rocky Mountain National Park, Colorado. We compared our results to PrPCWD IHC of rectoanal mucosa-associated lymphoid tissue and obex from the same animals. The PMCA successfully detected PrPCWD in feces from elk that were positive by IHC, with estimated prion loads from 100 to 5,000 pg PrPCWD/g of feces. These data show for the first time PrPCWD in feces from naturally exposed free-ranging elk and demonstrate the potential of PMCA as a new, noninvasive CWD diagnostic tool to complement IHC.

Epidemiology of chronic wasting disease in captive white-tailed and mule deer.

The natural occurrence of chronic wasting disease (CWD) in a 1993 cohort of captive white-tailed deer (Odocoileus virginianus) afforded the opportunity to describe epidemic dynamics in this species and to compare dynamics with those seen in contemporary cohorts of captive mule deer (O. hemionus) also infected with CWD. The overall incidence of clinical CWD in white-tailed deer was 82% (nine of 11) among individuals that survived >15 mo. Affected white-tailed deer died or were killed because of terminal CWD at age 49–76 mo (x̄=59.6 mo, SE=3.9 mo). Epidemic dynamics of CWD in captive white-tailed deer were similar to dynamics in mule deer cohorts. Incidence of clinical CWD was 57% (4/7) among hand-raised (HR) and 67% (4/6) among dam-raised (DR) mule deer; affected HR mule deer succumbed at 64–86 mo of age (x̄=72 mo; SE=5 mo), and affected DR mule deer died at age 31–58 mo (x̄=41.3 mo; SE=6.1 mo). Sustained horizontal transmission of CWD most plausibly explained epidemic dynamics, but the original source of exposures could not be determined. Apparent differences in mean age at CWD-caused death among these cohorts may be attributable to differences in the timing or intensity of exposure to CWD, and these factors appear to be more likely to influence epidemic dynamics than species differences. It follows that CWD epidemic dynamics in sympatric, free-ranging white-tailed and mule deer sharing habitats in western North American ranges also may be similar.

Wildlife Disease Management: A Manager's Model

Wildlife disease management (WDM) is one of the great challenges of contemporary wildlife management. Experience with chronic wasting disease (CWD) indicates the importance of human dimensions in WDM. Wildlife management and disease specialists created a concept map (managers model) of the WDM system that depicts the human dimensions considerations involved in WDM and how they may affect management objectives and actions. The WDM model includes risk perception, impact tolerance, and social acceptability of management actions that contribute to perceived impacts of wildlife disease and management responses. The managers model of WDM is an experience-grounded, normative framework for discussing management of CWD.

EFFICACY OF ANTEMORTEM RECTAL BIOPSIES TO DIAGNOSE AND ESTIMATE PREVALENCE OF CHRONIC WASTING DISEASE IN FREE-RANGING COW ELK (CERVUS ELAPHUS NELSONI)

A reliable antemortem test is needed to understand the ecology of chronic wasting disease (CWD) in elk (Cervus elaphus nelsoni). We measured the ability of antemortem biopsy samples from the rectal mucosa to detect the abnormal prion protein associated with CWD (PrPCWD), the relationship between test results from the obex and rectal biopsies at varying stages of CWD progression, and the prevalence of CWD in free-ranging elk from Rocky Mountain National Park, Colorado, USA. We sampled and placed radio collars on 136 adult female elk in the winter of 2007–08. Elk with biopsy samples found positive for PrPCWD by immunohistochemistry (IHC) were euthanized and the obex and retropharyngeal lymph nodes were examined with IHC. We resampled, euthanized, and necropsied 20, 25, and 34 of the remaining study elk in each of the three following winters, respectively. Sensitivity of rectal biopsy samples increased in an asymptotic fashion with follicle count and was maximized at 85% (95% credible limits [CL]=60, 98) in the beginning of the study, when a greater proportion of elk were in a detectable stage of prion infection. However, maximum sensitivity was reduced to 72% (CL=46, 94) when we included resampled elk, which included recently infected elk that were initially negative using rectal biopsies and IHC. Test results were similar between rectal biopsies and the obex, but the earliest stages of prion infection were only detected by using retropharyngeal lymph nodes. Minimum CWD prevalence was estimated to be 9.9% (CL=5.7, 15.7) using rectal biopsies, but this rose to 12.9% (CL=8.0, 19.1) when we included four elk that were likely misdiagnosed at initial capture. Our results indicate rectal biopsies can provide a useful research tool for CWD in elk populations, but should be used with caution because they can miss individuals in early stages of infection and underestimate prevalence. Prevalence estimates from this population are the highest reported to date in elk and indicate that under appropriate conditions, CWD may be able to affect the dynamics of high-density elk populations.

THE ROLE OF PREDATION IN DISEASE CONTROL: A COMPARISON OF SELECTIVE AND NONSELECTIVE REMOVAL ON PRION DISEASE DYNAMICS IN DEER

Effective measures for controlling chronic wasting disease (CWD), a contagious prion disease of cervids, remain elusive. We review theoretic relationships between predation and host-parasite dynamics and describe a mathematical model to evaluate the potential influence of random removal through harvest or culling and selective predation by wolves (Canis lupus) upon CWD dynamics in deer (Odocoileus spp.) populations. Imposing nonselective mortality representing a 15% annual harvest or cull 51 yr after CWD introduction lowered both deer population size and steady state CWD. Selective (4×) mortality at the same 15% predation rate caused a more modest reduction in deer population size accompanied by a relatively rapid decline in CWD prevalence and elimination of the disease from a closed population. The impacts of selective predation on epidemic dynamics were sensitive to assumptions on parameter estimates; however, within expected ranges, the results of selective predation were consistent and robust. We suggest that as CWD distribution and wolf range overlap in the future, wolf predation may suppress disease emergence or limit prevalence.

SAFETY AND EFFICACY OF BRUCELLA ABORTUS STRAIN RB51 VACCINE IN CAPTIVE PREGNANT ELK

Brucella abortus strain RB51 is a laboratory-derived rough mutant of virulent B. abortus strain 2308 used as a vaccine because it induces antibodies that do not react on standard brucellosis serologic tests. Strain RB51 vaccine was evaluated in pregnant captive elk (Cervus elaphus) to determine (1) if it induced abortion and (2) if it protected against abortion following subsequent challenge. The time period of this study (February–June, 1998) was similar to field conditions where elk are vaccinated and possibly exposed to B. abortus. Fourteen elk were randomly and equally divided into vaccinated and control groups. The vaccinated group was vaccinated intramuscularly with 1.03 × 1010 colony-forming units (CFU) of strain RB51 and seroconverted postvaccination. Antibodies to strain RB51 were detected by a modification of an existing dot-blot assay. Both groups were challenged 40 days postvaccination with 9.8 × 106 CFU of B. abortus strain 2308 administered intraconjunctivally. The first abortion occurred 38 days postchallenge. Abortion occurred in all control elk and in five of seven vaccinated elk 5 to 12 wk postchallenge (P = 0.23). Mixed strain RB51 and 2308 infections were present in fetuses and vaginas from the vaccinated group whereas only strain 2308 was cultured from control group fetuses and vaginal swabs. Further evaluation of strain RB51 will be necessary to determine if it will be safe and efficacious in free-ranging pregnant elk.