Figure 1

Figure 1. Immunohistochemical analysis for the prion protein showing scrapie prion protein (PrP^Sc) deposits in brains (A–D) and retinas (E, F) from reindeer (Rangifer tarandus tarandus) with chronic wasting disease. PrP^Sc immunodetection using...

In 2005, we challenged reindeer fawns from a farm in Alaska, USA, where CWD had never been reported, by intracranial inoculation (7) with pooled brain material from CWD-affected elk from South Dakota (CWD^elk), CWD-affected mule deer from Wyoming (CWD^md), or CWD from white-tailed deer from Wisconsin combined with brain material from experimentally challenged white-tailed deer (CWD^wtd) (Table 1; Technical Appendix). Additional uninoculated fawns served as negative controls, controls for indirect transmission, and controls for direct transmission (Table 1; online Technical Appendix). We determined the prion protein gene (PRNP) genotype of each fawn (Technical Appendix), and we tried to ensure that each PRNP genotype was present in each group (Table 2). Control reindeer were housed in the same barn as inoculated reindeer but in separate pens that prevented direct physical contact (i.e., nose-to-nose) between control and inoculated animals (Technical Appendix Figure 1). Indirect and direct contact control groups were formed 25 months after intracranially challenged reindeer were inoculated (Technical Appendix Figure 1, panel B).

Clinical signs consistent with CWD were first observed 20.9 months after inoculation (Table 2). Common clinical features included found dead without clinical signs noted, loss of body condition, recumbency, and lethargy (Table 2; online Technical Appendix).

At death, a full necropsy was performed on all reindeer. Two sets of tissue samples were collected: 1 set was fixed in 10% buffered formalin, embedded in paraffin wax, sectioned at 5 μm for microscopy examination after hematoxylin and eosin staining or immunohistochemical staining using primary antibody F99/96.7.1 (Technical Appendix). A second set of tissues was frozen, and selected tissues were used for immunodetection of scrapie prion protein (PrP^Sc) by Western blot (brain tissue only) as described previously (7) but with some modifications, or an ELISA (brainstem and/or retropharyngeal lymph node) using a commercial kit (IDEXX HerdChek BSE-Scrapie Antigen ELISA; IDEXX, Westbrook, ME, USA) according to the manufacturers’ instructions (Technical Appendix).

In the intracranially inoculated groups, when intercurrent deaths were excluded, reindeer with the NN138 polymorphism (reindeer nos. 2, 6, and 12) had the shortest survival times in each group (Table 2). Different inocula did not produce significantly different survival times (log-rank test, p = 0.0931), but we observed differences in the amount of vacuolation and PrP^Sc in the brain at the clinical stages of disease in CWD^wtd- and CWD^elk-inoculated reindeer, compared with CWD^md-inoculated reindeer (Table 2; Technical Appendix). In the indirect contact animals, PrP^Sc was present in the brain but restricted to the dorsal motor nucleus of the vagus nerve and area postrema.

We observed different patterns of PrP^Sc deposition in the brain (Figure 1, panels A–D; Technical Appendix), the most striking of which was dominated by aggregated deposits of various sizes, including plaque-like deposits (Figure 1, panels A,B). This pattern was seen in reindeer with the NS138 NN176 (no. 8, CWD^elk; no. 13, CWD^md) or SS138 DD176 (no. 4, CWD^wtd) genotypes. With regard to immunoreactivity in the retina (Figure 1, panels E, F; online Technical Appendix), in 2 of 3 reindeer with aggregated deposits in the brain (nos. 8 and 13), aggregated immunoreactivity also was observed in the inner plexiform layer of the retina (Figure 1, panel f).

Figure 2

Figure 2. Western blot characterization of the inocula used to inoculate reindeer and brainstem samples from representative reindeer from each experimental group in study of chronic wasting disease transmission. Scrapie prion protein (PrP^Sc)...

Reindeer that were negative by immunohistochemical analysis in brain also were negative by Western blot and ELISA. Different Western blot migration patterns were observed in PrP^Sc-positive animals (Figure 2), but we found no clear association between migration pattern and challenge group or PRNP genotype.

PrP^Sc was widespread in lymphoid tissues from most reindeer (Table 2; online Technical Appendix). Reindeer with the NS138 genotype had a significantly lower average percentage of lymphoid follicles positive than did reindeer with NN138 (analysis of variance, p = 0.003) or SS138 (p = 0.003) deer. Excluding intercurrent deaths, PrP^Sc was detected in all 4 CWD^wtd-challenged reindeer, all 5 CWD^elk-challenged reindeer, all 4 CWD^md-challenged reindeer, both indirect contact reindeer, and 2 of 4 direct contact reindeer (Table 2).

Potential sources of infectivity for direct contact animals include urine, feces, and saliva from their CWD^wtd-challenged pen-mates, as has been shown for CWD-affected white-tailed deer (6,8,9). Pinpointing the source of infectivity in the indirect contact group is more difficult. Infectious prions can travel at least 30 m in airborne particulate (10), but because the negative control reindeer in the pen adjacent to the indirect contact reindeer did not become positive, a more direct route of transmission is likely in this case. Penning, feeding, and watering protocols were designed to prevent exposure of negative control and indirect contact reindeer to potential infectivity on feed and water buckets, bedding, or fencing (6,11). However, reindeer might have had access to bedding from adjacent pens that had spread into the central alleyway.

During the 5-year course of this study, reindeer were moved between pens several times to maintain an optimal number of animals per pen (Technical Appendix Figure 1). Prolonged persistence of prion infectivity in the natural environment has been documented for both CWD (2 years [5]) and scrapie (up to 16 years [12]). In addition, thorough cleaning and disinfection might not be sufficient to remove all infectivity from the environment, leading to persistence of infectivity under experimental housing conditions (13).

In reindeer challenged orally with the agent of CWD, the SS138 genotype (serine/serine at PRNP codon 138) has been associated with susceptibility to disease and the NS138 (asparagine/serine) genotype with resistance (1). In the study we report, disease developed in reindeer with the NS138 genotype after intracranial inoculation, although the extent of lymphoreticular system involvement was significantly lower than in NN138 and SS138 reindeer. The potential association of the NN138 polymorphism with shorter survival times is interesting. However, as with all potential genotype versus phenotype interactions, care should be taken not to over-interpret these results given the small group sizes and the large number of PRNP genotype groups in this study.

Our results demonstrate that reindeer are susceptible to the agent of CWD from white-tailed deer, mule deer, and elk sources after intracranial inoculation. Furthermore, naive reindeer are susceptible to the agent of CWD after direct and indirect exposure to CWD-infected reindeer, suggesting a high potential for horizontal transmission of CWD within and between farmed and free-ranging reindeer (and caribou) populations.