Horizontal Transmission of Chronic Wasting Disease in Reindeer

We challenged reindeer by the intracranial route with the agent of chronic wasting disease sourced from white-tailed deer, mule deer, or elk and tested for horizontal transmission to naive reindeer. Reindeer were susceptible to chronic wasting disease regardless of source species. Horizontal transmission occurred through direct contact or indirectly through the environment.

The inocula were prepared from pooled brain material from chronic wasting diseaseaffected elk from South Dakota, USA (CWD elk ), and mule deer from Wyoming, USA (CWD md ), as described previously (2). The inoculum from CWD-infected white-tailed deer (CWD wtd ) was prepared from pooled brainstems from 3 white-tailed deer; 1 deer had been inoculated intracranially with the CWD elk inoculum, the second deer with the CWD md inoculum, and the third deer with pooled brain material from CWD-affected white-tailed deer from Wisconsin (2).
All deer from which the inocula were prepared were positive for the pathogenic form of the scrapie prion protein (PrP Sc ) by immunohistochemistry (IHC), and the pooled inocula were positive by Western blot. The brain material for the inocula was homogenized to 10% (w/v) in phosphate-buffered saline; pH 6.15), and gentamicin was added (to 100 g/mL).

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Animals Twenty-three 3-month-old reindeer fawns were obtained from a farm in Alaska, USA, that had never had a reported case of CWD. Reindeer were used in preference to caribou because they are semidomesticated and so more appropriate for experimental studies. The fawns were divided into 4 groups: group 1 (n = 5) was inoculated with CWD wtd ; group 2 (n = 5) was inoculated with CWD elk ; group 3 (n = 5) was inoculated with CWD md ; and group 4 (n = 8) remained as negative controls (n = 2) or were placed in direct (n = 4) or indirect (n = 2) contact groups.

Genotyping
Genotype analysis was conducted on nucleic acid extracted from blood samples from live animals, as described previously (3). The nucleotide numbering for the prion protein gene (PRNP) sequence is based on the GenBank Rangifer tarandus tarandus sequence (accession no.

Animal Housing and Pen-to-Pen Movement
All reindeer were housed in an animal biosafety level 2 containment facility at the National Animal Disease Center, US Department of Agriculture (Ames, IA, USA), and were monitored twice daily during the experiment. 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,  Twenty-five months after intracranially challenged reindeer were inoculated, 4 control reindeer (reindeer #16, #17, #18, #19) were moved into the same pen as CWD wtd -inoculated reindeer (Technical Appendix Figure, panel B, pen 5+7) to form the direct contact control group (Table 1, group 4 direct). At the same time, 2 control reindeer (#20, #21) were moved into pen 6, which was adjacent to pen 4 which housed the CWD md -inoculated reindeer (Technical Appendix Figure, panel B, pen 4, pen 6) to form the indirect contact control group. Solid partitions between pen 4 and pen 6 prevented physical contact between the reindeer in these 2 pens; however, reindeer in both pens could reach through the front of the pen to access a central alleyway that could contain bedding or water from adjacent pens generated during daily cleaning. The 2 control reindeer (#22, #23) in pen 8 formed the negative control group (Technical Appendix Figure, panel B, pen 8).
Forty-four months later, CWD md -inoculated reindeer #15 was moved from pen 4 to join reindeer #13 in pen 2 (Technical Appendix Figure, panel C). Pen 4 was cleaned by manual sweeping to remove bedding, etc., followed by pressure washing with water, followed by decontamination with 20,000 ppm sodium hypochlorite for a 1h contact time. Group 4 indirect contact reindeer were moved from pen 6 to pen 4. Pen 6 was cleaned and decontaminated, and then group 4 control reindeer were moved from pen 8 to pen 6. Reindeer remained in these pens until the end of the study.

Survival Times
Survival times for intracranially challenged reindeer are expressed as months postinoculation (MPI) and are calculated from the day the reindeer were inoculated. Survival times for direct and indirect contact reindeer are expressed as months postchallenge and are calculated from the date that direct contact control reindeer were mixed with CWD wtd -inoculated reindeer.

Immunohistochemistry
All paraffin-embedded tissues were stained by an automated IHC method for detection of PrP Sc as described previously (3) using an automated immunostainer (Ventana Medical System Inc, Tucson, AZ, USA). The primary antibody was F99/97.6.1 (4), used at a concentration of 10 µg/mL, and incubation was carried out at 37C for 32 min. ELISA Frozen brainstem (at the level of the obex) and/or retropharyngeal lymph node were used for detection of PrP Sc using the IDEXX HerdChek BSE-Scrapie Antigen ELISA (IDEXX, Westbrook, ME, USA). For animals that were negative by IHC, brainstem and retropharyngeal lymph node samples were processed as per kit instructions. For animals that were positive by IHC, brainstem samples were homogenized in phosphate-buffered saline to enable testing of the same sample by both ELISA and Western blot.
ELISA was performed according to the kit instructions (short protocol) with the following modifications: capture plate incubation was performed for 1.5 h; conjugate incubation was performed for 1 h. Samples were run as singles with kit-provided negative and positive controls included in each run. Small ruminant brain conjugate was used for all samples.

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Absorbance was measured using a SpectraMax 190 plate reader at 450 nm with a reference wavelength of 620 nm.

Clinical Presentation
Clinical signs included loss of body condition (n = 5), recumbency (n = 4), and lethargy (n = 2). Seven reindeer were found dead without clinical signs noted. Seizures developed in 1 CWD wtd -inoculated reindeer (#4), and it was euthanized. Bloat that was not responsive to treatment developed in a reindeer (#21) from the indirect contact group, and it was euthanized.

Vacuolation and PrP Sc Distribution in Central Nervous System Tissues
Neither neuropil nor neuronal vacuolation was seen in direct or indirect contact animals.
PrP Sc was not detected in the brains from any direct contact animals, including the 2 reindeer that had PrP Sc in non-central nervous system (CNS) tissues (#17 and #18).

All 4 CWD wtd -inoculated reindeer had both CNS vacuolation and PrP Sc accumulation at
clinical stages (20.9-53.3 MPI), except for 1 intercurrent death (#1) at 2.6 MPI. Both vacuolation and PrP Sc accumulation were seen in the brain of the CWD elk -inoculated reindeer that was found dead at 24.7 MPI (#6). A second CWD elk -inoculated reindeer was found dead at 36.4 MPI (#7) without microscopic evidence of spongiform encephalopathy. The remaining 3 CWD elkinoculated reindeer had widespread CNS vacuolation and PrP Sc accumulation at death. In the CWD md -inoculated group, widespread vacuolation and PrP Sc accumulation were present in Page 5 of 7 reindeer that survived up to 30 months (n = 2, #12 and #13). In contrast, reindeer with survival times >30 months showed widespread PrP Sc accumulation but minimal (#15, thalamus and frontal cortex only) or no (#14) vacuolation.
PrP Sc was present in the brains of intracranially inoculated and indirect contact reindeer.
The most striking pattern of PrP Sc deposition in the brain was dominated by aggregated deposits of various sizes, including plaques, distributed throughout the neuroaxis (Figure 1, panels A,B).
The fourth pattern comprised neuropil labeling confined to the brainstem at the level of the obex (hypoglossal nucleus, dorsal motor nucleus of the vagus nerve, area postrema) and midbrain (trochlear nuclei) that was observed in 3 reindeer of the SS138 NN176 genotype (#10, #14, and #20).
All animals with PrP Sc immunoreactivity in brainstem also had PrP Sc in the retina ( Table   2). In the optic fiber layer, PrP Sc immunoreactivity was observed as mild punctate deposits and/or rare intramicroglial deposits ( commonly observed PrP Sc immunolabeling pattern in the inner plexiform layer was mild to moderate punctate deposits with occasional intramicroglial deposits (Figure 1, panel E). In 2 reindeer (#8 and #13) moderate particulate to coalescing immunolabeling was observed in the inner plexiform layer (Figure 1, panel F); notably, both of these animals had aggregated and plaque-like PrP Sc deposits in the brain. PrP Sc in the outer plexiform layer was present as very mild to moderate granular labeling.
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PrP Sc Distribution in Non-CNS Tissues
The tissue distribution of PrP Sc was similar for all intracranially inoculated reindeer ( The retropharyngeal lymph node, pharyngeal tonsil, palatine tonsil, mesenteric lymph node, spleen, prescapular lymph node and popliteal lymph node were IHC positive in the majority (55-67%) of reindeer. The gut-associated lymphoid tissue of the ileum (Peyer's patches) and recto-anal junction (RAMALT) were IHC positive in 29.4% and 61.9% of reindeer, respectively. Twelve reindeer that had PrP Sc in the RAMALT were overall IHC positive, and 2 reindeer (both direct contact group) were negative in the RAMALT and overall negative ( Table   2). Samples of RAMALT from the remaining 5 reindeer were IHC negative, but PrP Sc was demonstrated in other tissues ( Table 2).

ELISA
Reindeer that were IHC negative in brain also were negative by Western blot and ELISA.

Western Blot
The nonglycosylated (lowest) band from the CWD md , CWD elk , and CWD wtd inocula migrated at 21.4 kDa ( Figure 2). In some instances, the migration patterns of the nonglycosylated band of challenged reindeer varied relative to the source inoculum ( Figure 2).
There was not a clear association between a higher or lower position of the nonglycosylated band and either challenge group or PRNP genotype.