Experimental transmission of chronic wasting disease (CWD) from elk and white-tailed deer to fallow deer by intracerebral route: Final report

Abstract

Final observations on experimental transmission of chronic wasting disease (CWD) from elk (Cervus elaphus nelsoni) and white-tailed deer (Odocoileus virginianus) to fallow deer (Dama dama) are reported herein. During the 5-year study, 13 fawns were inoculated intracerebrally with CWD-infected brain material from white-tailed deer (n = 7; Group A) or elk (n = 6; Group B), and 3 other fawns were kept as uninoculated controls (Group C). As described previously, 3 CWD-inoculated deer were euthanized at 7.6 mo post-inoculation (MPI). None revealed presence of abnormal prion protein (PrP^d) in their tissues. At 24 (Group A) and 26 (Group B) MPI, 2 deer were necropsied. Both animals had a small focal accumulation of PrP^d in their midbrains. Between 29 and 37 MPI, 3 other deer (all from Group A) were euthanized. The 5 remaining deer became sick and were euthanized between 51 and 60 MPI (1 from Group A and 4 from Group B). Microscopic lesions of spongiform encephalopathy (SE) were observed in only these 5 animals; however, PrP^d was detected in tissues of the central nervous system by immunohistochemistry, Western blot, and by commercial rapid test in all animals that survived beyond 24 MPI. This study demonstrates that intracerebrally inoculated fallow deer not only amplify CWD prions, but also develop lesions of spongiform encephalopathy.

Chronic wasting disease (CWD), a prion disease, is a neurode-generative transmissible spongiform encephalopathy (TSE) that has been identified in captive and free-ranging cervids (1,2) and has been experimentally transmitted by intracerebral inoculation of infected brain material into a variety of domestic, wild, and laboratory animal species (2). The origin of CWD is not known; however, it has been proposed that the prion agent may have originated from sheep scrapie (2). Experimental inoculation of elk with scrapie resulted in a TSE that was neuropathologically indistinguishable from CWD in that species (3). In a previous publication, we reported preliminary findings of an experimental CWD inoculation of fallow deer via the intracerebral route up to 37 mo post-inoculation (4). The present communication describes final results of that study, which was terminated 5 years after the study was initiated.

Sixteen 7- to 8-month-old fallow deer fawns were purchased from a breeder outside of the CWD endemic area and were assigned to Group A inoculated with CWD^wtd (n = 7), Group B inoculated with CWD^elk (n = 6), and Group C uninoculated controls (n = 3) (4). The experimental design, inoculation procedure, inoculums used, and the husbandry of these animals have been described previously (4).

Animals were euthanized with pentobarbital sodium overdose and a complete necropsy was conducted on each of the carcasses. Representative samples of all organs were fixed by immersion in 10% neutral-buffered formalin. One eye (retina) was fixed in Bouin’s solution. The brain was cut longitudinally; one half was fixed in formalin for not less than 3 wk and the other half was frozen at −70°C. The formalin-fixed brain was cut into 2- to 4-mm wide coronal sections and a minimum of 5 hemi-sections of brain per animal (Figure 1) were processed, embedded in paraffin wax, and sectioned at 5 μm. Sections were stained with hematoxylin and eosin (H&E). Serial sections were subject to immunohistochemistry (IHC) for detection of abnormal prion protein (PrP^d) with the monoclonal antibody F99/97.6.1 (5) used at a final concentration of 1.0 μg/mL. Central nervous system tissues of one positive animal (No. 3661) were also labelled by IHC with monoclonal antibody 6H4 (0.2 μg/mL).

Figure 1.

Diagrammatic representation of the distribution of lesions of spongiform encephalopathy (black dots) and the presence of PrP^d (red) in 5 areas of the brain and at 3 locations of spinal cord of fallow deer No. 3641 (59.2 mo post-inoculation) inoculated with CWD from elk. A — Frontal cerebral cortex. B — Hippocampus. C — Midbrain at the level of superior colliculus. D — Cerebellum. E — Medulla oblongata at the level of obex. F — Cervical spinal cord. G — Thoracic spinal cord. H — Lumbar spinal cord.

For detection of PrP^d by western blotting, a method approved by the Office international des épizooties (OIE) (6) was employed with frozen brain (brainstem) material.

As described previously (4), at 7.6 mo post-inoculation (MPI), 3 deer (2 from group A and 1 from group B; Nos. 3649, 3656, 3654; Table I) were euthanized (Table I). At 24 MPI one deer died (No. 3530, group A, Table I). Shortly thereafter (26 MPI), a non-clinical inoculated deer from group B (No. 3463, Table I) was euthanized to see if this animal was also positive by IHC. No gross or microscopic lesions were seen in this animal, but like in animal No. 3530, there was presence of PrP^d by IHC in the section of colliculus (Table I).

Table I.

Findings in fallow deer intracerebrally inoculated with chronic wasting disease (CWD) agent from white-tailed deer (CWD^wtd) and elk (CWD^elk)

Group	Number	Ear tag number	Sex	Inoculum	Survival time (MPI)	Histopathology (SE)/PrPd Immunohistochemistry								WB rapid tests brainstem	BD	IX
						Cerebrum	Colliculus	Hippocampus	Cerebellum	Brainstem	Cervical SC	Thoracic SC	Lumbar SC
A	1*	3649	F	CWDwtd	7.6**	−/−	−/−	−/−	−/−	−/−	−/−	NE	NE	−	−	−
	2*	3656	F	CWDwtd	7.6**	−/−	−/−	−/−	−/−	−/−	−/−	NE	NE	−	−	−
	3*	3530	MC	CWDwtd	24.8	−/−	−/+	−/−	−/−	−/−	NE	NE	NE	+	+	+
	4*	3659	MC	CWDwtd	29.2	−/−	−/+	−/−	−/−	−/+	−/+	−/+	−/+	+	+	+
	5*	3652	MC	CWDwtd	34.9	−/−	−/+	−/−	−/−	−/+	−/+	−/−	−/−	+	+	+
	6*	3661	MC	CWDwtd	37.0	−/−	−/+	−/−	−/−	−/+	−/+	−/+	−/+	+	+	+
	7	3629	F	CWDwtd	53.7	−/+	+/+	−/+	−/+	+/+	+/+	+/+	+/+	+	NE	+
Mean 36.0 ± 5
B	8*	3654	F	CWDelk	7.6**	−/−	−/−	−/−	−/−	−/−	−/−	NE	NE	−	−	−
	9*	3463	MC	CWDelk	26.0	−/−	−/+	−/−	−/−	−/−	−/−	−/−	−/−	−	−	−
	10	3658	F	CWDelk	51.1	+/+	+/+	−/+	−/+	+/+	+/+	−/+	+/+	+	NE	+
	11	3470	MC	CWDelk	56.0	+/+	+/+	−/+	+/+	+/+	+/+	+/+	+/+	+	NE	+
	12	3643	MC	CWDelk	58.3	+/+	+/+	+/+	−/+	+/+	+/+	+/+	+/+	+	NE	+
	13	3641	MC	CWDelk	59.2	+/+	+/+	+/+	−/+	+/+	−/+	−/+	−/+	+	NE	+
Mean 50.0 ± 6
C	14	3630	MC	Control	41.0	−/−	−/−	−/−	−/−	−/−	−/−	−/−	−/−	−	−	−
	15	3631	MC	Control	60.4	−/−	−/−	−/−	−/−	−/−	−/−	−/−	−/−	−	NE	−
	16	3640	MC	Control	60.4	−/−	−/−	−/−	−/−	−/−	−/−	−/−	−/−	−	NE	−

MPI — months post-inoculation; SE — spongiform encephalopathy; WB — Western blot; BD — Biorad; IX — Idexx; CWD^wtd — chronic wasting disease of white-tailed deer; CWD^elk — chronic wasting disease of elk; SC — spinal cord; F — female; MC — castrated male; + — lesion/antigen present; − — lesion/antigen absent; NE — not examined;

^**Euthanized at 7.6 MPI (not included in the mean).

^*[Data documented previously in a preliminary publication (Hamir et al., J Comp Pathol 138:121–130, 2008)].

Between 29 and 37 MPI, 3 other deer from group A (Nos. 3659, 3652, 3661; Table I) developed clinical signs of progressive chronic illness. All had shown gradual decrease in appetite and some loss of body weight over a period of 2 to 3 wk. Also, all 3 deer had shown intermittent drooling of saliva and sometimes dropping of feed from the mouth. During the last week of their lives, hind leg weakness was noticed which had led to difficulty in getting up. At necropsy the carcasses were in fair nutritional state. Significant gross lesions were not observed in any of the carcasses and microscopic lesions of spongiform encephalopathy (SE) were not seen.

The 5 remaining experimental animals (Nos. 3629, 3658, 3470, 3643, and 3641; Table I) showed clinical signs and were euthanized between 51 and 60 MPI. At this time the study was terminated and the remaining 2 control deer were euthanized (Table I). Mean survival times of group A was shorter (36 MPI) than group B (50 MPI), however, this was not statistically significant (P = 0.11).

The 5 deer that survived ≥ 51 mo revealed morphologic lesions of spongiform encephalopathy (SE; Table I; Figures 1–3). The lesions were most severe in sections of colliculi. Although lesions of SE were present in the brainstems of these deer, they were rather mild and were mostly confined to the area of dorsal motor ganglion of the vagus (Figures 1 and 3). The SE lesions consisted of vacuolation of neuropil and an occasional vacuole within neuronal perikarya (Figures 1–3). At the affected sites neither gliosis nor neuronal degeneration was seen. Florid plaques as observed in experimental CWD in white-tailed deer (7) were not seen in any of the fallow deer.

Figure 3.

Fallow deer; brain of animal No. 3641 (59.2 mo post-inoculation) inoculated with CWD from elk. H&E. Obex. There are moderate numbers of vacuoles in the neuropil and occasional small vacuoles in neuronal perikarya. Bar = 60 μm.

There was wider distribution of PrP^d labeling in these fallow deer relative to the initial animals that were examined previously (4). PrP^d labeling was present throughout the brain and spinal cord (Table I; Figures 1, 4, and 5). The labeling was present in sections examined from brainstem (obex; Figure 5), cerebellum, colliculus, hippocampus (Figure 4), rostral cerebrum, and spinal cord at the cervical, thoracic, and lumbar levels from all inoculated deer. PrP^d labeling in the eye occurred in all deer and was confined to retina and optic nerve and ranged from granular staining of the optic nerve and optical fiber layer at the optic nerve head to uniform labeling throughout the plexiform layers and with lesser amounts in the ganglion cell cytoplasm and between nuclei in the inner nuclear layer.

Figure 4.

Fallow deer; animal No. 3641 (59.2 months post-inoculation) inoculated with CWD from elk. Stained for PrP^d (red) by IHC (immunoalkaline phosphatase) and counter-stained with hematoxylin. Hippocampus. There is extensive diffuse presence of PrP^d staining in the neuropil. Bar = 270 μm.

Figure 5.

Fallow deer; animal No. 3641 (59.2 months post-inoculation) inoculated with CWD from elk. Stained for PrP^d (red) by IHC (immunoalka-line phosphatase) and counter-stained with hematoxylin. Obex. There are locally extensive areas of granular staining in the neuropil. Bar = 270 μm.

All IHC positive animals were determined to be positive by Western blot (WB; Table I) and rapid enzyme immunoassays (Table I). No differences were observed in molecular profile between the experimental animals and the positive control, a white-tailed deer CWD positive animal. The 3 control deer were negative on WB test.

Although naturally occurring CWD is widespread in cervid populations in certain areas of United States, the disease has not been documented in fallow deer under natural conditions. In this regard, an attempt to transmit CWD to fallow deer by exposing them to infected white-tailed deer and to contaminated pastures in Colorado has also been unsuccessful (Dr. Jack Rhyan, United States Department of Agriculture, personal communication, 2008).

Experimental transmission of prion agents between different host species can provide valuable clues as to the potential of prion diseases to cross the species barrier (8). In non-fallow deer, clinical CWD is characterized by emaciation, changes in behavior, and excessive salivation (2). In the present study, there was decreased appetite and moderate weight loss but none of the affected deer were emaciated. Also, the behavioral changes were rather subtle and intermittent salivation was observed in only 3 deer (Nos. 3659, 3652, 3661; group A).

There was no significant difference in survival times and clinico-pathological findings in fallow deer that were inoculated with CWD from WTD (group A) and CWD from elk (group B). In general the fallow deer in group A succumbed to disease earlier (most between 24 to 37 MPI; Table I) than fallow deer in group B (most between 51 to 59 MPI; Table I). However, as a group this difference was not statistically significant (P = 0.11).

Morphologic lesions of SE were observed in only the 5 animals that survived over 51 MPI (Table I). Although lesions of SE are considered the hallmark of TSEs (9), in some cross-species transmission experiments of TSEs, such as scrapie and CWD in cattle, the subjects did not show characteristic lesions of SE (10–13). A similar lack of morphologic lesions was observed in cattle inoculated with CWD^{mule deer} (second passage) (14). It is speculated that the neurologic signs in the affected animals were most likely due to excessive accumulation of PrP^d in the CNS tissues.

Figure 2.

Fallow deer; brain of animal No. 3641 (59.2 mo post-inoculation) inoculated with CWD from elk. H&E. Hippocampus. There is presence of moderate vacuolation of the neuropil. Bar = 100 μm.