“Atypical” Chronic Wasting Disease in PRNP Genotype 225FF Mule Deer Author(s): Lisa L. Wolfe, Karen A. Fox, and Michael W. Miller Source: Journal of Wildlife Diseases, 50(3):660-665. Published By: Wildlife Disease Association DOI: http://dx.doi.org/10.7589/2013-10-274 URL: http://www.bioone.org/doi/full/10.7589/2013-10-274
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.
BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.
DOI: 10.7589/2013-10-274
Journal of Wildlife Diseases, 50(3), 2014, pp. 660–665 # Wildlife Disease Association 2014
‘‘Atypical’’ Chronic Wasting Disease in PRNP Genotype 225FF Mule Deer Lisa L. Wolfe,1,2 Karen A. Fox,1 and Michael W. Miller1 1Colorado Division of Parks and Wildlife, Wildlife Research Center, 317 West Prospect Road, Fort Collins, Colorado 80526-2097, USA; 2Corresponding author (email: [email protected])
to, and slower progression of, clinical CWD (Johnson et al. 2006, 2011; Miller et al. 2012). In mule deer (Odocoileus hemionus), S/phenylalanine (F) dimorphism at codon 225 (Brayton et al. 2004; Jewell et al. 2006) appears to have a similar influence, although S homozygosity at codon 225 leads to a more-rapid disease course (Fox et al. 2006). The time course of CWD infection after oral inoculation appeared prolonged, and deposition of CWD-associated PrP (PrPCWD) in the central nervous system tissues was delayed in 225SF mule deer compared with 225SS individuals (Fox et al. 2006). Moreover, 225SF and 225FF individuals may be underrepresented in mule deer populations as a whole (9.3%) but are even rarer among CWD-positive deer (0.3%; Jewell et al. 2006). Although PrPCWD has been detected in 225FF mule deer (Jewell et al. 2006), no clinical CWD cases have been reported in deer with this genotype. Collectively, these observations suggest the possibility of variable susceptibility associated with PRNP genotype in mule deer. To compare the susceptibility of 225SS and 225FF mule deer to CWD under conditions approximating natural transmission via environmental exposure, we exposed six yearling mule deer (three 225SS and three 225FF) to the CWD agent, by holding them together in a common, contaminated 0.5-ha paddock, and observed the outcome. Our study was conducted at the Colorado Parks and Wildlife Foothills Wildlife Research Facility (FWRF; Fort Collins, Colorado, USA) after institutional animal care and use review and approval (file 07-2007). The hand-raised 225SS deer were acquired at about 10 mo of age from a captive herd at
We compared mule deer (Odocoileus hemionus) of two different PRNP genotypes (225SS, 225FF) for susceptibility to chronic wasting disease (CWD) in the face of environmental exposure to infectivity. All three 225SS deer had immunohistochemistry (IHC)positive tonsil biopsies by 710 days postexposure (dpe), developed classic clinical signs by 723–1,200 dpe, and showed gross and microscopic pathology, enzyme-linked immunosorbent assay (ELISA) results, and IHC staining typical of prion disease in mule deer. In contrast, although all three 225FF deer also became infected, the two individuals surviving .720 dpe had consistently negative biopsies, developed more-subtle clinical signs of CWD, and died 924 or 1,783 dpe. The 225FF deer were ‘‘suspect’’ by ELISA postmortem but showed negative or equivocal IHC staining of lymphoid tissues; both clinically affected 225FF deer had spongiform encephalopathy in the absence of IHC staining in the brain tissue. The experimental cases resembled three cases encountered among five additional captive 225FF deer that were not part of our experiment but also died from CWD. Aside from differences in clinical disease presentation and detection, 225FF mule deer also showed other, more-subtle, atypical traits that may help to explain the rarity of this genotype in natural populations, even in the presence of enzootic CWD. Key words: Chronic wasting disease, genotype, mule deer, Odocoileus hemionus, prion, PRNP.
ABSTRACT:
Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) found in North American cervids. Susceptibility to CWD and other TSEs appears to be affected by various polymorphisms in the gene (PRNP) encoding the host’s cellular prion protein (PrP) (Goldmann 2008; Robinson et al. 2012). For example, white-tailed deer (Odocoileus virginianus) that are heterozygous or homozygous for serine (S) at codon 96 of the PRNP gene showed reduced susceptibility 660
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Washington State University (Pullman, Washington, USA) with no history of CWD and were held indoors at the FWRF before this study began; the 225FF deer were born at the FWRF and dam-raised in outdoor paddocks that had not been used in CWD experimentation. All six individuals were negative for PrPCWD via tonsil biopsy immunohistochemistry (IHC; Wolfe et al. 2002) before being placed in the contaminated paddock. The paddock serving as an exposure source had been contaminated by use between December 2004 and February 2007 to house 20 orally inoculated 225SS mule deer (Miller et al. 2012), the last 14 of which were euthanized with clinical CWD between August 2006 and February 2007. Experimental exposure of subject deer began on 15 May 2007 (day 0). Deer were fed high-quality alfalfa, grass hay, and a pelleted nutritional supplement ad libitum throughout the study; water was from an automatic dispenser, which was cleaned regularly. We sedated deer for sampling with a combination of butorphanol (30 mg/ deer), azaperone (12.5 mg/deer), and medetomidine (10 mg/deer) and tested them for CWD using tonsil and rectal biopsy IHC (Wolfe et al. 2008) beginning after about 8 mo of exposure and every 6–8 mo thereafter. All deer were observed daily by animal caretakers for general health and were scored subjectively (05not shown, 15subtle, 25obvious) for behavioral changes, loss of body condition, ataxia, and ptyalism or polyuria/polydipsia about every 6–8 wk by a veterinarian experienced at recognizing clinical CWD. Deer with a cumulative clinical score of .4 were euthanized and necropsied. We screened retropharyngeal lymph node tissue for evidence of PrPCWD using an enzyme-linked immunosorbent assay (ELISA; Bio-Rad Laboratories, Hercules, California, USA) following methods described by Hibler et al. (2003); samples with an optical density (OD) value $0.1 were considered ‘‘suspect.’’ We also
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screened paraffin-embedded sections of a larger set of formalin-fixed lymphoid and brain tissues by IHC for prion accumulation using monoclonal antibody (MAb) F99/97.6.1 and methods described by Spraker et al. (2002); in addition, we examined embedded tissues via light microscopy after H&E staining. Tissues examined by light microscopy and IHC included retropharyngeal lymph node, tonsil, spleen, adrenal gland, vagus nerve, ileum with Peyer patches, spiral colon with gut-associated lymphoid tissue, skeletal muscle, and multiple sections of brain (caudal medulla oblongata, medulla oblongata at the level of the dorsal motor nucleus of the vagus, cerebellum, thalamus at the level of the interthalamic adhesion, hypothalamus, basal nuclei, hippocampus, olfactory cortex, frontal cortex, temporoparietal cortex, and occipital cortex). One 225FF deer died from trauma 529 days postexposure (dpe); that deer was clinically normal, and although the ELISA OD value for retropharyngeal lymph node (0.47) was ‘‘suspect,’’ all tissues were IHC-negative, and there was no evidence of spongiform change in the obex or elsewhere in the brain (Table 1). The five remaining deer all appeared to die from CWD based on some commonality of clinical signs and laboratory findings, but differences between the genotypes emerged. All three 225SS deer had positive tonsil biopsies by 710 dpe, and all were euthanized after developing classic clinical CWD (clinical scores .4, including behavioral changes and emaciation; Table 1). In contrast, neither of the surviving 225FF deer yielded positive biopsies, and both were found dead unexpectedly while showing more subtle clinical signs (mild ataxia and weight loss without the pronounced behavioral changes typically seen; Table 1). At postmortem, the gross lesions in 225SS deer (Table 1) were typical of those described for CWD (Williams 2005) and
BR2 (f) ER2 (f) ME1 (cm) Wa06 (f) Wb06 (f) 11b06 (f)
225SS
dpe 5 days postexposure; T 5 tonsil biopsy; R 5 rectal mucosa biopsy.
723 1,058 1,200 529 (d) 924 (d) 1,783 (d)
Day euthanized or died (d)
E E, R, AP E, R, AP ngl (trauma) Me, AP E, SA, R, AP
Gross
d
2.55 2.7 2.52 0.47 3.01 1.44
ELISA OD
e
Obexg
+ + + 2 2 2
RLNf
+ + + 2 * *
Immunohistochemistry
Laboratory findings
ELISA 5 enzyme-linked immunosorbent assay; OD 5 optical density value.
RLN 5 retropharyngeal lymph node; + 5 positive staining; 2 5 no staining; * 5 equivocal staining (see Fig. 1B).
+ 5 positive staining; 2 5 no staining.
SE 5 spongiform encephalopathy.
e
f
g
h
E 5 emaciation; R 5 fluid-filled or frothy rumen; AP 5 aspiration pneumonia; ngl 5 no gross lesions associated with chronic wasting disease; Me 5 megaesophagus; SA 5 serous atrophy of fat.
SE SE SE no SE SE SE
Histopathologyh
d
E 5 emaciation; Pu 5 polyuria/polydypsia; Pt 5 ptyalism; AD 5 abdominal distension; D 5 depression; W 5 weight loss; At 5 ataxia (subtle).
f 5 female; cm 5 castrated male.
c
c
E, Pu, Pt E, Pu, AD E, Pu, D None W, At W, At
Clinical signs
b
T, R (710) T (472); R (710) T, R (710) all negative (472) all negative (892) all negative (1072)
Positive biopsy (dpe)
b
a
225FF
Animal (sex)
PRNP genotype
a
Disease course and clinical findings
TABLE 1. Disease course, clinical signs, and laboratory findings among mule deer of two PRNP genotypes after environmental exposure to the chronic wasting disease agent under controlled conditions.
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FIGURE 1. Influence of PRNP genotype on microscopic findings in mule deer that died from clinical chronic wasting disease. (A) Tonsil from a 225SS deer (ER2) with robust positive staining for PrPCWD by immunohistochemistry (IHC) at 1,058 days postexposure (dpe). Scale bar520 m. (B) Tonsil from a 225FF deer (11b06) with scant positive staining (see arrows) for PrPCWD by IHC at 1,783 dpe. Bar520 m. (C) Obex from a 225SS deer (ER2) with robust positive staining for PrPCWD by IHC. Bar5100 m. (D) Obex from a 225FF deer (Wb06) lacking positive staining for PrPCWD by IHC, despite spongiform changes shown in panel F (immediately below). Bar5100 m. (E) Obex from a 225SS deer (ER2) with dorsal motor nucleus of the vagus showing spongiform changes. H&E stain. Bar5100 m. (F) Obex from a 225FF deer (Wb06) with dorsal motor nucleus of the vagus showing spongiform changes. H&E stain. Bar5100 m.
included emaciation, fluid-filled or frothy rumen, megaesophagus, and aspiration pneumonia. The ELISA OD values for retropharyngeal lymph node tissue were $2.52 (Table 1). The IHC results also were
typical of clinical prion disease in mule deer, including robust positive staining within lymphoid follicles of the retropharyngeal lymph node, tonsil (Fig. 1A), spleen, Peyer patches, and gut-associated
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FIGURE 2. Clinical signs of chronic wasting disease in a 225FF mule deer included emaciation, distended abdomen, and megaesophagus. At postmortem, the deer had depleted fat stores, cachexia, a dilated esophagus, fluid-filled rumen, and aspiration pneumonia. Microscopic examination of brain tissue revealed lesions of spongiform encephalopathy in the absence of positive staining for PrPCWD by immunohistochemistry.
lymphoid tissue of the colon, scattered staining within the adrenal medulla and vagus nerve, robust staining within the dorsal motor nucleus of the vagus and adjacent nuclei (Fig. 1C), and variably prominent staining within the thalamus, hypothalamus, basal nuclei, hippocampus, and cerebral cortex. Spongiform changes (Fig. 1E) were predominantly limited to the dorsal motor nucleus of the vagus and adjacent nuclei, although the olfactory cortex was also affected in one deer (ME1). The poor body condition, fluid rumen content, megaesophagus and aspiration pneumonia in the two longer-lived 225FF deer also were consistent with clinical CWD. However, the ELISA OD
value (1.44) was lower than expected in end-stage CWD (usually .2.5) for one 225FF deer, and IHC findings were equivocal for both (Table 1). Postmortem IHC evaluation of the tonsils revealed markedly diminished staining that was restricted to the central lymphoid follicle (Fig. 1B). One 225FF deer (Wb06) also had similar minimal staining in the retropharyngeal lymph node, spleen, and Peyer patches. Neither 225FF deer had positive IHC staining at any level of brain (Fig. 1D), in contrast to the robust positive staining seen in 225SS deer. However, both 225FF deer showed spongiform encephalopathy predominantly limited to the dorsal motor nucleus of the vagus and adjacent nuclei (Fig. 1F) comparable to changes seen in the 225SS deer; spongiform changes also were seen in the hypothalamic nucleus of one 225FF deer (11b06). Although only two 225FF deer survived long enough to develop clinical disease in our controlled experiment, these cases resembled three additional CWD cases we have encountered during the past decade among five other captive 225FF mule deer that were held at the FWRF but that were not part of our experiment. Those three cases also showed some clinical signs (Fig. 2), postmortem lesions, and histopathology consistent with CWD but yielded variable ELISA results postmortem and negative IHC results both before and after death. The prolonged incubation, more subtle signs, and incongruous laboratory findings in 225FF mule deer could be products of differences in the abnormal prion protein propagated in the course of disease or in host response to the abnormal protein. For example, atypical IHC findings could result from 225FF PrPCWD being more vulnerable to endogenous proteases, formalin fixation, or formic acid than 225SS PrPCWD, lessreadily bound by MAb F99/97.6.1, or some combination of these and other effects. In light of the similarities and differences between mule deer genotypes,
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understanding the nature of PrPCWD and its relationship to clinical disease in 225FF mule deer could help illuminate broader mechanisms underlying prion disease. Beyond the differences in prion disease manifestation described here, other relatively intangible traits that we have noted among 225FF mule deer held at the FWRF suggest these deer may be atypical in other ways that could merit more formal study. The few individuals we have observed tended to behave somewhat erratically and to have poorer hair coats, less-optimal body condition, and poorer fawn recruitment as compared with captive 225SS mule deer. Perhaps as a consequence, 225FF deer have, in our experience, been difficult to propagate in captivity. These traits, if representative of the genotype, may help explain the rarity of 225FF mule deer in natural populations even in the presence of enzootic CWD. Our work was supported by the Colorado Division of Parks and Wildlife. We thank T. Davis, I. LeVan, K. Griffin, M. Fisher, and many others for their assistance during the course of this study, and T. Spraker for consultation on histopathology and IHC interpretations. Anonymous reviewers provided helpful comments on an earlier manuscript draft. LITERATURE CITED Brayton KA, O’Rourke KI, Lyda AK, Miller MW, Knowles DP Jr. 2004. A processed pseudogene contributes to apparent mule deer prion gene heterogeneity. Gene 326:167–173. Fox KA, Jewell JE, Williams ES, Miller MW. 2006. Patterns of PrPCWD accumulation during the course of chronic wasting disease infection in orally inoculated mule deer (Odocoileus hemionus). J Gen Virol 87:3451–3461. Goldmann W. 2008. PrP genetics in ruminant transmissible spongiform encephalopathies. Vet Res 39:30. doi: 10.1051/vetres:2008010. Hibler CP, Wilson KL, Spraker TR, Miller MW, Zink RR, DeBuse LL, Andersen E, Schweitzer D, Kennedy JA, Baeten LA, et al. 2003. Field
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validation and assessment of an enzyme-linked immunosorbent assay for detecting chronic wasting disease in mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus), and Rocky Mountain elk (Cervus elaphus nelsoni). J Vet Diagn Invest 15:311–319. Jewell JE, Conner MM, Wolfe LL, Miller MW, Williams ES. 2005. Low frequency of PrP genotype 225SF among free-ranging mule deer (Odocoileus hemionus) with chronic wasting disease. J Gen Virol 86:2127–2134. Johnson C, Johnson J, Vanderloo JP, Keane D, Aiken JM, McKenzie D. 2006. Prion protein polymorphisms in white-tailed deer influence susceptibility to chronic wasting disease. J Gen Virol 87:2109–2114. Johnson CJ, Herbst A, Duque-Velasquez C, Vanderloo JP, Bochsler P, Chappell R, McKenzie D. 2011. Prion protein polymorphisms affect chronic wasting disease progression. PLoS One 6: e17450. doi:10.1371/journal.pone.0017450. Miller MW, Wolfe LL, Sirochman TM, Sirochman MA, Jewell JE, Williams ES. 2012. Survival patterns in white-tailed and mule deer after oral inoculation with a standardized, conspecific prion dose. J Wildl Dis 48:526–529. Robinson SJ, Samuel MD, O’Rourke KI, Johnson CJ. 2012. The role of genetics in chronic wasting disease of North American cervids. Prion 6:153– 162. Spraker TR, O’Rourke KI, Balachandran A, Zink RR, Cummings BA, Miller MW, Powers BE. 2002. Validation of monoclonal antibody F99/97.6.1 for immunohistochemical staining of brain and tonsil in mule deer (Odocoileus hemionus) with chronic wasting disease. J Vet Diagn Invest 14:3–7. Williams ES. 2005. Chronic wasting disease. Vet Path 42:530–549. Wolfe LL, Conner MM, Baker TH, Dreitz VJ, Burnham KP, Williams ES, Hobbs NT, Miller MW. 2002. Evaluation of antemortem sampling to estimate chronic wasting disease prevalence in free-ranging mule deer. J Wildl Manage 66:564– 573. Wolfe LL, Spraker TR, Gonza´lez L, Dagleish MP, Sirochman TM, Brown JC, Jeffrey M, Miller MW. 2007. PrPCWD in rectal lymphoid tissue of deer (Odocoileus spp.). J Gen Virol 88:2078– 2082.
Submitted for publication 21 October 2013. Accepted 22 January 2014.
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.
BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.
DOI: 10.7589/2013-10-274
Journal of Wildlife Diseases, 50(3), 2014, pp. 660–665 # Wildlife Disease Association 2014
‘‘Atypical’’ Chronic Wasting Disease in PRNP Genotype 225FF Mule Deer Lisa L. Wolfe,1,2 Karen A. Fox,1 and Michael W. Miller1 1Colorado Division of Parks and Wildlife, Wildlife Research Center, 317 West Prospect Road, Fort Collins, Colorado 80526-2097, USA; 2Corresponding author (email: [email protected])
to, and slower progression of, clinical CWD (Johnson et al. 2006, 2011; Miller et al. 2012). In mule deer (Odocoileus hemionus), S/phenylalanine (F) dimorphism at codon 225 (Brayton et al. 2004; Jewell et al. 2006) appears to have a similar influence, although S homozygosity at codon 225 leads to a more-rapid disease course (Fox et al. 2006). The time course of CWD infection after oral inoculation appeared prolonged, and deposition of CWD-associated PrP (PrPCWD) in the central nervous system tissues was delayed in 225SF mule deer compared with 225SS individuals (Fox et al. 2006). Moreover, 225SF and 225FF individuals may be underrepresented in mule deer populations as a whole (9.3%) but are even rarer among CWD-positive deer (0.3%; Jewell et al. 2006). Although PrPCWD has been detected in 225FF mule deer (Jewell et al. 2006), no clinical CWD cases have been reported in deer with this genotype. Collectively, these observations suggest the possibility of variable susceptibility associated with PRNP genotype in mule deer. To compare the susceptibility of 225SS and 225FF mule deer to CWD under conditions approximating natural transmission via environmental exposure, we exposed six yearling mule deer (three 225SS and three 225FF) to the CWD agent, by holding them together in a common, contaminated 0.5-ha paddock, and observed the outcome. Our study was conducted at the Colorado Parks and Wildlife Foothills Wildlife Research Facility (FWRF; Fort Collins, Colorado, USA) after institutional animal care and use review and approval (file 07-2007). The hand-raised 225SS deer were acquired at about 10 mo of age from a captive herd at
We compared mule deer (Odocoileus hemionus) of two different PRNP genotypes (225SS, 225FF) for susceptibility to chronic wasting disease (CWD) in the face of environmental exposure to infectivity. All three 225SS deer had immunohistochemistry (IHC)positive tonsil biopsies by 710 days postexposure (dpe), developed classic clinical signs by 723–1,200 dpe, and showed gross and microscopic pathology, enzyme-linked immunosorbent assay (ELISA) results, and IHC staining typical of prion disease in mule deer. In contrast, although all three 225FF deer also became infected, the two individuals surviving .720 dpe had consistently negative biopsies, developed more-subtle clinical signs of CWD, and died 924 or 1,783 dpe. The 225FF deer were ‘‘suspect’’ by ELISA postmortem but showed negative or equivocal IHC staining of lymphoid tissues; both clinically affected 225FF deer had spongiform encephalopathy in the absence of IHC staining in the brain tissue. The experimental cases resembled three cases encountered among five additional captive 225FF deer that were not part of our experiment but also died from CWD. Aside from differences in clinical disease presentation and detection, 225FF mule deer also showed other, more-subtle, atypical traits that may help to explain the rarity of this genotype in natural populations, even in the presence of enzootic CWD. Key words: Chronic wasting disease, genotype, mule deer, Odocoileus hemionus, prion, PRNP.
ABSTRACT:
Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) found in North American cervids. Susceptibility to CWD and other TSEs appears to be affected by various polymorphisms in the gene (PRNP) encoding the host’s cellular prion protein (PrP) (Goldmann 2008; Robinson et al. 2012). For example, white-tailed deer (Odocoileus virginianus) that are heterozygous or homozygous for serine (S) at codon 96 of the PRNP gene showed reduced susceptibility 660
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Washington State University (Pullman, Washington, USA) with no history of CWD and were held indoors at the FWRF before this study began; the 225FF deer were born at the FWRF and dam-raised in outdoor paddocks that had not been used in CWD experimentation. All six individuals were negative for PrPCWD via tonsil biopsy immunohistochemistry (IHC; Wolfe et al. 2002) before being placed in the contaminated paddock. The paddock serving as an exposure source had been contaminated by use between December 2004 and February 2007 to house 20 orally inoculated 225SS mule deer (Miller et al. 2012), the last 14 of which were euthanized with clinical CWD between August 2006 and February 2007. Experimental exposure of subject deer began on 15 May 2007 (day 0). Deer were fed high-quality alfalfa, grass hay, and a pelleted nutritional supplement ad libitum throughout the study; water was from an automatic dispenser, which was cleaned regularly. We sedated deer for sampling with a combination of butorphanol (30 mg/ deer), azaperone (12.5 mg/deer), and medetomidine (10 mg/deer) and tested them for CWD using tonsil and rectal biopsy IHC (Wolfe et al. 2008) beginning after about 8 mo of exposure and every 6–8 mo thereafter. All deer were observed daily by animal caretakers for general health and were scored subjectively (05not shown, 15subtle, 25obvious) for behavioral changes, loss of body condition, ataxia, and ptyalism or polyuria/polydipsia about every 6–8 wk by a veterinarian experienced at recognizing clinical CWD. Deer with a cumulative clinical score of .4 were euthanized and necropsied. We screened retropharyngeal lymph node tissue for evidence of PrPCWD using an enzyme-linked immunosorbent assay (ELISA; Bio-Rad Laboratories, Hercules, California, USA) following methods described by Hibler et al. (2003); samples with an optical density (OD) value $0.1 were considered ‘‘suspect.’’ We also
661
screened paraffin-embedded sections of a larger set of formalin-fixed lymphoid and brain tissues by IHC for prion accumulation using monoclonal antibody (MAb) F99/97.6.1 and methods described by Spraker et al. (2002); in addition, we examined embedded tissues via light microscopy after H&E staining. Tissues examined by light microscopy and IHC included retropharyngeal lymph node, tonsil, spleen, adrenal gland, vagus nerve, ileum with Peyer patches, spiral colon with gut-associated lymphoid tissue, skeletal muscle, and multiple sections of brain (caudal medulla oblongata, medulla oblongata at the level of the dorsal motor nucleus of the vagus, cerebellum, thalamus at the level of the interthalamic adhesion, hypothalamus, basal nuclei, hippocampus, olfactory cortex, frontal cortex, temporoparietal cortex, and occipital cortex). One 225FF deer died from trauma 529 days postexposure (dpe); that deer was clinically normal, and although the ELISA OD value for retropharyngeal lymph node (0.47) was ‘‘suspect,’’ all tissues were IHC-negative, and there was no evidence of spongiform change in the obex or elsewhere in the brain (Table 1). The five remaining deer all appeared to die from CWD based on some commonality of clinical signs and laboratory findings, but differences between the genotypes emerged. All three 225SS deer had positive tonsil biopsies by 710 dpe, and all were euthanized after developing classic clinical CWD (clinical scores .4, including behavioral changes and emaciation; Table 1). In contrast, neither of the surviving 225FF deer yielded positive biopsies, and both were found dead unexpectedly while showing more subtle clinical signs (mild ataxia and weight loss without the pronounced behavioral changes typically seen; Table 1). At postmortem, the gross lesions in 225SS deer (Table 1) were typical of those described for CWD (Williams 2005) and
BR2 (f) ER2 (f) ME1 (cm) Wa06 (f) Wb06 (f) 11b06 (f)
225SS
dpe 5 days postexposure; T 5 tonsil biopsy; R 5 rectal mucosa biopsy.
723 1,058 1,200 529 (d) 924 (d) 1,783 (d)
Day euthanized or died (d)
E E, R, AP E, R, AP ngl (trauma) Me, AP E, SA, R, AP
Gross
d
2.55 2.7 2.52 0.47 3.01 1.44
ELISA OD
e
Obexg
+ + + 2 2 2
RLNf
+ + + 2 * *
Immunohistochemistry
Laboratory findings
ELISA 5 enzyme-linked immunosorbent assay; OD 5 optical density value.
RLN 5 retropharyngeal lymph node; + 5 positive staining; 2 5 no staining; * 5 equivocal staining (see Fig. 1B).
+ 5 positive staining; 2 5 no staining.
SE 5 spongiform encephalopathy.
e
f
g
h
E 5 emaciation; R 5 fluid-filled or frothy rumen; AP 5 aspiration pneumonia; ngl 5 no gross lesions associated with chronic wasting disease; Me 5 megaesophagus; SA 5 serous atrophy of fat.
SE SE SE no SE SE SE
Histopathologyh
d
E 5 emaciation; Pu 5 polyuria/polydypsia; Pt 5 ptyalism; AD 5 abdominal distension; D 5 depression; W 5 weight loss; At 5 ataxia (subtle).
f 5 female; cm 5 castrated male.
c
c
E, Pu, Pt E, Pu, AD E, Pu, D None W, At W, At
Clinical signs
b
T, R (710) T (472); R (710) T, R (710) all negative (472) all negative (892) all negative (1072)
Positive biopsy (dpe)
b
a
225FF
Animal (sex)
PRNP genotype
a
Disease course and clinical findings
TABLE 1. Disease course, clinical signs, and laboratory findings among mule deer of two PRNP genotypes after environmental exposure to the chronic wasting disease agent under controlled conditions.
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FIGURE 1. Influence of PRNP genotype on microscopic findings in mule deer that died from clinical chronic wasting disease. (A) Tonsil from a 225SS deer (ER2) with robust positive staining for PrPCWD by immunohistochemistry (IHC) at 1,058 days postexposure (dpe). Scale bar520 m. (B) Tonsil from a 225FF deer (11b06) with scant positive staining (see arrows) for PrPCWD by IHC at 1,783 dpe. Bar520 m. (C) Obex from a 225SS deer (ER2) with robust positive staining for PrPCWD by IHC. Bar5100 m. (D) Obex from a 225FF deer (Wb06) lacking positive staining for PrPCWD by IHC, despite spongiform changes shown in panel F (immediately below). Bar5100 m. (E) Obex from a 225SS deer (ER2) with dorsal motor nucleus of the vagus showing spongiform changes. H&E stain. Bar5100 m. (F) Obex from a 225FF deer (Wb06) with dorsal motor nucleus of the vagus showing spongiform changes. H&E stain. Bar5100 m.
included emaciation, fluid-filled or frothy rumen, megaesophagus, and aspiration pneumonia. The ELISA OD values for retropharyngeal lymph node tissue were $2.52 (Table 1). The IHC results also were
typical of clinical prion disease in mule deer, including robust positive staining within lymphoid follicles of the retropharyngeal lymph node, tonsil (Fig. 1A), spleen, Peyer patches, and gut-associated
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FIGURE 2. Clinical signs of chronic wasting disease in a 225FF mule deer included emaciation, distended abdomen, and megaesophagus. At postmortem, the deer had depleted fat stores, cachexia, a dilated esophagus, fluid-filled rumen, and aspiration pneumonia. Microscopic examination of brain tissue revealed lesions of spongiform encephalopathy in the absence of positive staining for PrPCWD by immunohistochemistry.
lymphoid tissue of the colon, scattered staining within the adrenal medulla and vagus nerve, robust staining within the dorsal motor nucleus of the vagus and adjacent nuclei (Fig. 1C), and variably prominent staining within the thalamus, hypothalamus, basal nuclei, hippocampus, and cerebral cortex. Spongiform changes (Fig. 1E) were predominantly limited to the dorsal motor nucleus of the vagus and adjacent nuclei, although the olfactory cortex was also affected in one deer (ME1). The poor body condition, fluid rumen content, megaesophagus and aspiration pneumonia in the two longer-lived 225FF deer also were consistent with clinical CWD. However, the ELISA OD
value (1.44) was lower than expected in end-stage CWD (usually .2.5) for one 225FF deer, and IHC findings were equivocal for both (Table 1). Postmortem IHC evaluation of the tonsils revealed markedly diminished staining that was restricted to the central lymphoid follicle (Fig. 1B). One 225FF deer (Wb06) also had similar minimal staining in the retropharyngeal lymph node, spleen, and Peyer patches. Neither 225FF deer had positive IHC staining at any level of brain (Fig. 1D), in contrast to the robust positive staining seen in 225SS deer. However, both 225FF deer showed spongiform encephalopathy predominantly limited to the dorsal motor nucleus of the vagus and adjacent nuclei (Fig. 1F) comparable to changes seen in the 225SS deer; spongiform changes also were seen in the hypothalamic nucleus of one 225FF deer (11b06). Although only two 225FF deer survived long enough to develop clinical disease in our controlled experiment, these cases resembled three additional CWD cases we have encountered during the past decade among five other captive 225FF mule deer that were held at the FWRF but that were not part of our experiment. Those three cases also showed some clinical signs (Fig. 2), postmortem lesions, and histopathology consistent with CWD but yielded variable ELISA results postmortem and negative IHC results both before and after death. The prolonged incubation, more subtle signs, and incongruous laboratory findings in 225FF mule deer could be products of differences in the abnormal prion protein propagated in the course of disease or in host response to the abnormal protein. For example, atypical IHC findings could result from 225FF PrPCWD being more vulnerable to endogenous proteases, formalin fixation, or formic acid than 225SS PrPCWD, lessreadily bound by MAb F99/97.6.1, or some combination of these and other effects. In light of the similarities and differences between mule deer genotypes,
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understanding the nature of PrPCWD and its relationship to clinical disease in 225FF mule deer could help illuminate broader mechanisms underlying prion disease. Beyond the differences in prion disease manifestation described here, other relatively intangible traits that we have noted among 225FF mule deer held at the FWRF suggest these deer may be atypical in other ways that could merit more formal study. The few individuals we have observed tended to behave somewhat erratically and to have poorer hair coats, less-optimal body condition, and poorer fawn recruitment as compared with captive 225SS mule deer. Perhaps as a consequence, 225FF deer have, in our experience, been difficult to propagate in captivity. These traits, if representative of the genotype, may help explain the rarity of 225FF mule deer in natural populations even in the presence of enzootic CWD. Our work was supported by the Colorado Division of Parks and Wildlife. We thank T. Davis, I. LeVan, K. Griffin, M. Fisher, and many others for their assistance during the course of this study, and T. Spraker for consultation on histopathology and IHC interpretations. Anonymous reviewers provided helpful comments on an earlier manuscript draft. LITERATURE CITED Brayton KA, O’Rourke KI, Lyda AK, Miller MW, Knowles DP Jr. 2004. A processed pseudogene contributes to apparent mule deer prion gene heterogeneity. Gene 326:167–173. Fox KA, Jewell JE, Williams ES, Miller MW. 2006. Patterns of PrPCWD accumulation during the course of chronic wasting disease infection in orally inoculated mule deer (Odocoileus hemionus). J Gen Virol 87:3451–3461. Goldmann W. 2008. PrP genetics in ruminant transmissible spongiform encephalopathies. Vet Res 39:30. doi: 10.1051/vetres:2008010. Hibler CP, Wilson KL, Spraker TR, Miller MW, Zink RR, DeBuse LL, Andersen E, Schweitzer D, Kennedy JA, Baeten LA, et al. 2003. Field
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Submitted for publication 21 October 2013. Accepted 22 January 2014.
