USDA, Agricultural Research Service, Animal Disease Research Unit, 337 Bustad Hall WSU, Pullman, WA 99164-7030, USA1
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040, USA2
Colorado Division of Wildlife, 317 W. Prospect, Fort Collins, CO 80526, USA3
South Dakota Animal Industry Board, 411 S. Fort St, Pierre, SD 57501, USA4
Diagnostic Laboratories, College of Veterinary Medicine and Biomedical Sciences, Fort Collins, CO 80523, USA5
USDA, APHIS National Veterinary Services Laboratories, Ames, IA 50010, USA6
Elk Research Council, North American Elk Breeders' Association, 3901 Hwy 29, Alexandria, MN 56308, USA 7
Wyoming State Veterinary Laboratory, University of Wyoming, 1174 Snowy Range Road, Laramie, WY 82070, USA8
Author for correspondence: Katherine O'Rourke.Fax +1 509 335 8328. e-mail korourke{at}vetmed.wsu.edu
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Abstract |
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Introduction |
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Relative susceptibility to sporadic, iatrogenic and nvCJD is associated with the primary sequence of the host PrP-C. A nonpathogenic polymorphism at codon 129 of the human PrP gene encodes Met or Val. Homozygosity at codon 129 predisposes individuals to iatrogenic and sporadic CJD (Collinge et al., 1991 ; Palmer et al., 1991
). nvCJD has been diagnosed to date only in 129 Met/Met homozygotes (Zeidler et al., 1997
). We have described a polymorphism (Met to Leu) at the equivalent PrP site in elk, cervid codon 132 (O'Rourke et al., 1998
). In this study, we determined the PrP genotypes of CWD-affected elk and of unaffected elk in several free-ranging and captive populations to determine whether this codon modulates susceptibility to CWD.
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Methods |
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Group 2 consisted of 88 healthy, hunter-harvested elk from Larimer County, CO. The incidence of CWD in elk in this region, based on examination of brain tissue from hunter-harvested elk, is less than 1% (M. W. Miller, unpublished data). Group 3 consisted of healthy, hunter-harvested elk collected near Jackson, WY, an area with no reported cases of CWD. Brain tissue collected from elk in groups 2 and 3 showed neither histological nor immunohistochemical evidence of CWD.
Group 4 included live, healthy elk from South Dakota (SD) that were anaesthetized, radiocollared and released for a habitat evaluation project by the US Department of Agriculture, Forest Service, Rocky Mountain Research Station. Brain tissue will be collected if a radiocollared elk is subsequently hunter-harvested. There are no reported cases of CWD in free-ranging elk in SD. The elk in group 4 range the entire Black Hills region of SD and WY encompassing an area of 4500 square miles. This area includes a privately owned game farm with a high incidence of CWD (groups 5 and 6, as follows).
Groups 5 and 6 included farm-raised elk originating from a single SD facility that houses elk in several different subpopulations, grouped by sex, breeding status and age. CWD was diagnosed in an elk in this herd in 1997. Examination of archived tissues suggests that a case occurred as early as 1995. Forty-two cases have been confirmed subsequently. Frozen tissues or blood samples collected at the time of euthanasia were submitted from 23 elk subsequently diagnosed with CWD by histology and/or immunohistochemistry (group 5). The youngest CWD- affected animal was 17 months old when diagnosed; all other CWD- positive elk were 2 years of age or older. Blood samples were collected from 59 elk (group 6) which had been penned with the CWD-affected elk. Group 6 elk are alive at the time of this writing or were healthy and free of microscopic lesions and PrP-Sc immunostaining in brainstem tissue when culled.
Blood samples were collected from healthy, live elk from a farm in Minnesota with no history of CWD (group 7). These animals are observed regularly and necropsies will be performed on elk that are culled or die from injury or illness.
Samples were collected from 47 elk on a game farm in Montana (MT) (group 8). A single animal originating from this herd was diagnosed with CWD in 1998 after being housed in two facilities in other states between 1996 and 1998. CWD has not been diagnosed in any MT game farm animals or MT wildlife at this time.
Group 9 consisted of 30 samples from farm-raised elk originating in North Dakota and Nebraska and transported to a facility in SD. These animals shared a short length (approximately 150 m) of fenceline with a paddock housing 10 elk from the infected herd (groups 5 and 6). Elk were housed in these adjoining paddocks for 90 days. A separation of groups was re-established so that no fenceline contact was possible. Ninety days of separation had occurred when a group 5 elk died from CWD. The remaining nine elk from group 5 were removed from the facility. The 30 elk in group 9 remain in SD under quarantine with a mandatory CWD laboratory diagnosis on all adult cervid deaths. No cases of CWD have been diagnosed in this herd. Samples were collected from animals that are alive at the time of this writing or were free of histological lesions and PrP-Sc immunostaining when culled.
CWD diagnosis.
Free-ranging affected elk were usually observed by local residents who reported dead or sick animals. Clinical signs included emaciation, loss of fear of humans, ataxia, inability to stand, dehydration, excessive salivation, drooping of the head and ears and rough dull haircoat. Captive elk were observed regularly by caretakers familiar with the animals' behaviour and euthanized when they exhibited clinical signs of CWD (Miller et al., 1998 ). CWD was confirmed by histopathological lesions, most notably spongiform encephalopathy characterized by microcavitation, primarily of the grey matter, with single or multiple intracytoplasmic vacuoles in neuronal perikarya and neuronal degeneration (Spraker et al., 1997
; Miller et al., 1998
). Diagnosis was confirmed by immunohistochemistry using MAb F89/160.1.5 (VMRD and ABR) (O'Rourke et al., 1998
) (Fig. 1
B) or rabbit polyclonal anti-PrP antiserum R78295, provided by R. Rubenstein to the National Veterinary Services Laboratories (Miller et al., 1994
). When fresh brain was available, tissues were examined for the presence of the disease-associated isoform of PrP (PrP-Sc or scrapie-associated fibrils) via negative stain electron microscopy (Guiroy et al. , 1991
) or by Western immunoblot (Rubenstein et al. , 1986
) using a polyclonal anti-mouse (ME7) serum provided by R. Rubenstein (Fig. 1A
).
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Statistical analysis.
The 2 test was used with the Yates correction for continuity to compare genotype frequencies of animals with and without CWD. For these analyses, animals homozygous for Met at codon 132 were compared with the combined frequencies of animals homozygous for Leu and heterozygous Met/Leu animals, in order to eliminate most instances of analyses containing cells with no observations or with more than 20% of the cells containing less than five expected observations, situations where the
2 test is considered unreliable. For those comparisons where there were less than five expected observations in one cell of a 2x2 table even after combining Met/Leu and Leu/Leu animals, genotype frequencies were compared by determining whether or not there was overlap between the exact 95% confidence intervals of both groups' collapsed genotype frequencies based on the binomial distribution. Genotype frequencies of CWD-affected elk born in the wild in CO and WY (group 1) were compared to those of CWD-affected elk of game farm origin (group 5). Genotype frequencies of the two CWD-affected groups were compared separately to their appropriate control groups. Group 1 elk, born wild in CO or WY, were compared to CWD-unaffected elk from herds in CO, WY and SD (groups 2, 3 and 4 pooled). Genotype frequencies of CWD-affected elk born and raised on game farms (group 5) were compared to unaffected elk from the same facility (group 6) and from pooled data from four game farms (groups 6, 7, 8 and 9). Genetic frequencies of CWD-affected elk in free- ranging and game farm populations compared using Fisher's exact test differed significantly (P=0·026).
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Results |
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Genotypic frequencies varied widely among the farm-raised, unaffected elk (Table 1, groups 6, 7, 8, 9). Codon 132 genotype frequencies were significantly different among the four captive elk herds sampled (
2=5·8, P <0·05). Pairwise testing showed that, with the exception of group 8, the genotypes in each herd differed significantly from the genotypes of the healthy free-ranging elk (pooled data from groups 2, 3 and 4) (
2=5·8, P<0·05). Therefore, the genotype frequencies of farm-raised elk with CWD were compared to non-affected elk from the same facility (group 5 vs group 6) rather than elk from the other game farms. The frequency of Met/Met elk with CWD differed significantly from that of non-affected elk from the same farm (
2=8, P<0·01) (Table 1
). Met/Met homozygosity was therefore significantly over-represented in the CWD-affected farm-raised elk at this facility.
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Discussion |
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In this study, we first analysed samples from free-ranging elk with CWD. All 20 cases were homozygous for Met/Met132, although the mean genotypic frequency of Met/Met132 was 0·75 in the free-ranging populations. Twenty-three cases of CWD in elk on a game farm in SD provided a second sample group. A total of 74% of the captive CWD- affected elk sampled were homozygous for Met/Met132 although only 47% of the sampled elk from this herd were Met/Met132 homozygotes. Taken together, these data suggest that Met/Met132 homozygosity predisposes exposed elk to CWD.
CWD was diagnosed in Met/Leu132 heterozygotes in the game farm population but not in free-ranging elk. This observation may be an artefact of small sample size of free-ranging CWD-affected elk and the low frequency of the 132Leu allele in free-ranging elk. Alternatively, this difference may reflect varying routes or doses of the transmissible agent under the game farm husbandry conditions or the presence of a novel CWD strain. Biochemical profiles of PrP-Sc from different populations and different genetic groups will provide additional insight into the range of CWD disorders in elk. The relationship between heterozygosity and incubation time in natural disease cannot be determined in CWD-infected herds because the time of infection is not known. However, two of the CWD-affected Met/Leu elk were diagnosed at age 2 years, indicating that heterozygosity is not invariably associated with prolonged incubation time.
The absence of CWD in the Leu/Leu132 elk population is of interest. Homozygosity for PrP Ala136 is associated with resistance to strain A scrapie in sheep (Goldmann et al., 1994a ) and a single copy of the ovine PrP allele encoding Arg171 is sufficient to protect most sheep from strain C scrapie (Goldmann et al., 1994b
). Insufficient numbers of elk with the Leu/Leu genotype were available to allow evaluation of their relative susceptibility to CWD. Extended observations on the surviving elk from the CWD-affected herd and experimental infection of Leu/Leu132 elk with infectious material from Met/Leu132 elk will be necessary to determine the relative resistance of Leu/Leu132 elk to CWD.
Selective breeding for TSE resistance is a valuable adjunct to epidemiology and diagnostic testing in control of sheep scrapie (Dawson et al., 1998 ) if the scrapie strains endemic in the population are known. Our data suggest that large proportions of many free-ranging and farmed Rocky Mountain elk populations are susceptible to CWD. The interaction of PrP genetic polymorphisms and CWD strain types in clinical disease progression, relative susceptibility, transmission efficiency and incubation time in elk warrants further investigation.
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Acknowledgments |
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Footnotes |
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References |
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Received 23 March 1999;
accepted 1 July 1999.