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Short Communication |
1 Roslin Institute, Neuropathogenesis Unit, Edinburgh, UK
2 Wildlife Research Center, Colorado Division of Wildlife, Fort Collins, CO, USA
Correspondence
Wilfred Goldmann
wilfred.goldmann{at}roslin.ed.ac.uk
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ABSTRACT |
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0.24) and all three genotypes showed equivalent susceptibility to infection. The GenBank/EMBL/DDBJ accession numbers for the wapiti PrP haplotype sequences determined in this study are EU032288–EU032294.
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), white-tailed deer (Odocoileus virginianus) (Spraker et al., 1997), wapiti (also called Rocky Mountain elk; Cervus elaphus nelsoni) (Williams & Young, 1982) and moose (Alces alces) (Baeten et al., 2007). The origin of CWD, its relationship to other prion diseases of mammalian species and factors influencing CWD epidemiology and susceptibility are not completely understood (Williams, 2005).
In prion diseases of domestic ruminant hosts, PrP (prion protein) gene polymorphisms are associated with susceptibility and with the length of incubation periods (Baylis & Goldmann, 2004). A number of amino acid polymorphisms have been identified in the PrP gene from the four CWD-affected cervid species. Polymorphisms in the mule and white-tailed deer PrP gene have been found at codons 20, 65, 95, 96, 116, 225 and 226 (Heaton et al., 2003; Johnson et al., 2003; Jewell et al., 2005). Low levels of PrP variability have been identified in moose (at codon 209) (Huson & Happ, 2006) and wapiti (at codon 132) (O'Rourke et al., 1999).
Wapiti PrP is polymorphic at codon 132, which corresponds to codon 129 in humans. The amino acid change in wapiti is from methionine (M) to leucine (L), whereas in humans it is to valine (V). The M129V polymorphism in the human PrP gene has been shown to have a major effect on susceptibility to, and phenotypic expression of, human prion diseases (Mead, 2006). Similarily, association with CWD susceptibility in free-ranging and farmed wapiti has been suggested, based on the findings that animals homozygous for methionine at codon 132 (MM132) were over-represented in both free-ranging and farmed CWD-affected wapiti compared with healthy-control groups (O'Rourke et al., 1999; Spraker et al., 2004). We investigated genotype profiles and their association with CWD infection in recent (years 2002–2005) CWD cases and apparently uninfected controls from three free-ranging wapiti populations in Colorado, USA.
The Colorado Division of Wildlife (CDOW) manages wildlife populations on a local biological-unit basis, the Data Analysis Unit (DAU). DAUs are geographical areas where a particular herd resides; DAU boundaries encompass a herd's entire range throughout the year. DAUs are composed of one or more Game Management Units (GMUs), which subdivide the DAU based on road and other geographical boundaries to distribute hunting pressure (Conner & Miller, 2004; Miller & Conner, 2005). Wapiti samples were collected in nine different GMUs located within three different DAUs (E6, E8 and E9): GMUs 11, 12, 13, 23, 24 and 231 are part of E6, GMUs 18 and 181 are part of E8 and GMU 20 is E9. A previous study by O'Rourke et al. (1999) also examined samples from GMU 20 (DAU E9). All samples analysed here were collected after 2002. Of the three DAUs studied, E9 had the highest estimated CWD prevalence (95 % confidence interval) at 1.7 % (0.9–2.5 %), followed by E8 at 1.2 % (0.4–2.1 %) and E6 at 0.3 % (0.1–0.4 %) (CDOW, unpublished data). We used 171 samples from free-ranging wapiti, including 47 samples from confirmed CWD cases and 124 from apparently uninfected animals, distributed as follows: 51 from DAU E6 (17 CWD), 53 from DAU E8 (11 CWD) and 67 from DAU E9 (19 CWD). For all wapiti, retropharyngeal lymph-node tissue samples were initially screened by using an ELISA for abnormal PrP (Bio-Rad; Hibler et al., 2003); light absorbance of samples was measured by using a Bio-Rad model 550 microplate reader with 450 and 620 nm filters. Samples identified as ‘suspect’ through ELISA (absorbance0.1) were then analysed by immunohistochemistry (IHC) using antibody F99/97.6.1 to confirm disease status (Hibler et al., 2003). Samples that were negative by ELISA were regarded as negative for CWD and were not examined by IHC.
All DNA was extracted from tissues by using a DNeasy Tissue kit (Qiagen) according to the manufacturer's instructions. DNA was then treated twice with phenol followed by precipitation with ethanol. All samples were genotyped at codons 104 [lysine (AAA)/lysine (AAG)] and 132 [methionine (ATG)/leucine (TTG)] by direct sequence analysis of DNA fragments (2066, 2032 or 1317 bp) generated by PCR performed with oligonucleotide pair MP+690u (5'-CAGATTTGCTCTCAGTGTCTAGAA-3') and MP-540d (5'-TGATCTCAGCACCTACCTTGG-3'), pair MP+690u and MP-574d (5'-GAGGATGTGAGCCAATATTAG-3') or pair MP252u (5'-TTGCCCCTATCCTACTATGAGA-3') and MP-540d. PCRs were set up with decreasing annealing temperatures from 66 to 59 °C over seven cycles, followed by 26 cycles with annealing at 59 °C. PCR fragments were sequenced with oligonucleotides MP196u (5'-GGTGAAGTTCTCCCCCTTGGT-3') and MP50d (5'-CCGCTATCCACCTCAGGGA-3') using BigDye reagent (Applied Biosystems) and 25 cycles, each consisting of 10 s at 96 °C, 5 s at 50 °C and 4 min at 60 °C. Sequence reactions were loaded on an ABI Prism 377 automated sequencer (Applied Biosystems) and traces were analysed visually. PrP haplotype sequences have been deposited in GenBank under accession numbers EU032288 [GenBank] –EU032294. Genotypes are presented as methionine/methionine (MM132), methionine/leucine (ML132) or leucine/leucine (LL132). Codon numbers are equivalent to the sheep PrP sequence. Wapiti PrP is uniformly alanine136–arginine154–glutamine171 in comparison with sheep PrP.
We used the Freeman–Halton extension of Fisher's exact probability test to compare genotype frequencies of different groups of animals. The test was carried out for two-row by three-column contingency tables; each table contained counts of the three genotypes (the two homozygotes and the heterozygote) among CWD cases and uninfected animals. In addition, we compared genotype frequencies among uninfected wapiti between the three populations that we studied, as well as between previous (O'Rourke et al., 1999) and recent (
1999 versus 2002) sampling years in E9 to ensure that genotype frequencies had not changed between study periods.
All populations and subpopulations analysed here were found to be in Hardy–Weinberg equilibrium. Genotype frequencies among healthy-control samples from the three studied DAUs did not differ (E6 versus E8, P=0.17; E6 versus E9, P=0.09; E8 versus E9, P=0.93); although frequencies did not differ among DAUs, we noticed a trend toward divergence with geographical distance among sampled populations. Similarly, genotype frequencies did not differ (P=0.5) between previous (1999) and recent (2002) sampling years for E9.
Overall genotype frequencies for uninfected wapiti in the three DAUs were 65.3 % MM132, 32.3 % ML132 and 2.4 % LL132 (group 1, Table 1). For CWD-infected animals, these frequencies were 70.2 % MM132, 27.7 % ML132 and 2.1 % LL132 (group 2, Table 1). MM132 animals were not over-represented among CWD-infected individuals, either overall (P=0.82, group 1 versus group 2) or within individual DAUs (P0.24; Table 1). These results were surprising because, based on data from O'Rourke et al. (1999) (groups A and B, Table 1), we expected to see an over-representation of methionine homozygotes in CWD-infected wapiti, at least in E9.
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). Most of the animals genotyped in this study were adults (155 of 171) and only two CWD-infected yearlings were available, thus precluding analysis of genotype frequencies in relation to different age groups. However, a practically equal number of males (45.6 %) and females (54.4 %) enabled comparison of genotype frequencies in relation to sex, which did not differ significantly for any of the DAUs (P0.13; Table 2).
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A) of codon 104 (Lys). The mutation was found on a haplotype with Met132. Based on 108 analysed samples, the haplotype frequencies were 88 % 104G–Met132 and 12 % 104A–Met132. There was no association between susceptibility and these two haplotypes (P0.54); the codon 104 polymorphism is therefore unlikely to be a useful marker for disease.
O'Rourke et al. (1999) showed that MM132 wapiti were over-represented in both farmed and free-ranging CWD-affected animals. All of the free-ranging CWD-infected animals were MM132 homozygotes, but some CWD-infected ML132 individuals were identified among the captive animals. Later, over-representation of methionine homozygotes was also reported in a captive wapiti population from Saskatchewan, Canada (Balachandran et al., 2002). Subsequently, CWD susceptibility of the LL132 genotype was described in captive wapiti from the USA and Canada (Spraker et al., 2004), thus showing that, at least in captivity, animals of all genotypes are susceptible to disease. The lack of CWD diagnosis in any of the free-ranging wapiti carrying the L132 allele was believed to be the result either of small sample size or of factors such as dose, route of infection or strain type (O'Rourke et al., 1999).
Data from the three foregoing studies, together with the analysis of PrPSc deposition, suggested that heterozygosity at codon 132, similar to heterozygosity at codon 129 in human TSEs, could have a protective, although incomplete, role against CWD and that the low number of CWD-infected LL132 homozygotes was probably due to the rarity of this genotype (Spraker et al., 2004). Oral CWD-transmission studies by Hamir et al. (2006) reported a mean incubation period of 23 months for two MM132 and 39 months for two ML132 animals; LL132 animals succumbed at about 60 months (O'Rourke et al., 2007).
Our findings contradict previous results of genotyping (O'Rourke et al., 1999) by showing a lack of over-representation of MM132 in CWD-infected wapiti and the relatively equivalent susceptibility of all genotypes in free-ranging animals. However, our findings do not preclude the possibility that heterozygosity at this position modulates CWD incubation time. The sample sizes of CWD-infected wapiti for DAU E9 were comparable between the current and a previous study. However, O'Rourke et al. (1999) collapsed the ML132 and LL132 groups and ran two-row by two-column tables to handle the small sample sizes; this was not necessary in the current study, making implementation of Fisher's exact test possible and the analysis more reliable. In addition to this, the genotyping carried out here represents three different wapiti populations, thus eliminating the possibility of a geographical or population artefact.
The detection of one CWD-infected LL132 homozygous wapiti in E9 confirms that all three genotypes are susceptible to naturally acquired CWD. Diseased animals carrying this genotype had previously only been reported in captivity (Spraker et al., 2004).
It is unclear whether changes in diagnostic methods during the time between the 1999 study (O'Rourke et al., 1999) and the collection of samples used for the current analysis may have contributed to the over-representation of MM132 individuals in the first study. More contemporary CWD surveillance and screening has focused on sampling of harvested animals, and a larger proportion of the CWD cases included here were apparently healthy (i.e. preclinical) individuals. In contrast, most of the infected wapiti studied by O'Rourke et al. (1999) either showed clinical signs at the time of death or were found dead and submitted as ‘CWD suspects’ (Miller et al., 2000). It follows that the shorter incubation times of MM132 animals might have inadvertently biased the sample of cases used for the earlier study because, on average, these individuals would have been further along in the disease course and thus their infections would have been detected more readily. We also assume that the screening by histopathology of brain tissue used by O'Rourke et al. (1999) was more likely to miss infected but preclinical animals than the screening of retropharyngeal lymph node by ELISA used in this study (Hibler et al., 2003).
The MM132 genotype over-representation identified by O'Rourke et al. (1999) in captive wapiti could simply be the result of an artefact related to husbandry or the timing of the sample collections: it is unclear whether, in all cases, CWD-negative controls were animals penned together with the infected animals and therefore were uniformly exposed before being culled. The same could be true in the studies carried out by Spraker et al. (2004) and by Balachandran et al. (2002); moreover, both of the latter studies failed to report genotype frequencies of uninfected animals and it is possible that the genotype patterns in the infected samples could be the result of a pre-existing bias in underlying population genotype frequencies.
The existence of several strains (Bruce, 1993) or the adaptation of existing strains to animals with different PRNP genotypes could explain the differences in the genotype frequencies among diseased individuals seen between the current study and that carried out by O'Rourke et al. (1999). To date, most research on CWD supports the existence of only one strain, which is able to cause disease in different cervid species (Race et al., 2002; Browning et al., 2004; Tamguney et al., 2006); however, several strains have recently been proposed in CWD transmissions to rodents (Raymond et al., 2007). Lack of variation between the genotype patterns of healthy animals indicated that the frequencies of codon 132 genotypes in DAU E9 have remained constant during the time separating the two studies. One could speculate that the prion strain that was affecting the wapiti population in E9 adapted over time to infect ML132 and LL132 animals. However, it is hard to see how the low CWD prevalence within E9 (1.7 %; 95 % confidence interval, 0.9–2.5 %) could support this form of selection over such a short period of time. It is also possible that, in the course of the epidemic, the dose of the agent has increased or the route of infection has changed, with consequences for CWD susceptibility. These parameters may warrant further investigation in suitable models, possibly transgenic mice.
In summary, the results reported here indicate that, for recent CWD cases in free-ranging wapiti from Colorado, USA, no codon 132 genotype-related infection patterns can be identified and that all genotypes are similarly susceptible to infection. Despite this, a modulatory role of this residue in the incubation time of CWD in wapiti cannot be excluded. The discrepancies between the current investigation and data from a genotyping study carried out on free-ranging wapiti at the end of the 1990s (O'Rourke et al., 1999) could be the result of small sample size, sampling bias and/or diagnostic methods, although other biological factors cannot be completely excluded.
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ACKNOWLEDGEMENTS |
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Received 5 September 2007;
accepted 14 January 2008.
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