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1 Department of Animal Health and Biomedical Sciences, University of Wisconsin, 1656 Linden Drive, Madison, WI 53706, USA
2 Wisconsin Veterinary Diagnostic Laboratory, Madison, WI 53705, USA
Correspondence
Debbie McKenzie
mckenzie{at}svm.vetmed.wisc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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K polymorphism at codon 226 and a single octapeptide repeat insertion into the pseudogene, have not been reported previously. The predominant alleles – wild-type (Q95, G96 and Q226) and a G96S polymorphism – comprised almost 98 % of the Prnp alleles in the Wisconsin white-tailed deer population. Comparison of the allelic frequencies in the CWD-positive and CWD-negative deer suggested that G96S and a Q95H polymorphism were linked to a reduced susceptibility to CWD. The G96S allele did not, however, provide complete resistance, as a CWD-positive G96S/G96S deer was identified. The G96S allele was also linked to slower progression of the disease in CWD-positive deer based on the deposition of PrPCWD in the obex region of the medulla oblongata. Although the reduced susceptibility of deer with at least one copy of the Q95H or G96S allele is insufficient to serve as a genetic barrier, the presence of these alleles may modulate the impact of CWD on white-tailed deer populations. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Westaway et al., 1987), sheep (Tranulis, 2002) and humans (Prusiner & Scott, 1997). Variability in Prnp probably affects CWD susceptibility in cervid populations. In elk, variability at PrP codon 132 was found to have a significant influence on susceptibility to CWD (O'Rourke et al., 1999). Mule deer heterozygous for serine and phenylalanine (S/F heterozygous) or F/F homozygous at codon 225 were underrepresented in the infected population, suggesting decreased susceptibility to CWD (Jewell et al., 2005). Genetic analysis also suggests that the susceptibility of white-tailed deer to CWD is influenced by polymorphisms at codon 96 (Johnson et al., 2003; O'Rourke et al., 2004) and codon 116 (O'Rourke et al., 2004), although the relatively small sample sizes in both studies allowed only limited conclusions.
Our initial assessment of the sequence of the Prnp gene of white-tailed deer revealed three PrP alleles (Johnson et al., 2003). The most common allele, referred to as wild-type (wt), encoded Q95 and G96. Alleles encoding a Q95H polymorphism and a G96S polymorphism were also present in this population. White-tailed deer populations in the western region of the USA show additional Prnp polymorphisms of G65E and A116G (Heaton et al., 2003). A previously characterized polymorphism at codon 138 (S138N; Raymond et al., 2000; Johnson et al., 2003) was determined to be a processed pseudogene in both mule deer (Brayton et al., 2004) and white-tailed deer (O'Rourke et al., 2004).
In this study, a larger number of infected and uninfected free-ranging deer was analysed. Significantly fewer Q95H and G96S PrP alleles were found in the CWD-positive population compared with the CWD-negative population. Immunohistochemical scoring of CWD progression supported a reduced rate of disease progression in deer heterozygous for PrP allele G96S. We also identified two additional Prnp polymorphisms present in both infected and uninfected deer populations.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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150 square miles) in Wisconsin from 2001 to 2004. Another set of samples was obtained from confirmed-negative deer harvested in the wDEZ, during the 2002 autumn hunting season, from regions throughout northern and central Wisconsin where CWD was not detected in the free-ranging deer population by state-wide surveys. CWD was diagnosed by testing the RPLNs (or the obex if RPLNs were not available) for the presence of PrPCWD using immunohistochemistry (IHC) at the Wisconsin Veterinary Diagnostic Laboratory in Madison, Wisconsin. Deer defined as CWD-positive were positive for PrPCWD in the RPLNs (or obex) and CWD-negative animals were those that had no staining for PrPCWD. Testing for CWD in these deer populations was performed primarily on the RPLNs. In a separate study (unpublished observations), obex tissue from several thousand deer that were CWD-negative in the RPLNs was stained for PrPCWD deposition. All RPLN-negative animals were also obex-negative.
Amplification and sequencing of the white-tailed deer Prnp.
The Prnp gene was sequenced from 624 Wisconsin white-tailed deer: 292 CWD-positive and 153 CWD-negative deer from the south-central CWD-affected region and 179 CWD-negative deer from outside the CWD-affected region. Genomic DNA was isolated from ear or muscle tissue and the coding region of the Prnp gene was amplified as described previously (Johnson et al., 2003). The primer set used was: CWD-13 (5'-TTTTGCAGATAAGTCATCATGGTGAAA-3', nt 13–39) and CWD-LA (5'-AGAAGATAATGAAAACAGGAAGGTTGC-3', nt 830–804). The nucleotide numbering for Prnp is based on the cervid sequence in GenBank (GenBank accession no. AF156185
[GenBank]
). PCR amplification and sequencing reactions were performed as described previously (Johnson et al., 2003). Primer sets CWD-13, CWD-LA, CWD-161 (5'-AGGGAAGTCCTGGAGGCAACCGCTATCC-3', nt 161–188) and CWD-418 (5'-CACCAAGGCCCCCTACCACTGCTCCAGC-3', nt 418–391) were used for sequencing. All samples that appeared to be heterozygous with respect to Prnp alleles were cloned and sequenced to determine the allele with which the polymorphism was associated. Sequences were analysed using DNAStar or EditView and MacVector 6.5 software.
Identification of the S138N pseudogene.
Two methods were employed to determine whether the S138N change detected in the Wisconsin white-tailed deer population was a processed pseudogene. A step-down PCR genome-walking protocol, described by Zhang & Gurr (2000), was adapted to characterize the 5' and 3' flanking regions of the white-tailed deer Prnp open reading frame (ORF). Using a white-tailed deer Prnp-specific and an adaptor-specific primer, nested PCR was subsequently performed to amplify the 5' and 3' regions flanking the ORF. The primers used for specific amplification of white-tailed deer Prnp were selected from regions not expected to have polymorphisms and are shown in Table 1. PCR products were gel purified, cloned and sequenced using T7- and SP6-specific primers (Promega), as described previously (Johnson et al., 2003).
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IHC for PrPCWD scoring in the obex.
Deer that tested positive for CWD in the RPLNs were analysed for the level of PrPCWD deposition in the obex using IHC as described previously (Spraker et al., 2002), but omitting the initial formic acid treatment. Scoring was performed blind, i.e. without prior knowledge of the Prnp genetics. Briefly, obex tissues from 139 CWD-positive animals were fixed in 10 % neutral buffered formalin, dehydrated and embedded in paraffin. Tissue sections (5 µm) were mounted on positively charged slides, deparaffinized and exposed to hydrated autoclaving in antigen retrieval buffer. The tissue sections were exposed to anti-PrP mAb F99/97.6.1, which recognizes the conserved QYQRES peptide (Spraker et al., 2002), using a NexES automated immunostainer (Ventana Medical Systems). Primary antibody binding was detected using a biotinylated secondary anti-mouse antibody, followed by horseradish peroxidase–streptavidin conjugate and chromagen substrate. Obex tissue was assessed for the distribution of PrPCWD deposition and scored with a value of between 0 and 4 as follows: 0, no staining in the obex; 1, a low level of staining in the obex dorsal motor nucleus of the vagus nerve (DMNV); 2, positive staining in the entire DMNV; 3, staining beyond the DMNV; 4, a high level of staining throughout the obex. The PrPCWD staining data were aligned with the Prnp genotype data.
Statistical analysis.
To estimate the proportion of the white-tailed deer population in the CWD-affected region that were genetically susceptible to CWD, we constructed a 95 % confidence interval based on a large-sample approximation to the binomial distribution using the allelic combination frequencies in the CWD-negative population. 
2 analysis was used to determine whether differences in allelic combination percentages between CWD-positive and CWD-negative deer populations were significant. PrP deposition in the obex was analysed using the GLM procedure (SAS). A one-way analysis of variance was used to compare mean obex scores for deer with different Prnp alleles. Individual genotype means were compared using Fisher's least significant difference (P<0.05).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Analysis of the 5' region flanking the Prnp ORF of the wt, Q95H, G96S and Q226K alleles suggested that they were all functional genes.
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). In the CWD-positive deer population, 81.5 % of the animals were wt/wt compared with 51 % of the CWD-negative deer (Fig. 2). The wt/wt genotype was significantly overrepresented in the infected deer population (P<0.0001). Conversely, 17.4 % of CWD-positive deer had at least one copy of the G96S allele, compared with 44.4 % of CWD-negative deer. The wt/G96S and G96S/G96S genotypes represented 39.2 and 5.2 % of the CWD-negative animals, respectively; however, significantly fewer CWD-positive deer were identified with these alleles [17.1 % (P<0.0001) and 0.3 % (P=0.0005), respectively) (Fig. 2). In addition to the G96S allele, deer heterozygous for the Q95H allele were also significantly underrepresented in the CWD-positive population (P=0.007). No Q95H allele was identified in the CWD-positive deer population, whilst 3.9 % of the CWD-negative deer assessed from the affected region were heterozygous for the Q95H allele.
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) and captive white-tailed deer (O'Rourke et al., 2004). Since our initial primers did not differentiate between functional and non-functional genes (amplifying only the ORF), we used two approaches to characterize further the S138N allele in Wisconsin white-tailed deer. All amplification products from heterozygous deer were cloned and sequenced. Three unique Prnp clones were isolated and characterized from a single animal, G96S, S138N and wt, indicating the presence of at least one additional copy of Prnp. Following step-down genome walking, the 5' upstream region of Prnp was also sequenced. A BLAST search against GenBank, using the Prnp sequence from the 5' untranslated region of the allele with the S138N change, showed 98 % sequence similarity to the mule deer processed pseudogene (GenBank accession no. AY371694
[GenBank]
). There was no significant difference between CWD-positive and CWD-negative deer for the presence of the pseudogene (20.9 % and 18.3 %, respectively; P=0.5165).
Prnp allele frequencies are similar inside and outside the wDEZ
White-tailed deer in Wisconsin are non-migratory. To determine whether the allele frequencies observed within the wDEZ were specific to the endemic area, Prnp genotypes were also determined for free-ranging white-tailed deer located 100–300 miles outside the wDEZ. All of these deer tested negative by IHC. The three major Prnp alleles identified in deer from the CWD-affected region also comprised the majority of the alleles in these deer. The frequency of alleles was similar to the allele frequencies observed in uninfected animals in the CWD-affected region: 76.8 % wt, 21.8 % G96S and 1.4 % Q95H.
Analysis of disease progression in the obex of infected animals
Since the G96S allele was less prevalent in the infected population, suggesting reduced susceptibility to CWD, we hypothesized that CWD would progress at a slower rate in infected deer with the G96S allele. To determine whether the presence of the G96S allele affected progression of the disease, the intensity and distribution of PrPCWD staining in the obex of the medulla oblongata was assessed, without prior knowledge of the Prnp genotypes, and correlated with identified Prnp genotypes (Fig. 3). The mean stage of obex disease progression, as determined by PrPCWD staining, was 2.13±0.05 (n=86) in wt/wt deer and 2.29±0.07 (n=31) in wt/wt/pseudogene deer, compared with only 1.32±0.11 (n=22) in wt/G96S deer. The mean obex staining level in wt/G96S deer was significantly less than in animals with the other genotypes (P<0.05), whereas no significant difference was identified in CWD progression in wt/wt deer with or without the pseudogene. The only CWD-positive deer homozygous for G96S was positive for the presence of PrPCWD in the RPLN but not in the obex, precluding an analysis of disease progression in the animal.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The mean level of PrPCWD staining in the obex of CWD-positive deer was lower in animals with the G96S allele, suggesting that disease progression is retarded in these animals. CWD-positive white-tailed deer that were homozygous for the G96S have been reported in a captive population (O'Rourke et al., 2004). The one homozygous G96S CWD-positive Wisconsin white-tailed deer was weakly positive in its RPLNs (unpublished data), with no PrPCWD deposition observed in other peripheral lymph nodes tested or in the obex of this animal. This deer was harvested from a wild population and thus the dose and time of infection were unknown. Controlled oral inoculations will be required to determine the degree to which the G96S allele affects the susceptibility of white-tailed deer to CWD.
The association of the G96S allele with reduced susceptibility has been noted in captive white-tailed deer (O'Rourke et al., 2004), although with less impact on susceptibility than is suggested by our data. Several factors may contribute to this apparent difference in genetic effect on susceptibility. The strain of agent in the two populations may be different. The high percentage of positive deer in the captive population would suggest that CWD was present in that population for several years and may have adapted to passage in deer with at least one copy of the G96S allele. Conversely, the density of deer and the high prevalence of infected animals in the captive population could result in high levels of exposure to the agent. If the G96S allele provides moderate resistance to CWD, high titres of agent may overcome genetic resistance.
An in vitro means of determining the molecular compatibility between PrPC and PrPSc, the cell-free conversion assay, has been used to estimate genetic susceptibility to CWD (Raymond et al., 2000). When PrPC from different alleles of white-tailed deer Prnp was combined with CWD agent from white-tailed deer in the cell-free conversion assay, it appeared that both wt (GMSQ in Raymond et al., 2000) and G96S (SMSQ in Raymond et al., 2000) were converted to PrPres with similar efficiencies. Although these data appear to contradict our population genetics study, there are several explanations for the differences. The cell-free conversion assay is indicative of the molecular interaction between the normal and abnormal forms of PrP and does not account for the dose, strain of agent and route of infection, all of which can affect the likelihood of PrPSc and PrPC interacting. The study by Raymond et al. (2000) used CWD agent from western white-tailed deer and it is not yet known whether the agent from Colorado/Wyoming is the same as the Wisconsin strain. In vivo strain differences are not necessarily reflected in the cell-free conversion assay. For example, both the HY and DY strains of hamster-adapted transmissible mink encephalopathy convert hamster PrPC to PrPres in a strain-specific manner in cell-free conversion assays (Bessen et al., 1995; Iniguez et al., 2000). Although HY and DY agents result in clinical disease when the hamsters are infected by the intracerebral route, DY does not cause disease when the route of infection is intraperitoneal or oral (Bartz et al., 2004). Cell-free conversion assays have also suggested that different PrP isoforms from elk are converted equivalently by infectious agent from elk (Raymond et al., 2000). However, Hamir et al. (2006) demonstrated that, in experimental oral infection experiments, the 132LL genotype is much less susceptible, with 132MM animals becoming clinically positive at 23 months post-infection, whilst 132LL animals are still clinically normal at 48 months.
The deer in the wDEZ were randomly sampled and thus it is possible that the number of CWD-positive deer with at least one G96S allele was artificially low due to variability in exposure to CWD. Therefore, we assessed the distribution of deer within the wDEZ. The location of harvest for each CWD-positive deer was determined using the deer harvest database maintained by the Wisconsin Department of Natural Resources. The same database was then used to identify CWD-negative animals (n=107) harvested within 1 square mile of CWD-positive deer (data not shown). At least one copy of the G96S allele was present in 51.5 % of these animals, compared with 46.4 % of all sequenced CWD-negative deer in the wDEZ, suggesting that lack of exposure to CWD was not a reason for the underrepresentation of G96S in the CWD-positive population.
Sequencing of Prnp from white-tailed deer outside the CWD-affected region of Wisconsin was performed to identify potential genetic barriers to disease spread and potential reservoirs for strain development and to establish basal allelic levels for future studies of disease impact on genetic drift. Given the homogeneous distribution of white-tailed deer Prnp alleles between the deer populations of the CWD-affected region and deer 100–300 miles distant from that population, it is likely that our results in the CWD-affected region are indicative of what can be expected for a much greater regional deer population. Localized areas of different Prnp allelic profiles may exist; however, our data suggest that there is no genetic barrier to CWD spread through the regional deer population. Previously, we estimated that 91 % of the CWD-negative deer had allelic combinations present in CWD-positive deer, suggesting that there is no major genetic barrier to transmission in the deer population (Johnson et al., 2003). From the results of this increased analysis of Prnp alleles in CWD-positive deer, we estimate that 91–98 % of white-tailed deer are genetically susceptible to CWD.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Heaton, M. P., Leymaster, K. A., Freking, B. A. & 7 other authors (2003). Prion gene sequence variation within diverse groups of U.S. sheep, beef cattle, and deer. Mamm Genome 14, 765–777.[CrossRef][Medline]
Iniguez, V., McKenzie, D., Mirwald, J. & Aiken, J. M. (2000). Strain-specific propagation of PrPSc properties into baculovirus-expressed PrPC. J Gen Virol 81, 2565–2571.
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O'Rourke, K. I., Spraker, T. R., Hamburg, L. K., Besser, T. E., Brayton, K. A. & Knowles, D. P. (2004). Polymorphisms in the prion precursor functional gene but not the pseudogene are associated with susceptibility to chronic wasting disease in white-tailed deer. J Gen Virol 85, 1339–1346.
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Received 17 October 2005;
accepted 10 March 2006.
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