J. Anim Sci. 2007. 85:3142-3147. doi:10.2527/jas.2007-0208
© 2007 American Society of Animal Science
Polymorphisms of the prion gene promoter region that influence classical bovine spongiform encephalopathy susceptibility are not applicable to other transmissible spongiform encephalopathies in cattle1,2
B. W. Brunelle*,
A. N. Hamir*,
T. Baron
,
A. G. Biacabe,
J. A. Richt*,
R. A. Kunkle*,
R. C. Cutlip*,
J. M. Miller* and
E. M. Nicholson*,3
* Virus and Prion Diseases of Livestock Research Unit, National Animal Disease Center, USDA, ARS, Ames, IA 50010; and and
Agence Française de Sécurité Sanitaire des Aliments (AFSSA), Unité ATNC, Lyon Cedex 07, France
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Abstract
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Two regulatory region polymorphisms in the prion gene of cattle have been reported to have an association with resistance to classical bovine spongiform encephalopathy (BSE). However, it is not known if this association also applies to other transmissible spongiform encephalopathies (TSE) in cattle. In this report, we compare the relationship between these 2 polymorphisms and resistance in cattle affected with naturally occurring atypical BSE as well as in cattle experimentally inoculated with either scrapie, chronic wasting disease, or transmissible mink encephalopathy. Our analysis revealed no association between genotype and resistance to atypical BSE or experimentally inoculated TSE. This indicates the promoter polymorphism correlation is specific to classical BSE and that atypical BSE and experimentally inoculated TSE are bypassing the site of influence of the polymorphisms. This genetic discrepancy demonstrates that atypical BSE progresses differently in the host relative to classical BSE. These results are consistent with the notion that atypical BSE originates spontaneously in cattle.
Key Words: atypical • bovine spongiform encephalopathy • genotype • prion
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INTRODUCTION
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Prions are the causative agents of a class of fatal neurodegenerative diseases known collectively as transmissible spongiform encephalopathies (TSE; Prusiner, 1998
). Transmissible spongiform encephalopathies have been identified in numerous species, including scrapie in sheep, chronic wasting disease (CWD) in cervids, transmissible mink encephalopathy (TME) in mink, and bovine spongiform encephalopathy (BSE) in cattle. Bovine spongiform encephalopathy is differentiated into 2 strains, classical and atypical BSE, based upon Western blot and in vivo analysis; atypical BSE can be further subdivided into high and low subtypes based upon apparent molecular weight on a Western blot (Biacabe et al., 2004; Casalone et al., 2004; Buschmann et al., 2006).
Resistance to classical BSE in cattle has been found to be modulated by 2 nucleotide polymorphisms in regulatory regions of the prion gene (PRNP; Figure 1; Sander et al., 2004; Juling et al., 2006). The first is an insertion-deletion (indel) in the promoter region, where the 23-bp deletion removes a binding site for the repressor protein RP58. The second polymorphism is an indel in the first intron, where the 12-bp deletion removes a binding site for transcription factor SP1. Insertion variants of either regulatory element have the potential to lower host prion protein expression levels (Sander et al., 2005), thus providing a biological basis for BSE resistance in cattle homozygous for the presence of the insertions (Carlson et al., 1994; Manson et al., 1994; Juling et al., 2006).
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Figure 1. Bovine PRNP gene organization. Introns, exons, and coding sequence (CDS) are indicated. The entire 795-bp CDS is located within exon 3. The 12-bp insertion-deletion (indel) polymorphism is located within intron 1, and the 23-bp indel polymorphism is located within the upstream promoter region. Scale is in kilobases (kb).
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This work addresses whether the influence of the homozygous insertion genotypes extends beyond that observed for classical BSE in cattle. Specifically, the genetic relationship of the 23-bp and 12-bp indel was investigated in cattle naturally affected with atypical BSE as well as in cattle that have been experimentally inoculated with the non-BSE TSE scrapie, TME, or CWD. Identification of genotypic influences among various TSE would give greater insight into TSE disease progression in cattle.
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MATERIALS AND METHODS
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All experimental animals were housed under approved National Animal Disease Center animal care and use committee protocols.
Samples
Postmortem tissue samples (brain, spleen, liver, or tonsil) for retrospective analysis were available from previous studies that involved intracerebral (IC) inoculation of 52 cattle with a TSE: 8 with TME (Hamir et al., 2006a), 9 with scrapie (Cutlip et al., 1994), 14 with CWD from white-tailed deer (CWDwhite-tailed; Hamir et al., 2006b), and 21 with CWD from mule deer (CWDmule; Hamir et al., 2005, 2006b). Deoxyribonucleic acid was extracted from 50 to 80 mg of tissue collected using the Tissue Lysis protocol from the High Pure PCR Template Preparation Kit (Roche Applied Science, Indianapolis, IN). The same protocol was used to extract DNA from tissue of 12 cattle orally inoculated with scrapie (Cutlip et al., 2001). Deoxyribonucleic acid was provided from 1 naturally occurring classical BSE case (US 2003 imported) and 9 naturally occurring, unrelated atypical BSE cases, 7 of which were of the high molecular weight phenotype (US 2004, US 2006, and 5 French samples) and 2 of which were of the low molecular weight phenotype.
Genotyping Assay
Based on previous reports (Hills et al., 2001; Sander et al., 2004), PCR primer pairs were designed to amplify a 130-bp to 153-bp region surrounding the 23-bp promoter indel and a 190- to 202-bp region containing the 12-bp intron 1 indel in cattle (Table 1). Each 25-µL PCR reaction contained 
50 ng of genomic DNA, 1x PCR buffer with 1.5 mM of MgCl2, 1 mM each deoxy-nucleoside triphosphate, 0.8 µM each primer, and 1.5 units of Taq polymerase. An Applied Biosystems 2720 thermal cycler (Applied Biosystems, Foster City, CA) was used to amplify the PCR reactions under the following conditions: 94°C for 1 min; 35 cycles of 94°C for 30 s, 56°C for 30 s, 72°C for 30 s; 72°C for 2 min; 4°C hold. The PCR products were run on 4% NuSieve gels (Cambrex, Rockland, ME), and the genotypes were readily distinguished by visual inspection of molecular mass verified against a 100-bp DNA size standard (Pro-mega, Madison, WI). Several PCR products were selected at random and sequenced to verify that the correct regions had been amplified (data not shown).
PRNP Gene Sequence
Primers for amplification and sequencing of the PRNP coding region are listed in Table 1
. Using the PCR conditions described in the previous section, the coding region of the PRNP gene was amplified. After verification on a 2% NuSieve gel, the PCR products were treated with ExoSAP-IT (GE Healthcare, Piscata-way, NJ) and sequenced using an ABI 3100 sequencer in conjunction with ABI Big-Dye v3.1 chemistry (Applied Biosystems). Sequence data were analyzed using Lasergene 6 (DNASTAR Inc., Madison, WI) and submitted to GenBank (accession numbers DQ875147 through DQ875207, EF541152 through EF541163).
Statistical Analyses
Based upon suitability for small data sets and the calculation of an exact probability, Fisher’s exact test was used to test for statistical associations. Differences in the frequencies of the homozygous insertion for each 23-bp and 12-bp genotype were tested among various healthy, classical BSE-affected, and atypical BSE-affected populations of cattle using the PROC FREQ procedure in SAS (SAS Inst. Inc., Cary, NC). This was accomplished by comparing the frequencies of the homozygous insertion and the nonhomozygous insertion (the combined heterozygous and homozygous deletion) between the 2 populations of interest.
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RESULTS
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Orally Inoculated TSE
A previous study found that cattle orally inoculated with scrapie were not susceptible to infection (Cutlip et al., 2001). To ascertain if the indel polymorphisms were correlated with resistance to scrapie via the oral route, we determined the 23-bp and 12-bp genotypes for the 12 cattle that were euthanized 8 yr postinoculation (Table 2). Of the 12 cattle, 5 were homozygous for both the 23-bp and 12-bp insertions, 5 were heterozygous at both sites, and 2 were homozygous for deletions at both loci. Given the distribution of genotypes, resistance to orally inoculated scrapie in cattle cannot be attributed to the 23-bp or 12-bp polymorphisms.
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Table 2. Number of cattle affected vs. number of cattle orally inoculated with scrapie for each 23-bp and 12-bp genotype1
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IC Inoculated TSE
Several studies have shown that cattle are not always 100% susceptible to TSE disease by IC inoculation (Hamir et al., 2005, 2007). To establish if there was an association between the indel polymorphisms and susceptibility or resistance to TSE infection via the IC route, we determined the 23-bp and 12-bp genotypes for 52 cattle previously inoculated with either scrapie (Cutlip et al., 1994), TME (Hamir et al., 2006a), CWDmule (Hamir et al., 2005, 2006b), or CWDwhite-tailed (Hamir et al., 2006b; Table 3). If an association was present, there should have been a higher prevalence of the resistant homozygous insertion genotypes in the cattle that did not develop a TSE disease.
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Table 3. Number of cattle affected vs. number of cattle intracerebrally inoculated with a transmissible spongiform encephalopathy (TSE) for each 23-bp and 12-bp genotype1
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In contrast to the cattle inoculated orally with scrapie, all 9 cattle IC inoculated with scrapie developed disease. Four of these cattle had the homozygous insertion genotypes, and these were not resistant. Intracerebral scrapie infection was not influenced by the indel polymorphisms.
All 4 cattle inoculated in a first passage of TME developed disease, and 3 of these cattle were homozygous for the insertion genotypes. Material from these 4 TME-affected cattle was subsequently used to inoculate 4 additional cattle. The 4 cattle in the second passage all developed a TSE, and 2 of these were homozygous for the insertion genotypes. The indel genotype did not have an effect on either the first or second passage of TME in cattle.
Fourteen cattle were inoculated with CWDwhite-tailed, and 12 became affected with a TSE (86%). Although 1 of the unaffected cattle was homozygous for the 23-bp and 12-bp insertions, 4 other cattle of that genotype developed disease (80%). Resistance in the 2 cattle that did not develop disease cannot be ascribed to the indel polymorphisms, because the 80% attack rate in the 5 cattle with the homozygous insertion genotype was comparable to the 89% attack rate in the 9 cattle that did not have the homozygous insertion genotype.
Chronic wasting disease from mule deer was used to inoculate 13 cattle, 5 of which became affected (38%). This resistance was not due to the homozygous insertion genotype, because none were present in the experimental group. Material from the 5 CWDmule-positive cattle was then used to inoculate an additional 8 cattle, all of which became affected. Of these 8 cattle, 3 were homozygous for the insertion genotype. Genotype did not influence resistance or susceptibility in either the first or second passage of CWDmule in cattle.
In total, 17 cattle IC inoculated with a TSE were homozygous for the 23-bp and 12-bp insertion, and 16 (94%) developed disease. This includes the 5 cattle inoculated with material in second-passage experiments (i.e., challenged with cattle-derived prions), all of which developed disease. Additionally, there was no correlation between incubation time and genotype (data not shown). Therefore, no association was present between genotype and resistance in cattle IC inoculated with a non-BSE TSE derived from sheep, deer, mink, or cattle.
Classical BSE
A previously published study demonstrated a significant difference in the 23-bp and 12-bp genotype frequencies between BSE-affected cattle and healthy cattle from Germany and the United Kingdom (Juling et al., 2006). To this German and United Kingdom data, we incorporated published data from healthy US sires (Seabury et al., 2004b) as well as the lone imported US classical BSE case. Entirely consistent with Juling et al. (2006), our analyses showed a difference in the frequency of the homozygous 23-bp and 12-bp insertion genotype between healthy and BSE-affected cattle (P< 0.001; Tables 4 and 5).
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Table 4. The 23-bp and 12-bp genotype frequencies in healthy, bovine spongiform encephalopathy(BSE)-affected, and atypical BSE-affected cattle1
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Table 5. Statistical analysis of the homozygous insertion genotype frequency among healthy, classical bovine spongiform encephalopathy-affected (cBSE), and atypical bovine spongiform encephalopathy-affected (aBSE) cattle using Fisher’s exact test
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Atypical BSE
Samples were obtained from 9 unrelated cattle identified by Western blot analysis as being positive for atypical BSE, which included 7 high and 2 low molecular weight phenotypes. These cattle were then genotyped for the 23-bp and 12-bp polymorphisms, and 2 of the 9 (1 high, 1 low) were found to be homozygous for insertions at both loci, a prevalence of 22% (Table 4). In contrast to classical BSE, the atypical BSE frequencies are consistent with the homozygous insertion frequencies observed in healthy cattle (Table 5). In addition, the 23-bp homozygous insertion frequency in the atypical BSE-affected cattle tended to differ from that of the classical BSE cases (22% vs. 6%; P = 0.093).
No Bos indicus breeds were included in the published analyses of classical BSE, and it is apparent that B. indicus and B. indicus crossbred cattle have distinct allele frequencies concerning the 23-bp and 12-bp indel polymorphisms when compared with Bos taurus cattle (Seabury et al., 2004b). Because both US atypical cases used in this study were B. taurus-B. indicus crossbreds, and these crossbreds may not accurately reflect either the healthy or classical BSE data sets in this study, we elected to evaluate the data excluding all B. indicus genetic influence. Including only B. taurus cattle, our analyses showed a more marked difference between the atypical and classical BSE-affected cattle with regards to the homozygous 23-bp insertion frequency (29% vs. 6%; P = 0.059). Overall, the frequency of the 23-bp homozygous insertion is similar between healthy and atypical BSE-affected cattle, and both differ from that seen in classical BSE-affected cattle.
The 9 atypical BSE samples used in this study represent approximately half of all reported atypical BSE cases worldwide. Given the rare nature of atypical BSE, a substantial increase in sample number is not currently possible and is not feasible within a reasonable time frame. However, evaluation of the data using Fisher’s exact test is valid for these samples, because it does not use assumptions based on large sample distributions and, therefore, does not necessitate a minimum sample size for any data group.
PRNP Coding Region
The observed association between genotype and susceptibility to TSE infection cannot be attributed to AA changes in the host prion protein. The coding region of PRNP was sequenced for 12 cattle orally inoculated with scrapie, 52 cattle IC inoculated with a TSE, the 2003 imported US classical BSE case, the 2004 US atypical BSE case, and 7 atypical BSE cases from France. A total of 4 nucleotide sites contained a substitution within the 795-bp coding region (nucleotides 234 A/G, 339 C/T, 555 C/T, 576 C/T). None of these changes led to a replacement in the corresponding AA sequence, and all have been reported previously (Seabury et al., 2004a). Two samples were heterozygous for the 5/6 octa-peptide repeat, whereas the remainder were homozygous for 6 repeats. None of these polymorphisms are associated with susceptibility or resistance to TSE in cattle (Hunter et al., 1994; Neibergs et al., 1994).
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DISCUSSION
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Previous studies have shown an association between a 23-bp and 12-bp indel polymorphism and resistance to classical BSE disease in cattle. By comparing genotypic data from healthy, oral TSE-inoculated, IC TSE-inoculated, atypical BSE-affected, and classical BSE-affected cattle, we found that only classical BSE maintained an association between the homozygous insertion genotypes and resistance to a TSE.
Cattle orally challenged with scrapie were 100% resistant, which was not due to genotype but was most likely due to a species barrier effect. It appears IC inoculations can bypass at least some portion of the species barrier, because only 19% of the cattle were resistant to TSE disease. The indel genotypes also showed no effect, because there was no association between the polymorphisms and susceptibility or resistance to disease. From this, we conclude that the IC route of TSE inoculation is bypassing the normal route of infection and is therefore bypassing the site of influence of the insertion genotype.
The results indicate that the frequency of the 23-bp homozygous insertion in cattle affected with atypical BSE reflects that found in the general healthy cattle population, both of which exhibit a prevalence greater than that observed in cattle affected with classical BSE. This lack of association between genotype and resistance is consistent with the pathogenesis of atypical BSE bypassing the site of influence of the 23-bp insertion polymorphism, similar to that observed in the IC inoculated TSE. Thus, we conclude the pathogenesis of atypical BSE is occurring via an alternative mode relative to classical BSE.
Atypical and classical BSE are different strains based upon Western blot profiles (Hill, 2004; Normile, 2004; Baron et al., 2006), and this study indicates that disease progresses via different routes for these strains. The disparate routes of pathogenesis in atypical BSE can occur by 1 of 2 means. One possibility is that the source of infectivity in atypical BSE is exposure to contaminated feedstuffs, as is the case for classical BSE, but progression occurs in a disparate manner that bypasses the influence of the indel polymorphisms. The other possibility is that atypical BSE is occurring spontaneously in the host. Support for atypical BSE occurring spontaneously are the parallels to sporadic TSE in humans, specifically, occurrence in older hosts and a comparable low incidence rate (Baron and Biacabe, 2006). Furthermore, atypical BSE occurs as isolated, sporadic cases in contrast to the clustering of cases observed for feedborne classical BSE (Donnelly et al., 1997). Interestingly, the only native-born cases of BSE in the United States identified to date have been classified as atypical BSE.
No experiment can conclusively confirm a spontaneous nature for atypical BSE. However, all available data are consistent with the widely made supposition that atypical BSE is a spontaneous TSE of cattle. Recognition of a spontaneous TSE in cattle, coupled with evidence indicating that low molecular weight atypical BSE can convert to classical BSE upon serial passage in mice (Capobianco et al., 2007), has broad implications for our understanding of the origins of both classical and atypical BSE as well as how we address the emergence of novel TSE in other host species.
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Footnotes
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1 We thank D. E. Palmquist, ARS Midwest Area statistician, for assistance in planning and conducting statistical analysis. We would also like to thank T. L. Nicholson, M. E. Kehrli, and L. A. Hobbs for critical evaluation of the manuscript and S. Lebepe-Mazur, D. Clouser, and K. Hassall (National Animal Disease Center, USDA, ARS, Ames, IA) for technical assistance. 
2 Mention of trade names or commercial products in this article is solely to provide specific information and does not imply recommendation or endorsement by the USDA.
3 Corresponding author: eric.nicholson{at}ars.usda.gov
Received for publication April 10, 2007.
Accepted for publication August 6, 2007.
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Abstract
INTRODUCTION
