Potential Pronghorn (Antilocapra americana) Susceptibility to Chronic Wasting Disease Through PRNP Sequencing

ABSTRACT

Prion diseases such as Chronic Wasting Disease (CWD) are significant concerns for wildlife managers, public health officials, and hunters. Given the complexity of prion transmission and emergence of novel prion diseases, interfamilial transmission represents a potential challenge to CWD management. Pronghorn (Antilocapra americana) use similar habitats and are geographically sympatric with some CWD-susceptible cervids. However, no published research exists specifically focused upon pronghorn susceptibility to prion diseases, namely CWD, as their membership in a non-cervid ungulate family provides the conceptual framework to assume resistance. Yet, pronghorn are sympatric with free-ranging white-tailed deer (Odocoileus virginianus), elk (Cervus canadensis), and mule deer (O. hemionus) throughout their geographic range. We sequenced PRNP exon 3 in pronghorn from Texas (n = 47) and New Mexico (n = 28) to compare amino acid sequences of known susceptible cervids and assess if pronghorn could be susceptible to prion diseases. Pronghorn possess one additional octapeptide repeat relative to the five repeats observed in cervids, and while whole-gene phylogenetic analyses place pronghorn outside Cervidae. Pronghorn share amino acids with cervids at loci 95, 96, 116, 132, 225 and 226 – the positions most strongly associated with CWD susceptibility. At locus 173, pronghorn share the bovid residue (serine) rather than the cervid residue (asparagine), further illustrating that pronghorn occupy a genuinely intermediate molecular position between these lineages. Although no CWD-positive pronghorn detections have been reported in the literature, the assumption of resistance based on taxonomic placement is not supported by site-specific sequence data, and this study provides a molecular basis for reassessing that assumption.

Key words: Antilocapra americana, chronic wasting disease, prion protein, PRNP, pronghorn

This paper is based on Chapter 1 of the author's Masters thesis.

INTRODUCTION

Over the last century, pronghorn (Antilocapra americana) have experienced dramatic reductions in range and population size (Seton 1909, McCabe et al. 2004, White et al. 2012, French 2015) largely attributed to habitat loss, fencing, livestock grazing, and overhunting (O’Gara and Yoakum 2004, Simpson et al. 2010). Beyond these factors, pronghorn are susceptible to disease outbreaks and die-offs due to their grouping and social cohesion, and disease can have major negative effects on population stability and recruitment (Thorne et al. 1988, White et al. 2012, Weaver 2013, Jones et al. 2023). Pronghorn have experienced localized extirpations due to epizootic hemorrhagic disease (EHD), blue-tongue virus (BTV), haemonchosis, and parainfluenza virus type 3 (PI3) in some years (Weaver 2013, Jones et al. 2023, Dubay et al. 2006). Similarly, each of these diseases can impact free-ranging white-tailed deer (Odocoileus virginianus) and mule deer (O. hemionus) and trigger significant mortality events in some years (Baygents and Bani-Yaghoub 2018, Cain et al. 2018, Conner and Miller 2004). Pronghorn are commonly sympatric with white-tailed deer, elk (Cervus canadensis), and mule deer, yet there is limited published research on either verified or assumed pathogen transmission among sympatric Cervids and pronghorn (see Cullingham et al. 2020).

Little is known about pronghorn in relation to prion diseases, particularly Chronic Wasting Disease (CWD), and there are few references that have posited that wild ungulates other than Cervids may be susceptible to prion disease, aside from Cullingham et al. (2020). To date, no studies have investigated pronghorn susceptibility to or pathology of prion diseases, including CWD. Cullingham et al. (2020) used the cytb (cytochrome B) gene to assemble a phylogeny of mammalian taxa, including cattle (Bos taurus, susceptible to Bovine Spongiform Encephalopathy, ergo BSE) hypothesized to be (a) susceptible, (b) resistant, or (c) unknown and compared PrP^C amino acid polymorphisms to identify susceptibility probability. Using this cytb phylogeny, they found that CWD susceptibility is highly correlated with phylogenetic history. By including species like pronghorn who have unknown susceptibility to CWD, Cullingham et al. (2020) suggested that pronghorn potentially have a “lowered” species barrier to prion disease. If pronghorn are indeed susceptible, then potential transmission among known-susceptible, sympatric species and pronghorn represents a potential risk throughout their range, as pronghorn occur near the primary CWD endemic region in the United States (O’Gara and Yoakum 2004, USGS 2025, Figure 1). The increasing regional prevalence of CWD in these sympatric Cervids with pronghorn underscores a need for further research to understand pronghorn susceptibility to prion diseases, namely CWD.

This study had three primary objectives: (a) PRNP characterization in pronghorn, (b) phylogenetic analyses to understand evolutionary relationships, and (c) comparative analyses with native, known CWD-susceptible Cervids. We sequenced PRNP in pronghorn from Texas and New Mexico to determine the specific amino acid sequence of prion protein (PrP^C) to identify any genetic variations within pronghorn PRNP. By comparing the PRNP amino acid sequences of pronghorn to those of native, sympatric Cervids like white-tailed deer, mule deer, and elk, we aimed to assess genetic similarity at loci associated with susceptibility to prion diseases.

METHODS

Sample acquisition

Wild Pronghorn samples (n = 73) consisted of 0.1 g of ear tissue clips (n = 45) from Texas pronghorn and whole blood samples (n = 28) from New Mexico pronghorn. The Texas samples consisted of adult males (n = 8), adult females (n = 37), and two additional adults (of unknown sex) during 2019 capture and translocation efforts in Irion, Culberson, and Hudspeth counties by Texas Parks and Wildlife Department (TPWD; acquired from Shawn Gray, Pronghorn and Mule Deer Program Leader, TPWD). These samples were stored in liquid nitrogen at the Natural Science Research Laboratory (NSRL) at Texas Tech University (Loan #OL2021-080; Patrick 2023). The New Mexico samples were whole blood, stored in anticoagulant-treated (ethylenediaminetetraacetic acid; EDTA) tubes collected from male (n = 7) and female (n = 21) juvenile and adult pronghorn from a 2015 capture and translocation effort in Colfax County, New Mexico (acquired from Tony Opatz, Pronghorn Biologist, New Mexico Department of Game and Fish); see Patrick (2023). Pronghorn in the study areas were not screened for prion infection. All samples were collected during capture events by TPWD and NMDGF staff following approved guidelines and were reviewed by the Texas Tech University Institutional Animal Care and Use Committee (IACUC Protocol Numbers 13011-01, 17037-04C, and 2024-1602).

DNA Amplification and Sequencing

Amplification, extraction, and sequencing methods followed those established by Buchholz et al. (2021), with some deviations detailed below; more methodological details can be found in Patrick (2023). We isolated genomic DNA (gDNA) using the Qiagen DNeasy kit (Qiagen Inc., Valencia, CA, USA) from 0.1 g ear tissue samples and from 100 µL anticoagulant-treated blood. The full length of PRNP exon 3 (771 bp in Cervids, 854 bp in pronghorn) and up to 53 base pairs (bp) of the 5’ flanking sequence and 83 bp of the 3’ flanking sequence was amplified with polymerase chain reaction methods (PCR, Saike et al. 1988), using the amplification primers MD582F and MD1479RC, established by Jewell et al. (2006). PCR reactions for the ear tissue samples followed the standard HotStarTaq (Qiagen Inc., Valencia, CA, USA) protocol. The thermal profile followed that of Buchholz et al. (2021) with annealing temperature modified to 54°C for 45s (Patrick 2023). Due to poor amplification with HotStarTaq likely due to sample type and age, reactions for the New Mexico blood samples followed the standard CYTIVA BeadKit (Global Life Sciences Solutions USA, Marlborough, MA, USA) protocol, details of which can be found in Patrick (2023). No differences attributable to extraction or amplification methods were observed in the resulting sequences.

All PCR products were purified with ExoSAP-IT PCR Product Cleanup and cycle-sequenced using BigDyeTerminator v3.1 (Applied Biosystems, Foster City, CA, USA) with the primers from Jewell et al. (2006; MD582F, MD1479RC, 12, and 3FL1) used to amplify fragments on both the forward and reverse strands (Patrick 2023). Sequencing products were then purified with Sephadex columns (Princeton Separation, Adelphia, NJ, USA) and centrifugation, followed by dehydration and resuspension in formamide prior to shipping to Eurofins Genomics where sequences were analyzed on an ABI Prism 3730xl automated sequencer (Biotechnology Resource Center, Institute of Biotechnology, Cornell University, Ithaca, New York; Eurofins Genomics, Louisville, Kentucky). Raw sequence reads returned were visually inspected, and chromatograms were inspected using Sequencher 4.10.1 software to verify all base changes (Gene Codes Corporation, Ann Arbor, Michigan).

Sequence analysis of PRNP Exon 3

Nucleotide sequences were aligned and trimmed to the open reading frame (ORF) of PRNP exon 3 using MEGA-X 11.0.13 following Kumar et al. (2018). The trimmed nucleotide sequences were then translated into protein using the standard genetic code and inspected for any non-synonymous substitutions at the nucleotide and amino acid level. Additional nucleotide sequences of PRNP exon 3 representing taxa from all species of Ruminantia (n = 589), as well as one sequence from Przewalski’s horse (Equus przewalksii), were obtained from GenBank and served as comparative sequences to ensure all substitutions, if any, were observed in pronghorn (Table 1). The sequence from Przewalski’s horse was used as the outgroup for the phylogenetic analyses as a basal extant member of Perissodactyla (Der Sarkissian et al. 2015). Pronghorn PRNP exon 3 sequences were aligned against PRNP exon 3 sequences from white-tailed deer (GenBank: AY286008); mule deer (GenBank: MT709572); elk (GenBank: U21210); reindeer (Rangifer tarandus, GenBank: MN052820); the wild type domestic sheep (Ovis aries, PRNP-B allele, GenBank: DQ885787); cattle (Bos taurus, GenBank: DQ112003.1); and zebu (Bos indicus, GenBank: AY720680.1).

Phylogenetic analyses

To identify the phylogenetic placement of pronghorn for PRNP, an initial dataset of the 589 individual sequences from Ruminantia were obtained from GenBank in addition to the 73 sequences of Texas and New Mexico pronghorn used in this study (see Table 1). A set of 88 maximum likelihood models were used to evaluate DNA sequences using jModelTest-2.1.7 with the Akaike Information Criterion with a correction for small sample sizes (AICc) and identified the General Time Reversible plus Inverse Gamma (GTR+I+G) model of evolution (-lnL = 2,326.0861) as the most appropriate for the dataset. These models were then verified utilizing the ModelFinder tool in W-IQ-TREE (Trifiniopoulos et al. 2016, Kalyaanamoorthy et al. 2017). A maximum likelihood analysis using a Bayesian inference model was conducted in BEAST2 and BEAUTi2 packages to calculate posterior probability values (PPV; Bouckaert et al. 2019; Bouckaert 2022; Douglas et al. 2021). The GTR+I+G (general time reversible plus inverse gamma) nucleotide substitution model (Rodríguez et al. 1990) and the following parameters were used: ten initialization attempts with four Markov-chains (one cold and three heated; MCMCMC), 1.0 x 10⁷ generations, and sampling every 10,000 generations over the previous nine million generations. The Yule birth model was used as an additional tree prior to account for birth rates and the substitution parameters of partition rate parameters (AC, AG, AT, CG and GT) were assigned Gamma priors (0.05-10.0). A visual inspection of likelihood scores in TRACER with the first 1,000,000 trees (10% burn-in) and a consensus tree (50% majority rule) constructed from the remaining trees, where PPV ≥ 0.95 were used to designate nodal support (Huelsenbeck et al. 2002, Bouckaert et al. 2019).

RESULTS

The PRNP exon 3 in pronghorn was 854 bp long with an extra octapeptide repeat compared to Cervid PRNP exon 3 for a total of six repeats. Four octapeptide repeats were flanked by nonapeptide repeats on either side from 73 individual pronghorn (Texas n = 45, New Mexico n = 28; Figure 2; see Patrick 2023). Cervid PRNP was 771 bp long and contained five total repeats, having only three octapeptide repeats and two nonapeptide repeats. Because pronghorn possess this additional octarepeat, pronghorn loci sites are adjusted to the right by eight amino acids. For example, pronghorn loci 140 would be equivalent to Cervid loci 132 . Three individuals were heterozygous at synonymous sites; however, no nonsynonymous variation was observed. Sequences from all 73 individuals were submitted to the GenBank databases under accession numbers PQ880041-PQ880113.

Characterization of exon 3 of PRNP in Pronghorn

Codons at loci 103, 104, 124, 132, 233, and 234 (Cervid loci 95, 96, 116, 132, 225, and 226) in pronghorn were identical to consensus Cervid amino acids at these loci (Robinson et al. 2012). Codons at pronghorn loci 108, 166, 297 (Cervid loci 100, 158, and 289) matched the consensus Cervid sequence rather than the consensus Bovid sequence. The presence of serine (S) at site 181 (Cervid locus173) links pronghorn to Bovids at this site, while Cervids instead possess an asparagine (N). The primary observed difference between pronghorn and Cervid PRNP sequences is the number of octapeptide repeats. At locus 56, pronghorn exhibited a nonapeptide repeat containing the amino acid sequence of PHGGGGWGQ followed by four octapeptide repeats containing the amino acid sequence of PHGGGWGQ and ending with the nonapeptide repeat at loci 106 (Figure 2).

Sequence Comparison with Ruminants

Pronghorn amino acid sequences of PRNP compared with sympatric, known CWD-susceptible Cervids suggest that they are most similar to mule deer, particularly at locus 132 (methionine), and white-tailed deer. Elk possess a glutamic acid (E) at locus 226 (glutamic acid vs. glutamine in mule deer, pronghorn and white-tailed deer), where this substitution is reported to have limited biochemical distinction despite the change from acidic, electrically charged side chain (glutamic acid) to an uncharged, polar side chain (glutamine; Table 2). Comparisons with sheep and cattle revealed that at locus 173, pronghorn possess an S (serine) instead of an N (asparagine), and both amino acids are uncharged and hydrophilic. The molecular similarity between pronghorn and mule deer, combined with extensive ecological overlap, is consistent with mule deer representing a plausible source of exposure; however, transmission pathways remain

Phylogenetic Analyses:

Phylogenetic analyses yielded two clades (A and B), where Clade A consisted of known prion disease-susceptible Ruminants, while Clade B consisted of pronghorn and giraffe (Figure 3). Support for divergence of pronghorn (≥ 0.9 posterior probability, Huelsenbeck et al. 2002) from Bovids, Ovids, Caprids and Cervids with the DNA sequences was limited to Clade A. Despite limited support for internal structure within Clade B, pronghorn grouped as expected with giraffe, their most recent common ancestor (Figure 3, O’Gara and Yoakum 2004).

DISCUSSION

The third exon of PRNP was successfully sequenced in pronghorn from Texas and New Mexico, and they share the same amino acid residues as Cervids at loci 95, 96, 116, 132, 225 and 226; and then possess an additional octapeptide repeat (Figures 2 and 3). These specific sites selected for comparison were based on the work of Robinson et al. (2012) and Buchholz et al. (2021), as these have been previously linked to CWD susceptibility and resistance in Cervids. No prior studies have explicitly identified these specific characteristics of PRNP in pronghorn. Pronghorn PRNP sequences occupy a genuinely intermediate position between Cervid and Bovid sequences by sharing characteristics with both lineages at key residues implicated in prion disease susceptibility. This site-specific similarity to Cervids at functionally relevant positions is the critical finding, and it persists despite whole-gene phylogenetic analyses placing pronghorn in Clade B, alongside giraffe. The discordance between phylogenetic placement and site-specific similarity underscores that taxonomic distance alone is not a reliable proxy for prion disease resistance, and that the assumption of pronghorn resistance based on family-level classification is not supported by these sequence data. The functional implications of these shared residues remain untested in pronghorn, but the molecular profile is inconsistent with confidently ruling out susceptibility.

Should pronghorn develop a prion disease, mule deer represent a plausible source of exposure given ecological overlap, and similarity of amino acids at the most common loci that correspond to susceptibility in mule deer if cross-species transmission were to occur (Table 2). Wyoming is considered the national stronghold for pronghorn (Jones et al. 2013) and does have high CWD prevalence in mule deer (Robinson et al. 2012, Fisher et al. 2022). For example, CWD prevalence rates in mule deer in Wyoming have been hovering around 30% since at least 2005, with some areas having a prevalence rate of up to 48% in male mule deer, while prevalence may be greater in sympatric areas with both elk and white-tailed deer (Robinson et al. 2012, Fisher et al. 2022). Pronghorn and mule deer are sympatric throughout their entire North American ranges, particularly in migration corridors, where they commonly forage in close proximity (O’Gara and Yoakum 2004, IUCN 2016, Mysterud 2023, Heffelfinger and Krausman 2023). The notion that pronghorn could develop their own prion disease remains untested, particularly under a scenario that could emerge from the quotidian overlap of their sympatric migration patterns.

The amino acid sequence of PrP^C has been linked to intra- and interspecies transmission of, and susceptibility to, prion disease through the sequence similarity between an infected individual and a recipient (Angers et al. 2014, Arifin et al. 2021). Resistance to prion pathology can occur through PrP^C amino acid sequence diversity among species, potentially at the familial level, and can reduce the probability of a conformational change of PrP^C to the misfolded isoform, which has been described as a component of an interspecies barrier to prion transmission (Prusiner 1995, Hill and Collinger 2004, Mead 2006, Buchanan et al. 2012). When considering the amino acid sites associated with resistance in deer, evolutionarily deer and bovines are equidistant from humans (Robinson et al. 2012). As bovine prions are known to subvert human PrPC as seen in BSE, then the same could be possible with Cervid prions (Zink 2020), as evolutionary distance alone may not provide enough evidence of, or exist as, a reliable species barrier (Zink et al. 2020). Other changes in amino acids at various loci are hypothesized to prevent different prion diseases in other species. For example, in domestic sheep, changes at locus 136 of alanine (A) to valine (V), locus 154 of arginine (R) to histidine (H) and at locus 171 of glutamine (Q) to either arginine or histidine, provide the molecular basis for genotypic resistance to scrapie (Hagenaars et al. 2018, Osterholm et al. 2019). Currently, in pronghorn, specific sequences of resistance or susceptibility using these PrP approaches remain unknown, but we have demonstrated there are similarities in molecular susceptibility to prion disease in pronghorn that align closely with mule deer.

PRNP Genetics and Evolution

Certain alleles and genotypes provide reduced susceptibility, to some degree, to various TSEs (Mead 2006, Buchanan and Zinke 2012, Zink 2020, Zink et al. 2020), and identification of the additional octapeptide repeat in pronghorn is unusual and previously unreported. This octapeptide repeat follows the amino acid sequence of PHGGGWGQ and most mammals are known to have three or four copies of this repeat (Figure 2; see van Rheede et al. 2003). Sheep, deer, and cattle have 3 tandem repeats of this octapeptide sequence while humans and pronghorn have 4 tandem repeats (Puckett et al. 1991, Schätzl et al. 1995, Goldmann et al. 1998, van Rheede et al. 2003, Buchanan and Zink 2012, Hagenaars et al. 2018, Zink 2020). While the number of repeats does not typically follow any phylogenetic pattern and is a frequent mutation in the Eutherian prion protein gene, balancing the number of repeats via selection can contribute to having too many repeats and therefore promoting the early onset of prion disease (Hagenaars et al. 2018). Variations in the number of repeats with the addition of 2-9 octarepeats in humans can result in an inherited prion disease (Puckett et al. 1991). Also, in goats, individuals with 3 repeats succumbed to prion disease after an unusually long incubation period (Schätzl et al. 1995). Neither humans nor pronghorn have known instances of contracting prion disease from a CWD-positive source (either environmentally or directly) which might be a potential indicator of a species barrier, however, variation in the number of octapeptide repeats seems to play little to no role in CWD expression in North American Cervids (Buchanan and Zink 2012, Zink 2020).

Fox et al. (2021) emphasizes the importance of publishing long-term surveillance data for pronghorn (if they exist) even in the absence of CWD-positive detections. Given the predicted potential susceptibility of pronghorn based on prion protein genetics, reporting negative surveillance results is critical to furthering management of CWD in all ungulate species. Reporting lack of detection in surveillance data for CWD and other diseases is arguably as important as reporting prevalence data, particularly if or when surveillance eventually reveals positive trends, and there are no baselines to refer to when developing epidemiological timelines and management actions. Such approaches provide clarity regarding lack of detection and tend to firmly support hypotheses examining either resistance or exposure risk, rather than perpetuating notions of resistance due to lack of published data or surveillance effort. As highlighted by the opportunistic surveillance efforts undertaken by Fox et al. (2021) in bighorn sheep (Ovis canadensis), the lack of detection should not be interpreted as evidence of resistance. Without systematic surveillance and reporting, even for negative, or non-detect tests for CWD and other prion diseases, we risk conflating non-detection with non-susceptibility, particularly when non-detection is due to lack of surveillance in the first place.

DECLARATION OF INTEREST STATEMENT

No potential conflict of interest is reported by the authors.

DECLARATION OF FUNDING STATEMENT

This research was funded by the Rumsey Research and Development Fund and the Bricker Endowment for Wildlife Management within the Department of Natural Resources Management, Texas Tech University.

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Table 1.PRNP sequences utilized in phylogenetic analyses. Aside from the 73 pronghorn (Antilocapra americana) sequenced from Texas and New Mexico, all other sequences were sourced from GenBank. Except for Przewalksi’s horse (Equus przewalskii), all other sequences occurred within Ruminantia and were sourced from GenBank. Species and number count of individuals.

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Table 2. Comparison of pronghorn PRNP to sympatric, known CWD-susceptible Cervids. Each locus is at its approximate location on the gene. For each locus, the amino acid that confers susceptibility is listed first and the amino acid that confers resistance is listed at the end. For example, at locus 226, pronghorn are more like white-tailed deer or mule deer rather than elk with a Q (glutamine) instead of an E (glutamic acid).

[Table]

Figures and legends:

Figure 1.[Image] Overlap among the known national Chronic Wasting Disease (CWD) endemic areas, including detections in wild and captive Cervids (red, green and blue polygons, Richards 2021) and pronghorn distribution (dotted polygons, USGS 2018).

Figure 2. Visual representation of the differences in the number of repeats in pronghorn (top, 2 nonapeptide repeats and 4 octapeptide repeats for a total of 6 repeats) and Cervids (bottom, 2 nonapeptide repeats and 3 octapeptide repeats for a total of 5 repeats). Nonapeptide repeats contain the sequence P(H/Q)GGGGWG and octapeptide repeats contain the sequence PHGGGGWGQ.

[Image]

Figure 3.[Image] Phylogenetic tree from DNA sequences of the prion protein gene (PRNP) from suborder Ruminantia with Bayesian inference analyses (BEAST2 and BEAUTi2, Bouckaert et al. 2019). Nodal support is indicated by asterisks (*, ≥0.9 posterior probability, Huelsenbeck et al. 2002).