First report of a novel 108 bp deletion and five novel SNPs in PRNP gene of stray cats and in silico analysis of their possible relation with feline spongiform encephalopathy.

Prion diseases are fatal neurodegenerative diseases affecting humans and animals. A relationship between variations in the prion gene of some species and susceptibility to prion diseases has been detected. However, variations in the prion protein of cats that have close contact with humans and their effect on prion protein are not well-known. Therefore, this study aimed to investigate the variations of prion protein-encoding gene (PRNP gene) in stray cats and to evaluate variants detected in terms of genetic factors associated with susceptibility or resistance to feline spongiform encephalopathy using bioinformatics tools. For this, cat DNA samples were amplified by a PCR targeting PRNP gene and then sequenced to reveal the variations. Finally, the effects of variants on prion protein were predicted by bioinformatics tools. According to the obtained results, a novel 108 bp deletion and nine SNPs were detected. Among SNPs, five (c314A>G, c.454T>A, c.579G>C, c.642G>C and c.672G>C) were detected for the first time in this study. Bioinformatics findings showed that c.579G>C (Q193H), c.454T>A (Y152N) and c.457G>A (E153K) variants have deleterious effects on prion protein and c.579G>C (Q193H) has high amyloid propensities. This study demonstrates prion protein variants of stray cats and their deleterious effects on prion protein for the first time.

Single nucleotide polymorphisms (SNPs) in the open reading frame (ORF) of prion protein gene (PRNP) in Nigerian livestock species.

BACKGROUND: Prion diseases, also known as transmissible spongiform encephalopathies (TSEs) remain one of the deleterious disorders, which have affected several animal species. Polymorphism of the prion protein (PRNP) gene majorly determines the susceptibility of animals to TSEs. However, only limited studies have examined the variation in PRNP gene in different Nigerian livestock species. Thus, this study aimed to identify the polymorphism of PRNP gene in Nigerian livestock species (including camel, dog, horse, goat, and sheep). We sequenced the open reading frame (ORF) of 65 camels, 31 village dogs and 12 horses from Nigeria and compared with PRNP sequences of 886 individuals retrieved from public databases. RESULTS: All the 994 individuals were assigned into 162 haplotypes. The sheep had the highest number of haplotypes (n = 54), and the camel had the lowest (n = 7). Phylogenetic tree further confirmed clustering of Nigerian individuals into their various species. We detected five non-synonymous SNPs of PRNP comprising of G9A, G10A, C11G, G12C, and T669C shared by all Nigerian livestock species and were in Hardy-Weinberg Equilibrium (HWE). The amino acid changes in these five non-synonymous SNP were all "benign" via Polyphen-2 program. Three SNPs G34C, T699C, and C738G occurred only in Nigerian dogs while C16G, G502A, G503A, and C681A in Nigerian horse. In addition, C50T was detected only in goats and sheep. CONCLUSION: Our study serves as the first to simultaneously investigate the polymorphism of PRNP gene in Nigerian livestock species and provides relevant information that could be adopted in programs targeted at breeding for prion diseases resistance.

Detection of classical BSE prions in asymptomatic cows after inoculation with atypical/Nor98 scrapie.

The emergence of bovine spongiform encephalopathy (BSE) prions from atypical scrapie has been recently observed upon experimental transmission to rodent and swine models. This study aimed to assess whether the inoculation of atypical scrapie could induce BSE-like disease in cattle. Four calves were intracerebrally challenged with atypical scrapie. Animals were euthanized without clinical signs of prion disease and tested negative for PrPSc accumulation by immunohistochemistry and western blotting. However, an emergence of BSE-like prion seeding activity was detected during in vitro propagation of brain samples from the inoculated animals. These findings suggest that atypical scrapie may represent a potential source of BSE infection in cattle.

Novel polymorphisms in the prion protein gene (PRNP) and stability of the resultant prion protein in different horse breeds.

Prion diseases are fatal neurodegenerative disorders in which the main pathogenic event is the conversion of the cellular prion protein (PrPC) into an abnormal and misfolded isoform known as PrPSc. Most prion diseases and their susceptibility and pathogenesis are mainly modulated by the PRNP gene that codes for PrP. Mutations and polymorphisms in the PRNP gene can alter PrPC amino acid sequence, leading to a change in transmission efficiency depending on the place where it occurs. Horses are animals that are considered to be highly resistant to prions. Several studies have attempted to identify polymorphisms in the PRNP gene that explain the reason for this high resistance. In this study, we have analysed 207 horses from 20 different breeds, discovering 3 novel PRNP polymorphisms. By using computer programmes such as PolyPhen-2, PROVEAN, PANTHER, Meta-SNP and PredictSNP, we have predicted the possible impact that these new polymorphisms would have on the horse prion protein. In addition, we measured the propensity for amyloid aggregation using AMYCO and analysed the lack of hydrogen bridges that these changes would entail together with their electrostatic potentials using Swiss-PdbViewer software, showing that an increased amyloid propensity could be due to changes at the level of electrostatic potentials.

Inter- and intra-species conversion efficacies of Norwegian prion isolates estimated by serial protein misfolding cyclic amplification.

Prion diseases, including chronic wasting disease (CWD) in cervids, are fatal neurodegenerative disorders caused by the misfolding of cellular prion proteins. CWD is known to spread among captive and free-ranging deer in North America. In 2016, an outbreak of contagious CWD was detected among wild reindeer in Norway, marking the first occurrence of the disease in Europe. Additionally, new sporadic forms of CWD have been discovered in red deer in Norway and moose in Fennoscandia. We used serial protein misfolding cyclic amplification to study the ability of Norwegian prion isolates from reindeer, red deer, and moose (two isolates), as well as experimental classical scrapie from sheep, to convert a panel of 16 brain homogenates (substrates) from six different species with various prion protein genotypes. The reindeer CWD isolate successfully converted substrates from all species except goats. The red deer isolate failed to convert sheep and goat substrates but exhibited amplification in all cervid substrates. The two moose isolates demonstrated lower conversion efficacies. The wild type isolate propagated in all moose substrates and in the wild type red deer substrate, while the other isolate only converted two of the moose substrates. The experimental classical scrapie isolate was successfully propagated in substrates from all species tested. Thus, reindeer CWD and classical sheep scrapie isolates were similarly propagated in substrates from different species, suggesting the potential for spillover of these contagious diseases. Furthermore, the roe deer substrate supported conversion of three isolates suggesting that this species may be vulnerable to prion disease.

Distribution of the misfolded isoform of the prion protein in peripheral tissues and spinal cord of Rocky Mountain elk (Cervus elaphus nelsoni) with naturally occurring chronic wasting disease.

Chronic wasting disease (CWD) is an infectious transmissible spongiform encephalopathy of cervids associated with the presence of a misfolded prion protein (PrPCWD). Progression of PrPCWD distribution has been described using immunohistochemistry and histologic changes in a single section of brain stem at the level of the obex resulting in scores from 0 (early) to 10 (terminal) in elk with naturally occurring CWD. Here we describe the spread and distribution of PrPCWD in peripheral tissues and spinal cord in 16 wild and 17 farmed Rocky Mountain elk (Cervus elaphus nelsoni) with naturally occurring CWD and correlate these findings with obex scores. Spinal cord and approximately 110 peripheral tissues were collected, processed, stained with hematoxylin and eosin, and immunolabeled with the anti-prion protein monoclonal antibody F99/97.6.1. The medial retropharyngeal and tracheobronchial lymph nodes were the first tissues to accumulate PrPCWD, followed by other lymphoid tissues, myenteric plexus, spinal cord, and finally tissues outside of the lymphatic and neural systems. However, the only significant histological lesion observed was mild spongiform encephalopathy in the dorsal column of the lower spinal cord in elk with an obex score of ≥9. Initial exposure to CWD prions may be through the respiratory system and spread appears to occur primarily via the autonomic nervous system. Therefore, we suggest using obex scores as a proxy for stage of disease progression and verifying with key peripheral tissues.

Estimating sequence diversity of prion protein gene (PRNP) in Portuguese populations of two cervid species: red deer and fallow deer.

Among the transmissible spongiform encephalopathies (TSEs), chronic wasting disease (CWD) in cervids is now a rising concern in wildlife within Europe, after the detection of the first case in Norway in 2016, in a wild reindeer and until June 2022 a total of 34 cases were described in Norway, Sweden and Finland. The definite diagnosis is post-mortem, performed in target areas of the brain and lymph nodes. Samples are first screened using a rapid test and, if positive, confirmed by immunohistochemistry and Western immunoblotting. The study of the genetics of the prion protein gene, PRNP, has been proved to be a valuable tool for determining the relative susceptibility to TSEs. In the present study, the exon 3 of PRNP gene of 143 samples from red deer (Cervus elaphus) and fallow deer (Dama dama) of Portugal was analysed. Three single nucleotide polymorphisms (SNPs) were found in red deer - codon A136A, codon T98A, codon Q226E - and no sequence variation was detected in fallow deer. The low genetic diversity found in our samples is compatible with previous studies in Europe. The comparison with results from North America suggests that the free-ranging deer from our study may present susceptibility to CWD, although lack of experimental data and the necessity of continuous survey are necessary to evaluate these populations.

Sheep scrapie and deer rabies in England prior to 1800.

Eighteenth-century England witnessed the emergence of two neurological diseases in animals. Scrapie, a transmissible spongiform encephalopathy, is a fatal neurodegenerative disease of sheep and goats that appears in classical and atypical forms. Reports of classical scrapie in continental Europe with described symptoms date back to 1750 in what is now western Poland. However, two major outbreaks of scrapie appeared in England prior to the 1800s. References to a sheep disease with a resemblance to scrapie first appear in Southwestern England between 1693 and 1722 and in the East Midlands between 1693 and 1706. Concurrent with the descriptions of scrapie in sheep was a neurological disease of deer first appearing in the East of England. Two 18th-century writers remarked on the symptomatic similarities between the sheep and deer neurological diseases. Multiple outbreaks of the unknown deer disease existing as early as 1772 are examined and are identified as rabies.

Characterization of Classical Sheep Scrapie in White-tailed Deer after Experimental Oronasal Exposure.

BACKGROUND: Classic scrapie is a prion disease of sheep and goats that is associated with accumulation of abnormal prion protein (PrPSc) in the central nervous and lymphoid tissues. Chronic wasting disease (CWD) is the prion disease of cervids. This study was conducted to determine the susceptibility of white-tailed deer (WTD) to the classic scrapie agent. METHODS: We inoculated WTD (n = 5) by means of a concurrent oral/intranasal exposure with the classic scrapie agent from sheep or oronasally with the classic scrapie agent from goats (n = 6). RESULTS: All deer exposed to the agent of classic scrapie from sheep accumulated PrPSc. PrPSc was detected in lymphoid tissues at preclinical time points, and necropsies in deer 28 months after inoculation showed clinical signs, spongiform lesions, and widespread PrPSc in neural and lymphoid tissues. Western blots on samples from the brainstem, cerebellum, and lymph nodes of scrapie-infected WTD have a molecular profile similar to CWD and distinct from samples from the cerebral cortex, retina, or the original classic scrapie inoculum. There was no evidence of PrPSc in any of the WTD inoculated with classic scrapie prions from goats. CONCLUSIONS: WTD are susceptible to the agent of classic scrapie from sheep, and differentiation from CWD may be difficult.

In-depth examination of PrPSc in Holstein cattle carrying the E211K somatic mutation of the bovine prion protein gene (PRNP).

Prion diseases are transmissible spongiform encephalopathies caused by deleterious prion protein (PrPSc ) derived from normal prion protein (PrPC ), which is encoded by the prion protein gene (PRNP). We performed an in-depth examination to detect PrPSc by using enzyme immunoassay (EIA), real-time quaking-induced conversion reactions (RT-QuIC) and protein misfolding cyclic amplification (PMCA) in nine brain tissues derived from three Holstein cattle carrying the E211K somatic mutation of the bovine PRNP gene. The EIA, RT-QuIC and PMCA analyses were not able to detect the PrPSc band in any tested samples. To the best of our knowledge, this report is the first to describe an in-depth examination of PrPSc in cattle carrying the E211K somatic mutation of the bovine PRNP gene.

RT-QuIC detection of pathological prion protein in subclinical goats following experimental oral transmission of L-type BSE.

OBJECTIVE: The spread of bovine spongiform encephalopathy (BSE) agent to small ruminants is still a major issue in the surveillance of transmissible spongiform encephalopathies (TSEs). L-type bovine spongiform encephalopathy (L-BSE) is an atypical form of BSE with an unknown zoonotic potential that is transmissible to cattle and small ruminants. Our current knowledge of bovine atypical prion strains in sheep and goat relies only on experimental transmission studies by intracranial inoculation. To assess oral susceptibility of goats to L-BSE, we orally inoculated five goats with cattle L-BSE brain homogenates and investigated pathogenic prion protein (PrPsc) distribution by an ultrasensitive in vitro conversion assay known as Real-Time Quaking Induced Conversion (RT-QuIC). RESULTS: Despite a prolonged observation period of 80 months, all these animals and the uninfected controls did not develop clinical signs referable to TSEs and tested negative by standard diagnostics. Otherwise, RT-QuIC analysis showed seeding activity in five out of five examined brain samples. PrPsc accumulation was also detected in spinal cord and lymphoreticular system. These results indicate that caprine species are susceptible to L-BSE by oral transmission and that ultrasensitive prion tests deserve consideration to improve the potential of current surveillance systems against otherwise undetectable forms of animal prion infections.

Evaluation of Winter Ticks (Dermacentor albipictus) Collected from North American Elk (Cervus canadensis) in an Area of Chronic Wasting Disease Endemicity for Evidence of PrPCWD Amplification Using Real-Time Quaking-Induced Conversion Assay.

Chronic wasting disease (CWD) is a progressive and fatal spongiform encephalopathy of deer and elk species, caused by a misfolded variant of the normal prion protein. Horizontal transmission of the misfolded CWD prion between animals is thought to occur through shedding in saliva and other forms of excreta. The role of blood in CWD transmission is less clear, though infectivity has been demonstrated in various blood fractions. Blood-feeding insects, including ticks, are known vectors for a range of bacterial and viral infections in animals and humans, though to date, there has been no evidence for their involvement in prion disease transmission. In the present study, we evaluated winter ticks (Dermacentor albipictus) collected from 136 North American elk (Cervus canadensis) in an area where CWD is endemic for evidence of CWD prion amplification using the real-time quaking-induced conversion assay (RT-QuIC). Although 30 elk were found to be CWD positive (22%) postmortem, amplifiable prions were found in just a single tick collected from an elk in advanced stages of CWD infection, with some evidence for prions in ticks collected from elk in mid-stage infection. These findings suggest that further investigation of ticks as reservoirs for prion disease may be warranted. IMPORTANCE This study reports the first finding of detectable levels of prions linked to chronic wasting disease in a tick collected from a clinically infected elk. Using the real-time quaking-induced conversion assay (RT-QuIC), "suspect" samples were also identified; these suspect ticks were more likely to have been collected from CWD-positive elk, though suspect amplification was also observed in ticks collected from CWD-negative elk. Observed levels were at the lower end of our detection limits, though our findings suggest that additional research evaluating ticks collected from animals in late-stage disease may be warranted to further evaluate the role of ticks as potential vectors of chronic wasting disease.

OPPORTUNISTIC SURVEILLANCE OF CAPTIVE AND FREE-RANGING BIGHORN SHEEP (OVIS CANADENSIS) IN COLORADO, USA, FOR TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES.

Bighorn sheep (Ovis canadensis) are predicted to have a degree of susceptibility to the transmissible spongiform encephalopathies (TSE) chronic wasting disease and scrapie. We opportunistically screened 127 captive bighorn sheep and 152 free-ranging bighorn sheep in Colorado, US for the presence of TSE over a period of 35 yr. None of the animals demonstrated clinical signs, gross pathology, histopathology, or immunohistochemical staining patterns suggestive of TSE.

Animal prion diseases: A review of intraspecies transmission.

Animal prion diseases are a group of neurodegenerative, transmissible, and fatal disorders that affect several animal species. The causative agent, prion, is a misfolded isoform of normal cellular prion protein, which is found in cells with higher concentration in the central nervous system. This review explored the sources of infection and different natural transmission routes of animal prion diseases in susceptible populations. Chronic wasting disease in cervids and scrapie in small ruminants are prion diseases capable of maintaining themselves in susceptible populations through horizontal and vertical transmission. The other prion animal diseases can only be transmitted through food contaminated with prions. Bovine spongiform encephalopathy (BSE) is the only animal prion disease considered zoonotic. However, due to its inability to transmit within a population, it could be controlled. The emergence of atypical cases of scrapie and BSE, even the recent report of prion disease in camels, demonstrates the importance of understanding the transmission routes of prion diseases to take measures to control them and to assess the risks to human and animal health.

Novel Polymorphisms and Genetic Features of the Prion Protein Gene (PRNP) in Cats, Hosts of Feline Spongiform Encephalopathy.

Prion diseases are fatal neurodegenerative disorders characterized by vacuolation and gliosis in the brain. Prion diseases have been reported in several mammals, and genetic polymorphisms of the prion protein gene (PRNP) play an essential role in the vulnerability of prion diseases. However, to date, investigations of PRNP polymorphisms are rare in cats, which are the major host of feline spongiform encephalopathy (FSE). Thus, we investigated the genetic polymorphisms of the cat PRNP gene and analyzed the structural characteristics of the PrP of cats compared to those of dog, prion disease-resistant animal. To investigate the genetic variations of the cat PRNP gene in 208 cats, we performed amplicon sequencing and examined the genotype, allele and haplotype frequencies of cat PRNP polymorphisms. We evaluated the influence of cat PRNP polymorphisms using PolyPhen-2, PANTHER, PROVEAN and AMYCO. In addition, we carried out structural analysis of cat PrP according to the allele of nonsynonymous single nucleotide polymorphism (SNP) (c.457G > A, Glu153Lys) using Swiss-PdbViewer. Finally, we compared the structural differences between cat and canine PrPs for SNPs associated with prion disease resistance in dogs. We identified a total of 15 polymorphisms, including 14 novel SNPs and one insertion/deletion polymorphism (InDel). Among them, Glu153Lys was predicted to affect the structural stability and amyloid propensity of cat PrP. In addition, asparagine at codon 166 of cat PrP was predicted to have longer hydrogen bond than aspartic acid at codon 163 of canine PrP. Furthermore, substitution to dog-specific amino acids in cat PrP showed an increase in structural stability. To the best of our knowledge, this is the first study regarding the structural characteristics of cat PRNP gene.

Novel Polymorphisms and Genetic Characteristics of the Prion Protein Gene (PRNP) in Dogs-A Resistant Animal of Prion Disease.

Transmissible spongiform encephalopathies (TSEs) have been reported in a wide range of species. However, TSE infection in natural cases has never been reported in dogs. Previous studies have reported that polymorphisms of the prion protein gene (PRNP) have a direct impact on the susceptibility of TSE. However, studies on polymorphisms of the canine PRNP gene are very rare in dogs. We examined the genotype, allele, and haplotype frequencies of canine PRNP in 204 dogs using direct sequencing and analyzed linkage disequilibrium (LD) using Haploview version 4.2. In addition, to evaluate the impact of nonsynonymous polymorphisms on the function of prion protein (PrP), we carried out in silico analysis using PolyPhen-2, PROVEAN, and PANTHER. Furthermore, we analyzed the structure of PrP and hydrogen bonds according to alleles of nonsynonymous single nucleotide polymorphisms (SNPs) using the Swiss-Pdb Viewer program. Finally, we predicted the impact of the polymorphisms on the aggregation propensity of dog PrP using AMYCO. We identified a total of eight polymorphisms, including five novel SNPs and one insertion/deletion polymorphism, and found strong LDs and six major haplotypes among eight polymorphisms. In addition, we identified significantly different distribution of haplotypes among eight dog breeds, however, the kinds of identified polymorphisms were different among each dog breed. We predicted that p.64_71del HGGGWGQP, Asp182Gly, and Asp182Glu polymorphisms can impact the function and/or structure of dog PrP. Furthermore, the number of hydrogen bonds of dog PrP with the Glu182 and Gly182 alleles were predicted to be less than those with the Asp182 allele. Finally, Asp163Glu and Asp182Gly showed more aggregation propensity than wild-type dog PrP. These results suggest that nonsynonymous SNPs, Asp182Glu and Asp182Gly, can influence the stability of dog PrP and confer the possibility of TSE infection in dogs.

Identification of the novel polymorphisms and potential genetic features of the prion protein gene (PRNP) in horses, a prion disease-resistant animal.

Prion diseases, a protein misfolded disorder (PMD) caused by misfolded prion protein (PrPSc), present in a wide variety of hosts, ranging from ungulates to humans. To date, prion infections have not been reported in horses, which are well-known as prion disease-resistant animals. Several studies have attempted to identify distinctive features in the prion protein of horses compared to prion disease-susceptible animals, without the study on polymorphisms of the horse prion protein gene (PRNP). Since single nucleotide polymorphisms (SNPs) of PRNP in prion disease-susceptible animals are major susceptibility factors, the investigation of SNPs in the horse PRNP gene is important; however, only one study investigated a single horse breed, Thoroughbred. Thus, we investigated genetic polymorphisms and potential characteristics of the PRNP gene in 2 additional horse breeds. To this end, we performed amplicon sequencing of the horse PRNP gene and investigated SNPs in Jeju and Halla horses. We compared genotype, allele and haplotype frequencies among three horse breeds, namely, Thoroughbred, Jeju and Halla horses. In addition, we evaluated the potential influence of the identified nonsynonymous SNPs on the prion protein using PolyPhen-2, PROVEAN, and PANTHER. Furthermore, we measured the aggregation propensity of prion proteins using AMYCO and analyzed linkage disequilibrium (LD) between PRNP and prion-like protein gene (PRND) SNPs. A total of 4 SNPs were found, including two nonsynonymous SNPs (c.301 T > A, c.525 C > A) and three novel SNPs (c.-3A > G, c.301 T > A and c.570 G > A). There were significant differences in genotype, allele and haplotype frequencies among the three horse breeds. The nonsynonymous SNP, c.301 T > A (W101R), was predicted to be benign, deleterious, and possibly damaging by PolyPhen-2, PROVEAN and PANTHER, respectively. In addition, the amyloid propensity of horse prion protein according to 4 haplotypes of nonsynonymous SNPs was predicted to be benign by AMYCO. Finally, we identified weak LD between PRNP and PRND SNPs.

MicroRNA Alterations in a Tg501 Mouse Model of Prion Disease.

MicroRNAs (miRNAs) may contribute to the development and pathology of many neurodegenerative diseases, including prion diseases. They are also promising biomarker candidates due to their stability in body fluids. We investigated miRNA alterations in a Tg501 mouse model of prion diseases that expresses a transgene encoding the goat prion protein (PRNP). Tg501 mice intracranially inoculated with mouse-adapted goat scrapie were compared with age-matched, mock inoculated controls in preclinical and clinical stages. Small RNA sequencing from the cervical spinal cord indicated that miR-223-3p, miR-151-3p, and miR-144-5p were dysregulated in scrapie-inoculated animals before the onset of symptoms. In clinical-stage animals, 23 significant miRNA alterations were found. These miRNAs were predicted to modify the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways including prion disease, extracellular matrix interactions, glutaminergic synapse, axon guidance, and transforming growth factor-beta signaling. MicroRNAs miR-146a-5p (up in cervical spinal cord) and miR-342-3p (down in cervical spinal cord, cerebellum and plasma), both indicated in neurodegenerative diseases earlier, were verified by quantitative real-time polymerase chain reaction (qRT-PCR). Minimal changes observed before the disease onset suggests that most miRNA alterations observed here are driven by advanced prion-associated pathology, possibly limiting their use as diagnostic markers. However, the results encourage further mechanistic studies on miRNA-regulated pathways involved in these neurodegenerative conditions.

Absence of Strong Genetic Linkage Disequilibrium between Single Nucleotide Polymorphisms (SNPs) in the Prion Protein Gene (PRNP) and the Prion-Like Protein Gene (PRND) in the Horse, a Prion-Resistant Species.

Prion disease is a fatal neurodegenerative disorder caused by a deleterious prion protein (PrPSc). However, prion disease has not been reported in horses during outbreaks of transmissible spongiform encephalopathies (TSEs) in various animals in the UK. In previous studies, single nucleotide polymorphisms (SNPs) in the prion protein gene (PRNP) have been significantly associated with susceptibility to prion disease, and strong linkage disequilibrium (LD) between PRNP and prion-like protein gene (PRND) SNPs has been identified in prion disease-susceptible species. On the other hand, weak LD values have been reported in dogs, a prion disease-resistant species. In this study, we investigated SNPs in the PRND gene and measured the LD values between the PRNP and PRND SNPs and the impact of a nonsynonymous SNP found in the horse PRND gene. To identify SNPs in the PRND gene, we performed direct sequencing of the PRND gene. In addition, to assess whether the weak LD value between the PRNP and PRND SNPs is a characteristic of prion disease-resistant animals, we measured the LD value between the PRNP and PRND SNPs using D' and r2 values. Furthermore, we evaluated the impact of a nonsynonymous SNP in the Doppel protein with PolyPhen-2, PROVEAN, and PANTHER. We observed two novel SNPs, c.331G > A (A111T) and c.411G > C. The genotype and allele frequencies of the c.331G > A (A111T) and c.411G > C SNPs were significantly different between Jeju, Halla, and Thoroughbred horses. In addition, we found a total of three haplotypes: GG, AG, and GC. The GG haplotype was the most frequently observed in Jeju and Halla horses. Furthermore, the impact of A111T on the Doppel protein was predicted to be benign by PolyPhen-2, PROVEAN, and PANTHER. Interestingly, a weak LD value between the PRNP and PRND SNPs was found in the horse, a prion disease-resistant animal. To the best of our knowledge, these results suggest that a weak LD value could be one feature of prion disease-resistant animals.