PrP gene polymorphisms in Cyprus goats and their association with resistance or susceptibility to natural scrapie

In contrast to scrapie in sheep, the genetic basis of susceptibility to scrapie in goats is not well understood. To study the association of prion protein (PrP) alleles with susceptibility to scrapie in goats in Cyprus, the coding sequence of the caprine PrP gene was determined in 717 goats, including 218 scrapie positive animals. Several novel polymorphisms were detected, such as a novel octarepeat variant and a stop codon mutation. Amino acids at codons 146 and 154 were associated with susceptibility to goat scrapie. Animals heterozygous for serine (S) and aspartate (D) at codon 146 were significantly under-represented in scrapie positive animals and no positive animals were found that were homozygous for these amino acids at codon 146. These results might provide the basis for genetic control of scrapie in Cypriot goats.     

Novel polymorphisms at codons 146 and 151 in the prion protein gene of Cyprus goats, and their association with natural scrapie

To discern whether an association exists between specific combinations of polymorphisms of the prion protein (PrP) and natural scrapie in Cyprus goats, 250 goats were examined, including 164 histologically positive cases. Previously reported amino acid polymorphisms were detected at codons 154 (R-->H), 168(P-->Q), 220(Q-->H) and 240 (S-->P) and nucleotide alterations at codons 42 (a-->g) and 138 (c-->t). Additionally, novel amino acid polymorphisms were detected at codons 146 (N-->S or D) and 151 (R-->H) and new "silent" mutations were found at codons 179 (V,g-->t), 181 (D,c-->t) and 219 (T,c-->t). The two novel polymorphisms at codon 146 were found only in the healthy control and scrapie-negative goats. By comparison, none of the scrapie-affected goats encoded these polymorphisms.     

A histopathologic and immunohistochemical review of archived UK caprine scrapie cases

In 2005, a prion disease identified in a goat from France was reported to be consistent with disease from the bovine spongiform encephalopathy (BSE) agent. Subsequent retrospective examination of UK goat scrapie cases led to the identification of one potentially similar, but as yet unconfirmed, case from Scotland. These findings strengthened concerns that small ruminant populations exposed to the BSE agent have become infected. The lack of data relating specifically to scrapie in goats has been contributory to past assumptions that, in general, sheep and goats respond similarly to prion infections. In this study, brain material from 22 archived caprine scrapie cases from the UK was reviewed by histopathology and by immunohistochemical examination for accumulations of disease-specific prion protein (PrP(Sc)) to provide additional data on the lesions of caprine scrapie and to identify any BSE-like features. The vacuolar change observed in the goats was characteristic of transmissible spongiform encephalopathies in general. PrP(Sc) immunohistochemical morphologic forms described in scrapie and experimental BSE infections of sheep were demonstrable in the goats, but these were generally more extensive and variable in PrP(Sc) accumulation. None of the cases examined showed a PrP(Sc) immunohistochemical pattern indicative of BSE.     

Effects of polymorphisms in ovine and caprine prion protein alleles on cell-free conversion

ABSTRACT: In sheep polymorphisms of the prion gene (PRNP) at the codons 136, 154 and 171 strongly influence the susceptibility to scrapie and bovine spongiform encephalopathy (BSE) infections. In goats a number of other gene polymorphisms were found which are suspected to trigger similar effects. However, no strong correlation between polymorphisms and TSE susceptibility in goats has yet been obtained from epidemiological studies and only a low number of experimental challenge data are available at present. We have therefore studied the potential impact of these polymorphisms in vitro by cell-free conversion assays using mouse scrapie strain Me7. Mouse scrapie brain derived PrPSc served as seeds and eleven recombinant single mutation variants of sheep and goat PrPC as conversion targets. With this approach it was possible to assign reduced conversion efficiencies to specific polymorphisms, which are associated to low frequency in scrapie-affected goats or found only in healthy animals. Moreover, we could demonstrate a dominant-negative inhibition of prion polymorphisms associated with high susceptibility by alleles linked to low susceptibility in vitro.     

State-of-the-art review of goat TSE in the European Union, with special emphasis on PRNP genetics and epidemiology

Scrapie is a fatal, neurodegenerative disease of sheep and goats. It is also the earliest known member in the family of diseases classified as transmissible spongiform encephalopathies (TSE) or prion diseases, which includes Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy (BSE), and chronic wasting disease in cervids. The recent revelation of naturally occurring BSE in a goat has brought the issue of TSE in goats to the attention of the public. In contrast to scrapie, BSE presents a proven risk to humans. The risk of goat BSE, however, is difficult to evaluate, as our knowledge of TSE in goats is limited. Natural caprine scrapie has been discovered throughout Europe, with reported cases generally being greatest in countries with the highest goat populations. As with sheep scrapie, susceptibility and incubation period duration of goat scrapie are most likely controlled by the prion protein (PrP) gene (PRNP). Like the PRNP of sheep, the caprine PRNP shows significantly greater variability than that of cattle and humans. Although PRNP variability in goats differs from that observed in sheep, the two species share several identical alleles. Moreover, while the ARR allele associated with enhancing resistance in sheep is not present in the goat PRNP, there is evidence for the existence of other PrP variants related to resistance. This review presents the current knowledge of the epidemiology of caprine scrapie within the major European goat populations, and compiles the current data on genetic variability of PRNP.     

Immunohistochemical detection and distribution of prion protein in a goat with natural scrapie.

Formalin-fixed, paraffin-embedded tissue sections from a 3-year-old female Angora goat suffering from clinical scrapie were immunostained after hydrated autoclaving using a monoclonal antibody (mAb, F99/97.6.1; IgG1) specific for a conserved epitope on the prion protein. Widespread and prominent deposition of the scrapie isoform of the prion protein (PrPSc) was observed in the brain, brainstem, spinal cord, retina, postganglionic neurons associated with parasympathetic ganglia of myenteric and submucosal plexuses, Peyer's patches, peripheral lymph nodes, and pharyngeal and palatine tonsils. The goat was homozygous for PrP alleles encoding 5 octapeptide repeat sequences in the N-terminal region of the prion protein and isoleucine at codon 142, a genotype associated with high susceptibility and short incubation times in goats. The results of this study indicate that mAb F99/97.6.1 is useful for detection of PrPSc deposition, and this is a specific and reliable immunohistochemical adjunct to histopathology for diagnosis of natural caprine scrapie, although precise determination of the diagnostic sensitivity and specificity of the assay as a diagnostic test for scrapie in goats will require examination of a sufficiently large sample size. As with ovine scrapie, prion protein is widely distributed in the central and peripheral nervous systems, gastrointestinal tract, and lymphoid tissues in natural caprine scrapie.     

Unique amino acid polymorphisms of PrP genes in Mongolian sheep breeds

To characterize amino acid polymorphisms of sheep prion protein (PrP) gene, DNA from 740 sheep of nine breeds raised in Mongolia was isolated and analyzed. A total of 16 genotypes and seven allelic variants of the PrP gene at codons 112, 136, 154, and 171 were found. The MARQ/MARQ genotype associated with susceptibility to scrapie was found in 82.6% of the sheep while the MARR/MARR genotype associated with resistance to scrapie was found in 1.8% of the sheep. The polymorphisms of valine and serine at codon 127, and leucine and arginine at codon 189 were detected in eight Mongolian sheep breeds, suggesting that these polymorphisms are a common feature among Mongolian sheep breeds.     

Protective effect of the AT137RQ and ARQK176 PrP alleles against classical scrapie in Sarda breed sheep

The susceptibility of sheep to scrapie is under the control of the host’s prion protein (PrP) gene and is also influenced by the strain of the agent. PrP polymorphisms at codons 136 (A/V), 154 (R/H) and 171 (Q/R/H) are the main determinants of susceptibility/resistance of sheep to classical scrapie. They are combined in four main variants of the wild-type ARQ allele: VRQ, AHQ, ARH and ARR. Breeding programmes have been undertaken on this basis in the European Union and the USA to increase the frequency of the resistant ARR allele in sheep populations. Herein, we report the results of a multi-flock study showing the protective effect of polymorphisms other than those at codons 136, 154 and 171 in Sarda breed sheep. All ARQ/ARQ affected sheep (n = 154) and 378 negative ARQ/ARQ controls from four scrapie outbreaks were submitted to sequencing of the PrP gene. The distribution of variations other than those at the standard three codons, between scrapie cases and negative controls, was statistically different in all flocks. In particular, the AT₁₃₇RQ and ARQK₁₇₆ alleles showed a clear protective effect. This is the first study demonstrating a protective influence of alleles other than ARR under field conditions. If further investigations in other sheep breeds and with other scrapie sources confirm these findings, the availability of various protective alleles in breeding programmes of sheep for scrapie resistance could be useful in breeds with a low frequency of the ARR allele and would allow maintaining a wider variability of the PrP gene.     

PRNP gene polymorphisms in main indigenous Turkish goat breeds

Tropical Animal Health and Production - The polymorphisms of the PRNP gene influence the susceptibility to scrapie in goats. In this study, caprine PRNP gene was analysed in a total of 249...     

Amino acid polymorphisms of PrP gene in Mongolian sheep

To characterize amino acid polymorphisms in sheep prion protein (PrP), we analyzed the PrP genes from 271 sheep of 4 breeds (Khalkh, Yeroo, Orkhon and Khangai) raised in central Mongolia (Tuv, Uvurkhangai and Selenge prefectures). A total of 16 genotypes and 8 allelic variants of the PrP gene at codons 112, 136, 154 and 171 were found. At codon 171, 1.8% of the sheep had arginine/arginine (R/R) (resistant to scrapie) and 66.8% had glutamine/glutamine (Q/Q) (susceptible to scrapie). Several Yeroo and Orkhon sheep raised in Selenge prefecture had valine at codon 136 (136V) (highly susceptible to scrapie). Several Yeroo, Orkhon and Khangai sheep raised in Selenge prefecture had histidine at codon 154 (154H). Novel polymorphisms of valine (V) and serine (S) at codon 127, lysine (K) at codon 171, and leucine (L) and arginine (R) at codon 189 were also found in Khalkh, Yeroo and Orkhon sheep. It is not known whether these novel polymorphisms affect scrapie susceptibility.     

Determination of sheep prion gene polymorphisms from paraffin-embedded tissue.

Amino acid polymorphisms of the prion protein (PrP) greatly influence the susceptibility of sheep to scrapie. Selective breeding to increase the prevalence of PrP gene alleles associated with scrapie resistance is a flock management practice that is important for scrapie control programs. Determination of sheep PrP alleles typically has required extraction of DNA from host tissues that are freshly derived or stored frozen. We describe application of a DNA extraction procedure for formalin-fixed, paraffin-embedded tissues (PET) for the purpose of PCR amplification and nucleotide sequencing of relevant codons (136-171) of the sheep PrP gene. Tissues derived from 96 sheep were studied. The DNA sequence identity was confirmed in 87 of 94 matched samples of PET and frozen tissue specimens. DNA from brainstem PET of 2 sheep, from which fresh tissue was not available, was amplified and sequenced after formalin fixation for 7-70 days. This method will allow retrospective analysis of PrP genetics of sheep subsequent to postmortem diagnosis of scrapie when nonfixed tissue is unavailable for DNA extraction; however, it is not recommended that submission of fixed tissue supplant collection of fresh tissues for the purpose of determining PrP gene polymorphisms.     

Linkage of the gene for the scrapie-associated fibril protein (PrP) to the Sip gene in Cheviot sheep

The gene Sip with two alleles, sA and pA, is the major gene determining the incubation period of scrapie in its natural host, sheep. Two lines of Cheviot sheep have been bred which differ in their response to experimental infection with SSBP/1 scrapie. The negative line have a decreased incidence of disease caused by SSBP/1 and are SippApA. The positive line have an increased incidence of disease and the majority are either SipsAsA or SipsApA; it is not possible to distinguish between the two genotypes on the basis of scrapie incubation time because the sA allele is fully dominant with SSBP/1 scrapie. There are also rare SippApA segregants in the positive line. The major protein (PrP) of scrapie-associated fibrils is encoded by a cellular gene and a cDNA copy of the hamster PrP mRNA has been used to analyse the restriction fragment length polymorphism of the two lines of Cheviot sheep. Two polymorphisms of the sheep PrP gene were found, by using HindIII and EcoRI, which appear to act as markers for the alleles of Sip. Using these polymorphisms it is now possible to assign a Sip genotype to the sheep in the Cheviot flock. Preliminary results from Anglo-Nubian goats and a cow are also reported.     

Pathogenesis of natural goat scrapie: modulation by host PRNP genotype and effect of co-existent conditions

After detection of a high prevalence of scrapie in a large dairy goat herd, 72 infected animals were examined by immunohistochemistry with prion protein (PrP) antibody Bar224 to study the pathogenesis of the infection. Tissues examined included the brain and thoracic spinal cord (TSC), a wide selection of lymphoreticular system (LRS) tissues, the distal ileum and its enteric nervous system (ENS), and other organs, including the mammary gland. The whole open reading frame of the PRNP gene was sequenced and antibodies to caprine arthritis-encephalitis virus (CAEV) infection were determined. Unexpectedly, accumulation of disease-associated PrP (PrP^d) in the brain was more frequent in methionine carriers at codon 142 (24/32, 75.0%) than amongst isoleucine homozygotes (14/40, 35.0%). The latter, however, showed significantly greater amounts of brain PrP^d than the former (average scores of 9.3 and 3.0, respectively). A significant proportion of the 38 goats that were positive in brain were negative in the ENS (44.7%) or in the TSC (39.5%). These results, together with the early and consistent involvement of the circumventricular organs and the hypothalamus, point towards a significant contribution of the haematogenous route in the process of neuroinvasion. Chronic enteritis was observed in 98 of the 200 goats examined, with no association with either scrapie infection or presence of PrP^d in the gut. Lymphoproliferative interstitial mastitis was observed in 13/31 CAEV-positive and scrapie-infected goats; PrP^d in the mammary gland was detected in five of those 13 goats, suggesting a possible contribution of CAEV infection in scrapie transmission via milk.     

Identification of new allelic variants in the ovine prion protein (PrP) gene

The association between scrapie and polymorphisms of the prion protein (PrP) gene was studied in 1108 German sheep of 33 different breeds. The aim of the investigation was the determination of the codons 136, 154 and 171 of the PrP gene, which are important for scrapie susceptibility. In addition to the published allelic variants ARR, ARQ, AHQ, ARH and VRQ, two novel, rare haplotypes (AHR and VRR) were found in the breeds of Texel, Nolana and Suffolk. A comparison of PrP genotype frequencies among the analysed different breeds revealed distinct variations. Breeds such as Texel showed a complex genotype distribution over 17 variants, while breeds such as Friesian Milk Sheep indicated only seven different genotypes.     

Characterization of the prion protein gene (PRNP) region in Swiss sheep breeds

Polymorphisms in the PRNP gene have both an influence on the progress of scrapie and also of bovine spongiform encephalopathy (BSE) in sheep. In the light that sheep are potentially susceptible to BSE the goal of this project was to characterize 50 animals of each of the four major Swiss sheep breeds, namely Swiss Oxford Down, Swiss Black‐Brown Mountain, Valais Blacknose and Swiss White Alpine with respect to polymorphisms in the PRNP region. A 628‐bp fragment of exon 3 of PRNP comprising codons 112–211 was amplified by PCR and analysed by direct sequencing. Heterozygous genotypes were further verified by restriction fragment length polymorphism analyses. Polymorphisms could be observed at codons 112, 136, 137, 141, 143, 154 and 171 but not at codons 138, 151 and 211. One of the scrapie cases in Switzerland, a sheep of Swiss White Alpine, was also genotyped. Based on the polymorphisms observed at codons 136, 154 and 171 all four sheep breeds should be considered potentially susceptible to scrapie and BSE.     

Prion protein gene polymorphism in four West African sheep populations

A total of 162 individuals, belonging to three Burkinabé and one Niger sheep populations, were analysed for prion protein (PrP) gene polymorphism at codons 136, 154 and 171. The ARQ allele was the most frequent in both the Burkinabé (86.7%) and the Niger (67.5%) sheep populations. The highly sensitive allele VRQ was not found in the sampled individuals. The highly resistant ARR allele was in very low frequency in the Burkina-Sahel (4.4%) and Mossi (3.2%) populations and was not present in the Djallonké and Touareg populations. Only 4 out of 15 possible PrP genotypes were identified in the sampled individuals. No favourable ARR/ARR genotypes were found in either of the breeds. Sequencing a subgroup of the samples allowed the identification of other five polymorphisms on the PrP gene sequence at codons 116, 138, 151, 237 and 240. The very low frequency of the ARR allele in the West African sheep should dissuade the implementation of a preventive selection programme aimed to increase resistance to scrapie, to avoid an extreme erosion of the genetic stock.     

PrP polymorphisms in Basque sheep breeds determined by PCR-restriction fragment length polymorphism and real-time PCR

Two new PCR-based methods were developed to decode prion protein (PrP) gene polymorphisms at codons 136, 154 and 171: a PCR-restriction fragment length polymorphism (RFLP) analysis consisting of two PCR reactions followed by three enzymatic digestions, and a real-time PCR consisting of four reactions with seven fluorogenic probes. Both methods were used to study the distribution of PrP gene polymorphisms in a representative sample (1297 animals) of the populations of the two native breeds of sheep of the Spanish Basque Country, Latxa and Carranzana. Fourteen genotypes were found in the Latxa breed, in which ARQ/ARQ was the genotype most frequently observed (49.3 per cent), followed by ARR/ARQ (32.6 per cent) and ARQ/ARH (5.8 per cent). The genotype associated with the highest resistance to scrapie (ARR/ARR) was present in 5 per cent of the animals analysed. Similar results were observed in the Carranzana sheep.     

Caprine prion gene polymorphisms are associated with decreased incidence of classical scrapie in goat herds in the United Kingdom

ABSTRACT: The application of genetic breeding programmes to eradicate transmissible spongiform encephalopathies in goats is an important aim for reasons of animal welfare as well as human food safety and food security. Based on the positive impact of Prnp genetics on sheep scrapie in Europe in the past decade, we have established caprine Prnp gene variation in more than 1100 goats from the United Kingdom and studied the association of Prnp alleles with disease phenotypes in 150 scrapie-positive goats. This investigation confirms the association of the Met142 encoding Prnp allele with increased resistance to preclinical and clinical scrapie. It reveals a novel association of the Ser127 encoding allele with a reduced probability to develop clinical signs of scrapie in goats that are already positive for the accumulation of disease-specific prion protein in brain or periphery. A United Kingdom survey of Prnp genotypes in eight common breeds revealed eleven alleles in over thirty genotypes. The Met142 encoding allele had a high overall mean allele frequency of 22.6%, whereas the Ser127 encoding allele frequency was considerably lower with 6.4%. In contrast, a well known resistance associated allele encoding Lys222 was found to be rare (0.9%) in this survey. The analysis of Prnp genotypes in Mexican Criollas goats revealed nine alleles, including a novel Phe to Leu substitution in codon 201, confirming that high genetic variability of Prnp can be found in scrapie-free populations. Our study implies that it should be feasible to lower scrapie prevalence in goat herds in the United Kingdom by genetic selection.     

Detection of prion protein gene polymorphisms in Baltic breeds of sheep

A total of 167 sheep belonging to the Estonian whiteheaded mutton, Estonian blackheaded mutton, Lithuanian coarsewool native, Lithuanian blackface and Latvian darkheaded mutton breeds, and a population of sheep kept isolated on the Estonian island of Ruhnu, were sequence-analysed for polymorphisms in the prion protein (PrP) gene, to determine their genotype and the allele frequencies of polymorphisms in PrP known to confer resistance to scrapie. A 939 base pair fragment of exon 3 from the PrP gene was amplified by pcr and analysed by direct sequencing. For animals showing polymorphism at two nucleotide positions, both haplotypes of these double-heterozygous genotypes were further verified by pcr cloning and sequence analysis. Known polymorphisms were observed at codons 136, 154 and 171, and six different haplotypes (arr, ahq, arh, ahr, arq and vrq) were determined. On the basis of these polymorphisms, the six populations of sheep possessed the resistant arr haplotype at different frequencies. The high-risk arq haplotype occurred in high frequencies in all six populations, but vrq, the haplotype carrying the highest risk, occurred at low frequencies and in only three of the populations.     

Natural scrapie in British sheep: breeds, ages and PrP gene polymorphisms.

One hundred and sixty-seven sheep of 32 breeds and crossbreeds affected by natural scrapie throughout Britain were tested for the presence of restriction fragment length polymorphisms of the PrP gene observed when their DNA was digested with EcoRI or HindIII. These polymorphisms have already been associated with different susceptibilities to experimental scrapie (controlled by alleles of the Sip gene) in a flock of Cheviot sheep. In two studies 86 to 92 per cent of the sheep were found to carry the PrP gene EcoRI fragment e1 which is associated with high susceptibility (or the sA allele of Sip) to experimental scrapie. The PrP gene HindIII genotypes of the natural scrapie sheep were not apparently associated with differences in susceptibility to scrapie. There was no link between the polymorphisms and the age or breed of the affected sheep.