Animal transmissible spongiform encephalopathies and genetics

The genotype of the host plays a crucial role in the pathogenesis of transmissible spongiform encephalopathies (TSEs). In this respect, the most important factor is represented by the gene of the prion protein (PrP). The present work summarizes the currently available knowledge on the genetic basis of TSEs focusing, in particular, on sheep scrapie. Interest in this disease has grown markedly following the discovery of bovine spongiform encephalopathy, both for scientific and health reasons. In Italy, specific research grants from the Ministry of Health and the National Research Council (CNR), together with cooperation between the Istituto Superiore di Sanità and the Istituti Zooprofilattici Sperimentali, have allowed us to study the PrP genotype and to investigate the genetic susceptibility to scrapie in the most important Italian sheep breeds, with special reference to Sarda, Comisana and Massese. The PrP genotype in relation to scrapie susceptibility was also studied in goats of Ionica breed.     

Animal Transmissible Spongiform Encephalopathies and Genetics

The genotype of the host plays a crucial role in the pathogenesis of transmissible spongiform encephalopathies (TSEs). In this respect, the most important factor is represented by the gene of the prion protein (PrP). The present work summarizes the currently available knowledge on the genetic basis of TSEs focusing, in particular, on sheep scrapie. Interest in this disease has grown markedly following the discovery of bovine spongiform encephalopathy, both for scientific and health reasons. In Italy, specific research grants from the Ministry of Health and the National Research Council (CNR), together with cooperation between the Istituto Superiore di Sanità and the Istituti Zooprofilattici Sperimentali, have allowed us to study the PrP genotype and to investigate the genetic susceptibility to scrapie in the most important Italian sheep breeds, with special reference to Sarda, Comisana and Massese. The PrP genotype in relation to scrapie susceptibility was also studied in goats of Ionica breed.     

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.     

Frequencies of PrP genotypes in meat breeds of Japanese sheep and trail of selective breeding in experimental sheep flock

The selection of sheep with scrapie-resistant PrP genotypes is one of the control measures for transmissible spongiform encephalopathies in ruminants. In this study, we investigated the frequencies of PrP genotypes in meat breeds in Japan. The nationwide surveillance revealed that nearly half of the Suffolk sheep, a major meat breed in Japan, carried scrapie-susceptible AQ/AQ and AQ/VQ genotypes. In addition, the VQ haplotype, which confers high susceptibility to scrapie within sheep, was also found in Poll Dorset sheep. A trial of selective breeding using sires with scrapie-resistant PrP genotypes AQ/AR and AR/AR could raise the ratio of scrapie-resistant sheep from less than 50% to 80% within 3 years. However, the use of sires with the AR/AR genotype and the selection of ewes would be required to achieve a higher ratio of scrapie-resistant sheep.     

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.     

Epidemiological and genetical differences between classical and atypical scrapie cases

The aim of this study was to analyze the epidemiology and prion protein (PrP) genetics in scrapie-affected sheep flocks in Germany. For this purpose, 224 German scrapie cases in sheep diagnosed between January 2002 and February 2006 were classified as classical or atypical scrapie and the amino acids at codons 136, 141, 154 and 171 were determined. Likewise, representative numbers of flock mates were genotyped. Significant epidemiological differences were observed between classical and atypical scrapie cases in regard to the numbers of scrapie-affected sheep within a flock, the sizes of flocks with only a single scrapie-positive sheep or more than one scrapie-positive sheep and the age distribution of the scrapie-positive sheep. Sheep with the ARQ/ARQ genotype had by far the highest risk for acquiring classical scrapie, but the risk for atypical scrapie was the highest for sheep carrying phenylalanine (F) at position 141 (AF(141)RQ) and/or the AHQ haplotype. However, atypical scrapie also occurred with a notable frequency in sheep with the PrP haplotypes ARR and/or ARQ in combination with Leucine at position 141 (AL(141)RQ). Furthermore, six atypical scrapie-positive sheep carried the PrP genotype ARR/ARR. The high proportion of sheep flocks affected by atypical scrapie underscores the importance of this scrapie type.     

PrP genotype frequencies of Quebec sheep breeds determined by real-time PCR and molecular beacons

The allele and genotype frequencies of the prion protein gene (PrP), known to have an impact on scrapie susceptibility, were determined by real-time PCR for 500 Quebec purebred rams. Molecular beacons were very efficient in discriminating the 5 alleles investigated. Polymorphisms at coding positions 136, 154, and 171 of the PrP gene were analyzed using 3 separate real-time PCR reactions and a total of 7 molecular beacons. A total of 4 different alleles (ARQ, ARR, AHR, and VRQ) were observed at different frequencies among the 7 breeds of sheep investigated. Results show that more than 50% of the rams in every breed carried at least one ARR allele, which is considered the most resistant to scrapie. The susceptibility ARQ allele was also present in every breed and together with the ARR allele, they were the most frequent alleles found in Quebec rams. The VRQ allele associated with the highest susceptibility to scrapie occurred in 5 of the 7 breeds, although at low frequencies. Overall, the results indicate that the frequencies of PrP alleles and genotypes in common breeds of sheep in Quebec make it feasible to reduce scrapie risk by selective breeding.     

Clinical and pathological features of experimental scrapie in Irish Blackface Mountain sheep

There have been no reports of natural scrapie in Irish Blackface Mountain (BM) sheep which account for approximately 16% of the Irish national sheep flock. The aim of this study was to determine if Irish BM sheep had unusual clinical and/or pathological features of scrapie which would account for failure to diagnose the disease in this breed. BM (n=7), Texel (n=3) and Suffolk sheep (n=1) of scrapie-susceptible PrP genotypes (ARQ/ARQ and VRQ/ARQ) were orally challenged with scrapie-infected brain inoculum. The incubation period, clinical signs, pathology and distribution of disease specific prion protein (PrP(d)) in scrapie-affected BM sheep were similar to scrapie in the Texel and Suffolk sheep. It was concluded that there was no evidence to suggest that scrapie in BM sheep differs clinicopathologically from scrapie in other breeds of sheep.     

Postmortem diagnosis of preclinical and clinical scrapie in sheep by the detection of disease-associated PrP in their rectal mucosa

Samples of tissue from the central nervous system (cns), the lymphoreticular system (lrs) and the rectal mucosa of a large number of scrapie-exposed sheep, with and without signs of clinical disease, were examined immunohistochemically for evidence of disease-associated prion protein (PrP(d)). The rectal mucosa has received almost no attention so far in scrapie diagnosis, despite its abundant rectoanal mucosa-associated lymphoid tissue, and its accessibility. The scrapie-confirmed cases included 244 with clinical disease, of which 237 (97.1 per cent) were positive in the rectal mucosa, and 121 apparently healthy sheep, of which 104 (86 per cent) were positive in the rectal mucosa. PrP(d) was detected in 86.4 to 91.5 per cent of the other lrs tissues of the healthy sheep examined and in 77.7 per cent of their cns tissues. The stage of infection, therefore, affected the probability of a positive result in the rectal mucosa, whereas the breed, PrP genotype, age and sex had little or no independent effect. Accumulations of PrP(d) were observed in the rectal mucosa and other lrs tissues of vrq/arr sheep with preclinical and clinical scrapie, albeit with a lower frequency and magnitude than in sheep of other PrP genotypes. Western immunoblotting analyses of samples of rectal mucosa gave the characteristic PrP glycoprofile, with a sensitivity similar to that of immunohistochemistry.     

The use of pestivirus antigen ELISA currently available for the detection of hairy shaker disease/border disease virus in sheep

AIMS: To estimate the number of cases of scrapie that would occur in sheep of different prion protein (PrP) genotypes if scrapie was to become established in New Zealand, and to compare the performance of two commercially available, rapid ELISA kits using ovine retro-pharyngeal lymph nodes (RLN) from non-infected and infected sheep of different PrP genotypes.

METHODS: Using published data on the distribution of PrP genotypes within the New Zealand sheep flock and the prevalence of cases of scrapie in these genotypes in the United Kingdom, the annual expected number of cases of scrapie per genotype was estimated, should scrapie become established in New Zealand, assuming a total population of 28 million sheep. A non-infected panel of RLN was collected from 737 sheep from New Zealand that had been culled, found in extremis or died. Brain stem samples were also collected from 131 of these sheep. A second panel of infected samples comprised 218 and 117 RLN from confirmed scrapie cases that had originated in Europe and the United States of America, respectively. All samples were screened using two commercial, rapid, transmissible spongiform encephalopathy ELISA kits: Bio-Rad TeSeE ELISA (ELISA-BR), and IDEXX HerdChek BSE-Scrapie AG Test (ELISA-ID).

RESULTS: If scrapie became established in New Zealand, an estimated 596 cases would occur per year; of these 234 (39%) and 271 (46%) would be in sheep carrying ARQ/ARQ and ARQ/VRQ PrP genotypes, respectively. For the non-infected samples from New Zealand the diagnostic specificity of both ELISA kits was 100%. When considering all infected samples, the diagnostic sensitivity was 70.4 (95% CI=65.3–75.3)% for ELISA-BR and 91.6 (95% CI=88.2–94.4)% for ELISA-ID. For the ARQ/ARQ genotype (n=195), sensitivity was 66.2% for ELISA-BR and 90.8% for ELISA-ID, and for the ARQ/VRQ genotype (n=107), sensitivity was 81.3% for ELISA-BR and 98.1% for ELISA-ID.

CONCLUSIONS: In this study, the ELISA-ID kit demonstrated a higher diagnostic sensitivity for detecting scrapie in samples of RLN from sheep carrying scrapie-susceptible PrP genotypes than the ELISA-BR kit at comparable diagnostic specificity.

CLINICAL RELEVANCE: The diagnostic performance of the ELISA-ID kit using ovine RLN merits the consideration of including this assay in the national scrapie surveillance programme in New Zealand.     

Naturally Occurring Scrapie in Southdown Sheep

The purpose of this study was to characterize naturally occurring scrapie in the Southdown breed of sheep. Experimental subjects included 4 Southdown ewes admitted to the University of Missouri, College of Veterinary Medicine Large Animal Clinic. All 4 sheep had signs compatible with clinical scrapie. Cerebrospinal fluid (CSF) cell counts ranged from a low of 1 nucleated cell/microL to high of 4 cells/microL with a median of 3 cells/microL. Cerebrospinal protein concentrations ranged from 26 to 78 mg/dL with a median of 53 mg/dL. Immunoassay of the CSF for the 14-3-3 protein yielded positive results in 3 of the 4 sheep. Sequencing of the prion protein (PrP) gene revealed that all 4 sheep were homozygous for glutamine at codon 171 and, hence, were of the QQ genotype. Histopathologic examination of brain stem tissue sections revealed intracytoplasmic neuronal vacuolation and mild spongiform changes in the gray matter neuropil in all 4 ewes. The diagnosis of scrapie was confirmed by immunohistochemical staining for the abnormal PrP Our results suggest that the genetics of scrapie susceptibility are probably similar in Suffolk and Southdown sheep. Positive immunoassay results for the 14-3-3 protein were observed in 3 of the 4 sheep.     

Clinical signs, histopathology and genetics of experimental transmission of BSE and natural scrapie to sheep and goats

This paper compares the dinical signs, histopathology, detection of PrPSc protein and PrP genetics of the transmission of BSE to sheep and goats, with the effects of the transmission of natural scrapie from a brain homogenate from a single sheep. After intracerebral and oral inoculations there were similarities in the clinical signs due to the two sources of infection, but there were differences in pathology at the end stage of disease and in the genotypes of the sheep which succumbed to the challenges. The incubation period of BSE was associated with the sheep PrP codon 171 genotype, but the natural scrapie source, despite inducing disease only in known susceptible genotypes, showed no clear association with PrP genotype.     

Susceptibility of transgenic mice expressing chimeric sheep,bovine and human PrP genes to sheep scrapie

The use of Transgenic (Tg) mice expressing chimeric sheep/mouse (Sh/Mo) prion protein (PrP) and chimeric bovine/mouse (Bo/Mo) PrP genes was evaluated as a sheep scrapie model. We also investigated the potential for the transmission of sheep scrapie to a human/mouse (Hu/Mo) PrP Tg mouse line. The Sh/Mo PrP and Bo/Mo PrP Tg Prnp(+/+) or Prnp(0/0) mouse lines were inoculated intracerebrally with brain homogenates from three sheep with natural scrapie (KU, Y5 or S2). Incubation periods were slightly shorter in Sh/Mo PrP Tg Prnp(+/+), than in non-Tg mice inoculated with KU brain homogenate. In contrast, the incubation period was significantly prolonged (p<0.05) in Bo/Mo PrP Tg Prnp(+/+) mice inoculated with KU brain homogenate. The incubation period was significantly longer in all Tg Prnp(+/+) and Prnp(0/0), than in non-Tg mice (p<0.01) inoculated withY5 brain homogenate. None of the Tg Prnp(0/0) mice inoculated with S2 brain homogenate developed clinical signs and PrP(Sc) was undetectable in their brains. These results suggested that expression of the Sh/Mo PrP or Bo/Mo PrP transgenes does not confer susceptibility to sheep prions upon mice, and thus none of the Tg mouse lines could be a suitable model of sheep scrapie. Hu/Mo PrP Tg Prnp(0/0) mice inoculated with natural and experimental scrapie or mouse prions did not develop clinical signs of scrapie and PrP(Sc) was undetectable. These results suggested that neither sheep nor mouse strains of scrapie are highly transmissible to humans.     

Active surveillance for scrapie in the Netherlands: effect of a breeding programme on the prevalence of scrapie in sheep (2002-2010)?

The susceptibility of sheep to scrapie is modulated by the prion protein (PrP) genotype of the animal. An ambitious voluntary scrapie control programme was started in the Netherlands in 1998, based on selection of rams with theARR/ARR genotype for breeding. This programme was followed by an obligatory programme in 2004; the programme has been voluntary since 2007. We monitored the prevalence of PrP genotype frequencies and the prevalence of scrapie in the Dutch sheep population between 2002 and June 2010. Results showed that selection for scrapie-resistant sheep resulted in an increase in the ARR allele frequency in the Dutch national flock from 37.5% in 2005 to 61.4% in 2009. Moreover, surveillance data showed that there was a significant decrease in the prevalence of scrapie a few years after the start of the obligatory breeding programme, from more than 0.2% in 2004 to 0.015% in 2009. This decrease is a consequence of the increased number of scrapie-resistant sheep in the Dutch sheep population. To date, the results and the models based on the data show that the selective breeding programme should be continued for several years in order to successfully eradicate scrapie. It will be important to monitor the PrP frequency and scrapie prevalence in the Dutch sheep population in the coming years.     

Association between incubation time and genotype in sheep experimentally inoculated with scrapie-positive brain homogenate

OBJECTIVE: To compare incubation time and clinical signs of scrapie in codon 136/171 alanine-valine/glutamine-glutamine (AVQQ) experimentally inoculated sheep with that in sheep with the more common 136/171 AAQQ genotype. ANIMALS: 60 Suffolk sheep. PROCEDURE: Twenty-seven 171 QQ ewes purchased from 2 private flocks were bred with a 171 QQ Suffolk ram before being inoculated with a 20% solution of scrapie-positive brain homogenate (5 mL, PO) from sheep containing genotypes 136/154/171 AA/arginine-arginine (RR)/QQ, AVRRQQ, and VVRRQQ that had died of scrapie. Ewes had 33 lambs, which were inoculated in the same manner on the day of birth. RESULTS: All 16 genotype 136/154/171 AVRRQQ sheep that died of scrapie were 9 to 11 months of age; clinical signs lasted 1 day to 3 weeks with no wasting and only mild pruritus. The first AARRQQ sheep died with typical clinical signs of scrapie 27 months after inoculation, and 14 were still alive 37 to 42 months after inoculation. The 136/171 AVQQ sheep had minimal accumulation of modified cellular protein (PrP(SC)) as determined by immunohistochemical (IHC) staining within affected cells; thus the severity of clinical signs and time of death were not associated with brain lesions or the amount of PrP(SC) in brain TISSUE OF 136/154/171 AVRRQQ sheep as determined by IHC staining. CONCLUSIONS AND CLINICAL RELEVANCE: The rapid incubation time may have been influenced by the codon 136 genotype, a new unreported valine (V)-dependent strain of scrapie similar to strain SSBP/1, or the inoculum may have contained a traditional strain and a V-dependent or SSBP/1-like strain of 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.     

Assessing the efficacy of a ram-genotyping programme to reduce susceptibility to scrapie in Great Britain

Susceptibility to clinical scrapie is associated with polymorphisms in the PrP gene. The 'ARR' allele of this gene reduces susceptibility to clinical disease caused by all known strains of the transmissible spongiform encephalopathy (TSE) agent. The British government proposes to use a ram-genotyping scheme to breed genetic resistance to clinical scrapie into the national sheep population. We considered how best to target limited genotyping resources to achieve the maximum rate of genotype evolution. We created a metapopulation model of the British sheep industry, which includes the major pure-breeds of sheep and the cross-breeds produced by crossing these pure-bred animals. The main criterion for assessing the efficacy of different strategies was the time taken to increase the prevalence of the ARR allele in the slaughter-lamb population. Our model predicted that the most-effective strategy would be to target genotyping to those rams used for pure-breeding (i.e. mated with the same breed of ewe). This strategy was compared to two further strategies, in which the proportion of rams genotyped in each breed depended on the prevalence of the ARR/ARR genotype in that breed. A policy in which the proportion of animals genotyped is reduced as the ARR prevalence in that breed increases is efficient. The most-effective policy was targeting the hill sector in the early years and gradually switching to genotyping more terminal-sire and longwool rams as the resistance of the hill sector increases.     

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.     

PrP genotyping of Austrian sheep breeds

Scrapie, an ovine and caprine transmissible spongiforme encephalopathy, is widely spread among sheep populations in many European countries. As it is known that susceptibility to scrapie is determined genetically, breeding programmes aiming at providing scrapie-resistant flocks have been established. Selection is based on the prion protein (PrP) genotype, which is used to classify animals into risk groups of susceptibility (R1-R5) according to the amino acids encoded by codons at positions 136, 154 and 171, respectively. At position 136 (136V-->136A) alanine and at position 154 (154H-->154R) as well as 171 (171Q-->171R) arginine are the favoured amino acids. Whereas PrP genotyping data are available for many of the European sheep breeds, comparable data for local Austrian sheep breeds are missing. The most known among these are Tyrolean mountain sheep, forest sheep. Tyrolean stone sheep and Carynthian sheep. The genotypes of 112 sheep from these four local breeds were determined. In terms of PrP genetics, Austrian breeds belong to the group of non-valine-breeds, with the exception of the Carynthian sheep, that exhibited a frequency of 136V of 4.2%. The most frequent allele was ARQ with 64.6-71.2% (depending on the breed), followed by ARR (14.8-25.8%). In contrast to the above-mentioned findings, scrapie has never been diagnosed in any of the Austrian sheep breeds. Native Austrian sheep breeds exhibit a very robust constitution, a pronounced adaptation to harsh climates and good reproduction parameters as well as a marked mother instinct. Therefore, these breeds are often used in crossbreeding programmes. Beside the above-mentioned characteristics, our results indicate that the investigated breeds may be effectively used in crossing-out breeding programmes for eliminating valine at position 136 of PrP.