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.     

Atypical scrapie in a Swiss goat and implications for transmissible spongiform encephalopathy surveillance.

Different types of transmissible spongiform encephalopathies (TSEs) affect sheep and goats. In addition to the classical form of scrapie, both species are susceptible to experimental infections with the bovine spongiform encephalopathy (BSE) agent, and in recent years atypical scrapie cases have been reported in sheep from different European countries. Atypical scrapie in sheep is characterized by distinct histopathologic lesions and molecular characteristics of the abnormal scrapie prion protein (PrP(sc)). Characteristics of atypical scrapie have not yet been described in detail in goats. A goat presenting features of atypical scrapie was identified in Switzerland. Although there was no difference between the molecular characteristics of PrP(sc) in this animal and those of atypical scrapie in sheep, differences in the distribution of histopathologic lesions and PrP(sc) deposition were observed. In particular the cerebellar cortex, a major site of PrP(sc) deposition in atypical scrapie in sheep, was found to be virtually unaffected in this goat. In contrast, severe lesions and PrP(sc) deposition were detected in more rostral brain structures, such as thalamus and midbrain. Two TSE screening tests and PrP(sc) immunohistochemistry were either negative or barely positive when applied to cerebellum and obex tissues, the target samples for TSE surveillance in sheep and goats. These findings suggest that such cases may have been missed in the past and could be overlooked in the future if sampling and testing procedures are not adapted. The epidemiological and veterinary public health implications of these atypical cases, however, are not yet known.     

Breeding programmes for TSE resistance in British sheep. I. Assessing the impact on prion protein (PrP) genotype frequencies

From April 2005, member states of the European Union were required to implement a compulsory breeding programme for resistance to transmissible spongiform encephalopathies (TSEs) in sheep as part of measures to eradicate TSEs from national flocks. In this paper, we assessed the impact of four different breeding strategies on prion protein (PrP) genotype frequencies using a mathematical model which describes in detail gene flow in the British sheep flock. These strategies ranged from the minimum requirements laid down in by EU legislation to compulsory implementation of the current National Scrapie Plan for Great Britain (NSP) ram genotyping scheme. All four strategies were predicted to substantially reduce the frequency of the VRQ allele, which is associated with the highest risk of scrapie, although schemes with more stringent requirements produced a larger reduction. However, there were marked differences in the impact of the strategies on the frequency of other PrP alleles. In particular, restrictions beyond those required by EU legislation were necessary to change the frequency of other PrP alleles substantially. Consequently, a breeding programme which aims to reduce the risk to human health by reducing the frequency of the ARQ allele (associated with the highest risk of BSE in sheep) must place restrictions on ARQ-bearing animals. Similarly, a programme which seeks to increase the frequency of the ARR allele (associated with the lowest risk of TSE) must favour ARR-bearing animals.     

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.     

Lack of PrP(sc) immunostaining in intracranial ectopic lymphoid follicles in a sheep with concomitant non-suppurative encephalitis and Nor98-like atypical scrapie: a case report

During active surveillance for transmissible spongiform encephalopathies (TSEs) in sheep, an initial reactor was detected using a rapid test on a brain sample. Immunohistochemistry confirmed an atypical TSE presentation that closely resembled the previously described Nor98 cases. Sequencing of the prnp gene confirmed the ARQ/AHQ genotype with the L141F mutation at codon 141 associated with this phenotype. The head, including the brain and cranial lymphoid tissues, was sampled and examined thoroughly. Non-purulent encephalitis, with ectopic lymphoid follicle formation within the brain, was diagnosed concomitant to the TSE. When scrapie-associated prion protein (PrP(sc)) deposition was studied by immunohistochemistry there was a noticeable lack of lymphotropism. The distribution of PrP(sc) in the brain differed considerably from that of classical scrapie cases. Astrogliosis and microgliosis were demonstrated by histochemical procedures.     

Chronic wasting disease

Chronic wasting disease (CWD) is a unique transmissible spongiform encephalopathy (TSE) of mule deer (Odocoileus hemionus), white-tailed deer (O. virginianus), and Rocky Mountain elk (Cervus elaphus nelsoni). The natural history of CWD is incompletely understood, but it differs from scrapie and bovine spongiform encephalopathy (BSE) by virtue of its occurrence in nondomestic and free-ranging species. CWD has many features in common with scrapie, including early widespread distribution of disease-associated prion protein (PrP(d)) in lymphoid tissues, with later involvement of central nervous system (CNS) and peripheral tissues. This distribution likely contributes to apparent efficiency of horizontal transmission and, in this, is similar to scrapie and differs from BSE. Clinical features and lesions of CWD are qualitatively similar to the other animal TSEs. Microscopically, marked spongiform lesions occur in the central nervous system (CNS) after a prolonged incubation period and variable course of clinical disease. During incubation, PrP(d) can be identified in tissues by antibody-based detection systems. Although CWD can be transmitted by intracerebral inoculation to cattle, sheep, and goats, ongoing studies have not demonstrated that domestic livestock are susceptible via oral exposure, the presumed natural route of exposure to TSEs. Surveillance efforts for CWD in captive and free-ranging cervids will continue in concert with similar activities for scrapie and BSE. Eradication of CWD in farmed cervids is the goal of state, federal, and industry programs, but eradication of CWD from free-ranging populations of cervids is unlikely with currently available management techniques.     

Evidence for maternal transmission of scrapie in naturally affected flocks

It has been known for many years that the offspring of scrapie affected ewes are at increased risk of developing scrapie but whether this is simply the result of an increased genetic susceptibility or transmission of infection has always been unclear. To contribute to clarify this we analysed the data collected in a detailed study of scrapie occurrence in a number of naturally affected commercial sheep flocks in Great Britain (GB) to investigate the association between PrP genotype and parental scrapie status and the incidence of scrapie. Our analyses confirmed the strong association between PrP genotype and the incidence of scrapie found in previous studies and a low incidence of scrapie in animals carrying the ARR allele and a high risk in homozygous VRQ animals. However, we also demonstrate an increased incidence of scrapie in the offspring of scrapie affected ewes controlling for the confounding effect of PrP genotype, but no increased scrapie incidence in the offspring of scrapie affected sires. Our results suggest that some of the increased incidence of scrapie in the offspring of scrapie affected ewes is the result of transmission of infection from mother to offspring. Our results confirm that a breeding policy aimed at decreasing the genetic susceptibility of the population should decrease the incidence of scrapie and that removing the offspring of scrapie affected animals from affected flocks could contribute to the control of this disease.     

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.     

Prion protein self-interaction in prion disease therapy approaches

Transmissible spongiform encephalopathies (TSEs) or prion diseases are unique disorders that are not caused by infectious micro-organisms (bacteria or fungi), viruses or parasites, but rather seem to be the result of an infectious protein. TSEs are comprised of fatal neurodegenerative disorders affecting both human and animals. Prion diseases cause sponge-like degeneration of neuronal tissue and include (among others) Creutzfeldt-Jacob disease in humans, bovine spongiform encephalopathy (BSE) in cattle and scrapie in sheep. TSEs are characterized by the formation and accumulation of transmissible (infectious) disease-associated protease-resistant prion protein (PrP(Sc)), mainly in tissues of the central nervous system. The exact molecular processes behind the conversion of PrP(C) into PrP(Sc) are not clearly understood. Correlations between prion protein polymorphisms and disease have been found, however in what way these polymorphisms influence the conversion processes remains an enigma; is stabilization or destabilization of the prion protein the basis for a higher conversion propensity? Apart from the disease-associated polymorphisms of the prion protein, the molecular processes underlying conversion are not understood. There are some notions as to which regions of the prion protein are involved in refolding of PrP(C) into PrP(Sc) and where the most drastic structural changes take place. Direct interactions between PrP(C) molecules and/or PrP(Sc) are likely at the basis of conversion, however which specific amino acid domains are involved and to what extent these domains contribute to conversion resistance/sensitivity of the prion protein or the species barrier is still unknown.     

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.     

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.     

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.     

The evidence of associations between prion protein genotype and production, reproduction, and health traits in sheep

The EU Commission issued a regulation in 2003, which requires all member states to implement a breeding programme for resistance to transmissible spongiform encephalopathies in sheep by selecting for specific alleles of the prion protein (PrP) gene. A key concern with regard to this regulation was that the intensive selection programmes, designed to increase resistance to scrapie, may have a negative impact on a range of other economically important production, reproduction, and disease traits in sheep. Such problems could arise for a number of reasons. Firstly, a number of breeds have a low frequency of the resistant PrP allele. Secondly, there may be a negative association between the resistant allele and animal performance. Thirdly, selection for scrapie resistance may reduce the rate of improvement towards current breeding goals. The evidence concerning the relationship between PrP genotype and reproduction, production, and disease traits is the subject of this review. We conclude that there is no evidence for a negative association between PrP genotype and reproduction traits (e.g. litter size), lamb performance traits (e.g. growth rate, conformation, carcass composition) or milk production. There is, however, a distinct paucity of information on the relationship between the PrP gene and disease traits. In this context it is noted that there are a number of genes located on chromosome 13, in close proximity to the PrP gene, that are involved in intracellular cell signalling, apoptosis, phagocytosis, and immune function. Thus further direct studies of key disease traits associated with sheep production systems are warranted.     

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 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.     

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.     

Biology of PrPsc accumulation in two natural scrapie-infected sheep flocks.

Sheep scrapie is a prion disease that requires interaction of exogenous prions with host prion protein (PrP) supporting prion formation. Disease is associated with deposition of a host-generated conformational variant of PrP, PrPsc, in a variety of tissues, including brain, resulting in fatal spongiform encephalopathy. Efficiency of PrPsc formation is determined by polymorphisms in the PrP-coding sequence. This article adds to previous data of natural sheep scrapie, concentrating on the effect of host genotype and age on PrPsc accumulation patterns during preclinical and clinical disease. Two entire scrapie-infected, predominantly Suffolk-cross, sheep flocks euthanized for regulatory purposes were genotyped and analyzed for PrPsc deposition in various tissues using single- and dual-label immunohistochemistry. Scrapie, as defined by PrPsc deposition, occurred in 13/80 sheep. Preclinical disease was evident in nearly 70% of infected sheep, ranging in age from 14 months to 7 years. PrPsc accumulated systemically in the nervous tissue, various lymphoid tissues, both alimentary tract related and non-alimentary tract related, and the placenta. Clinical neurological illness was always associated with spongiform encephalopathy and PrPsc deposition in the brain. Only 6 of 9 sheep with preclinical scrapie had PrPsc deposition in the brain but widespread PrPsc deposition in peripheral lymphoid tissue, supporting previous data showing peripheral PrPsc accumulation preceding deposition in the brain. PrPsc colocalized with a marker for follicular dendritic cells throughout the lymphoid system. PrPsc also accumulated in the peripheral nervous system, particularly the nervous supply of the gastrointestinal tract. Abundant PrPsc was evident in trophoblast cells of placentomes but not in the endometrium, myometrium, or associated nervous plexus. PrPsc deposits were not observed in the mammary parenchyma or bone marrow. Scrapie susceptibility was defined genetically by PrP codon 171: PrPsc deposition was restricted to PrP genotype AA136RR154QQ171 in 12/13 cases or AV136RR154QQ171 in 1/13 cases. The earliest accumulation was observed in the single VRQ/ARQ heterozygous animal, consistent with the reported high scrapie susceptibility and brief incubation period observed in breeds with predominance of the V136R154Q171 allele. Disease occurred within, as well as independent of, mother-daughter lines, suggesting both maternal and nonmaternal transmission in the flocks.     

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.     

Derivation of a scrapie-free sheep flock from the progeny of a flock affected by scrapie

The Cheviot flock at the Institute for Animal Health's Neuropathogenesis Unit (npu) has endemic scrapie, which affects primarily vrq/vrq sheep and at high frequency. A new flock with a full range of PrP genotypes, including the highly susceptible vrq/vrq, has been produced on a separate site, from animals in the npu breeding flock, and it remains scrapie-free after eight years. In contrast, in a parallel flock at the npu farm, scrapie has reappeared after five years, although the animals were kept in separate accommodation from the scrapie-affected sheep. During this time the npu breeding flock continued to have scrapie cases. Although it is known that highly susceptible sheep can remain free of infection in a clean environment, this is the first report of the infection being removed successfully from the bloodlines of scrapie-affected sheep. The results confirm that scrapie is not a genetic disease dependent only on the PrP gene sequence, but requires both genetic susceptibility and an infectious agent.