PrP Genotype Research Articles

Assessment of the Genetic Susceptibility of Sheep to Scrapie by Protein Misfolding Cyclic Amplification and Comparison with Experimental Scrapie Transmission Studies

The susceptibility of sheep to scrapie is influenced mainly by the prion protein polymorphisms A136V, R154H, and Q171R/H. Here we analyzed the ability of protein misfolding cyclic amplification (PMCA) to model the genetic susceptibility of sheep to scrapie. For this purpose, we studied the efficiency of brain homogenates from sheep with different PrP genotypes to support PrP(Sc) amplification by PMCA using an ARQ/ARQ scrapie inoculum. The results were then compared with those obtained in vivo using the same sheep breed, genotypes, and scrapie inoculum. Genotypes associated with susceptibility (ARQ/ARQ, ARQ/AHQ, and AHQ/ARH) were able to sustain PrP(Sc) amplification in PMCA reactions, while genotypes associated with resistance to scrapie (ARQ/ARR and ARR/ARR) were unable to support the in vitro conversion. The incubation times of the experimental infection were then compared with the in vitro amplification factors. Linear regression analysis showed that the efficiency of in vitro PrP(Sc) amplification of the different genotypes was indeed inversely proportional to their incubation times. Finally, the rare ARQK₁₇₆/ARQK₁₇₆ genotype, for which no in vivo data are available, was studied by PMCA. No amplification was obtained, suggesting ARQK₁₇₆/ARQK₁₇₆ as an additional genotype associated with resistance, at least to the isolate tested. Our results indicate a direct correlation between the ability of different PrP genotypes to undergo PrP(C)-to-PrP(Sc) conversion by PMCA and their in vivo susceptibility and point to PMCA as an alternative to transmission studies and a potential tool to test the susceptibility of numerous sheep PrP genotypes to a variety of prion sources.

Characterization of the effect of heat on agent strains of the transmissible spongiform encephalopathies

The causal agents of the transmissible spongiform encephalopathy (TSE) diseases, sometimes called prion diseases, are characterized by high resistance to inactivation with heat. Results from thermal inactivation experiments on nine TSE strains, seven passaged in two PrP genotypes, showed differences in sensitivity to heat inactivation ranging over 17 °C. In addition, the rate of inactivation with increasing temperature varied between TSE models. In some cases passage in an alternative PrP genotype had little effect on the resulting inactivation properties, but for others the infectious agent was inactivated at lower temperatures. No strain with higher thermostability properties was selected. The effect of mixing two TSE strains, to see whether their properties were affected through interaction with each other, was also examined. The results showed that both strains behaved as expected from the behaviour of the unmixed controls, and that the strain responsible for inducing TSE disease could be identified. There was no evidence of a direct effect on intrinsic strain properties. Overall, the results illustrate the diversity in properties of TSE strains. They require intrinsic molecular properties of TSE agents to accommodate high resistance to inactivation and a mechanism, independent of the host, to directly encode these differences. These findings are more readily reconciled with models of TSE agents with two separate components, one of which is independent of the host and comprises a TSE-specific nucleic acid, than with models based solely on conformational changes to a host protein.

Impairment of Erythropoiesis In Inbred Cellular Prion Protein Deficient Mice.

AbstractAbstract 2032Cellular prion protein (PrPc) plays a key role in pathogenesis of prion diseases, however, its physiologic function remains unclear. The involvement of PrPc in hematopoiesis was suggested by importance of its expression for self renewal and survival of long term repopulating hematopoietic stem cells. Prion diseases were shown to deregulate transcription of several erythroid genes and we have demonstrated reduction of erythroid cell and erythropoietin production in FVB PrP-/- (Zurich I) mice in response to acute anemia (Zivny J. et al. Blood Cells Mol Dis. 2008;40:302-307). In this study, we exploited different mouse models with manipulated level of PrPc expression to verify the role of PrPc in erythropoiesis. First set of experiments was carried out on PrP-/- (Zurich I) and Tga20 PrP over-expressing mice on a mixed C57Bl6/129Sv genetic background. Inbred C57Bl6 mice served as a wild type control (WT). Induction of acute anemia by phenylhydrazine (PHZ) in PrP-/- and WT mice (n=18) led to drop in the hematocrit (HCT) from 52.5±1.5 and 49.8±2.5% to 37.9± 1.0 and 41.9±3.0% after 24 h, respectively. The course of anemia was significantly deeper in PrP-/- mice at 72 h, 96 h and 120 h (p < 0.01) after PHZ administration. Plasma levels of erythropoietin (Epo) in response to anemia reached higher maximum levels in PrP-/- than WT mice (2250 vs. 1810 pg/mL) although rose more slowly. The level of Epo mRNA in kidneys increased approximately 30-fold in both, WT and PrP-/- mice, however, in WT mice peaked at 24 h whereas in KO mice at 96 h. We repeated the study with smaller groups of PrP-/- and Tga20 mice (n=9) and analysed samples 24 h and 96 h post anemia induction. Random PrP gene re-introduction in Tga20 mice rescued the animals from severe anemia. Decrease in HCT after PHZ administration was significantly lower in Tga20 comparing to PrP-/- mice and was accompanied by less elevated reticulocyte (RTC) count, plasma Epo level and level of Epo mRNA in kidneys. Next we studied the dynamics of unchallenged erythropoiesis in PrP-/-, Tga20 and WT mice by in vivo labelling of blood cells with NHS-biotin and subsequent flow cytometric analysis of relative numbers of newly produced non-labelled RBC. WT mice had significantly enhanced turnover of RBC with higher counts of non-labelled RBC comparing to PrP-/- during 46 days of chase (p < 0.05). Half- life of labeled RBC in WT mice was 20 days, but 32 and 30 days in PrP-/- and Tga20 mice, respectively. Tga20 mice displayed tendency to increased RBC turnover over PrP-/- mice, but the difference was significant only 2 and 33 days after initiation of the experiment. Having in mind possible limitations of experiments conducted in genetically modified inbred mice we have designed second set of experiments in more stringent animal models. We mated C57Bl6/129Sv PrP-/- mice with inbred C57Bl6 and outbred CD-1 mice. Heterozygotes in F1 generation were mated and their PrP -/-, PrP -/+ and PrP +/+ offspring used in the experiments. Anemia was induced by PHZ and blood was sampled from tail vein at defined time points and HCT and RTC count were analysed. In C57Bl6 crossbreeds we observed significantly higher starting HCT level in PrP-/- mice (p < 0.05) compared to PrP-/+ and PrP+/+ mice reaching 53.2±2.3, 50.0±2.1 and 49±2.9%, respectively. Similar decrease in HCT was observed for all PrP groups 24 h after PHZ administration, however, significant differences between PrP-/- and PrP+/+ mice were recorded at 48 h and 72 h. The recovery to normal HCT was again retarded in PrP-/- mice. RTC counts rose more rapidly in PrP+/+ mice after PHZ administration and declined to basal levels faster than in PrP-/- mice, the difference reached significance at 24 h, 48 h and 96 h. Dynamics of unchallenged erythropoiesis in C57Bl6 crossbreeds was similar in all three PrP genotypes with no significant differences in numbers of newly produced RBC during 57 days of the experiment. In CD-1 crossbreed mice no significant differences in HCT and RTC counts were detected after PHZ induced anemia among PrP-/-, PrP-/+ and PrP+/+ siblings. Also the dynamics of unchallenged erythropoiesis was similar in all PrP genotypes. To sum up, our data confirmed the role of PrPc in stress erythropoiesis in studied inbred mouse models. In outbred model the effect of PrP deletion on erythropoiesis seems to be compensated. (GACR310/08/0878, GAUK86408) Disclosures:No relevant conflicts of interest to declare.

Molecular biology and pathology of prion strains in sporadic human prion diseases.

Prion diseases are believed to propagate by the mechanism involving self-perpetuating conformational conversion of the normal form of the prion protein, PrP(C), to the misfolded, pathogenic state, PrP(Sc). One of the most intriguing aspects of these disorders is the phenomenon of prion strains. It is believed that strain properties are fully encoded in distinct conformations of PrP(Sc). Strains are of practical relevance to human prion diseases as their diversity may explain the unusual heterogeneity of these disorders. The first insight into the molecular mechanisms underlying heterogeneity of human prion diseases was provided by the observation that two distinct disease phenotypes and their associated PrP(Sc) conformers co-distribute with distinct PrP genotypes as determined by the methionine/valine polymorphism at codon 129 of the PrP gene. Subsequent studies identified six possible combinations of the three genotypes (determined by the polymorphic codon 129) and two common PrP(Sc) conformers (named types 1 and 2) as the major determinants of the phenotype in sporadic human prion diseases. This scenario implies that each 129 genotype-PrP(Sc) type combination would be associated with a distinct disease phenotype and prion strain. However, notable exceptions have been found. For example, two genotype-PrP(Sc) type combinations are linked to the same phenotype, and conversely, the same combination was found to be associated with two distinct phenotypes. Furthermore, in some cases, PrP(Sc) conformers naturally associated with distinct phenotypes appear, upon transmission, to lose their phenotype-determining strain characteristics. Currently it seems safe to assume that typical sporadic prion diseases are associated with at least six distinct prion strains. However, the intrinsic characteristics that distinguish at least four of these strains remain to be identified.

Effect of PrP genotype on milk traits in Bovška sheep

Abstract. The aim of this study was to evaluate the effect of PrP genotype on milk traits in the Slovenian autochthonous Bovška sheep breed. The estimated population size is 3 700 animals, half of which are included in the selection programme. Genetic resistance for scrapie, a well known prion disease, is determined by the genotype at the PrP locus. In 2003, the Slovenian National Scrapie Programme following EU Regulations was introduced to control scrapie by selecting out the less resistant genotypes and until 2008, a total of 1 875 ewes were genotyped. This study determined frequencies of PrP alleles in Bovška sheep, AHQ 17.32 %, ARH 7.30 %, ARQ 57.14 %, ARR 17.12 % and VRQ 1.12 %, which were very similar to frequencies in other European breeds. The PrP genotype had a significant effect on milk, protein and lactose yield produced in lactation, but the productivity of the most resistant ARR/ARR homozygotes was similar to breed average and did not statistically differ from other genotypes. Heterozygotes AHQ/ARH, AHQ/ARQ and ARH/ARQ exhibited significantly higher productivity compared to respective homozygotes but not from the most resistant ARR/ARR genotype. It is expected therefore that increased frequency of the ARR allele or even its exclusivity will not directly influence the productivity of the breed. However, a careful management programme favouring resistant PrP genotypes should be implemented to reduce potential negative effects of productivity due to reduced selection potential or inbreeding.

Quantitative estimation of genetic risk for atypical scrapie in French sheep and potential consequences of the current breeding programme for resistance to scrapie on the risk of atypical scrapie

BackgroundSince 2002, active surveillance programmes have detected numerous atypical scrapie (AS) and classical scrapie cases (CS) in French sheep with almost all the PrP genotypes. The aim of this study was 1) to quantify the genetic risk of AS in French sheep and to compare it with the risk of CS, 2) to quantify the risk of AS associated with the increase of the ARR allele frequency as a result of the current genetic breeding programme against CS.MethodsWe obtained genotypes at codons 136, 141, 154 and 171 of the PRNP gene for representative samples of 248 AS and 245 CS cases. We used a random sample of 3,317 scrapie negative animals genotyped at codons 136, 154 and 171 and we made inferences on the position 141 by multiple imputations, using external data. To estimate the risk associated with PrP genotypes, we fitted multivariate logistic regression models and we estimated the prevalence of AS for the different genotypes. Then, we used the risk of AS estimated for the ALRR-ALRR genotype to analyse the risk of detecting an AS case in a flock homogenous for this genotype.ResultsGenotypes most at risk for AS were those including an AFRQ or ALHQ allele while genotypes including a VLRQ allele were less commonly associated with AS. Compared to ALRQ-ALRQ, the ALRR-ALRR genotype was significantly at risk for AS and was very significantly protective for CS. The prevalence of AS among ALRR-ALRR animals was 0.6‰ and was not different from the prevalence in the general population.ConclusionIn conclusion, further selection of ALRR-ALRR animals will not result in an overall increase of AS prevalence in the French sheep population although this genotype is clearly susceptible to AS. However the probability of detecting AS cases in flocks participating in genetic breeding programme against CS should be considered.

Inference of genotype probabilities and derived statistics for PrP locus in the Jezersko–Solcava sheep

The aim of this work was to infer PrP genotype probabilities and derived statistics in sheep to provide additional information for selection against susceptibility to classical scrapie. The method was demonstrated with data from Jezersko–Solcava sheep breed. Pedigree data consisted of 10429 animals of which 3669 animals had known PrP genotype and 2673 were alive and not genotyped. Five PrP alleles were present with the following frequencies: ARR 0.174, AHQ 0.074, ARH 0.083, ARQ 0.632, and VRQ 0.037. Iterative allelic peeling with incomplete penetrance model as implemented in the GenoProb program was used to infer genotype probabilities. There were only some additional identifications of PrP genotype and NSP (national scrapie plan) type with high probability. The main reasons for a low number of additional identifications can be attributed to the large number of alleles with moderate frequencies, incomplete penetrance model, uniform prior, and inherent pedigree and genotype data structure. In order to overcome the limits of additional genotype identifications novel statistics were derived (maximal NSP type, average NSP value and its variance and reliability) to facilitate practical implementation of selection for scrapie resistance based on NSP types. Maximal NSP type can be used to infer maximal potential scrapie susceptibility of individual animals as well as for the entire flocks. The average NSP value encompasses all information contained in PrP genotype probabilities and is the most useful statistic for the selection on NSP type and therefore PrP genotype. These novel statistics can be used as a criterion for the selection against scrapie susceptibility for the whole population taking into the account possible errors in genotype and/or pedigree data.

Prions and neuronal death

Dear Editor, It is widely accepted that a prominent neuron cell loss occurs in the brain in the course of prion diseases (PDs) or transmissible spongiform encephalopathies (TSEs),1 which are a group of slowly progressive and invariably fatal neurological disorders affecting man and animals.2 Despite this, the inner mechanisms underlying prion-induced neuronal death at the central nervous system (CNS) level are still largely unclear.3 A number of peripheral tissues of the host are colonized by the infectious agent during the preclinical phase of sheep scrapie, the TSE ‘prototype'. These include several lymphoreticular system districts, such as palatine tonsils, ileal Peyer's patches (PPs), and enteric nervous system (ENS) plexuses, from which ‘neuroinvasion' – the long journey of prions to the CNS – is believed to take place.1 In this respect, calbindin (CALB)-immunoreactive (IR), neuronal nitric oxide synthase (nNOS)-IR (Figure 1), and somatostatin-IR neurons have been recently identified as three distinct cell populations residing within ileal ENS plexuses that are selectively targeted during natural and oral experimental sheep scrapie infection.4, 5 However, differently from that reported at the CNS level,1 we did not observe significant differences in total (HuC/D-IR) as well as in CALB-IR and nNOS-IR neuron cell counts between the ileal ENS plexuses from scrapie-affected sheep and those from uninfected control sheep carrying an identical ‘susceptible' (ARQ/ARQ) PrP genotype.4 Furthermore, no evidence of gut dysfunction and/or functional loss was found in these scrapie-affected sheep, which is also in open contrast with that constantly observed in the brain of any PD-affected individual.1, 2, 3 Figure 1 Sheep. Tangential histological sections from an ileal myenteric plexus (MP) of a natural scrapie-affected Sarda breed sheep carrying the homozygous (scrapie-susceptible) ARQ/ARQ PrP genotype. Specific anti-nNOS (neuronal nitric oxide synthase) (a) and ... These striking results may argue in favour of an ‘interaction pattern' between the sheep scrapie agent (and, presumably, also between other TSE agents), on one side, and the host's neurons, on the other, which is different when dealing with either CNS or ENS neurons. Alternatively, a regenerative activity allowing damaged ENS neuronal cells to be numerically restored (at least in part) should not be ruled out. In conclusion, there is no doubt that this highly fascinating and still largely ‘mysterious' topic will continue to be a real challenge for neuropathologists and neuroscientists in the years to come.

Eradication of scrapie with selective breeding: are we nearly there?

BackgroundFollowing EU decision 2003/100/EC Member States have recently implemented sheep breeding programmes to reduce the prevalence of sheep with TSE susceptible prion genotypes. The present paper investigates the progress of the breeding programme in the Netherlands. The PrP genotype frequencies were monitored through time using two sets of random samples: one set covers the years 2005 to 2008 and is taken from national surveillance programme; the other is taken from 168 random sheep farms in 2007. The data reveal that although the level of compliance to the breeding programme has been high, the frequency of susceptible genotypes varies substantially between farms. The 168 sheep farms are a subset of 689 farms participating in a postal survey inquiring about management and breeding strategies. This survey aimed to identify how much these strategies varied between farms, in order to inform assessment of the expected future progress towards eradication of classical scrapie.ResultsOn the one hand, we found that compliance to the national breeding program has been high, and the frequency of resistant genotypes is expected to increase further in the next few years. On the other hand, we observed a large variation in prevalence of the scrapie resistant PrP genotype ARR between farms, implicating a large variation of genetic resistance between farms. Substantial between-flock differences in management and breeding strategies were found in the postal survey, suggesting considerable variation in risk of scrapie transmission between farms.ConclusionsOur results show that although there has been a good progress in the breeding for scrapie resistance and the average farm-level scrapie susceptibility in the Netherlands has been significantly reduced, still a considerable proportion of farms contain high frequencies of susceptible genotypes in their sheep population. Since 2007 the breeding for genetic resistance is voluntarily again, and participation to selective breeding can decrease as a result of this. This, together with the patterns of direct and indirect contact between sheep farms, might present a challenge of the aim of scrapie eradication. Communication to sheep owners of the effect of the breeding programme thus far, and of the prospects for classical scrapie eradication in The Netherlands might be essential for obtaining useful levels of participation to the voluntary continuation of the breeding programme.

Scrapie prevalence in sheep of susceptible genotype is declining in a population subject to breeding for resistance

BackgroundSusceptibility of sheep to scrapie infection is known to be modulated by the PrP genotype of the animal. In the Netherlands an ambitious scrapie control programme was started in 1998, based on genetic selection of animals for breeding. From 2002 onwards EU regulations required intensive active scrapie surveillance as well as certain control measures in affected flocks.Here we analyze the data on genotype frequencies and scrapie prevalence in the Dutch sheep population obtained from both surveillance and affected flocks, to identify temporal trends. We also estimate the genotype-specific relative risks to become a detected scrapie case.ResultsWe find that the breeding programme has produced a steady increase in the level of genetic scrapie resistance in the Dutch sheep population. We also find that a significant decline in the prevalence of scrapie in tested animals has occurred a number of years after the start of the breeding programme. Most importantly, the estimated scrapie prevalence level per head of susceptible genotype is also declining significantly, indicating that selective breeding causes a population effect.ConclusionsThe Dutch scrapie control programme has produced a steady rise in genetic resistance levels in recent years. A recent decline in the scrapie prevalence per tested sheep of susceptible prion protein genotype indicates that selective breeding causes the desired population effect.

Co-existence of classical scrapie and Nor98 in a sheep from an Italian outbreak

Nor98 is an atypical scrapie strain characterized by a molecular pattern and brain distribution of the pathological prion protein (PrP(Sc)) different from classical scrapie. In Italy, 69 atypical cases have been identified so far and all were characterized as Nor98 strain. In this paper we report an unusual case in a sheep which showed immunohistochemical and molecular features of PrP(Sc) different from the other atypical cases. The sheep was from an outbreak where the index and the other four cases were affected by classical scrapie. Histopathological, immunohistochemical and Western blot analyses on the brain of the unusual case revealed the simultaneous presence of pathological features characteristic of Nor98 and classical scrapie. Interestingly, the prevalent disease phenotype in the brainstem was classical scrapie-like, while in the cerebral cortex and cerebellum the Nor98 phenotype was dominant. The sub-mandibular lymph node was positive and showed a PrP(Sc) molecular pattern referable to classical scrapie. The PrP genotype was AL(141)RQ/AF(141)RQ. Taken together, the occurrence of classical scrapie in the outbreak, the PrP genotype, the involvement of different cellular targets in the brain and the pathological and molecular PrP(Sc) features observed suggest that this unusual case may result from the co-existence of Nor98 and classical scrapie.

Visual pathology in animal prion diseases.

Prion diseases, also known as the transmissible spongiform encephalopathies (TSEs), are a group of slowly developing neurodegenerations occurring in human and animals. Prion diseases can be transferred between animals, humans, from humans to animals, and from animals to humans. As a result, the central nervous system is attacked, resulting in microglia activation, astrocytosis, prion plaque deposition, and neuronal degeneration. Prion also targets on the eye and brain visual system. In scrapie-infected sheep, chronic wasting disease (CWD)-infected mule deer, and experimental animals infected with scrapie, transmissible mink encephalopathy (TME), and Creutzfeldt-Jakob disease (CJD), damage has been found in the outer and inner nuclear layers of the retina, brain stem, optic nerve, optic tract, optic radiation and visual cortex. This article reviews the prion agent and infectivity in the eye and brain visual system, and the visual and oculomotor pathology in animal prion diseases. Effects of PrP genotypes and PrPSc types on visual and oculomotor disorders will be discussed.

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.

A Retrospective Immunohistochemical Study Reveals Atypical Scrapie has Existed in the United Kingdom since at Least 1987

Atypical scrapie is a relatively recent discovery, and it was unknown whether it was a new phenomenon or whether it had existed undetected in the United Kingdom national flock. Before 1998, the routine statutory diagnosis of transmissible spongiform encephalopathy (TSE) in sheep relied on the presence of TSE vacuolation in the brainstem. This method would not have been effective for the detection of atypical scrapie. Currently, immunohistochemistry (IHC) and Western blot are commonly used for the differential diagnosis of classical and atypical scrapie. The IHC pattern of PrPd deposition in atypical scrapie is very different from that in classical scrapie using the same antibody. It is thus possible that because of a lack of suitable diagnostic techniques and awareness of this form of the disease, historic cases of atypical scrapie remain undiagnosed. Immunohistochemistry was performed on selected formalin-fixed, paraffin-embedded (FFPE) blocks of ovine brain from the Veterinary Laboratories Agency archives that were submitted for various reasons, including suspect neurological disorders, between 1980 and 1989. It was found that PrPd deposits in a single case were consistent with atypical scrapie. A method was developed to obtain a PrP genotype from FFPE tissues and was applied to material from this single case, which was shown to be AHQ/AHQ. This animal was a scrapie suspect from 1987, but diagnosis was not confirmed by the available techniques at that time.

Mapping of quantitative trait loci affecting classical scrapie incubation time in a population comprising several generations of scrapie-infected sheep

Although susceptibility to scrapie is largely controlled by the PrP gene, the role of other genes that affect scrapie resistance in sheep is now confirmed. Following the detection of quantitative trait loci (QTL) on chromosomes 6 and 18 in a half-sib family with an ARQ/VRQ susceptible PrP genotype, the whole pedigree of a naturally infected flock was investigated to confirm these QTL regions in different PrP genotypes. The present study has allowed us to confirm the QTL on chromosome 18, and to demonstrate the QTL effects in several PrP genotypes.

Associations of PrP genotype with lamb production traits in four commercial breeds of British upland and crossing sheep

Selection of sheep on PrP genotype to reduce risks of classical scrapie outbreaks is now widespread in the British sheep industry. However a recurring concern from breeders is that PrP genotype may be unfavourably associated with lamb performance. In this study we report the results from our investigations into this claim using performance data from 12,673 PrP genotyped lambs in four breeds of upland and crossing sheep: Beulah (4014), Blue Faced Leicester (725), Lleyn (5208) and North Country Cheviot (Park) (2726). We also included in the analyses performance data from around 19,000 ungenotyped lambs from the same flocks. The data were supplied by 36 breeders and comprised weights at birth, eight and twenty weeks, and ultrasonic measurements for muscle and fat depth, and deep pedigrees for all animals. Animal (direct) genetic effects and up to three maternal effects were fitted in linear mixed models for each trait. Potential associations with the PrP gene were assessed by fitting either PrP genotype or number of copies of individual alleles carried, as fixed effects. Significant associations between weight traits and PrP genotype were seen only in the North Country Cheviot (Park) breed, and were attributable to the number of copies of VRQ alleles carried. Although there was evidence of significant associations between the number of copies of individual alleles carried and weight traits at different ages in the other three breeds, these associations varied from breed to breed and were not consistent in either their direction, their magnitude or the allele concerned, and none survived Bonferroni corrections for multiple testing. The Blue Faced Leicester breed showed significant associations with PrP genotype and ultrasonic muscle depth, even after Bonferroni correction, due to the number of ARQ alleles carried. In the other breeds, associations between the number of allele copies carried and ultrasonic muscle depth were generally weak and inconsistent. No significant associations were seen for fat depth. In conclusion, few significant associations were seen. Selection on PrP genotype is unlikely to have any noticeable impact on the measured traits in these breeds.

Quantifying diversity losses due to selection for scrapie resistance in three endangered Spanish sheep breeds using microsatellite information

The effect of selection for scrapie resistance on genetic variability in three endangered Spanish sheep breeds (Colmenareña, Mallorquina and Rubia de El Molar) was studied using two different criteria for quantifying contributions to genetic variability: (a) molecular coancestry or genetic identity; and (b) average number of alleles per locus or allelic richness. A total of 236 (81 Colmenareña, 76 Mallorquina and 79 Rubia de El Molar) individuals were genotyped for the PrP gene and for 22 microsatellite markers. The analyses assumed a selective policy aimed at the elimination of the VRQ allele and the reduction of the frequency of the ARQ/ARQ genotype. These goals are approached by rejecting for breeding those individuals with the highest susceptibility for scrapie (risk groups R4 and R5) in a genetic scenario with no previous selection programmes considering the PrP gene polymorphism carried out. When all the individuals classified into risk groups R4 and R5 were removed from the dataset, the total molecular coancestry slightly increased in the Colmenareña breed illustrating that the carriers of undesirable PrP genotypes are not essential to maintain its overall gene diversity. When the allelic richness was considered, the removal of the R4 and R5 individuals gave high losses in the Rubia de El Molar breed. The analyses carried out considering the sex of the individuals informed that most increases in genetic identity in the Colmenareña breed resulted from the removal of the R4 and R5 males while in the Mallorquina breed resulted from the removal of the undesirable females. Losses of diversity in the Rubia de El Molar breed were basically independent of the sex of the individuals due to the balanced contributions to diversity of both sexes. As a general recommendation, not all the individuals of undesirable risk groups should be rejected for reproduction at the same time to avoid irretrievable loses of genetic diversity but according to the sex of the individuals.

Neuroinvasion in sheep transmissible spongiform encephalopathies: the role of the haematogenous route

It is generally believed that after oral exposure to transmissible spongiform encephalopathy (TSE) agents, neuroinvasion occurs via the enteric nervous system (ENS) and the autonomic nervous system. As a result, the dorsal motor nucleus of the vagus nerve is the initial point of disease-associated prion protein (PrP(d)) accumulation in the brain. HYPOTHESIS AND AIM: If direct ENS invasion following oral infection results in an early and specific brain targeting for PrP(d) accumulation, such topographical distribution could be different when other routes of infection were used, highlighting distinct routes for neuroinvasion. An immunohistochemical study has been conducted on the brain of 67 preclinically infected sheep exposed to natural scrapie or to experimental TSE infection by various routes. Initial PrP(d) accumulation consistently occurred in the dorsal motor nucleus of the vagus nerve followed by the hypothalamus, regardless of the breed of sheep, PrP genotype, TSE source and, notably, route of infection; these factors did not appear to affect the topographical progression of PrP(d) deposition in the brain either. Moreover, the early and consistent appearance of PrP(d) aggregates in the circumventricular organs, where the blood-brain barrier is absent, suggests that these organs can provide a portal for entry of prions when infectivity is present in blood. The haematogenous route, therefore, can represent a parallel or alternative pathway of neuroinvasion to ascending infection via the ENS/autonomic nervous system.

Ileal tract and Peyer’s patch innervation in scrapie-free versus scrapie-affected ovines

Ileal Peyer's patches (PPs) are involved early during sheep scrapie infection. This study qualitatively and semi-quantitatively evaluated ileal tract and PP innervation in 29 Sarda ovines of different age, PrP genotype and scrapie status. A prominent network of fibres was detected within PPs, mainly located in interfollicular lymphoid and stromal components. Intrafollicular fibres were rarely observed, with no apparent differences between scrapie-free and scrapie-affected animals, or among ovines carrying different PrP genotypes. In adult sheep, independent of their scrapie status, nerve fibres could be detected infrequently, close to the follicle-associated epithelium. Fibres were also detected within newly formed follicles and intrafollicular microgranulomas.

Associations of PrP genotype with lamb production traits in three commercial breeds of British lowland sheep

The Ram Genotyping Scheme was launched in Great Britain in 2001 as part of the National Scrapie Plan and was devised to reduce and eventually eradicate classical scrapie susceptible genotypes from the national pedigree flock. Anecdotal claims from breeders suggest that sheep with more resistant PrP genotypes may have inferior phenotypes. In this study, we test this possibility for lamb production traits in three breeds of lowland sheep: Charollais (22 752 lambs), Poll Dorset (22 589 lambs) and Texel (23 492 lambs). Data were received from 50 breeders and comprised weights at birth, 8 weeks and scanning (from which average daily weight gain was derived), and ultrasonic muscle and fat depths. Animal (direct) genetic effects and up to three maternal effects were fitted in linear mixed models for each trait. Fitting either PrP genotype or number of copies of individual alleles carried as fixed effects allowed potential associations with the PrP gene to be assessed. There were no significant associations seen in the Poll Dorset breed; however, significant associations were found with the number of allele copies carried in the other two breeds included in this study. Charollais lambs carrying one copy of the VRQ allele had significantly (P < 0.01) greater ultrasonic muscle depth (0.58 mm) and fat depth (0.2 mm) than non-carriers. In the Texel breed, lambs with one ARR allele were significantly heavier than those with two or zero ARR alleles; heterozygous ARR lambs were 0.07 kg heavier at birth (P < 0.05), 0.42 kg heavier at 8 weeks (P < 0.01) and 0.17 kg heavier at scan weight (P < 0.01), than non-carriers. After Bonferroni corrections to adjust significance thresholds to account for the large number of independent comparisons made, all significant results remained so at P < 0.05 or greater, except for the ARR allele effect on birth weight in the Texel breed, which was no longer significant. These results compare favourably with others from studies on many continental breeds of sheep, published in recent years, and add credence to the conclusion that selection on PrP genotype is unlikely to have any noticeable impact on the measured growth and carcass traits in sheep.