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.     

Breeding German sheep for resistance to scrapie

Susceptibility to clinical scrapie is associated with polymorphisms in the prion protein (PrP) gene. The ARR allele reduces susceptibility to clinical disease caused by all known strains of the transmissible spongiform encephalopathy (TSE) agents. For the economically important German breeds of sheep the PrP allele frequencies are well known, but this paper presents representative genotyping results for 1526 sheep from two smaller milk sheep breeds and 2446 sheep from 14 mostly indigenous land sheep breeds. The ARR allele was detected in each breed but the breed-specific ARR frequencies varied between 1 and 63 per cent. In small populations with a very low ARR frequency the ARR allele could be lost by genetic drift. A simulation study was therefore made to examine the effects of different breeding schemes in populations of different sizes on attempts to select for the ARR allele in an endangered population. In breeds in which no homozygous rams are available the breeding strategy would depend on the number of heterozygous rams, and the genotyping and selection of suitable breeding ewes would reduce the time required to achieve a highly resistant population. In general, in all the breeds a selection programme to achieve 99 per cent ARR homozygous genotypes would be feasible in six to nine generations, depending on the initial allele frequencies. In small populations the inbreeding rate may increase if no specific mating plans are developed by the breeding organisations.     

Breeding for scrapie resistance in the Hungarian sheep population

The first results of the Hungarian sheep prion protein (PrP) genotyping programme are discussed in this paper. To obtain initial genotype frequency data 10 commercial (Hungarian Merino, German Mutton Merino, Merino Landschaf, German Blackheaded, Suffolk, Texel, Ile de France, Charollais, Lacaune, British Milksheep) and 4 indigenous (Gyimes Racka, Hortobágy Racka, Tsigaja, Cikta) breeds were sampled in 2003 and 2004, and the PrP genotypes were determined by microsequencing analysis with capillary electrophoresis. In all commercial breeds, a higher number of sheep were genotyped in 2005 (3648) and in 2006 (3834) within the breeding programme to increase scrapie resistance, and the estimated frequency data were compared to the initial figures to evaluate the efficiency of selection. The new developments arising from the identification of the so-called 'atypical' scrapie cases are also discussed.     

Breeding programmes for smallholder sheep farming systems: II. Optimization of cooperative village breeding schemes

A simulation study was conducted to optimize a cooperative village‐based sheep breeding scheme for Menz sheep of Ethiopia. Genetic gains and profits were estimated under nine levels of farmers' participation and three scenarios of controlled breeding achieved in the breeding programme, as well as under three cooperative flock sizes, ewe to ram mating ratios and durations of ram use for breeding. Under fully controlled breeding, that is, when there is no gene flow between participating (P) and non‐participating (NP) flocks, profits ranged from Birr 36.9 at 90% of participation to Birr 21.3 at 10% of participation. However, genetic progress was not affected adversely. When there was gene flow from the NP to P flocks, profits declined from Birr 28.6 to Birr ?3.7 as participation declined from 90 to 10%. Under the two‐way gene flow model (i.e. when P and NP flocks are herded mixed in communal grazing areas), NP flocks benefited from the genetic gain achieved in the P flocks, but the benefits declined sharply when participation declined beyond 60%. Our results indicate that a cooperative breeding group can be established with as low as 600 breeding ewes mated at a ratio of 45 ewes to one ram, and the rams being used for breeding for a period of two years. This study showed that farmer cooperation is crucial to effect genetic improvement under smallholder low‐input sheep farming systems.     

A questionnaire survey of the prevalence of scrapie in sheep in Britain

An anonymous, self-administered questionnaire has been used in two independent surveys to try to determine the prevalence of scrapie in the national sheep flock. The disease was recorded in 35 counties in England and Wales. About a third (26.5 and 37.3 percent) of respondents owning 100 or more sheep indicated that they had seen sheep with scrapie in their flocks. The incidences of clinical cases recorded in affected flocks in the two surveys were 0.5 and 1.1 cases/100 ewes/year. At present there is no control over the disposal of these animals. If as has been suggested, an increase in the prevalence of scrapie was a contributory factor in the emergence of bovine spongiform encephalopathy, it would seem logical that measures should be introduced to monitor the prevalence and incidence of scrapie and to control the disposal of clinical cases.     

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.     

The impact of the genotype on the prevalence of classical scrapie at population level

ABSTRACT: Total number and genotypes of animals in holdings selected for the genotype & cull option in the Compulsory Scrapie Flock Scheme (CSFS) in Great Britain were extracted from the National Scrapie Plan data warehouse. The association between various genotype-related measures and scrapie prevalence infection was tested using zero-inflated negative binomial models with the counts of positive cases as dependent variable, and country, number of flocks in the scheme, flock size, surveillance source and the following genotype-related measurements: the centered-log ratios (clr) oof the 15 genotypes, of the proportions of the 5 alleles at codons 136, 154 and 171, of the proportions of the 5 NSP types, and two flock-susceptibility risk indicators, as explanatory variables. A total of 319341 genotyped animals from 168 holdings were included in the analysis. An increased proportion of the ARR/ARR genotype corresponded to a decrease in the number of scrapie cases. ARR/AHQ, AHQ/VRQ, ARH/VRQ and ARQ/VRQ genotypes, NSP type V, ARH, ARQ, AHQ and VRQ alleles and the low and high-susceptibility risk indicators are all associated with an increase risk in the number of scrapie cases.Regardless the management practices; the increased susceptibility that the non-ARR alleles confer on an individual could be extrapolated at the population level. Increasing prevalence of ARR allele reduces the overall risk of scrapie at population level. At genotype level, the VRQ/VRQ genotype, present a very low frequency in the study population, seems to play a residual effect in the overall risk of scrapie in a flock.     

Swiss scrapie surveillance. II. Epidemiologic aspects of the detection of neurological diseases in sheep and goats

Monitoring of transmissible spongiform encephalopathy (TSE) in Swiss sheep and goats is based on the examination of animals from different sources. In this study, frequencies and proportions of the different diagnoses were compared between routinely submitted sheep and goats, notified scrapie suspects as well as fallen stock. Meningitis/ encephalitis cases were significantly more frequent (OR = 2.2) in the scrapie suspect group when compared to the routine submissions. Metabolic-toxic encephalopathy was seen more frequently within the fallen stock. Rare neurological diagnoses were more frequent among scrapie suspects and routine submissions when compared to fallen stock. Listeriosis was diagnosed equally frequent among the scrapie suspects and routine submissions but less frequent in fallen stock. Scrapie prevalence among the fallen stock and the routine submissions was 0 (zero), with 95% certainty that prevalence is < 1%. The examined animals are representative for most of the Swiss regions with considerable sheep and goat production. Continuation of the detailed neuropathological examination of small ruminants from these three groups, substituted by actively testing a sufficiently large sample of fallen stock and possibly also healthy-slaughtered adult sheep and goats for transmissible spongiform encephalopathies would ensure a good surveillance within the small ruminant population.     

PrP genotype frequencies in German breeding sheep and the potential to breed for resistance to scrapie

Genetic susceptibility to scrapie is associated with polymorphisms in three different codons of the ovine prion protein (PrP) gene (136, 154, 171). Studies of PrP genotypes linked to scrapie have revealed the resistance of homozygous PrPARR/PrPARR animals and the high risk of PrPVRQ/PrPVRQ and PrPvRQ/PrPARQ animals in scrapie-affected flocks. The selection of PrPARR/PrPARR genotypes may therefore provide a strategy for controlling clinical scrapie. The genotypes of 1361 German breeding sheep from 15 different breeds in northern Germany were determined. Apart from the wildtype allele PrPARQ, at least four mutually exclusive allelic variants were found. The greatest variability within the PrP gene was encountered in texel sheep, in which 14 PrP genotypes were found. In the important meat breeds, Suffolk, German whiteheaded mutton and German blackheaded mutton, the PrPARR allele was predominant, and in these breeds the breeding of scrapie-resistant pedigree flocks within four generations seems to be a feasible option. In the texel sheep, the German merino, the German milk and the German land sheep breeds, the frequency of the PrPARR allele was much lower, and in several breeds no homozygous rams were available for breeding purposes. In these breeds the breeding strategy would depend on the number of heterozygous rams available, but resistant pedigree flocks could be achieved within nine generations.     

Rapid testing leads to the underestimation of the scrapie prevalence in an affected sheep and goat flock

To obtain a more detailed understanding of the prevalence of classical scrapie infections in a heavily affected German sheep flock (composed of 603 sheep and 6 goats), we analysed 169 sheep and 6 goats that carried the genotypes susceptible to the disease and that were therefore culled following discovery of the index case. The initial tests were performed using the Biorad TeSeE ELISA and reactive results were verified by official confirmatory methods (OIE-immunoblot and/or immunohistochemistry (IHC)) to demonstrate the deposition of scrapie-associated PrP(Sc) in the brain stem (obex). This approach led to the discovery of 40 additional subclinically scrapie-infected sheep. Furthermore, peripheral lymphatic and nervous tissue samples of the 129 sheep and 6 goats with a negative CNS result were examined by IHC in order to identify any preclinical infections which had not already spread to the central nervous system (CNS). Using this approach we found 13 additional sheep with PrP(Sc) depositions in the gut-associated lymph nodes (GALT) as well as in the enteric nervous system. Moreover, in most of these cases PrP(Sc) was also deposited in the spleen and in the retropharyngeal and superficial cervical lymph nodes. Taken together, these results show a 30.3% infection prevalence in this scrapie-affected flock. Almost 7.4% of the infected animals harboured PrP(Sc) exclusively in the peripheral lymphatic and nervous tissue and were therefore missed by the currently used testing strategy.     

Selection for scrapie resistance and simultaneous restriction of inbreeding in the rare sheep breed "Mergellander"

Scrapie is a fatal infectious neurodegenerative disease for which susceptibility is associated with polymorphisms in the ovine prion protein (PrP) gene. Scrapie-eradication programmes are based on eliminating the susceptible VRQ allele and/or breeding for the resistant ARR allele. In rare breeds or breeds with a low frequency of the ARR allele this can lead to unacceptably high inbreeding rates with associated increased risk of genetic defects and inbreeding depression. The conservation status of populations with inbreeding rates (DeltaF) above 1% is considered critical. In the Dutch rare sheep breed the Mergellander animals carrying ARR alleles are closely related to one another, and could reach 1.53% when only ARR/ARR animals are used as parents. Inbreeding rates can be reduced by selecting the set of parents according to their average co-ancestry. We minimised inbreeding rates by calculating the optimal contribution of each ram and selection of ewes. This resulted in inbreeding rates of -0.17% with exclusive use of homozygous ARR rams, and -0.38% if use of heterozygous rams was allowed as well. Thus sophisticated breeding programs can prevent unacceptably high inbreeding rates when breeding for scrapie resistance.     

Consequences of BSE/TSE for the clinical diagnostic in cattle and sheep

The first case of Bovine Spongiform Encephalopathy (BSE) in Germany induced a profound irritation not only of the consumers but also of the farmers and the veterinarians in Germany. The following BSE-crisis accelerated the structural changes in Beef and Dairy industries. The analysis of the detected BSE-cases of the last years in Germany and Switzerland shows that the sensitivity of BSE-tests is much higher in clinically preselected BSE-suspected cases compared to BSE-tests in normal slaughter cattle. Therefore it is necessary for an effective battle against BSE that clinical cases of CNS-diseases and disturbances of the locomotion especially in cows around partus should be clarified etiologically. If intra vitam the etiology of the disease can not be diagnosed by clinical and laboratory investigation of blood, urine and liquor samples and the therapy induces no improvement, the case has to be notified at the local veterinary officer. In the same way cases of Scrapie in sheep have to be handled. In sheep genotyping gives the opportunity to select for Scrapie resistance. In contrary to cattle with BSE Scrapie can be diagnosed intra vitam already before clinical symptoms occur by immunohistochemical investigations for Scrapie prion protein of tonsillar biopsies. These two diagnostic tools open new ways for fighting against Transmissible Spongiform Encephalopathies in sheep.     

Genetic diversity loss due to selection for scrapie resistance in the rare Spanish Xalda sheep breed

The effect of selection for scrapie resistance on genetic variability in the rare Xalda sheep breed was studied. Pedigree information comprised 1851 animals (1444 alive) at the moment of sampling. A total of 304 reproductive (or selected for reproduction) Xalda individuals were sampled and genotyped for 14 microsatellites. Genetic variability was assessed using: gene diversity (1 − average kinship, GD), mean average relatedness (AR) and self-coancestry (c_i) at the genealogical level; and expected heterozygosity (H_e), molecular mean kinship (Mk), molecular self-coancestry (s_i) and rarefacted average number of alleles per locus (A) at the molecular level. Two breeding strategies were evaluated: a) use of only young rams with genotype ARR/ARR and young ewes with low to moderate risk (risk groups R1 to R3); b) breeding without selection for PrP genotypes. The major cause of losses of genetic variability in the Xalda breed is the drift that occurs when a new group of reproductive individuals is selected. The loss of genetic variability is small in females compared to the males, where it is considerable. However, losses at the molecular level for young females with respect to adult females were above 5%. Young male individuals also retained most of the genetic variability assessed in adult rams. Selection against susceptibility to scrapie produced additional losses of more than 2% for H_e and A when rYF individuals are considered. As regards males, the situation becomes critical because of the scant number of available ARR/ARR young rams. The consequences for the management of the Xalda breed are discussed.     

Assessment of the genetic risk and impact of lateral transmission in a valine-associated scrapie outbreak in sheep

OBJECTIVE: To characterize an outbreak of valine-associated scrapie, assess the relative risk of scrapie infection in relation to allele frequency at codon 136, and investigate lateral transmission of infection in a sheep flock within the United States. ANIMALS: 1,006 sheep. PROCEDURE: To determine genotypes, blood or semen samples were assessed via commercial testing; in 190 slaughtered sheep, scrapie status was determined via immunohistochemical evaluation of tissues. Scrapie-positive sheep born to scrapie-negative dams and sheep infected after 1 year of age were identified to assess lateral transmission. RESULTS: Genotypes were determined for codon 171 (164 sheep) or codons 136 and 171 (842 sheep). Forty-four of 160 slaughtered sheep of known genotype were scrapie positive. In these sheep, the presence of at least 1 valine allele at codon 136 (V136) was highly correlated with scrapie-positive status. Lateral transmission was the probable source of infection for 4 scrapie-positive sheep born to scrapie-negative dams and for 11 sheep in which scrapie was diagnosed at > 50 months of age. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the outbreak of scrapie was associated with a relatively high frequency of V136 in the flock, introduction of a valine-dependent scrapie strain, and the occurrence of lateral transmission. Genotyping of sheep may assist management decisions following diagnosis of scrapie in a sheep with at least 1 V136. It may be prudent to remove sheep of the diploid genotype AVQR (at codons 136 and 171) from infected flocks.     

Breeding with resistant rams leads to rapid control of classical scrapie in affected sheep flocks

ABSTRACT: Susceptibility to scrapie, a transmissible spongiform encephalopathy in sheep, is modulated by the genetic make-up of the sheep. Scrapie control policies, based on selecting animals of resistant genotype for breeding, have recently been adopted by the Netherlands and other European countries. Here we assess the effectiveness of a breeding programme based on selecting rams of resistant genotype to obtain outbreak control in classical scrapie-affected sheep flocks under field conditions. In six commercially-run flocks following this breeding strategy, we used genotyping to monitor the genotype distribution, and tonsil biopsies and post-mortem analyses to monitor the occurrence of scrapie infection. The farmers were not informed about the monitoring results until the end of the study period of six years. We used a mathematical model of scrapie transmission to analyze the monitoring data and found that where the breeding scheme was consistently applied, outbreak control was obtained after at most four years. Our results also show that classical scrapie control can be obtained before the frequency of non-resistant animals is reduced to zero in the flock. This suggests that control at the national scale can be obtained without a loss of genetic polymorphisms from any of the sheep breeds.     

Studies on maternal transmission of scrapie in sheep by embryo transfer.

The technique of embryo transfer was used to investigate the maternal transmission of scrapie in sheep. Embryo donor ewes were experimentally infected with scrapie (all eventually developing the disease) and artificially inseminated six months later with semen from an uninfected scrapie-susceptible ram. Embryos were harvested five and six days after insemination and transferred by laparoscopy, unwashed, into recipient ewes which had been genetically selected for very low susceptibility to scrapie. Six of the 26 lambs born to these recipients developed scrapie.