PrP genotype in Sarda breed sheep and its relevance to scrapie

Summary.  Several PrP gene polymorphisms modulate sheep scrapie susceptibility. Recently, an increase of scrapie outbreaks has been reported in Italy. A vaccine containing sheep brain homogenate was used in most of the outbreaks. We investigated PrP gene polymorphisms in scrapie-affected and clinically healthy Sarda breed sheep from a flock exposed to the aforementioned vaccine, and in affected Sarda sheep from unexposed flocks. All affected animals were (Gln/Gln)₁₇₁ homozygous. Moreover, we observed no variation for Ala₁₃₆ and a new polymorphism (Lys to Asn) at codon 176. Our findings confirm the correlation between scrapie and (Gln/Gln)₁₇₁ in breeds with no variation for Ala₁₃₆. Accepted June 18, 2001 Recevied March 19, 2001     

PrP genotype frequencies of the most dominant sheep breed in a country free from scrapie

Summary.  Polymorphisms within the prion protein (PrP) gene are associated with scrapie susceptibility. We analysed the PrP genes of 140 Romney Marsh sheep, the dominant breed in New Zealand, a country free from scrapie. We found PrP alleles that are associated with a high susceptibility to scrapie. Sheep with these PrP genotypes would probably succumb to scrapie when born and raised in a scrapie endemic environment. These findings correspond to those obtained in minor breeds from New Zealand. We conclude that scrapie development not only depends on host genetic factors but also requires exogenous factors. Our findings demonstrate the effectiveness of the measures taken by New Zealand to maintain free from scrapie. Received July 20, 1998 Accepted October 1, 1998     

Strain-associated variations in abnormal PrP trafficking of sheep scrapie

Prion diseases are associated with the accumulation of an abnormal form of the host-coded prion protein (PrP). It is postulated that different tertiary or quaternary structures of infectious PrP provide the information necessary to code for strain properties. We show here that different light microscopic types of abnormal PrP (PrP^d) accumulation found in each of 10 sheep scrapie cases correspond ultrastructurally with abnormal endocytosis, increased endo-lysosomes, microfolding of plasma membranes, extracellular PrP^d release and intercellular PrP^d transfer of neurons and/or glia. The same accumulation patterns of PrP^d and associated subcellular lesions were present in each of two scrapie strains present, but they were present in different proportions. The observations suggest that different trafficking pathways of PrP^d are influenced by strain and cell type and that a single prion strain causes several PrP^d–protein interactions at the cell membrane. These results imply that strains may contain or result in production of multiple isoforms of PrP^d.     

Effects of agent strain and host genotype on PrP accumulation in the brain of sheep naturally and experimentally affected with scrapie

Different cellular and neuroanatomical types of disease-specific prion protein (PrP(d)) accumulation in the brain were identified in sheep of different breeds and PrP genotypes exposed to experimental or natural scrapie infection. Immunohistochemical examination of the brains of 43 sheep with clinical signs compatible with scrapie revealed 12 different PrP(d)types, which were subjectively quantified in eight different brain regions. The PrP(d)types were grouped into four PrP(d)patterns, the relative magnitude of which provided the PrP(d)profile of each sheep examined. The analysis of the differences in magnitude and relative proportion of each of these PrP(d)types and patterns indicated (1) an effect of the scrapie strain on the PrP(d)profile, and (2) a possible effect of the host genotype on the magnitude of PrP(d)accumulation in the brain, apparently related to the incubation period. Furthermore, intraneuronal deposition of PrP(d)was the type most closely associated with the development of clinical disease. We conclude that different scrapie strains can be distinguished by PrP immunohistochemical examination of brains of affected animals.     

PrP gene polymorphism in sheep breeds in Slovakia and susceptibility to scrapie

We analyzed the prion protein (PrP) genotype based on the codons 136, 154 and 171 and assigned to five risk groups (R1-R5) in healthy and scrapie-affected sheep in Slovakia. In healthy (asymptomatic) population, 119 Merino, 106 Improved Valachian, 117 Tsigai, and 48 Suffolk breeds were tested. Among the asymptomatic sheep, the low-risk genotypes R1 and R2 were most abundant in Suffolk (94%) and Merino (84%) breeds, followed by Tsigai (58%) and Improved Valachian (40%) breeds. The medium-risk group R3 was most frequent in Improved Valachian (31%) breed, followed by Tsigai (21%), Merino (10%), and Suffolk (6%) breeds. The occurrence of high-risk groups R4 and R5 was none in Suffolk breed, followed by Merino (6%), Tsigai (21%), and Improved Valachian (30%) breeds. Since 2003, altogether 48 cases of scrapie have been confirmed in Tsigai (38), Merino (4), Improved Valachian (2), Improved Valachian x Tsigai (3), and Suffolk (1) breeds. Among sheep with scrapie, Merino breed belonged to the medium-risk group R3. The majority of scrapie-affected Tsigai sheep were classified into high-risk R5 (50%) and medium-risk R3 (42%) groups. We showed an association of scrapie with medium- and high-risk groups of PrP genotype in Slovakia. In particular, the glutamine at position 171 appears to be of major importance for the susceptibility to scrapie.     

Natural scrapie in a closed flock of Cheviot sheep occurs only in specific PrP genotypes

Summary Natural scrapie in a closed flock of South Country Cheviot sheep has resulted in 45 deaths between 1986 and 1995. Of these cases, 35 sheep have been analysed for disease-linked PrP gene polymorphisms and all encode valine at codon 136 on at least one allele with 77% homozygous (VV₁₃₆) and 23% valine/alanine heterozygotes (VA₁₃₆). Mean survival time was 907 and 1482 days for VV₁₃₆ and VA₁₃₆ scrapie affected animals respectively. VV₁₃₆ animals were all at great risk of disease if allowed to live long enough. However scrapie occurred only in a specific subgroup of VA₁₃₆ sheep, survival advantage depending on VA₁₃₆ animals being heterozygous for other polymorphisms at codons 154 or 171. The flock history has been recorded in great detail since its foundation in 1960 however there was no strong evidence for simple maternal or paternal transmission of disease other than inheritance of PrP genotype.     

Diverse biological parameters in clinically healthy sheep from a flock with scrapie: variations, and correlations with OLA antigens.

A comparison was made in the blood levels of various cell types and biochemical substances and in lymphocyte antigens between 107 healthy sheep from a flock contaminated with scrapie (HC sheep) and 93 sheep from a noncontaminated flock (NC sheep), which served as a control population. Significant differences between the two groups of sheep were found in some of the levels, as had previously been found with lymphocyte antigens. The HC sheep, which included genetically resistant animals, could be distinguished from the NC sheep by their lower levels of various white cells, a noticeable decrease in urea, a moderate decrease in Mg2+ and Mn2+ ions, beta- and gamma-globulins, serotonin and vitamin B12, a strong increase in uric acid and a moderate increase in K+, Cl-, HCO3-, Zn2+, and Al3+ ions, as well as in total lipids and in the albumin to globulin ratio. In this HC population, the only enzyme with an increased level was aldolase; the levels of the other 7 enzymes measured were lowered. The observed modifications were considered to be signs of latent disturbances in the leukocyte system and in hepatic and renal functions, in spite of apparent resistance. Eleven lymphocyte antigens were studied. These antigens are not independent of the blood levels of the various substances measured, but are often correlated in a statistically significant manner with some of them. In the HC sheep, the lymphocyte antigens correlated with the modified levels in the blood were different from those in the control population.(ABSTRACT TRUNCATED AT 250 WORDS)     

Search for healthy carriers of scrapie: an assessment of subclinical infection of sheep in an Icelandic scrapie flock by three diagnostic methods and correlation with PrP genotypes

Summary.  Subclinical infection in scrapie of sheep, characterized by a long incubation period, may be of importance for the spread of the disease. We screened brain samples from all 65 sheep in a scrapie-affected flock for subclinical infection and correlated with results of PrP genotyping, which is of relevance for the epidemiology and the question, whether by breeding for resistant genotypes one would be breeding for healthy carriers. The sensitivity of three methods was compared, i.e. histopathological examination for vacuoles (HP), immunohistochemical staining (IHC) and Western blotting (WB) for PrP^Sc. Five sheep showed definite clinical signs and histological scrapie lesions, and signs of infection were detected in 25 of 60 asymptomatic sheep, by HP and/or IHC and WB. The IHC was slightly more sensitive than HP and WB. Sheep with subclinical infection were, with one exception, either homo- or heterozygotes for 136-V, as were four of the five sheep with clinical scrapie. The incidence of the VRQ allelic variant in the flock was unusually high compared to the Icelandic sheep population probably contributing to the high prevalence of both clinical and subclinical infection in the flock. Neither sheep with definite scrapie nor detectable subclinical infection, were of the resistant AHQ genotype, indicating that Icelandic AHQ sheep are not healthy carriers of scrapie infection. Received September 7, 2001 Accepted December 13, 2001     

Detection of PrP(Sc) in rectal biopsy and necropsy samples from sheep with experimental scrapie

Scrapie diagnosis is based on the demonstration of disease-associated prion protein (PrP(Sc)) in brain or, in the live animal, in readily accessible peripheral lymphoid tissue. Lymphatic tissues present at the rectoanal line were readily obtained from sheep without the need for anaesthesia. The presence of PrP(Sc) in such tissue was investigated in sheep infected orally with scrapie-infected brain material. The methods used consisted of immunohistochemistry and histoblotting on biopsy and post-mortem material. PrP(Sc) was detected in animals with PrP genotypes associated with high susceptibility to scrapie from 10 months after infection, i.e., from about the time of appearance of early clinical signs. In the rectal mucosa, PrP(Sc) was found in lymphoid follicles and in cells scattered in the lamina propria, often near and sometimes in the crypt epithelium. By Western blotting, PrP(Sc) was detected in rectal biopsy samples of sheep with the PrP genotype VRQ/VRQ, after electrophoresis of material equivalent to 8 mg of tissue. This study indicated that rectal biopsy samples should prove useful for the diagnosis of scrapie in sheep.     

Vacuolar lesion profile in sheep scrapie: factors influencing its variation and relationship to disease-specific PrP accumulation

Detailed neuropathological examination for vacuolar lesions was performed on the brains of 42 sheep with clinical signs compatible with scrapie. The sheep were grouped according to their breed (Poll-Dorset, Cheviot, Welsh Mountain, Shetland and Suffolk), their PrP genotype at codons 136, 154 and 171 (VRQ/VRQ, VRQ/ARQ, VRQ/ARR and ARQ/ARQ) and the type of infection (experimental infection with SSBP/1, or natural disease). Twenty-two neuroanatomical sites from seven brain regions were examined for vacuolation in the neuropil and five sites at the level of the obex were examined for intraneuronal vacuolation. In 36 sheep, immunohistochemical examination for disease-specific PrP (PrP(d)) accumulation had also been performed in the same brain regions in an earlier study. The magnitude of total neuropil vacuolation was highest in the naturally affected ARQ/ARQ Suffolk sheep and lowest in the experimentally infected VRQ/VRQ Cheviot sheep and VRQ/ARR Poll-Dorset sheep. The severity of neuropil vacuolation at nine of the 22 neuroanatomical sites examined was used to generate a vacuolar lesion profile, which showed variations between the different sheep groups. These variations could be attributed to both PrP genotype and sheep breed and also possibly to scrapie agent; there was, however, considerable individual variation in lesion profile within sheep groups. All groups showed a similar ratio of neuropil vacuolation to neuronal vacuolation at the level of the obex. Although a positive correlation between neuropil vacuolation and PrP(d) deposition was generally observed, it was low except for the astrocyte-associated pattern of PrP(d) accumulation. The study suggests that vacuolar lesion profiles in sheep are affected by several factors and, by comparison with lesion profiles in mice, are of no more than limited value for discriminating between scrapie strains.     

Occurrence and distribution of infection-specific PrP in tissues of clinical scrapie cases and cull sheep from scrapie-affected farms in Shetland

The prion protein (PrP) genotypes of all cull sheep originating from four scrapie-affected farms in Shetland in 1998-1999 were determined and a representative sample of the different genotypes was selected for necropsy. Samples of brain and selected viscera were removed from 159 such sheep aged 2-11 years. These samples were examined immunohistochemically and by Western blotting for infection-specific forms of PrP. None of the sheep bearing the following genotypes showed any evidence of PrP accumulation in brain, intestine, selected lymph nodes or the cranial mesenteric ganglia: ARQ/ARQ (n = 41), ARQ/ARH (n = 12), ARH/ARH (n = 2), ARQ/ARR (n = 24), ARR/ARR (n= 2). In five of 71 sheep bearing a single VRQ allele, PrP accumulation was detected immunohistochemically in viscera or brain, or both. These results suggested that only a small proportion of susceptible sheep showed evidence of infection (accumulation of PrP) on the farms studied, and that even sheep of the most susceptible genotype (VRQ/VRQ) did not invariably develop disease in an infected environment. Furthermore, there was no evidence that, in sheep of semi-resistant or fully resistant genotypes, infection could be sequestered within the lymphoreticular system or peripheral nervous system and thereby provide a possible "carrier" source of infection. Rather, the data suggested that some sheep, possibly because they had been exposed to a relatively low infective dose, became infected and accumulated the infective agent over a protracted pre-clinical phase of the disease. Such sheep might be potentially infective for many years. In two VRQ/ARR genotype sheep, PrP was confined to the brain. Infection-specific PrP was also confined to the brain in two of 24 clinical cases of VRQ/ARQ scrapie. Thus, direct neuroinvasion, apparently without a prior phase of replication in the lymphoreticular system, occurred in a proportion of VRQ/ARQ sheep. Possibly it may occur in all sheep of the VRQ/ARR genotype. The factors responsible for direct neuroinvasion are not understood. However, it cannot be attributed to genotype alone.     

Comparative performance of three TSE rapid tests for surveillance in healthy sheep affected by scrapie

Rapid tests specific for sheep and goats became part of European Union-wide active scrapie surveillance in 2006. Performance of three approved TSE rapid tests for the detection of sheep infected with scrapie in field cases in the pre-clinical stage of the disease was compared.The medulla oblongata of 969 asymptomatic sheep of various genotype and breed aged over 18 months from 23 Italian flocks affected with scrapie, were tested by the Bio-Rad TeSeE Sheep/Goat (A), the IDEXX HerdChek BSE-Scrapie Antigen Test Kit, EIA (B) and the Prionics^®-Check Western Small Ruminant (C) rapid tests.Of 136 positive samples of classical scrapie, as confirmed by Western blot assay, 132 were positive with test A (Se 97.06%, CI 95% 92.64-99.19); 135 with test B (Se 99.26%, 95% CI 95.97-99.98) and 128 with test C (Se 94.12%, 95% CI 88.74-97.43).Tests A and B showed the best performance on analytical sensitivity. All three systems demonstrated good reproducibility: being the intrarater and interrater kappa coefficients always over 0.83.The one available atypical scrapie sample was positive with tests A and B, negative with test C.Considering the discrepant results in the detection of low PrP^sc concentrations and of the atypical case, differences can be expected in the efficacy of an active surveillance system, depending on the test adopted.     

The association of a codon 136 PrP gene variant with the occurrence of natural scrapie

Summary Incidence of both experimental and natural scrapie in sheep has been associated with polymorphisms of the PrP gene. In case/ control studies the PrP allele which encodes valine at codon 136 (Val₁₃₆) is found in 96–100% of naturally infected scrapie sheep of Shetland, Scottish Halfbred and Bleu du Maine breeds. In contrast, in healthy animals from the same flocks, the most frequent allele encodes Ala₁₃₆ (91–100% of sheep). However Val₁₃₆ does not correlate with incidence of scrapie in two other flocks — Poll Dorsets and Suffolks — and there may therefore be breed differences in PrP genotypes affected by scrapie.     

Close vicinity of PrP expressing cells (FDC) with noradrenergic fibers in healthy sheep spleen

In naturally and experimentally occurring scrapie in sheep, prions invade the immune system and replicate in lymphoid organs. Here we analysed immunohistochemically, in seven spleens of 6-month-old healthy sheep, the nature of the cells expressing prion protein (PrP) potentially supporting prion replication, as well as their relationship with autonomic innervation. PrP was identified using either RB1 rabbit antiserum or 4F2 monoclonal antibody directed against AA 108-123 portion of the bovine and AA 79-92 of human prion protein respectively. Using double labelling analysis, we demonstrated that PrPc is expressed by follicular dendritic cells using a specific monoclonal antibody (CNA42). We also showed the close vicinity of these PrP expressing cells with noradrenergic fibers, using a polyclonal tyrosine hydroxylase antibody. Our results may help the study of the cellular requirements for the possible neuroinvasion from the spleen.     

Biological and biochemical characterization of sheep scrapie in Japan

Due to the apparent absence of an agent-specific nucleic acid genome, scrapie strains cannot be classified by genome characterization, which is commonly used for the classification of many viruses. However, scrapie strains can be distinguished to some extent by biological properties such as transmissibility to experimental animals and distribution of neuropathological lesions and by biochemical properties such as the molecular mass and relative protease-resistance of the disease-specific isoform of prion protein (PrP(Sc)). In order to preliminarily characterize the scrapie strains that are prevalent in Japan, we analyzed the transmissibility of sheep scrapie isolates to mice and the relative proteinase K (PK) resistance of the corresponding PrP(Sc). The results indicate that Japanese scrapie strains can be divided into at least three groups based on biological and biochemical properties. The first group includes isolates which cause disease in mice with an incubation period of approximately 400 days and possess PrP(Sc) with relatively high PK resistance. Isolates of the second group contain PrP(Sc) that is highly resistant to PK digestion but transmit poorly to mice. The final group consists of isolates that cause disease in mice with an incubation period of less than 300 days and are associated with PrP(Sc) with reduced PK resistance. Sheep scrapie has occurred sporadically in Japan since1982, with only approximately 60 officially reported cases so far. However, the diversity of scrapie strains in the field suggested by our data raises the concern that a scrapie strain similar to the parental agent of bovine spongiform encephalopathy could exist or emerge in Japan. Thus, continuous surveillance for scrapie will be required to prevent the further spread of scrapie, not only among the sheep population but also to other species, and to eliminate any potential risk of sheep scrapie to public health.     

Use of a new immunoassay to measure PrP Sc levels in scrapie-infected sheep brains reveals PrP genotype-specific differences

The diagnosis of prion diseases, such as scrapie and BSE, has traditionally relied upon the identification of the disease-associated form of the prion protein, PrP(Sc), based on its resistance to digestion by proteinase K (PK). A more recent development is the conformation-dependent immunoassay (CDI), which distinguishes between PrP Sc and normal PrP (PrP C) based on their differing solubility in guanidine hydrochloride rather than resistance or sensitivity to PK. We have developed a CDI-formatted sandwich immunoassay for the measurement of PrP Sc in sheep brain, which discriminates between clinically affected scrapie cases (natural or experimental) and uninfected controls of the same PrP genotype. Using this method, we have shown for the first time that, in sheep, the PrP genotype has a significant influence on the amount of PrP Sc deposited in the brains of animals experimentally infected with scrapie.     

Precise targeting of the pathology of the sialoglycoprotein, PrP, and vacuolar degeneration in mouse scrapie

Widespread immunostaining of PrP protein was demonstrated in scrapie mouse brain, distributed diffusely in the neuropil and focally in amyloid plaques, microglia and 2-5 microns structures resembling neuronal processes. With the 87V scrapie strain, which produces focal vacuolation in particular areas, PrP pathology was precisely targeted to these same areas, predating vacuolar degeneration by at least several weeks. On the other hand, both vacuolar and PrP changes were widely distributed throughout the brain with the ME7 scrapie strain. It is likely that the precise targeting of PrP pathology, followed by vacuolar degeneration, reflects an underlying targeting and localised replication of infectious agent.