Sigurdur Sigurdarson

PrP gene polymorphism and natural scrapie in Icelandic sheep.

The association between scrapie and polymorphism of the prion protein (PrP) gene was studied in the Icelandic sheep breed. Polymorphism of the three codons, 136, 154 and 171, that are important for scrapie susceptibility was determined. A BspHI restriction analysis was used to study the alleles of codons 136 and 154, while density gradient gel electrophoresis (DGGE) was used to analyse codon 171 and detect new polymorphisms. The PrP allelic variant, VRQ (amino acids at codons 136, 154 and 171), was found to be highly statistically associated with scrapie, whereas the allelic variant, AHQ, was never found in scrapie-affected animals, a finding that is statistically significant. Iceland has a few scrapie-free regions, which are a part of a quarantine network. Homozygotes for the VRQ variant were found there at a low frequency, indicating that genetic susceptibility is not enough for scrapie to develop and further evidence for the infectious nature of the disease. A comparison of PrP genotypes between sheep outside and within the scrapie-free zones revealed an increase in the AHQ allelic variant in the latter. No polymorphism was found at codon 171 in a total of 932 sheep studied, all individuals having the glutamine allele. Two novel, rare PrP alleles were found using DGGE at codons 138 and 151, i.e. S138N and R151C. Their relevance to scrapie is still unclear, but the former was found in scrapie-affected sheep as well as healthy sheep, whereas the latter was only found in healthy sheep.

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 PrPSc. 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.

Characteristics of scrapie isolates derived from hay mites.

Previous epidemiological evidence suggested that in some instances a vector and/or reservoir is involved in the occurrence and spread of transmissible spongiform encephalopathies (TSEs). In a preliminary study, hay mite preparations from five Icelandic farms with a history of scrapie were injected into mice, and some of these mice became sick after long incubation periods. To confirm that the disease was scrapie, subsequent passages in mice were performed. In addition, the characteristics of the disease process in these passages were assessed and the results compared to those findings with standard scrapie strains. As expected for scrapie, subsequent passages in the same host led to shortened incubation periods compared to those in primary isolate mice, and all mice had spongiform changes in brain. Results were similar for three of four isolates with regard to clinical manifestations, the incubation periods in mice of the three scrapie incubation-period genotypes (s7s7, s7p7, p7p7), and the PrPSc Western blot (WB) pattern. The characteristics of the fourth isolate were markedly different from the other three isolates with regard to these parameters. Comparison of the characteristics of standard mouse-adapted scrapie strains and the four isolates revealed differences; these differences were particularly pronounced for the fourth isolate.

Iron and iron/manganese ratio in forage from Icelandic sheep farms: relation to scrapie

This study was undertaken in order to examine whether any connection existed between the amounts of iron in forage and the sporadic occurrence of scrapie observed in certain parts of Iceland. As iron and manganese are considered antagonistic in plants, calculation of the Fe/Mn ratios was also included by using results from Mn determination earlier performed in the same samples. Forage samples (n = 170) from the summer harvests of 2001–2003, were collected from 47 farms for iron and manganese analysis. The farms were divided into four categories: 1. Scrapie-free farms in scrapie-free areas (n = 9); 2. Scrapie-free farms in scrapie-afflicted areas (n = 17); 3. Scrapie-prone farms (earlier scrapie-afflicted, restocked farms) (n = 12); 4. Scrapie-afflicted farms (n = 9). Farms in categories 1 and 2 are collectively referred to as scrapie-free farms. The mean iron concentration in forage samples from scrapie-afflicted farms was significantly higher than in forage samples from farms in the other scrapie categories (P = 0.001). The mean Fe/Mn ratio in forage from scrapie-afflicted farms was significantly higher than in forage from scrapie-free and scrapie-prone farms (P < 0.001). The results indicated relative dominance of iron over manganese in forage from scrapie-afflicted farms as compared to farms in the other categories. Thus thorough knowledge of iron, along with manganese, in soil and vegetation on sheep farms could be a pivot in studies on sporadic scrapie.

High incidence of subclinical infection of lymphoid tissues in scrapie-affected sheep flocks

Prion diseases are characterized by a long incubation period. In scrapie, sheep may incubate and spread the infection for several years before clinical signs evolve. We have previously studied the occurrence of subclincal infection in the brain. Now, we have studied the occurrence of subclinical infection in the brain and several lymphoid tissues in two scrapie-affected Icelandic sheep flocks by immunohistochemistry for PrPSc, a molecular marker for infectivity, and correlated this with results of PrP genotyping. At culling, one flock had one confirmed scrapie case, while the other flock had two. Analysis of 106 asymptomatic sheep by immunostaining for PrPSc revealed that the incidence of subclinical infection was 58.3% in one flock and 42.5% in the other. PrPSc was only detected in lymphoid tissues. The youngest positive sheep were 4 months old. PrP genotyping showed that over 90% of the sheep were of a genotype which is moderately sensitive to infection and may delay neuroinvasion. Our results show that asymptomatic sheep may spread the infection during the long incubation period of several years, which constitutes an important obstacle in the eradication of scrapie. Our findings indicate that contamination of the environment plays an important part in sustaining the infection.

Glutathione peroxidase (GPX) activity in blood of ewes on farms in different scrapie categories in Iceland

BackgroundPreliminary studies indicated decreased glutathione peroxidase (GPX) activity in blood of ewes on scrapie-afflicted farms. Other studies have shown decreased GPX activity in brain of prion-infected mice and in prion-infected cells in vitro. The aim of this study was to examine the GPX activity in blood as well as the distribution of GPX-activity levels from ewes on farms in scrapie-afflicted areas in Iceland.MethodsBlood samples were collected from 635 ewes (non-pregnant [n = 297] and pregnant [n = 338]) on 40 farms in scrapie-afflicted areas during the years 2001–2005, for analysis of GPX activity. The farms were divided into three categories: 1. Scrapie-free farms (n = 14); 2. Scrapie-prone farms (earlier scrapie-afflicted, restocked farms) (n = 12); 3. Scrapie-afflicted farms (n = 14). For comparison, 121 blood samples were also collected from non-pregnant ewes on one farm (farm A) in a scrapie-free area (scrapie never registered). Chi-square test was used to test for normal distribution of GPX-results, and Kruskal-Wallis test to compare GPX-results between categories.ResultsThe GPX-results appeared to be biphasically distributed in ewes in all three scrapie categories and on farm A. The presumptive breaking point was about 300 units g Hb-1. About 30–50% of the GPX-results from ewes in all three scrapie categories were below 300 units g Hb-1 but only about 13% of the GPX-results from ewes on farm A. The mean GPX activity was highest on farm A, and was significantly lower on scrapie-prone farms than on scrapie-free or scrapie-afflicted farms (non-pregnant and pregnant ewes: P < 0.005, respectively; non-pregnant and pregnant ewes combined: P < 0.0005).Conclusions1) the distribution of GPX-results in blood of Icelandic ewes apparently has a biphasic character; 2) the GPX-results were higher in ewes on one farm in a scrapie-free area than in ewes on farms in the scrapie-afflicted areas; 3) GPX-activity levels were significantly lowest on earlier scrapie-afflicted, restocked farms, which might have a bearing on the recurrence of sporadic scrapie on these farms; 4) further study on the possible role of GPX activity in the occurrence of scrapie in Iceland is warranted.