Lambs with scrapie susceptible genotypes have higher postnatal survival

Background

Prion protein (PrP) alleles associated with scrapie susceptibility persist in many sheep populations even with high frequencies despite centuries of selection against them. This suggests that scrapie susceptibility alleles have a pleiotropic effect or are associated with fitness or other traits that have been subject to selection.

Methodology/Principal Findings

We genotyped all lambs in two scrapie-free Scottish Blackface sheep flocks for polymorphisms at codons 136, 154 and 171 of the PrP gene. We tested potential associations of the PrP genotype with lamb viability at birth and postnatal survival using a complementary log-log link function and a Weibull proportional hazard model, respectively. Here we show there is an association between PrP genotype, as defined by polymorphisms at codons 154 ad 171, and postnatal lamb survival in the absence of scrapie. Sheep carrying the wild-type ARQ allele have higher postnatal survival rates than sheep carrying the more scrapie-resistant alleles (ARR or AHQ).

Conclusion

The PrP genotypes associated with higher susceptibility to scrapie are associated with improved postnatal survival in the absence of the disease. This association helps to explain the existence, and in many instances the high frequency, of the ARQ allele in sheep populations.     

Prions Are Secreted in Milk from Clinically Normal Scrapie-Exposed Sheep

The potential spread of prion infectivity in secreta is a crucial concern for prion disease transmission. Here, serial protein misfolding cyclic amplification (sPMCA) allowed the detection of prions in milk from clinically affected animals as well as scrapie-exposed sheep at least 20 months before clinical onset of disease, irrespective of the immunohistochemical detection of protease-resistant PrP^Sc within lymphoreticular and central nervous system tissues. These data indicate the secretion of prions within milk during the early stages of disease progression and a role for milk in prion transmission. Furthermore, the application of sPMCA to milk samples offers a noninvasive methodology to detect scrapie during preclinical/subclinical disease.PrP^Sc, a disease-specific marker for prion diseases and the likely infectious agent, is widely distributed within the central nervous system (CNS) and lymphoreticular tissues (LRS) in ovine scrapie, human variant Creutzfeldt-Jakob disease (vCJD), and cervine chronic wasting disease (CWD) during both clinical and preclinical stages (4, 11, 25). Furthermore, while the LRS distribution of PrP^Sc is much more restricted in bovine spongiform encephalopathy (BSE), sheep experimentally infected with BSE display a PrP^Sc tissue distribution more akin to that of ovine scrapie (11).For rodent-adapted scrapie and cervine CWD, the disease agent is detected in excreta when animals are in the clinical stages of disease, a process likely to contribute to environmental reservoirs of infectivity and lateral disease transmission (5, 13, 21). Within an experimental rodent model, it has also been demonstrated that the shedding of PrP^Sc and concomitant infectivity in feces occurs during preclinical scrapie (21).Evidence now also demonstrates that milk provides a vehicle for the transmission for prion diseases. Scrapie-free lambs fed milk from clinical scrapie-affected ewes propagate PrP^Sc within their LRS (8). Additionally, a recent study using a transgenic mouse bioassay demonstrated the secretion of infectivity in milk from preclinical animals where scrapie infectivity was found in milk months before the onset of clinical signs in animals with an ARQ/VRQ PrP genotype (10). The presence of scrapie infectivity within milk was irrespective of mammary gland pathology or PrP^Sc accumulation, and these animals were estimated to have considerable accumulation of immunohistochemically (IHC) detectable PrP^Sc within the LRS at the time of sampling.Here, we applied serial protein misfolding cyclic amplification (sPMCA) to the in vitro detection of PrP^Sc within sheep milk (Fig. ​(Fig.1)1) (Table ​(Table11).Open in a separate windowFIG. 1.sPMCA analysis of ovine milk samples. Milk was clarified and seeded into brain homogenate from sheep unexposed to the scrapie agent. Samples underwent sPMCA, and products were digested with proteinase K before analysis of 10 μl of each sample. PrP was detected with monoclonal antibodies SHA31 and P4; molecular mass markers are indicated (kDa). Milk was sampled from animals not exposed to the scrapie agent (U), those displaying clinical signs of scrapie (C), or those exposed to a scrapie-positive farm environment but not displaying clinical disease (S). NS, non-seeded PMCA brain substrate subjected to identical sPMCA conditions at the same time as positive samples were analyzed. Samples from the four nonexposed animals were analyzed 18 to 20 times each by sPMCA. Samples from clinically affected or clinically normal scrapie-exposed animals were analyzed in triplicate. For this triplicate analysis of each sample, the sPMCA round at which samples became positive is indicated under the appropriate lane. n, negative at round 12. Each sample was PrP^Sc negative until the stated round and thereafter was positive.

TABLE 1.

Timeline of exposure of animals to a scrapie-positive farm environment, sample collection, and scrapie status

Sheep with scrapie and mastitis transmit infectious prions through the milk

Prions are misfolded proteins that are infectious and naturally transmitted, causing a fatal neurological disease in humans and animals. Prion shedding routes have been shown to be modified by inflammation in excretory organs, such as the kidney. Here, we show that sheep with scrapie and lentiviral mastitis secrete prions into the milk and infect nearly 90% of naïve suckling lambs. Thus, lentiviruses may enhance prion transmission, conceivably sustaining prion infections in flocks for generations. This study also indicates a risk of prion spread to sheep and potentially to other animals through dietary exposure to pooled sheep milk or milk products.Prion diseases have emerged globally as a significant threat to human and animal health. Recently, human-to-human spread of prions is believed to have occurred through blood transfusions (9, 12, 16), underscoring the importance of understanding possible transmission routes. PrP^Sc, a misfolded, aggregated form of the normal prion protein, PrP^C, commonly accumulates in the follicles of lymphoid tissues, prior to entering the central nervous system (2, 11, 14). Inflammation can cause lymphoid follicles to form in other organs, such as liver and kidney, which leads to prion invasion of organs that are not typically prion permissive (1). In mice, prion infection in the inflamed kidney has the untoward consequence of prion excretion in urine (13). This finding, together with our report of sheep with PrP^Sc in the inflamed mammary gland (8), has raised concerns of prion secretion into milk.Here, we investigated whether PrP^Sc in the inflamed mammary gland leads to prion secretion in milk and infection of naïve lambs through suckling. Prion infectivity has been detected in the milk of sheep expressing a prion gene (Prnp) coding for VRQ/VRQ or VRQ/ARQ at polymorphic codons 136, 154, and 171 (3, 4). However, whether (i) sheep-to-lamb transmission of prions in milk leads to clinical prion disease or (ii) sheep with the common ARQ/ARQ Prnp genotype can infect lambs through milk is unknown. We induced a chronic lentiviral mastitis and inoculated ARQ/ARQ Sarda breed sheep with infectious prions. After 14 months, we bred the sheep and collected the milk. To avoid cross-contamination of newborn lambs, we fed the milk to imported known-naïve lambs and then monitored the lambs for signs of prion infection (Fig. ​(Fig.1A1A).Open in a separate windowFIG. 1.Sheep infected with prions and maedi-visna virus (MVV) develop lymphofollicular mastitis with PrP^Sc. (A) Experimental scheme. Sheep were inoculated with culture medium or MVV and were then orally exposed to scrapie prions and bred. Milk was collected near the time point that neurologic signs of scrapie developed and was fed to naïve lambs. The ratio of lambs with detectable PrP^Sc to lambs fed the indicated milk is shown for each experiment. (B) PrP immunohistochemistry assay of brain and tonsil from milk source sheep shows staining for PrP^Sc in the brainstem, particularly in the vagal nucleus (indicated by asterisks) and in the tonsillar follicles of scrapie-infected sheep (arrows). (C) Mammary gland (MG) of milk source sheep shows lymphoid follicles (arrowheads) with associated PrP^Sc (arrows) adjacent to milk ducts (md) in the MVV-inoculated sheep, whereas the medium-inoculated sheep had a histologically normal MG with no detectable PrP^Sc. Insets show a high magnification of follicles containing PrP^Sc. Scale bar = 100 μm; scale bar in inset = 25 μm. (D) Western blot analysis shows PrP^Sc detection in MG of sheep inoculated with MVV/scrapie agents but not in sheep inoculated with scrapie prions only. The sheep identification number is indicated for each lane. PK, proteinase K digested; pos. br, positive brain control; neg. br, negative brain control.To induce a chronic lymphofollicular mastitis, we exposed 7- to 10-day-old lambs (groups of 10) by intratracheal and intravenous routes to a common sheep lentivirus known as maedi-visna virus (MVV) or to cell culture medium only. To do this, lambs were inoculated with 3.5 ml intravenously and 0.5 ml intratracheally of MVV in culture supernatant containing 1.5 × 10⁶ tissue culture infectious doses/ml of the “rapid/high” MVV strain 85/34 (5, 15). Twenty days later, all lambs were orally inoculated with 25 ml of 10% scrapie-infected brain homogenate from a pool of naturally infected Sarda sheep.We sequenced the entire Prnp gene and found that all lambs expressed the ARQ/ARQ Prnp genotype, indicating that the sheep should be susceptible to scrapie. As negative controls, 2 lambs of Prnp genotype ARR/ARR and ARQ/ARQ were mock inoculated with cell culture medium and healthy brain homogenate. All lambs originated from scrapie-free flocks that had been monitored for clinical scrapie cases for at least 3 years.All inoculated sheep were naturally bred to rams at 15 months postinoculation (p.i.) and produced lambs at 20 months p.i. Sheep developed early signs of scrapie just after the lambs were born. Milk from each sheep was manually collected and frozen daily.Eight of 10 MVV-and-scrapie (denoted MVV/scrapie)-inoculated sheep and 9 of 10 scrapie-inoculated sheep showed clinical signs of scrapie, with mean incubation periods of 22 ± 1.4 and 23 ± 1.5 months postinoculation, respectively, and were euthanized. There was no significant difference in incubation period between the groups (Student''s t test, P = 0.5), indicating that inflammation associated with the MVV infection does not accelerate prion disease. This finding is consistent with the results of previous studies that showed that chronic pancreatitis or nephritis did not affect the scrapie incubation period (1). Scrapie infection was confirmed postmortem by the detection of PrP^Sc in brain and lymphoid tissues by Western blot and immunohistochemistry assays (Fig. ​(Fig.1B).1B). Interestingly, scrapie did not develop in 3 sheep with a Prnp gene encoding a rare polymorphism at codon 176 (K), consistent with recent reports describing scrapie resistance for this genotype (10).Antibodies to MVV were detected in serum of all the MVV-inoculated sheep by indirect enzyme-linked immunosorbent assay (ELISA) (Elitest kit; Hyphen BioMed). Five of 8 MVV/scrapie-infected sheep (63%) showed a lymphofollicular mastitis (Fig. ​(Fig.1C),1C), and 3 had a diffuse interacinar lymphoid infiltrate. Of the 5 sheep with lymphofollicular mastitis, 4 had PrP^Sc deposits detectable by immunohistochemistry and Western blot assays (Fig. 1C and D), whereas no sheep with diffuse lymphoid infiltrates had detectable PrP^Sc. Surprisingly, 2 of 9 sheep inoculated only with scrapie also had lymphofollicular mastitis and anti-MVV antibodies, one of which had visible PrP^Sc deposits. MVV is a common pathogen in Europe, and it is possible that these sheep were infected from the dam. The remaining 7 scrapie-inoculated sheep had histologically normal mammary glands (Fig. ​(Fig.1C)1C) and no detectable PrP^Sc (Fig. ​(Fig.1D)1D) or anti-MVV antibodies.We selected the stored milk from the 4 MVV/scrapie-infected sheep with PrP^Sc in the mammary glands and from the 7 scrapie-infected sheep with histologically normal mammary glands. Milk samples from the early, middle, and late stages of lactation were pooled for each group. We imported naïve Cheviot lambs (n = 9) from flocks that originated from scrapie-free New Zealand and had been bred and housed under strict biosecurity containment in the United Kingdom to ensure that the lambs had not been exposed to scrapie. The Sarda lambs (n = 4) originated from a scrapie-free flock in Sardinia. We then fed pooled milk from MVV/scrapie-infected sheep to each of 8 naïve ARQ/ARQ lambs and from scrapie-infected sheep to 3 naïve ARQ/ARQ lambs ad libitum. Each lamb ingested a total volume of 1 to 2 liters over a total period of 3 days (Table ​(Table1).1). Two lambs were orally inoculated with brain homogenate pooled from the scrapie-infected milk donors as positive controls. Groups of lambs were housed in separate stalls and subjected to isolation conditions.

TABLE 1.

Genotype, breed, and PrP^Sc detection in lambs fed milk from MVV/scrapie- or scrapie-infected sheep

Epidemiological Characteristics of Classical Scrapie Outbreaks in 30 Sheep Flocks in the United Kingdom

Background

Most previous analyses of scrapie outbreaks have focused on flocks run by research institutes, which may not reflect the field situation. Within this study, we attempt to rectify this deficit by describing the epidemiological characteristics of 30 sheep flocks naturally-infected with classical scrapie, and by exploring possible underlying causes of variation in the characteristics between flocks, including flock-level prion protein (PrP) genotype profile. In total, the study involved PrP genotype data for nearly 8600 animals and over 400 scrapie cases.

Methodology/Principal Findings

We found that most scrapie cases were restricted to just two PrP genotypes (ARQ/VRQ and VRQ/VRQ), though two flocks had markedly different affected genotypes, despite having similar underlying genotype profiles to other flocks of the same breed; we identified differences amongst flocks in the age of cases of certain PrP genotypes; we found that the age-at-onset of clinical signs depended on peak incidence and flock type; we found evidence that purchasing infected animals is an important means of introducing scrapie to a flock; we found some evidence that flock-level PrP genotype profile and flock size account for variation in outbreak characteristics; identified seasonality in cases associated with lambing time in certain flocks; and we identified one case that was homozygous for phenylalanine at codon 141, a polymorphism associated with a very high risk of atypical scrapie, and 28 cases that were heterozygous at this codon.

Conclusions/Significance

This paper presents the largest study to date on commercially-run sheep flocks naturally-infected with classical scrapie, involving 30 study flocks, more than 400 scrapie cases and over 8500 PrP genotypes. We show that some of the observed variation in epidemiological characteristics between farms is related to differences in their PrP genotype profile; although much remains unexplained and may instead be attributed to the stochastic nature of scrapie dynamics.     

Association between the prion protein genotype and animal performance traits in a large multibreed sheep population

Genetic susceptibility to scrapie, a fatal disease of sheep and goats, is modulated by polymorphisms in the prion protein (PrP). Neither the frequency of the PrP genotypes nor their association with animal performance has been investigated in a large multibreed Irish sheep population. Scrapie genotypes were available on 16 416 animals; the breeds represented included purebred Belclare (733), Charollais (333), Suffolk (739), Texel (1 857), Vendeen (191), and crossbreds (12 563). Performance data on lambing, lamb and ewe performance as well as health traits were available. The association between alternative approaches of describing the PrP genotype (i.e. 15 individually called PrP genotypes, five genotype classes representing susceptibility to scrapie, or number of ARR haplotypes) and animal performance were quantified using animal linear mixed models. All 15 of the possible scrapie genotypes were detected, although the frequency differed by breed. The frequency of the five PrP haplotypes in the entire population were 0.70 (ARR), 0.15 (ARQ), 0.11 (ARH), 0.02 (AHQ) and 0.01 (VRQ); the most susceptible haplotype (VRQ) was only detected in purebred Texels and crossbreds. No association was detected between the PrP genotype of either the animal or dam and any of the lambing traits (i.e. lambing difficulty score, perinatal mortality and birth weight). With the exception of ultrasound muscle depth, no association between the PrP genotype and any of the lamb performance traits (i.e. lamb BW and carcass) was observed. Lambs carrying the category four PrP genotype (i.e. ARR/VRQ) had 1.20 (SE = 0.45) mm, 1.38 (SE = 0.12) mm, 1.47 (S = 0.25) mm shallower ultrasound muscle depth relative to lambs of the less susceptible scrapie categories of 1, 2, 3, respectively (P < 0.05). Nonetheless, no association between PrP genotype and lamb carcass conformation, the ultimate end goal of producers, was detected. Ewe litter size, body condition score or lameness did not differ by PrP genotype of the ewe (P > 0.05). For ewe mature BW, ARH/VRQ ewes differed from most other ewe PrP genotypes and were, on average, 3.79 (SE = 1.66) kg heavier than ARR/ARR genotype ewes. Lamb dag score differed by dam PrP genotype (P < 0.05), although the differences were small. Results from this study show that scrapie is segregating within the Irish sheep population, but the PrP genotype was not associated with most traits investigated and, where associations were detected, the biological significance was minimal. This suggests minimal impact of selection on PrP genotype on performance, at least for the traits investigated in the present study.     

Selection of ovine housekeeping genes for normalisation by real-time RT-PCR; analysis of <Emphasis Type="Italic">PrP</Emphasis>gene expression and genetic susceptibility to scrapie

Background

Cellular prion protein expression is essential for the development of transmissible spongiform encephalopathies (TSEs), and in sheep, genetic susceptibility to scrapie has been associated to PrP gene polymorphisms. To test the hypothetical linkage between PrP gene expression and genetic susceptibility, PrP mRNA levels were measured by real-time RT-PCR in six ovine tissues of animals with different genotypes.

Results

Previous to the PrP gene expression analysis the stability of several housekeeping (HK) genes was assessed in order to select the best ones for relative quantification. The normalisation of gene expression was carried out using a minimum of three HK genes in order to detect small expression differences more accurately than using a single control gene. The expression stability analysis of six HK genes showed a large tissue-associated variation reflecting the existence of tissue-specific factors. Thereby, a specific set of HK genes was required for an accurate normalisation of the PrP gene expression within each tissue. Statistical differences in the normalised PrP mRNA levels were found among the tissues, obtaining the highest expression level in obex, followed by ileum, lymph node, spleen, cerebellum and cerebrum. A tendency towards increased PrP mRNA levels and genetic susceptibility was observed in central nervous system. However, the results did not support the hypothesis that PrP mRNA levels vary between genotypes.

Conclusion

The results on PrP gene expression presented here provide valuable baseline data for future studies on scrapie pathogenesis. On the other hand, the results on stability data of several HK genes reported in this study could prove very useful in other gene expression studies carried out in these relevant ovine tissues.

     

A transfectant RK13 cell line permissive to classical caprine scrapie prion propagation

To assess scrapie infectivity associated with caprine-origin tissues, bioassay can be performed using kids, lambs or transgenic mice expressing caprine or ovine prion (PRNP) alleles, but the incubation periods are fairly long. Although several classical ovine scrapie prion permissive cell lines with the ability to detect brain-derived scrapie prion have been available, no classical caprine scrapie permissive cell line is currently available. Therefore, the aims of this study were to generate a rabbit kidney epithelial cell line (RK13) stably expressing caprine wild-type PRNP (cpRK13) and then to assess permissiveness of cpRK13 cells to classical caprine scrapie prion propagation. The cpRK13 and plasmid control RK13 (pcRK13) cells were incubated with brain-derived classical caprine scrapie inocula prepared from goats or ovinized transgenic mice (Tg338, express ovine VRQ allele) infected with caprine scrapie. Significant PrP^Sc accumulation, which is indicative of scrapie prion propagation, was detected by TSE ELISA and immunohistochemistry in cpRK13 cells inoculated with classical caprine scrapie inocula. Western blot analysis revealed the typical proteinase K-resistant 3 PrP^res isoforms in the caprine scrapie prion inoculated cpRK13 cell lysate. Importantly, PrP^Sc accumulation was not detected in similarly inoculated pcRK13 cells, whether by TSE ELISA, immunohistochemistry, or western blot. These findings suggest that caprine scrapie prions can be propagated in cpRK13 cells, thus this cell line may be a useful tool for the assessment of classical caprine prions in the brain tissues of goats.     

Lack of Prion Accumulation in Lymphoid Tissues of PRNP ARQ/ARR Sheep Intracranially Inoculated with the Agent of Scrapie

Sheep scrapie is a transmissible spongiform encephalopathy that can be transmitted horizontally. The prion protein gene (PRNP) profoundly influences the susceptibility of sheep to the scrapie agent and the tissue levels and distribution of PrP^Sc in affected sheep. The purpose of this study was to compare the survival time and PrP^Sc tissue distribution in sheep with highly resistant and highly susceptible PRNP genotypes after intracranial inoculation of the agent of scrapie. Five sheep each of genotype VRQ/VRQ, VRQ/ARR or ARQ/ARR were inoculated. Sheep were euthanized when clinical signs of scrapie became severe. Clinical signs, microscopic lesions, and western blot profiles were uniform across genotypes and consistent with manifestations of classical scrapie. Mean survival time differences were associated with the 171 polymorphic site with VRQ/VRQ sheep surviving 18 months, whereas VRQ/ARR and ARQ/ARR sheep survived 60 and 56 months, respectively. Labeling of PrP^Sc by immunohistochemistry revealed similar accumulations in central nervous system tissues regardless of host genotype. Immunoreactivity for PrP^Sc in lymphoid tissue was consistently abundant in VRQ/VRQ, present but confined to tonsil or retropharyngeal lymph node in 4/5 VRQ/ARR, and totally absent in ARQ/ARR sheep. The results of this study demonstrate the susceptibility of sheep with the ARQ/ARR genotype to scrapie by the intracranial inoculation route with PrP^Sc accumulation in CNS tissues, but prolonged incubation times and lack of PrP^Sc in lymphoid tissue.     

Infection of Cell Lines with Experimental and Natural Ovine Scrapie Agents

Mouse bioassay remains the gold standard for determining proof of infectivity, strain type, and infectious titer estimation in prion disease research. The development of an approach using ex vivo cell-based assays remains an attractive alternative, both in order to reduce the use of mice and to hasten results. The main limitation of a cell-based approach is the scarcity of cell lines permissive to infection with natural transmissible spongiform encephalopathy strains. This study combines two advances in this area, namely, the standard scrapie cell assay (SSCA) and the Rov9 and MovS6 cell lines, which both express the ovine PrP VRQ allele, to assess to what extent natural and experimental ovine scrapie can be detected ex vivo. Despite the Rov9 and MovS6 cell lines being of different biological origin, they were both permissive and resistant to infection with the same isolates of natural sheep scrapie as detected by SSCA. Rov9 subclones that are 20 times more sensitive than Rov9 to SSBP/1-like scrapie infection were isolated, but all the subclones maintained their resistance to isolates that failed to transmit to the parental line. The most sensitive subclone of the Rov9 cell line was used to estimate the infectious titer of a scrapie brain pool (RBP1) and proved to be more sensitive than the mouse bioassay using wild-type mice. Increasing the sensitivity of the Rov9 cell line to SSBP/1 infection did not correlate with broadening susceptibility, as the specificity of permissiveness and resistance to other scrapie isolates was maintained.Prion diseases are a group of neurodegenerative diseases affecting humans and animals, including scrapie in sheep and goats and bovine spongiform encephalopathy (BSE) in cattle. A feature of prion diseases and, in particular, of scrapie, is the existence of different strains (6) which influence pathology and is most probably related to the conformation of the pathogenic form of the prion protein (PrP^Sc). The susceptibility of sheep to scrapie is determined by the PrP genotype; codons 136, 154, and 171 determine relative resistance and susceptibility, with amino acids valine (V), arginine (R), and glutamine (Q) at these positions (known as VRQ) being considered the sheep PrP allele most susceptible to classical scrapie (3).An array of diagnostic tests exist for prion diseases, aimed at the detection of the disease-associated protease-resistant form of the naturally occurring PrP^C protein, termed PrP^Sc or PrP^res after partial protease digestion. However, the level of detectable PrP^Sc does not quantitatively correlate with prion infectivity (2) and the current biochemical analysis of PrP^Sc cannot always determine the strain (6, 7).Mouse bioassay remains the gold standard for determining proof of infectivity, strain type, and infectious titer estimate in ruminant transmissible spongiform encephalopathy (TSE) research. Conventional mouse bioassays using wild-type mice are generally slow (>150 days, and considerably longer, >600, days for obtaining infectious titer information) and require multiple mice to be dosed (typically 6 or more) at each dilution of infectious material. Therefore, the development of an approach using ex vivo cell-based assays remains an ethically and economically desirable alternative. Using cell lines permissive to mouse-passaged scrapie strains, Klöhn et al. have developed a cell-based assay for measuring de novo infection and the titer of mouse-passaged scrapie (18).The main limitation of adopting a cell-based approach is the scarcity of cell lines permissive to infection with natural TSE strains (for a review, see references 31 and 34), as the majority of permissive cell lines can only be infected with rodent-adapted strains of scrapie and BSE (4, 9, 16, 20, 23, 24, 29, 33, 36). While there are currently no cell lines reported to be permissive to bovine BSE or human TSE diseases, there are cell lines which express ovine PrP that have been shown to be permissive to natural scrapie infection (1, 35). There is also one fibroblast-like deer cell line that is able to propagate chronic wasting disease (27).Two of the sheep scrapie-susceptible cell lines are the MovS6 cell line (1), a Schwann cell line derived from the tg301 transgenic mouse, and the Rov9 cell line (35), based on a stably transfected rabbit kidney epithelial cell line (RK13) that does not express endogenous PrP. Both express the VRQ allele of ovine PrP, the latter upon induction with doxycycline (35). These cell lines were found to be permissive to infection with a PrP genotype-matched VRQ homozygous scrapie field case, and de novo PrP^Sc maintained its phenotype when used as an inoculum in mouse bioassays (1, 35). Using fluorescence-activated cell sorting, Falanga et al. isolated Rov9 subclones that produce higher levels of PrP^C and PrP^Sc than the parental cell line when infected (11).The primary objective of this study was to assess the permissiveness of the Rov9 and MovS6 cell lines to a panel of scrapie isolates from a range of sheep breeds with a range of PrP genotypes. Second, subcloning of the Rov9 cell line was undertaken in an attempt to identify subclones with greater sensitivity and more diverse permissibility to ovine scrapie isolates.     

Signal transduction in neurons: effects of cellular prion protein on fyn kinase and ERK1/2 kinase

Background

It has been reported that cellular prion protein (PrP^c) co-localizes with caveolin-1 and participates to signal transduction events by recruiting Fyn kinase. As PrP^c is a secreted protein anchored to the outer surface membrane through a glycosylphosphatidylinositol (GPI) anchor (^secPrP) and caveolin-1 is located in the inner leaflet of plasma membrane, there is a problem of how the two proteins can physically interact each other and transduce signals.

Results

By using the GST-fusion proteins system we observed that PrP^c strongly interacts with caveolin-1 scaffolding domain and with a caveolin-1 hydrophilic C-terminal region, but not with the caveolin-1 N-terminal region. In vitro binding experiments were also performed to define the site(s) of PrP^c interacting with cav-1. The results are consistent with a participation of PrP^c octapeptide repeats motif in the binding to caveolin-1 scaffolding domain. The caveolar localization of PrP^c was ascertained by co-immunoprecipitation, by co-localization after flotation in density gradients and by confocal microscopy analysis of PrP^c and caveolin-1 distributions in a neuronal cell line (GN11) expressing caveolin-1 at high levels.

Conclusions

We observed that, after antibody-mediated cross-linking or copper treatment, PrP^c was internalized probably into caveolae. We propose that following translocation from rafts to caveolae or caveolae-like domains, ^secPrP could interact with caveolin-1 and induce signal transduction events.

     

Environmental Sources of Scrapie Prions

Ovine scrapie and cervine chronic wasting disease show considerable horizontal transmission. Here we report that a scrapie-affected sheep farm has a widespread environmental contamination with prions. Prions were amplified by protein-misfolding cyclic amplification (sPMCA) from seven of nine environmental swab samples taken, including those from metal, plastic, and wooden surfaces. Sheep had been removed from the areas from which the swabs were taken up to 20 days prior to sampling, indicating that prions persist for at least that long. These data implicate inanimate objects as environmental reservoirs for prion infectivity that are likely to contribute to facile disease transmission.Prion diseases are fatal neurological disorders. The archetypal prion disease is scrapie in sheep, and in the last few decades novel prion diseases have emerged in a range of species, including bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in deer, and variant Creutzfeldt-Jakob disease (vCJD) in humans. The “protein-only” hypothesis dictates that a pathological isoform, PrP^Sc, of the cellular prion protein (PrP^C) constitutes the infectious agent, or prion (13). A wide range of tissues from CWD- and scrapie-affected animals contain PrP^Sc, and affected animals have been shown to excrete or secrete prions in milk, saliva, urine, and feces (2, 3, 6, 7, 8, 10, 16). This finding led to the hypothesis that infectivity resides in the environment, thus explaining the facile transmission of CWD and scrapie. In support of this hypothesis, CWD infectivity has been transmitted from a combination of exposed bedding, water, and food of captive animals (9), and CWD PrP^Sc has been detected within a single environmental water sample (11).Environmental prions are likely to be present at very low levels. The most sensitive method available for the detection of PrP^Sc is serial protein-misfolding cyclic amplification (sPMCA) (1). This technique was pioneered by Soto and colleagues (15), has been used for the amplification of scrapie (17), CWD (4), and vCJD (5) within their natural hosts, and has facilitated the detection of prions within ovine milk (6) and saliva (7) and within cervine urine (4). Here we investigated sources of environmental scrapie prions by applying sPMCA to samples taken from a range of surfaces that were accessible to animals on a farm where scrapie is endemic.Environmental samples were taken from the Veterinary Laboratories Agency, United Kingdom, farm where natural scrapie is endemic, with a high incidence since 1996. Sheep within the flock were exposed to the scrapie agent by natural routes of transmission. Control samples were taken from a farm (ADAS, United Kingdom) that houses a New Zealand-derived scrapie-free flock kept under strict biosecurity conditions. Environmental swabs were taken by wetting foam swabs (Edson Electronics, Northumberland, United Kingdom) in sterile water and then gently swabbing five times in both directions across a surface approximately 10 cm by 2 cm. Two swabs were taken from each area, and all samples were stored at −80^°C. A total of nine environmental samples from a scrapie-affected farm and a scrapie-free farm were analyzed by sPMCA.Two swabs taken from each area were thawed to room temperature and placed in a single container to which 6 ml of 150 mM PO₄ buffer plus 0.5% (vol/vol) Nonidet P-40 and 0.5% (wt/vol) sodium deoxycholate were added. The container was rotated for 2 h. Prions released into this buffer were precipitated on silicon dioxide and then eluted in 200 μl of 0.1% (wt/vol) sodium dodecyl sulfate. Ten microliters of the eluate was amplified within PCR tubes by sPMCA exactly as previously described (7). Samples from both a scrapie-exposed environment and a non-scrapie-exposed environment were analyzed concurrently within the same run on the same sonicator. Each extract was amplified at least in triplicate within a single run and then analyzed by Western blotting (14).Samples from four metal surfaces from an indoor pen occupied by sheep for a few days each week—a gate, a water trough, a feed trough, and penning—were analyzed. Samples from an outdoor environment that had contained sheep 20 days previously—a metal fence, a metal gate, a metal water trough, a plastic post where sheep frequently scratched, and a wooden fence post (Table ​(Table1)—were1)—were also analyzed. After 8 rounds of amplification, PrP^Sc was detected in all samples with the exceptions of the outdoor water trough and gate (Figure ​(Figure11 and Table ​Table1).1). Equivalent samples from a farm housing a scrapie-free flock were also analyzed, and PrP^Sc was not amplified even after 10 rounds of sPMCA. For indoor surfaces from the scrapie-affected farm, 83% of the sPMCA reactions were positive (n = 12), and 0% were positive for equivalent samples from a scrapie-free farm (n = 24). Similarly, 27% of analyses were positive for samples from outdoor surfaces (n = 15), and again no prion was amplified from equivalent samples taken from a scrapie-free farm (n = 30). For comparison of the percentages of positive sPMCA reactions for different cohorts of samples, data were set up as 2 by 2 contingency tables, and Fisher''s exact test (one-tailed) was applied to derive P values. Overall, prions were significantly more likely to be present in the scrapie-affected farm on indoor (P < 0.001) and outdoor (P = 0.009) surfaces. Analyses of all samples were carried out in two independent experiments that gave equivalent results.Open in a separate windowFIG. 1.Amplification of prions from environmental samples. Prions were extracted from swabs taken of surfaces from a scrapie-free farm or a farm where scrapie is endemic. Swabs were taken from a wooden post (1), a plastic scratching post (2), and the following metal surfaces: fencing (3), gate (4 and 6), pen (5), feed trough (7), and water trough (8 and 9). Samples 1, 2, 3, 4, and 8 were taken from outdoor surfaces and 5, 6, 7, and 9 from indoor surfaces. Extracts were used as seeds for 8 rounds of sPMCA. Products were digested with proteinase K and analyzed by Western blotting. PrP was detected with monoclonal antibodies SHA31 and P4. Molecular-weight markers are shown.

TABLE 1.

sPMCA of prions found in the environment^a

Prions of Ruminants Show Distinct Splenotropisms in an Ovine Transgenic Mouse Model

Background

Transmissible agents involved in prion diseases differ in their capacities to target different regions of the central nervous system and lymphoid tissues, which are also host-dependent.

Methodology/Principal Findings

Protease-resistant prion protein (PrP^res) was analysed by Western blot in the spleen of transgenic mice (TgOvPrP4) that express the ovine prion protein under the control of the neuron-specific enolase promoter, after infection by intra-cerebral route with a variety of transmissible spongiform encephalopathies (TSEs) from cattle and small ruminants. Splenic PrP^res was consistently detected in classical BSE and in most natural scrapie sources, the electrophoretic pattern showing similar features to that of cerebral PrP^res. However splenic PrP^res was not detected in L-type BSE and TME-in-cattle, or in the CH1641 experimental scrapie isolate, indicating that some TSE strains showed reduced splenotropism in the ovine transgenic mice. In contrast with CH1641, PrP^res was also consistently detected in the spleen of mice infected with six natural “CH1641-like” scrapie isolates, but then showed clearly different molecular features from those identified in the brains (unglycosylated PrP^res at ∼18 kDa with removal of the 12B2 epitope) of ovine transgenic mice or of sheep. These features included different cleavage of the main PrP^res cleavage product (unglycosylated PrP^res at ∼19 kDa with preservation of the 12B2 epitope) and absence of the additional C-terminally cleaved PrP^res product (unglycosylated form at ∼14 kDa) that was detected in the brain.

Conclusion/Significance

Studies in a transgenic mouse model expressing the sheep prion protein revealed different capacities of ruminant prions to propagate in the spleen. They showed unexpected features in “CH1641-like” ovine scrapie suggesting that such isolates contain mixed conformers with distinct capacities to propagate in the brain or lymphoid tissues of these mice.     

A novel form of human disease with a protease-sensitive prion protein and heterozygosity methionine/valine at codon 129: Case report

Background

Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare neurodegenerative disorder in humans included in the group of Transmissible Spongiform Encephalopathies or prion diseases. The vast majority of sCJD cases are molecularly classified according to the abnormal prion protein (PrP^Sc) conformations along with polymorphism of codon 129 of the PRNP gene. Recently, a novel human disease, termed "protease-sensitive prionopathy", has been described. This disease shows a distinct clinical and neuropathological phenotype and it is associated to an abnormal prion protein more sensitive to protease digestion.

Case presentation

We report the case of a 75-year-old-man who developed a clinical course and presented pathologic lesions compatible with sporadic Creutzfeldt-Jakob disease, and biochemical findings reminiscent of "protease-sensitive prionopathy". Neuropathological examinations revealed spongiform change mainly affecting the cerebral cortex, putamen/globus pallidus and thalamus, accompanied by mild astrocytosis and microgliosis, with slight involvement of the cerebellum. Confluent vacuoles were absent. Diffuse synaptic PrP deposits in these regions were largely removed following proteinase treatment. PrP deposition, as revealed with 3F4 and 1E4 antibodies, was markedly sensitive to pre-treatment with proteinase K. Molecular analysis of PrP^Sc showed an abnormal prion protein more sensitive to proteinase K digestion, with a five-band pattern of 28, 24, 21, 19, and 16 kDa, and three aglycosylated isoforms of 19, 16 and 6 kDa. This PrP^Sc was estimated to be 80% susceptible to digestion while the pathogenic prion protein associated with classical forms of sporadic Creutzfeldt-Jakob disease were only 2% (type VV2) and 23% (type MM1) susceptible. No mutations in the PRNP gene were found and genotype for codon 129 was heterozygous methionine/valine.

Conclusions

A novel form of human disease with abnormal prion protein sensitive to protease and MV at codon 129 was described. Although clinical signs were compatible with sporadic Creutzfeldt-Jakob disease, the molecular subtype with the abnormal prion protein isoforms showing enhanced protease sensitivity was reminiscent of the "protease-sensitive prionopathy". It remains to be established whether the differences found between the latter and this case are due to the polymorphism at codon 129. Different degrees of proteinase K susceptibility were easily determined with the chemical polymer detection system which could help to detect proteinase-susceptible pathologic prion protein in diseases other than the classical ones.

     

No temporal trends in the prevalence of atypical scrapie in British sheep, 2002–2006

Background

So-called atypical scrapie was first identified in Great Britain (GB) in 2002 following the introduction of wide-scale scrapie surveillance. In particular, abattoir and fallen stock surveys have been carried out in GB since 2002, with a total of 147 atypical positives identified by the end of 2006. The results of these surveys provide data with which to assess temporal trends in the prevalence of atypical scrapie in sheep in Great Britain between 2002 and 2006.

Results

Using the results of abattoir and fallen stock surveys, the prevalence of atypical scrapie (percentage of samples positive) was estimated. The prevalence in the abattoir and fallen stock surveys, for all years combined, was 0.09% (95% confidence interval (CI): 0.08%–0.11%) and 0.07% (95% CI: 0.05%–0.11%), respectively. There were no significant temporal trends in either survey. Comparing the surveys' results, there were no significant differences in annual prevalence or the prevalence within PrP genotypes. For the abattoir survey, the PrP genotype with the highest prevalence was AHQ/AHQ, which was significantly higher than all other genotypes, except ARR/AHQ, AHQ/ARH and ARH/ARQ.

Conclusion

The estimated prevalence of atypical scrapie was similar in both the abattoir and fallen stock surveys. Our results indicate there was no significant temporal trend in prevalence, adding to evidence that this atypical form of scrapie may be a sporadic condition or, if it is infectious, that the force of infection is very low.

     

Prions in Milk from Ewes Incubating Natural Scrapie

Since prion infectivity had never been reported in milk, dairy products originating from transmissible spongiform encephalopathy (TSE)-affected ruminant flocks currently enter unrestricted into the animal and human food chain. However, a recently published study brought the first evidence of the presence of prions in mammary secretions from scrapie-affected ewes. Here we report the detection of consistent levels of infectivity in colostrum and milk from sheep incubating natural scrapie, several months prior to clinical onset. Additionally, abnormal PrP was detected, by immunohistochemistry and PET blot, in lacteal ducts and mammary acini. This PrP^Sc accumulation was detected only in ewes harbouring mammary ectopic lymphoid follicles that developed consequent to Maedi lentivirus infection. However, bioassay revealed that prion infectivity was present in milk and colostrum, not only from ewes with such lympho-proliferative chronic mastitis, but also from those displaying lesion-free mammary glands. In milk and colostrum, infectivity could be recovered in the cellular, cream, and casein-whey fractions. In our samples, using a Tg 338 mouse model, the highest per ml infectious titre measured was found to be equivalent to that contained in 6 µg of a posterior brain stem from a terminally scrapie-affected ewe. These findings indicate that both colostrum and milk from small ruminants incubating TSE could contribute to the animal TSE transmission process, either directly or through the presence of milk-derived material in animal feedstuffs. It also raises some concern with regard to the risk to humans of TSE exposure associated with milk products from ovine and other TSE-susceptible dairy species.     

Abnormalities in Stress Proteins in Prion Diseases

1. Prion diseases include kuru, Creutzfeldt–Jakob disease (CJD), Gerstmann–Sträussler–Scheinker disease (GSS), and fatal familia insomnia (FFI) of humans, as well as scrapie and bovine spongiform encephalopathy (BSE) of animals.2. All these disorders involve conversion of the normal, cellular prion protein (PrP^C) into the corresponding scrapie isoform (PrP^Sc). PrP^C adopts a structure rich in -helices and devoid of -sheet, in contrast to PrP^Sc, which has a high -sheet content and is resistant to limited digestion by proteases. That a conformational transition features in the conversion of PrP^C into PrP^Sc implies that prion diseases are disorders of protein conformation.3. This concept has been extended by our studies with heat shock proteins (Hsp), many of which are thought to function as molecular chaperones. We found that the induction of some Hsps but not others was profoundly altered in scrapie-infected cells and that the distribution of Hsp73 is unusual in these cells.4. Whether the conversion of PrP^C into PrP^Sc is assisted by molecular chaperones, or if the accumulation of the abnormally folded PrP^Sc is complexed with Hsps remains to be established.     

Sulfated Dextrans Enhance In Vitro Amplification of Bovine Spongiform Encephalopathy PrPSc and Enable Ultrasensitive Detection of Bovine PrPSc

Background

Prions, infectious agents associated with prion diseases such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep and goats, are primarily comprised of PrP^Sc, a protease-resistant misfolded isoform of the cellular prion protein PrP^C. Protein misfolding cyclic amplification (PMCA) is a highly sensitive technique used to detect minute amounts of scrapie PrP^Sc. However, the current PMCA technique has been unsuccessful in achieving good amplification in cattle. The detailed distribution of PrP^Sc in BSE-affected cattle therefore remains unknown.

Methodology/Principal Findings

We report here that PrP^Sc derived from BSE-affected cattle can be amplified ultra-efficiently by PMCA in the presence of sulfated dextran compounds. This method is capable of amplifying very small amounts of PrP^Sc from the saliva, palatine tonsils, lymph nodes, ileocecal region, and muscular tissues of BSE-affected cattle. Individual differences in the distribution of PrP^Sc in spleen and cerebrospinal fluid samples were observed in terminal-stage animals. However, the presence of PrP^Sc in blood was not substantiated in the BSE-affected cattle examined.

Conclusions/Significance

The distribution of PrP^Sc is not restricted to the nervous system and can spread to peripheral tissues in the terminal disease stage. The finding that PrP^Sc could be amplified in the saliva of an asymptomatic animal suggests a potential usefulness of this technique for BSE diagnosis. This highly sensitive method also has other practical applications, including safety evaluation or safety assurance of products and byproducts manufactured from bovine source materials.     

Nanopore analysis reveals differences in structural stability of ovine PrPC proteins corresponding to scrapie susceptible (VRQ) and resistance (ARR) genotypes

Species, as well as individuals within species, have unique susceptibilities to prion infection that are likely based on sequence differences in cellular prion protein (PrP^C). Species barriers to transmission also reflect PrP^C sequence differences. Defining the structure-activity relationship of PrP^C/PrP^Sc with respect to infectivity/susceptibility will benefit disease understanding and assessment of transmission risks. Here, nanopore analysis is employed to investigate genotypes of sheep PrP^C corresponding to differential susceptibilities to scrapie infection. Under non-denaturing conditions scrapie resistant (ARR) and susceptible (VRQ) genotypes display similar, type I (bumping) predominant event profiles, suggesting a conserved folding pattern. Under increasingly denaturing conditions both proteins shift to type II (intercalation/translocation) events but with different sensitivities to unfolding. Specifically, when pre-incubated in 2M Gdn-HCl, the VRQ variant had more of type II events as compared with the ARR protein, suggesting a more flexible unfolding pattern. Addition of PrP^Sc-specific polyclonal antibody (YML) to the ARR variant, pre-incubated in 2M Gdn-HCl, reduced the number of type II events with no clear intercalation/translocation peak, whereas for VRQ, type II events above blockades of 90 pA bound YML. A second PrP^Sc-specific antibody (SN6b) to a different cryptic epitope reduced type II events for VRQ but not the ARR variant. Collectively, the event patterns associated with sequential denaturation, as well as interactions with PrP^Sc-specific antibodies, support unique patterns and/or propensities of misfolding between the genotypes. Overall, nanopore analysis identifies intermediate conformations that occur during the unfolding pathways of ARR and VRQ genotypes and may help to understand the correlation of structural properties that induce protein misfolding.