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J Virol. Jan 2012; 86(1): 566–571.
PMCID: PMC3255911

The Oral Secretion of Infectious Scrapie Prions Occurs in Preclinical Sheep with a Range of PRNP Genotypes

Abstract

Preclinical sheep with the highly scrapie-susceptible VRQ/VRQ PRNP genotype secrete prions from the oral cavity. In order to further understand the significance of orally available prions, buccal swabs were taken from sheep with a range of PRNP genotypes and analyzed by serial protein misfolding cyclic amplification (sPMCA). Prions were detected in buccal swabs from scrapie-exposed sheep of genotypes linked to high (VRQ/VRQ and ARQ/VRQ) and low (ARR/VRQ and AHQ/VRQ) lymphoreticular system involvement in scrapie pathogenesis. For both groups, the level of prion detection was significantly higher than that for scrapie-resistant ARR/ARR sheep which were kept in the same farm environment and acted as sentinel controls for prions derived from the environment which might contaminate the oral cavity. In addition, sheep with no exposure to the scrapie agent did not contain any measurable prions within the oral cavity. Furthermore, prions were detected in sheep over a wide age range representing various stages of preclinical disease. These data demonstrate that orally available scrapie prions may be a common feature in sheep incubating scrapie, regardless of the PRNP genotype and any associated high-level accumulation of PrPSc within lymphoreticular tissues. PrPSc was present in buccal swabs from a large proportion of sheep with PRNP genotypes associated with relatively low disease penetrance, indicating that subclinical scrapie infection is likely to be a common occurrence. The significance of positive sPMCA reactions was confirmed by the transmission of infectivity in buccal swab extracts to Tg338 mice, illustrating the likely importance of orally available prions in the horizontal transmission of scrapie.

INTRODUCTION

The transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases affecting many mammalian species, for which there is 100% mortality. These include chronic wasting disease (CWD) in deer, bovine spongiform encephalopathy (BSE) in cattle, variant Creutzfeldt-Jakob disease (vCJD) in humans, and the prototype TSE, scrapie in sheep. Intensive research effort has been given to this group of diseases since the demonstration that the likely cause of vCJD in humans is the consumption of BSE agent-contaminated meat products (7). Central to the understanding of these diseases is the hypothesis that the infectious component is a conformer of a benign host protein known as the prion protein (PrPC). It is the infectious conformer of this ubiquitously expressed protein (PrPSc) that is responsible for the conversion of further healthy PrPC into PrPSc (21). This autocatalytic process leads to progressive neuronal loss, and ultimately death, with the duration of disease incubation ranging from months in some rodent models to decades in some human TSEs (1). While the levels of PrPSc accumulation are highest in the central nervous system (CNS), there can be appreciable accumulation in other tissue types. Within distinct TSE diseases, there are different levels of lymphoreticular system (LRS) involvement, which is dependent on both the strain and host species. This is well illustrated for the BSE agent, as in cattle there is little apparent involvement of the LRS, with restriction of prion replication mainly to the CNS. However, in sheep experimentally infected with the BSE agent and in human vCJD, there is marked involvement of the LRS (10, 27). In the case of scrapie in sheep, the genotype of the host can also have a strong influence on the tissue distribution of prion infectivity. In sheep with a V136R154Q171/A136R154R171 PRNP genotype, it has been reported that infection of the CNS can be seen without prior involvement of the LRS tissues, in contrast to disease pathogenesis within VRQ/VRQ animals, where there is sustained LRS involvement in PrPSc accumulation during the development of disease, both before and after prion invasion of the CNS (12).

Epidemiological and experimental data for both ovine scrapie and cervine CWD have shown both direct horizontal transmission of disease and environmental reservoirs of infectivity (2, 15, 19). The dissemination of infectivity from infected sheep into the environment and the direct animal-to-animal transmission of disease may be influenced by the levels of PrPSc within the LRS, which in turn may dictate the levels of PrPSc within secretory/excretory sites, for example, within skin (25), mammary glands (14), salivary glands (26), and the nasal mucosa (11).

We recently demonstrated the detection of scrapie prions in buccal swabs taken from sheep naturally exposed to and incubating scrapie. Prions were detected using an in vitro prion protein amplification technique known as serial protein misfolding cyclic amplification (sPMCA), a methodology conceptually analogous to nucleic acid amplification by PCR (23). Amplifiable prion material was detected in buccal swabs from exposed sheep from 9 months of age to the appearance of clinical disease in animals with a VRQ/VRQ PRNP genotype (17). It is likely that oral prion availability highlights an important factor in the horizontal spread of scrapie, both by direct sheep-to-sheep transmission of disease and via contribution to reservoirs of infectivity in the farm environment, including fomites (15). Here we analyzed buccal swabs taken from sheep that were incubating scrapie and heterozygous for PrP alleles representing different levels of LRS involvement during the infection process. Positive identification of prions within the oral cavity of sheep incubating scrapie was confirmed by the demonstration that PrPSc detected using this methodology correlated with biological infectivity transmitted to the Tg338 transgenic mouse line.

MATERIALS AND METHODS

All animal procedures were performed under Home Office (United Kingdom) and local ethical review committee approval and with compliance with the Animal (Scientific Procedures) Act 1986. Scrapie-exposed sheep were from the Animal Health and Veterinary Laboratories Agency (AHVLA; United Kingdom) Ripley experimental scrapie sheep flock, which has had a high incidence of naturally acquired scrapie for the last 15 years. Samples were taken from sheep born into the flock with the scrapie-susceptible genotypes ARR/VRQ, AHQ/VRQ, VRQ/VRQ, and ARQ/VRQ. In addition, swabs were taken from ARR/ARR sheep, associated with high resistance to scrapie infection, and these served as controls for the presence of prions in the oral cavity that may have been taken up from the farm environment (15). Non-scrapie-exposed sheep of the VRQ/VRQ, ARQ/ARQ, and ARR/ARR genotypes were obtained from a scrapie-free New Zealand-derived flock kept under strict biosecurity conditions (ADAS UK). Buccal swabs taken from the scrapie-exposed flock were from animals that were thought to be approaching the midpoint or in the latter half of disease incubation (based on historical disease incubation times in this flock). Following exposure from birth, disease incubation periods for sheep with distinct PRNP genotypes are in the following order: VRQ/VRQ < ARQ/VRQ < AHQ/VRQ < ARR/VRQ.

Buccal swab samples were collected by gently rubbing foam swabs (Edson Electronics) across both inner cheeks of each animal. Four swabs were collected from each animal at single time points and, as such, provide a snapshot of the likely presence of prions in the oral cavity. Two buccal swab samples were processed using a silicon dioxide (SiO2) enrichment step as previously described (17, 22). The SiO2-extracted material was then centrifuged for 3 min at 16,000 × g, and the supernatant was diluted 1:10 in PMCA brain homogenate substrate (10% [wt/vol]) from a VRQ/VRQ PRNP genotype animal (in phosphate-buffered saline [PBS]), 150 mmol/liter NaCl, 4 mmol/liter EDTA, pH 8.0, 1% (wt/vol) Triton X-100, and mini-protease inhibitor (Roche) to a final volume of 100 μl. All buccal samples were amplified in this single substrate, as positive buccal swab extracts were amplified most efficiently within VRQ/VRQ substrate rather than the homologous heterozygous substrate (data not presented). Samples contained in sealed 0.2-ml PCR tubes were placed in an ultrasonicating water bath (model S4000; Misonix) at 37°C, and sonications were performed for 40 s at 200 W. Sonications were repeated once every 30 min for 24 h (1 PMCA round), after which the amplified samples were diluted 1 in 3 with PMCA substrate in a final volume of 100 μl and the sample was subjected to additional rounds of PMCA. In the present study, the brain homogenate substrate required between five and nine rounds of sPMCA to achieve the same levels of amplification that had previously been achieved at round three or four with a different brain substrate (17). This estimation of the level of amplification efficiency between substrates was carried out using buccal extracts from VRQ/VRQ sheep. During each sPMCA experiment, samples from both the scrapie-exposed and unexposed cohorts were analyzed at the same time; typically, the sonicator horn contained 40% of one group and 60% of the other group in any given experiment.

Amplified samples were digested with 50 μg/ml proteinase K (PK) and 0.045% (wt/vol) SDS for 1 h at 37°C before Western blot analysis using 12% (wt/vol) NuPAGE precast Bis-Tris gels as described previously (22). A reaction was scored as positive if there was a defined PK-resistant triplet (~18 to 27 kDa) visible on the Western blot; in each instance, positive signals were determined to be at least 3 times the signal for the negative samples run on that particular Western blot, as determined by Quantiscan software.

Murine bioassay was carried out with the Tg338 mouse line, which expresses ovine VRQ PrPC at high levels and is sensitive to small amounts of scrapie infectivity (13). Briefly, swab extracts from 12 VRQ/VRQ animals (12 swabs), taken 9 months after exposure to environmental scrapie or at 12 months of age from a scrapie-free flock, were extracted as described for sPMCA. The extracts were pooled, and PrPSc was recovered by ultracentrifugation at 100,000 × g for 1 h at 18°C. Pelleted material was washed with sterile water and recentrifuged three more times before resuspension of pelleted material in PBS. Ten mice were inoculated with swab extract from each swab pool. Each mouse received an extract equivalent to half that extracted from a single swab, as a 20-μl sample in phosphate-buffered saline administered intracranially into the right-hand side of the brain. Mice were monitored daily for signs of clinical disease. At postmortem, brain and spleen tissues were taken and processed as 10% (wt/vol) homogenates as previously described (22). These samples were analyzed by both sPMCA (up to 9 rounds) and direct Western blot analysis of the proteinase K-digested tissues.

Statistical analysis.

In order to place significance on the data generated, amplification results were grouped in terms of numbers of positive and negative reactions per genotype. These data were analyzed in 2 × 2 contingency tables, and one-tailed Fisher's exact test was applied to compare the number of positive reactions for samples from sheep with distinct genotypes with that for samples from either unexposed control animals or the scrapie-exposed ARR/ARR sentinel controls.

RESULTS

PrPSc was detected in buccal swabs taken from sheep with the VRQ/VRQ, ARQ/VRQ, ARR/VRQ, and AHQ/VRQ genotypes (Table 1; Fig. 1). These animals were all in the preclinical stages of scrapie incubation following natural exposure to the disease agent. No sample from the non-scrapie-exposed control sheep amplified any PrPSc; this group included 27 individual animals of 3 homozygous genotypes and some 135 individual amplification reactions. All exposed sheep cohorts, including the ARR/ARR sentinel controls, had levels of positivity that were significantly above this background (Fig. 2). Swabs from the ARR/ARR sentinel control animals, which were flock-mates of the scrapie-susceptible exposed sheep and had therefore been housed within the same scrapie-affected environment, showed a low level of prion amplification (3 of 9 animals were positive; 5 of 27 individual reactions [19%] were positive). This is a measure of the levels of prions taken up from the farm environment, and the figure correlates well with that previously reported for a similar control group from the same farm (17). The animals of the genotypes with the highest disease penetrance and the most associated LRS involvement during infection, the VRQ/VRQ and ARQ/VRQ animals, showed detectable prion positivity in buccal swab extracts that was significantly above that for samples from the sentinel control sheep (Fig. 2). In total, 78% of individual reactions from the VRQ/VRQ sheep and 81% of individual reactions from the ARQ/VRQ sheep were positive (representing 19 of 20 and 7 of 7 sheep, respectively). Most ARQ/VRQ animals were estimated to be of an age representative of the latter half of disease incubation. Samples from VRQ/VRQ animals were taken at estimated midterm incubation (11 animals) or at time points near the end of disease incubation (9 animals). These data concur with our previous report where prions were readily detected in such samples taken from VRQ/VRQ sheep within the latter half of the disease incubation period. The ARR/VRQ and AHQ/VRQ groups of sheep represent cohorts with lower associated disease penetrance and little involvement of the LRS. For the small group of AHQ/VRQ animals, PrPSc was detected in swabs taken from all 3 animals, with 44% of reactions being positive. However, while this was higher than the rate of prion amplification for the sentinel animals (19%), it was not significant (P = 0.134). This is a likely reflection of the small sample size that was used in this instance. For ARR/VRQ animals, 8 of 12 sheep contained amplifiable PrPSc in the oral cavity, with 53% of the reactions being positive. This was significantly higher than the rate found for sentinel animals (P = 0.005). Considering the genotypes associated with low-level (ARR/VRQ and AHQ/VRQ) and high-level (VRQ/VRQ and ARQ/VRQ) LRS accumulation of PrPSc as groups, both cohorts were significantly different from the ARR/ARR control exposed group (P = 0.005 and 0.001, respectively). Interestingly, these two groups also differed from each other, with that associated with less LRS involvement yielding significantly less prion amplification (P = 0.003).

Table 1
Presence of prions in buccal swabs, length of exposure of sheep to scrapie-positive farm environment, and sample collection timesc
Fig 1
Prion secretion into the oral cavity in sheep with a range of PRNP genotypes. Prions extracted from buccal swabs were used as a seed for 9 rounds of sPMCA. Each sample was analyzed in triplicate. Products were digested with PK, and 10-μl samples ...
Fig 2
Comparison of prion secretion into the oral cavity between sheep of different PRNP genotypes. Reaction data were plotted as the percentage of positive reactions for each distinct PRNP genotype or for groups of genotypes associated with low-level (AHQ/VRQ ...

In order to determine the presence of infectivity within sPMCA-positive buccal swab extracts, such samples were used to inoculate 10 Tg338 mice. Ten more mice were inoculated with identical extracts derived from non-scrapie-exposed sheep. At 176 days postchallenge, one mouse receiving the positive buccal swab extract had shown clinical signs of disease. This mouse was negative by immunohistochemistry (IHC) of the brain; however, the left side of the brain and the spleen were used for further analysis by both sPMCA and direct Western blotting. Such analyses were also carried out on tissues from an additional 4 animals receiving the scrapie-exposed extract and 5 animals receiving the negative-control extract, which were sacrificed at 310 days. Although it was IHC negative in the brain, the mouse showing clinical signs at day 176 displayed PK-resistant PrPSc in the spleen by Western blotting (Fig. 3). None of the other 4 mice sacrificed at 310 days were PrPSc positive in either the brain or spleen by Western blotting (data not presented). However, both brain and spleen tissues were sPMCA positive for the mouse with clinical signs, and 2 of the other 4 mice sacrificed at 310 days demonstrated sPMCA positivity in the spleen (n = 2) or brain (n = 1) (Table 2). All samples from the mice in the control cohort were sPMCA negative. The remaining 5 mice in each group are currently alive and not displaying any clinical signs (day 369).

Fig 3
(A) Western blots of sPMCA products from brain and spleen tissue in murine bioassay. Triplicate sPMCA analyses of brain and spleen samples from a scrapie-challenged mouse displaying clinical symptoms and an unchallenged mouse are indicated as positive ...
Table 2
Tg338 bioassay of mice challenged with buccal swab extracts from clinically normal, scrapie-exposed sheep with VRQ/VRQ PRNP genotypea

DISCUSSION

The data presented in this paper strongly suggest that the presence of PrPSc in the oral cavity of sheep is a common feature of sheep scrapie, regardless of the genotype of the host and the associated level of LRS involvement in PrPSc replication and dissemination. Sheep with genotypes associated with “high LRS involvement” in pathogenesis displayed higher levels of oral PrPSc detection than animals with “low LRS involvement.” It was previously speculated that prions might enter the oral cavity based on the reported presence of PrPSc in the lumina of the salivary ducts (26). It is also possible, however, that the origin of the PrPSc collected in buccal swabs results from the tissue of the buccal epithelium and possibly derives from innervation of the oral buccal surfaces (9). Another possibility is that this material is derived from a hematogenous origin (8). The contribution of the LRS to the secretion of prions into the oral cavity seems to be supported by the current study and the lower overall positivity of the “low-LRS” cohort. However, the lower level of positivity in low-LRS sheep could also be explained by the lower penetrance of scrapie in these genotypes: 20% of sheep with the ARR/VRQ or AHQ/VRQ genotype did not yield any amplifiable prions in their buccal swabs. This is much lower than the rate for VRQ/VRQ and ARQ/VRQ animals, where PrPSc was absent in samples from only 3% of animals. In addition, the stage of scrapie incubation at the time of sampling influences the level of prions present within the oral cavity (17). Samples taken in the earlier stages of disease incubation may contain fewer prions than samples taken later, for example, from the midpoint of disease incubation onwards (17). Overall, almost all samples (13 of 15 samples) were taken from the low-LRS group before the likely midpoint of disease incubation, whereas for the high-LRS group, most samples (16 of 27 samples) were taken much later in disease incubation. However, what the data do establish is that PrPSc is present within the oral cavity in preclinical scrapie-infected sheep with a range of PRNP genotypes, including those associated with limited LRS involvement in pathogenesis. This is in agreement with data showing the secretion of prions in the milk of sheep with these PRNP genotypes (16).

The observed presence of sPMCA-amplifiable prions in the ARR/ARR sentinel control swabs may suggest that low-level prion replication occurs in these highly scrapie-resistant animals. There have been two published observations of ARR/ARR sheep with classical scrapie (4), but such cases are extremely rare, and this has never been observed in the studied flock. There is the possibility, however, that low-level replication of prions within ARR/ARR animals could occur and never develop into clinical scrapie during the lifetime of these animals. Another explanation could be that the presence of prions in the oral cavity in these animals is due to uptake of prions from the contaminated environment (17). Interestingly, immunohistochemical assay and enzyme-linked immunosorbent assay (ELISA) of CNS and LRS tissues have shown that the incidence of scrapie in the AHVLA Ripley flock is low for ARR/VRQ and AHQ/VRQ animals, at just 12% for the ARR/VRQ genotype and just 4% for the AHQ/VRQ genotype (our unpublished observations). Here we demonstrate a far higher frequency of PrPSc detection (52% of sheep of these genotypes), suggesting that there is low-level replication of prions in sheep that do not develop clinical disease or accumulate measurable PrPSc by conventional testing. The very high sensitivity of sPMCA may well factor into the increased frequency of observation of positive animals; these data indicate that subclinical infections may be common in scrapie-exposed animals, even for ARR and AHQ PRNP heterozygotes.

Preliminary data from a small number of transgenic mice inoculated with buccal swab extract demonstrated that the sPMCA-positive material represents infectivity in the oral cavity. One animal in the bioassay exhibited clinical symptoms and was clearly Western blot positive for PrPSc in spleen tissue at 176 days, and this animal was also sPMCA positive in both brain and spleen samples (Fig. 3). The sPMCA method was applied to brain material from all bioassay mice, and 1 additional mouse of 5 tested was also positive at 310 days postchallenge. These data imply that amplification from the brain tissue in 2 of these mice was the result of prion replication within the CNS. The presence of amplifiable prions within the spleens of 3 of the bioassay mice is further evidence for the propagation of scrapie in this bioassay. The analogous use of sPMCA to extend the sensitivity of prion bioassays has been described before in the context of CWD prions in white-tailed deer (6). This methodology appears to detect very early signs of infection and is pertinent for studying very small amounts of infectivity via mouse bioassay, a scenario where incubation times to clinical disease are longer than the time limits of the bioassay.

Scrapie and CWD are examples of TSEs where it is known that environmental reservoirs of infectivity are involved in the horizontal transmission of disease. The contamination of the environment by infectious prion material from all sources represents a considerable problem for the effective management of these diseases. A number of publications now highlight the potential for CWD infectivity to be transmitted via saliva (5, 6, 18). Georgsson et al. (2) documented the survival of scrapie infectivity on a farm for a period of at least 16 years, and while this contamination was from a variety of sources, such as urine (3), feces (24), and parturient material (20), it is likely that infectivity from the oral cavity can contaminate items such as feed and water troughs and items of penning, as well as potentially directly transmitting disease between sheep. A fuller understanding of all the routes of transmission of scrapie and CWD will greatly assist in the management of these diseases.

ACKNOWLEDGMENTS

We thank the staff at the Ripley Farm (AHVLA, Weybridge, United Kingdom) and at the Arthur Rickwood Sheep Unit (ADAS, Cambridge, United Kingdom) for sample collection, Richard Lockey and Ian Dexter for preparation and inoculation of the Tg338 mice, members of the animal services unit at the AHVLA for mouse monitoring and postmortem, Charlotte Cook for information on disease penetrance within the Ripley sheep flock, the histopathology department at the AHVLA for processing and IHC analysis of brains, and members of the mouse bioassay team for interpretation. We thank Hubert Laude for the Tg338 mice.

This work was funded by the UK Department for Food and Rural Affairs, under project SE2008.

Footnotes

Published ahead of print 19 October 2011

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