Edited by: Scott Napper, University of Saskatchewan, Canada
Reviewed by: Franck Carbonero, University of Arkansas, USA; Jason Kindrachuk, National Institutes of Health, USA
Specialty section: This article was submitted to Veterinary Infectious Diseases, a section of the journal Frontiers in Veterinary Science
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Classical scrapie is an environmentally transmissible prion disease of sheep and goats. Prions can persist and remain potentially infectious in the environment for many years and thus pose a risk of infecting animals after re-stocking.
Classical scrapie is a transmissible spongiform encephalopathy (TSE) of sheep and goats, which is caused by infection with a proteinacious infectious particle, the prion (
Soil has been implicated as plausible reservoir of scrapie prions because laboratory studies have shown that scrapie-associated prion protein (PrPSc) can bind to soil particles, which may remain close to the surface where it was released so that it is accessible to grazing animals (
While contamination of the environment had historically been mainly attributed to prions in placentae from scrapie-infected ewes (
Prions were detected by serial protein misfolding cyclic amplification (sPMCA) in swabs taken from water troughs and fences that had been present on a scrapie-infected sheep farm, and it was hypothesized that they could act as environmental reservoirs to contribute to scrapie transmission (
All procedures were in accordance with the United Kingdom (UK) Animal (Scientific Procedures) Act 1986, under license from the UK Government Home Office (project licenses 70/6774 and 70/7782) following approval by the institutional ethical review process.
A scrapie research flock was established in 1998 by purchasing clinically healthy sheep from scrapie-affected flocks with scrapie-susceptible genotypes and maintained through breeding sheep of susceptible genotypes (
Testing for infectivity was carried out using Cheviot sheep from a flock that was originally generated from imported sheep from New Zealand and kept free of classical scrapie through strict biosecurity measures (
Field furniture that had been in contact with the scrapie flock 8 weeks earlier was used. Selection of items was to some degree influenced by the results from an earlier study where environmental sources of scrapie prions were investigated by sPMCA and which had demonstrated the presence of PrPSc on a fire extinguisher box (referred to as plastic scratching post in Reference
For Pen 1. Five lambs in a pen with clean furniture (building controls). Pen 2. Six lambs in a pen with a water trough from a pasture used mainly by subclinically infected sheep of the scrapie flock. Pen 3. Six lambs in a pen with a water trough from a pasture, which was used by clinically affected sheep prior to cull. Pen 4. Seven lambs in a pen with a wooden fence post, fencing with traces of fleece from sheep attached to it and a fire extinguisher box. This box had been fixed outside an area where the sheep from the scrapie-infected flock would gather just before entering the handling area and had been used by sheep to rub their backs and head.
None of the items were cleaned but the water troughs were emptied and filled with new water in the animal accommodation. All items placed into pens 2–4 were swabbed and analyzed by sPMCA in triplicate before arrival of the sheep. Swabs were taken from analogous surfaces to the scrapie-contaminated farm, e.g., water troughs, metal hurdles, etc., at the farm housing the classical scrapie-free flock to serve as control samples, which were tested in parallel with the samples from the scrapie-contaminated objects.
The pens shared common air space but were separated by 3 m high concrete walls, and each pen had its own entrance and separate equipment and protective clothing to avoid cross-contamination.
Scrapie infection was monitored by rectal biopsy at approximately 6, 9, 13, and 19 months post exposure (mpe). Biopsies were taken under local anesthesia with a prilocaine and lidocaine mixture (EMLA cream; AstraZeneca, UK) and the recto-anal mucosa-associated lymphoid tissue (RAMALT) examined for presence of disease-associated prion protein (PrPSc) with rat monoclonal antibody R145 (APHA, UK) according to previously published methods (
Scrapie infection was determined postmortem by immunohistochemical examination of lymphoreticular tissue (RAMALT, distal ileum, mesenteric lymph node) in all animals and – in all sheep over 12 months of age – additional examination of brain tissue (right and left half of the obex) for PrPSc by immunohistochemistry (
For the field study, a 5.4 ha-sized pasture that had been occupied by scrapie sheep was divided into four equal paddocks, with two paddocks (termed 1 and 2) used for the reported study. Each paddock, measured approximately 13,000 m2, was double fenced with new fencing, with a minimum of 2 m between the fences to prevent nose-to-nose contact between sheep in different paddocks. Each paddock had its own set of protective clothing for farm workers and equipment.
Contamination of the soil in both paddocks used for the reported study was previously investigated by moving groups of six 2-day-old lambs with their dams to these paddocks, each equipped with a new water trough. None of the six lambs in each group had detectable PrPSc in a rectal biopsy taken at 6 and 9 mpe and although they were subsequently moved to a different paddock at 10 mpe, none of the sheep presented with detectable PrPSc in brain and lymphoid tissue when culled at 34 months of age.
Contaminated field furniture added to paddock 1, which by that time had not been grazed by scrapie-affected sheep for 40 months, were metal hurdles, a metal lamb creep and a water trough, replacing the previous one, which all had been in contact with the scrapie flock 8 weeks earlier.
The paddock was occupied by 24 lambs aged 1–4 days (67% aged 2 days) with their dams, with the exception of one ewe that arrived with a 14-day-old lamb delivered by cesarean section. Dams were removed 3–4 months later and not tested for scrapie. Rectal biopsies were taken at approximately 9 mpe and examined as described above. Based on the RAMALT results (see
Swabs were taken as described above from water trough, fence, wooden post, hurdles, and lamb creep 5 days after the first sheep were introduced in the paddock and examined for PrPSc by sPMCA in duplicate reactions.
Twelve 18- to 28-day-old lambs (median 21 days) were moved with their dams to paddock 1, which had been occupied up to 7 days previously by sheep to test for infectivity of contaminated furniture (see above), but clean furniture was now provided (water trough from a paddock not used by scrapie sheep before, fencing replaced). The increase in age was due to poor weather conditions at the time of lambing. Dams were removed as before after 3–4 months. The sheep were culled from 11 mpe and tissues examined by TSE postmortem tests, as described above.
Concurrently with the furniture replacement study, 13 12- to 25-day-old lambs (median 21 days) moved with their dams to paddock 2, which had been grazed by the natural scrapie flock 53 months previously, and contained a water trough and additional objects (lamb creep and hurdles used during lambing of the scrapie flock) from the scrapie-infected flock, which had not been used by these sheep for 18 months. Removal of dams and TSE testing protocol was identical to the furniture replacement study.
Where appropriate infection rates between groups were compared by Fisher’s exact test (Prism 6, GraphPad Software, USA), with
The results of the sheep studies are summarized in Table
Location | Exposure to | Rate of infection ( |
---|---|---|
Pen 1 | Clean furniture (control) | 0/5 |
Pen 2 | Water trough (used by predominantly pre-clinical scrapie sheep) | 0/6 |
Pen 3 | Water trough (used also by clinically affected scrapie sheep) | 4/5 |
Pen 4 | Scratch post, fence, fire extinguisher box | 4/7 |
Paddock 1 |
Metal hurdles, metal lamb creep, water trough not used by scrapie flock for 8 weeks on pasture not used by scrapie flock for 40 months | 20/23 |
Paddock 1 | Water trough not used by scrapie sheep before and new fencing on pasture not occupied by pre-clinical scrapie sheep for a week | 1/12 |
Paddock 2 |
Water trough, lamb creep and hurdles, not used by the scrapie flock for 18 months on pasture not occupied by the scrapie flock for 53 months | 0/13 |
Infection of sheep was first demonstrated in RAMALT at 9 mpe in a water trough-exposed group (water trough previously exposed to clinically infected sheep) and at 13 mpe in the rubbing objects-exposed group. Scrapie was confirmed in four sheep on brain examination by immunohistochemistry (three exposed to the water trough, one to rubbing objects) but only one was also confirmed by ELISA. This sheep was the only sheep that presented with clinical signs prior to cull at 23 mpe. Evidence of infection in the other sheep was based on presence of PrPSc in lymphoid tissue only.
Despite evidence of infection in the sheep exposed to one of the water troughs and the scratch objects, the difference compared to the control group was statistically not significant (
PrPSc was detected by sPMCA in swabs taken from water trough and fencing (see Table
Location | Object | sPMCA result ( |
---|---|---|
Pen 2 | Water trough used mainly by pre-clinical scrapie cases | |
Sample 1 | 0/3 | |
Sample 2 | 0/3 | |
Pen 3 | Water trough used by clinically affected scrapie cases | |
Sample 1 | 0/3 | |
Sample 2 | 1/3 | |
Pen 4 | Fire extinguisher box | 0/3 |
Fencing with traces of fleece attached to it | ||
Sample 1 | 0/3 | |
Sample 2 | 1/3 | |
Fence post | ||
Sample 1 | 0/3 | |
Sample 2 | 0/3 | |
Paddock 1 | Water trough | 0/2 |
Fence | 0/2 | |
Wooden post | 0/2 | |
Hurdles | 0/2 | |
Lamb creep | 0/2 |
PrPSc was first detected in RAMALT of 18 (78%) sheep. When the sheep were culled 3 months later at 11–12 mpe, scrapie was confirmed by postmortem tests on lymphoid tissue in 20 (87%), six of which also had PrPSc in the obex (no detectable PrPres by ELISA). Figure
None of the swabs taken from the furniture yielded detectable PrPSc by sPMCA (Table
Scrapie infection was confirmed in a single sheep (8%), which presented with PrPSc in lymphoid tissue only. Compared with exposure of sheep to contaminated furniture in the same paddock, this was a significant reduction in the infection rate (
Exposure to objects not used by scrapie-infected sheep for 18 months did not result in any infection.
Classical scrapie is an environmentally transmissible disease because it has been reported in naïve, supposedly previously unexposed sheep placed in pastures formerly occupied by scrapie-infected sheep (
Our study suggested that the risk of acquiring scrapie infection was greater through exposure to contaminated wooden, plastic, and metal surfaces via water or food troughs, fencing, and hurdles than through grazing. Drinking from a water trough used by the scrapie flock was sufficient to cause infection in sheep in a clean building. Exposure to fences and other objects used for rubbing also led to infection, which supported the hypothesis that skin may be a vector for disease transmission (
This study also indicated that the level of contamination of field furniture sufficient to cause infection was dependent on two factors: stage of incubation period and time of last use by scrapie-infected sheep. Drinking from a water trough that had been used by scrapie sheep in the predominantly pre-clinical phase did not appear to cause infection, whereas infection was shown in sheep drinking from the water trough used by scrapie sheep in the later stage of the disease. It is possible that contamination occurred through shedding of prions in saliva, which may have contaminated the surface of the water trough and subsequently the water when it was refilled. Contamination appeared to be sufficient to cause infection only if the trough was in contact with sheep that included clinical cases. Indeed, there is an increased risk of bodily fluid infectivity with disease progression in scrapie (
PrPSc detection by sPMCA is increasingly used as a surrogate for infectivity measurements by bioassay in sheep or mice. In this reported study, however, the levels of PrPSc present in the environment were below the limit of detection of the sPMCA method, yet were still sufficient to cause infection of in-contact animals. In the present study, the outdoor objects were removed from the infected flock 8 weeks prior to sampling and were positive by sPMCA at very low levels (2 out of 37 reactions). As this sPMCA assay also yielded 2 positive reactions out of 139 in samples from the scrapie-free farm, the sPMCA assay could not detect PrPSc on any of the objects above the background of the assay. False positive reactions with sPMCA at a low frequency associated with
In conclusion, the results in the current study indicate that removal of furniture that had been in contact with scrapie-infected animals should be recommended, particularly since cleaning and decontamination may not effectively remove scrapie infectivity (
TK analyzed the data and drafted the manuscript. HS designed and managed the study, supported by SH, LT, and TD who participated in the coordination of different parts of the study. BM and KG were responsible for the sPMCA investigations. All authors contributed to the final draft of this manuscript and read and approved the final manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The study was funded by the UK Department for Environment, Food and Rural Affairs (project SE1861).
We are grateful to present and past members of staff at the Animal Sciences Unit at APHA Weybridge who cared for the animals, and to staff at the Pathology Department and Prion unit at APHA Weybridge who provided the scrapie diagnostic results. We thank staff at ADAS-UK for provision of classical scrapie-free lambs.