Conceived and designed the experiments: MB AT MG AB BS RP KT. Performed the experiments: AT AB BS RP. Analyzed the data: MB AT MG AB BS RP KT. Contributed reagents/materials/analysis tools: MB AT. Wrote the paper: MB AT MG AB BS KT.
The authors have declared that no competing interests exist.
The persistence of infectious biomolecules in soil constitutes a substantial challenge. This holds particularly true with respect to prions, the causative agents of transmissible spongiform encephalopathies (TSEs) such as scrapie, bovine spongiform encephalopathy (BSE), or chronic wasting disease (CWD). Various studies have indicated that prions are able to persist in soil for years without losing their pathogenic activity. Dissemination of prions into the environment can occur from several sources, e.g., infectious placenta or amniotic fluid of sheep. Furthermore, environmental contamination by saliva, excrements or non-sterilized agricultural organic fertilizer is conceivable. Natural transmission of scrapie in the field seems to occur via the alimentary tract in the majority of cases, and scrapie-free sheep flocks can become infected on pastures where outbreaks of scrapie had been observed before. These findings point to a sustained contagion in the environment, and notably the soil. By using outdoor lysimeters, we simulated a contamination of standard soil with hamster-adapted 263K scrapie prions, and analyzed the presence and biological activity of the soil-associated PrPSc and infectivity by Western blotting and hamster bioassay, respectively. Our results showed that 263K scrapie agent can persist in soil at least over 29 months. Strikingly, not only the contaminated soil itself retained high levels of infectivity, as evidenced by oral administration to Syrian hamsters, but also feeding of aqueous soil extracts was able to induce disease in the reporter animals. We could also demonstrate that PrPSc in soil, extracted after 21 months, provides a catalytically active seed in the protein misfolding cyclic amplification (PMCA) reaction. PMCA opens therefore a perspective for considerably improving the detectability of prions in soil samples from the field.
Transmissible spongiform encephalopathies (TSEs) comprise a group of fatal neurodegenerative diseases such as bovine spongiform encephalopathy (BSE) in cows
Among the known TSEs, only scrapie and CWD are contagious diseases which show horizontal transmissibility under natural conditions
The contamination of soil with TSE infectivity can occur from several sources. Since recent studies could demonstrate that scrapie infectivity is present in various tissues and body fluids of infected animals
So far, the oral transmission efficacy of long-term prion contaminations in soil have not been investigated. Therefore, we have studied the persistence of PrPSc in the environment over time and measured its oral transmissibility by bioassay in Syrian hamsters. With outdoor lysimeter experiments we simulated the situation on pastures using soil spiked with scrapie-infected hamster brain homogenate over a period of 29 months and analyzed the fate of the prion proteins by sensitive Western blotting and, in part, also by protein misfolding cyclic amplification (PMCA). The infectivity of such contaminated soil samples and the respective aqueous soil extracts was tested in the hamster bioassay.
In the first phase of the study several buffers – known as standard buffers for protein extraction from mammalian cells – and other solutions were tested in order to find out the optimal extraction method for prion protein from contaminated soil (see
a) Western blot detection of PrPC extracted from soil mixed with non-infectious brain homogenate (5% pork brain in German standard soil). Several different buffers and solutions were used for extraction. Lane 1: water; lane 2: Triton X-100; lane 3: 1% urea; lane 4: 1% SDS; lane 5: Zwittergent; lane 6: RIPA buffer; lane 7: NP-40; lane 8: Na-sarcosyl. b) Western blot detection of PrP27-30, the proteinase K-resistant core of PrPSc, extracted by using 1% SDS from soil contaminated with 263 K scrapie brain homogenate from hamsters after 1 h of incubation (dilution series). PrPSc could be detected in soil samples containing 1.25 µg or higher amounts of scrapie brain tissue after extraction with 1% SDS-solution. Samples were digested with proteinase K prior to Western blotting.
The findings from our lysimeter experiments indicated a remarkable persistence of PrPSc in soil by clearly showing that - even after an incubation for 29 months - PrP
a) Western blot detection of PrP27-30 extracted from prion-contaminated soil after different time periods. Lane 1: PK-digested 263K scrapie hamster brain homogenate containing 5×10−7 g of brain tissue (positive control); lanes 2–10: PrP27-30 extracted at time point 0 (lane 2), after 1 month (lane 3), after 3 months (lane 4), after 6 months (lane 5), after 12 months (lane 6), after 18 months (lane 7), after 21 months (lane 8), after 26 months (lane 9) and after 29 months (lane 10). b) Deglycosylated PrP27-30 extracted from prion contaminated soil. Lane 1: PK-digested 263K scrapie hamster brain homogenate containing 5×10−7 g of brain tissue (positive control); lane 2: soil-extracted PrP27-30 after 21 months; lanes 3 and 4: deglycosylated soil-extracted PrP27-30 after 21 months (lane 3) and after 18 months (lane 4). c) PMCA amplification of PrPSc extracted from contaminated soil. Lane 1: PK-digested 263K scrapie hamster brain homogenate containing 5×10−7 g of brain tissue (positive control); lanes 2–6: sample signals after 0, 40, 80, 120 and 160 cycles of PMCA, respectively.
In order to check whether PrPSc extracted from soil can be used for the amplification of protease resistant prion protein by the protein misfolding cyclic amplification (PMCA) reaction
For analyzing the fate of the prion protein in more detail, soil samples in the vicinity of the gauze bag, as well as the gauze bag itself were analyzed by Western blotting. As shown in
M21: contaminated soil sample inside the gauze bag; lane 1: soil sample collected outside of the steel cage; lane 2: soil sample collected directly over the gauze bag; lane 3: analysis of the empty gauze bag; lane 4: soil sample collected underneath the steel cage; lane 5: soil sample collected directly next to the gauze bag; lane 6: roots collected next to the gauze bag; lane 7: soil sample collected underneath the gauze bag; lane 8: non-contaminated soil. Arrow head at lane 3 indicates a faint PrP27-30 signal, resulting from residual soil particles that remained attached to the gauze bag.
For a detailed risk assessment of scrapie-contaminated soil it was of major importance to analyze whether the detectable PrPSc in the soil extracts still exhibited oral infectivity after incubation times up to 29 months. Therefore, a bioassay with Syrian hamsters was performed by feeding the animals with contaminated soil or aqueous soil extracts that had been collected after soil incubation for 26 and 29 months, respectively. Hamsters fed with contaminated soil exhibited first scrapie-associated symptoms at 131±6 days [mean±SD] after the first application. The hamsters reached the terminal stage of scrapie at 162±12 days after the first feeding (
a) Western blot showing PrPSc in the brains of hamster orally challenged with contaminated soil samples and in the soil samples used for the bioassay. Lane 1: negative control hamster; lane 2: hamster H19 fed 12 times with aqueous extracts of soil samples from month 26 and 29; lane 3: hamster H4B fed 12 times with soil samples from month 26 and 29; lane 4: scrapie-contaminated soil (18 months); lane 5: scrapie-contaminated soil (21 months); lane 6: negative soil sample. b) Western blot typing of electrophoretic mobilities and glycosylation characteristics of PrP27-30 from different hamster-adapted TSE reference isolates, and from hamsters perorally challenged with 263K scrapie-contaminated soil. Lane 1: ME7-H scrapie agent; lane 2: hamster-adapted BSE-isolate (BSE-H); lane 3: 263K scrapie agent, lane 4: H10 (hamster 10, fed 12 times with soil samples from months 26 and 29); lane 5: H7 (hamster 7 fed 12 times with soil samples from month 26 and 29).
Date | amount of soil or aqueous extract administered orally | Collection time of soil sample |
31/01/06 | 100 mg or 100 µl | month 26 |
07/02/06 | 100 mg or 100 µl | month 26 |
14/02/06 | 100 mg or 100 µl | month 26 |
21/02/06 | 100 mg or 100 µl | month 26 |
28/02/06 | 100 mg or 100 µl | month 26 |
08/03/06 | 100 mg or 100 µl | month 26 |
14/03/06 | 100 mg or 100 µl | month 26 |
21/03/06 | 50 mg or 50 µl | month 26 |
28/03/06 | 100 mg or 100 µl | month 29 |
06/04/06 | 100 mg or 100 µl | month 29 |
11/04/06 | 100 mg or 100 µl | month 29 |
20/04/06 | 100 mg or 100 µl | month 29 |
mg relates to soil sample; µl relates to aqueous extract
Group | No. of animals infected with scrapie | Terminal stage of scrapie after the first oral application/Mean incubation period with the respective standard deviation in days |
Control | 0/5 | – |
Control soil | 0/5 | – |
Control aqueous extract | 0/5 | – |
Contaminated soil | 12/12 | 162 +/− 12 |
Contaminated aqueous soil extract | 4/11 | 256 +/− 41 |
In the marked groups one animal each died at the beginning of the bioassay because of digestive disorders unrelated to scrapie disease
Remarkably, not only the hamsters fed with contaminated soil but also four hamsters of the group fed with aqueous soil extracts developed terminal scrapie at 256±41 days after the first application (with incubation times of individual animals ranging from 201 to 321 days). Since this bioassay is ongoing, further animals of this group might still develop scrapie in the future.
The results of this research project show for the first time that the scrapie strain 263K remains persistent in soil over a period of at least 29 months and remains highly infectious after oral application to Syrian hamsters. It has to be pointed out that the key results of our time-course study on the fate of PrPSc in soil have been validated, in part by examining blinded samples, at independent laboratories.
Only a few studies have addressed the question of a persistence of prions in soil so far
In this study we show by Western blotting a strong decrease in the amount of extractable PrPSc over an incubation period of 29 months in soil. It is not yet clear whether this decrease resulted from a molecular degradation of PrPSc or a tighter binding to soil particles. Stronger binding of molecules to soil particles with increasing incubation time is a well-known phenomenon in soil chemistry – the so called “aging” – and influences bioavailability and re-mobilization significantly
Upon feeding hamsters with scrapie contaminated soil which had been incubated for over two years in outdoor lysimeters all animals developed terminal scrapie after relatively short incubation times (162 dpi). In other studies it has been well established that pure 10% (w/v) brain homogenates from 263K scrapie hamsters cause terminal scrapie in perorally challenged hamsters after mean incubation times of about 155–165 days with an attack rate of 100%
However, the relevance of the results obtained in this study for the field situation should be interpreted with some caution, since only one soil type was used and only a limited number of animals were challenged in the bioassay. Therefore, other soil types and a larger number of animals have to be tested in future studies to allow for a robust risk assessment. Furthermore the exact binding properties and degradation kinetics of PrPSc should be subject to further research. In addition, all published studies addressing the persistence of prion infectivity in soil were performed with scrapie prions while TSE agents causing BSE and especially CWD have not been analyzed so far.
An intensified monitoring of PrPSc (and possibly also prion infectivity) in the soil appears mandatory for a more precise assessment of the risks emanating for humans and animals from prions in the environment. As shown in this report, PrPSc extracted from soil can be used as a catalytically active seed in the protein misfolding cyclic amplification (PMCA) reaction. This opens a promising perspective for considerably improving the detectability of prions in the environment.
Assays with scrapie-infected hamster brain were performed under laboratory conditions according to bio safety level 3** and in protected outdoor lysimeters, respectively.
To identify a suitable buffer for extracting prion infectivity and PrPSc from soil, German standard soil (Lufa 2.2 and Borstel) was mixed with scrapie-infected hamster brain (strain 263K) provided by the TSE-Resource-Centre, Berkshire, Great Britain. To test the efficiency of this method the following solutions and buffers - especially non-ionic and ionic detergents - have been tested:
a) sterile water, b) 1% urea in sterile water c) 1% SDS in sterile water, d) 1% Zwittergent 3-08 in sterile water, e) 1% Triton X-100 sterile water, f) 10% Na-sarcosylate in sterile water, g) RIPA-buffer (0.25% Na-deoxycholate, 0.9% NaCl, 1% NOP-40, 0.8% Tris-HCl in sterile water (Carl Roth GmbH, Karlsruhe, Germany), h) 10% NOP-40 in sterile water, (Sigma-Aldrich, Steinheim, Germany).
Initially non-infectious pork brain was mixed with German standard soil and the efficiency of PrPC extraction using the above mentioned buffers was tested. In a second step, the optimal extraction procedure was verified by applying the procedure to soil samples mixed with 263K scrapie agent and monitoring the PrPSc retrieval.
The outdoor experiments and all other experiments were performed using brains of terminally-diseased hamsters challenged with scrapie strain 263K.
For each approach, 1 g infectious hamster brain material was homogenized in 10 ml PBS (phosphate buffered saline) and added to 20 g of standard German sandy loom soil (Borstel). The soil/brain mixture was filled into gauze bags and buried in lysimeters filled with the same soil at a depth between 15 and 20 cm. The gauze had a mesh size of 250 µm, which enables the contact with microorganisms and meso fauna with the soil/brain mixture but avoids the contact with macro fauna derived/related organisms. To protect the gauze bags from mice, the bags were put into steel cages. At defined time points (after 0, 1, 3, 6, 12, 18, 21, 26, 29 months) the steel cages were dug out and the gauze bags as well as the surrounding soil were taken for sampling and analyzing. The analyses on the presence of residual PrP27-30 in soil samples were performed with a different number of soil bags, depending on the incubation times to be tested. For “short” incubation times of <12 months, for which detectability of residual PrP appeared most likely when the experiments were designed, samples from three different soil bags were examined. For “intermediate” incubation times of 12–18 months samples from two different bags were tested, and for “long” incubation times of >18 months one soil bag was yet available for sampling. All bags were buried in close vicinity.
Western blot experiments were performed in independent runs at different laboratories, with each laboratory using its established techniques and procedures. The protocols are listed below. The different analytical protocols produced consistent results.
For the protein extraction from the contaminated soil as well as from surrounding soil samples, 20 ml of a 1%-SDS-solution (SDS; Sigma-Aldrich, Steinheim, Germany) were added to 20 g of testing material in a 50 ml tube. The suspension was vigorously shaken on a horizontal shaker for approx. 1 h, followed by a centrifugation step at 5,000 rpm for 20 min. 200 µl of the clear supernatant was incubated with proteinase K (50 µg/ml; 37°C; 1 h, Carl Roth GmbH, Karlsruhe, Germany) to eliminate non-resistant proteins. After digestion, the supernatants were boiled for 5 min in Laemmli's sampling buffer (50 mM Tris (pH 6.8), 2% SDS, 10% glycerol, 50 mM ß-mercaptoethanol and 0.001% bromphenol blue) in a 1.5 ml tube and analyzed by Western blotting. The prepared samples were stored at −80°C.
For screening, Western blot samples were boiled for 5 min and separated by polyacrylamide gel electrophoresis (SDS-PAGE) by using 8–16% Tris-Glycine-SDS precast gels (i-Gels, Gradipore, LTF-Labortechnik, Wasserburg, Germany) or 4–20% precast gels (Precise Protein Gels, Perbio Science GmbH, Heidelberg, Germany) according to standard procedures as described previously
For confirmation of these results, highly sensitive Western blotting was performed in independent experiments at different laboratories using their established protocols.
The protein extractions from contaminated soil samples were carried out by adding 2 ml 1%-SDS-solution to 2 g of testing material in a 15 ml tube. The suspension was vigorously shaken for 2 min, followed by a centrifugation step at 7,000 rpm for 10 min. 50 µl of the clear supernatant was used for proteinase K digestion (100 µg/ml; 37°C; 1 h). After digestion the supernatants were mixed with an equal volume of 2× sample loading buffer (50 mM Tris (pH 6.8), 2% SDS, 10% glycerol, 50 mM ß-mercaptoethanol and 0.001% bromphenol blue) and heated to 100°C for 5 min for PrP-Western blotting.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyzes of samples from hamsters were performed as described elsewhere
For deglycosylation, 500 µl of the soil extract solution were dialyzed in PBS for 16 h in order to remove the detergents, and 20 µl of the aliquot was digested using PNGase F (New England Biolabs, Ipswich, USA) according to the instructions of the manufacturer prior to Western blotting.
Sample preparation and extraction of the prion protein from soil samples as well as proteinase K digestion was performed as described in protocol I. After electrophoresis using 16% bis-acrylamide gels, proteins were transferred on a PVDF-membrane in a semi-dry chamber. The membranes were blocked in 5% dry fat milk in PBS (phosphate buffered saline) containing 0.1% Tween 20 (Merck, Darmstadt, Germany) (PBS-T) for 30 min and subsequently incubated with the PrP-specific monoclonal detection antibody 3F4 (Chemicon International, Inc., California) in a dilution of 1∶3,000 in 5% dry fat milk in PBS-T for 1 h 30 min. The membranes were washed 3 times for 10 min with PBS-T and then incubated with a secondary antibody bound to alkaline phosphatase in a dilution of 1∶2,000 in PBS-T (goat-anti-mouse-AP, Dianova, Hamburg, Germany) for 1 h. After again washing three times for 10 min, the membranes were incubated 2 times for 2 min in assay buffer containing 200 mM Tris-HCl and 10 mM MgCl2 (pH 9.8). Finally, the chemiluminescence substrate CDP-Star (Tropix, Bedford, USA) was applied and incubated on the membrane for 5 min before the light signals were detected in a camera using the analysis software Quantity One (Bio-Rad, Munich, Germany).
Forty two female Syrian hamsters, approx. 90 days old, were obtained from Charles River Laboratories, Germany. They were handled according to the regulations of the local authorities (Bezirksregierung Arnsberg, reference number 50.8735.1 Nr. 108/1) in a biosafety Level 3 containment facility.
12 animals were fed weekly over a period of 12 weeks; 11 times with 100 mg soil/brain mixture each and one time with 50 mg soil/brain mixture taken from the outdoor lysimeters after an incubation period of 26 and 29 months (
To analyze the remaining infectivity in the aqueous extract, 10 g contaminated soil was mixed with 10 ml water and was vigorously shaken on a horizontal shaker for approx. 1 h. The mixture was centrifuged with 5,000 rpm for 5 min and the supernatant was used for the bioassay. Oral application was performed weekly over a period of 12 weeks (1,150 µl in total) by mixing the extract with commercial hamster feed (ssniff, Soest, Germany). For this purpose, 12 additional animals were fed 11 times with 100 µl and 1 time with 50 µl aqueous extract from the soil/brain mixture taken from the outdoor lysimeters after an incubation period of 26 and 29 months.
The hamsters of both groups were monitored at least twice a week for the development of clinical signs of scrapie. Hamsters diseased with 263K scrapie showed head bobbing, ataxia of gait and generalized tremor. Such animals were frequently and persistently in motion, easily irritated by noise and touch, upon which they often twitch, and had difficulties maintaining balance and rising from a supine position. These clinical symptoms of hamsters are entirely consistent with those previously reported for the 263K scrapie agent
As a control, six animals were fed with non-contaminated standard soil and 6 animals were fed with aqueous extract from non-contaminated standard soil over a period of 12 weeks (1,150 mg or 1,150 µl in total). Additional 6 hamsters were fed only with commercial hamster feed (ssniff, Soest, Germany).
The PMCA method was carried out as reported previously
The PrPC substrate was mixed with 1/10 volume of the soil extract previously dialyzed in PBS, resulting in a total volume of 100 µl. PMCA amplification was performed by 40 cycles of sonication (40 sec. each) followed by incubation at 37°C for 1 h in the water-tank. The amplified product of the first round of amplification was diluted 1∶5 with normal brain homogenate and the second round was performed. This process was repeated 4 times to obtain 160 cycles of PMCA. From each PMCA-amplification round, aliquots of 50 µl were taken and digested with proteinase K (180 µg/ml) for 1 h at 56°C. Finally, the same volume of 2× sample buffer was added and heated for 10 min at 100°C prior to SDS-PAGE.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyzes of samples from hamsters for characterization of the PrPSc glycosylation and migration pattern were performed as described elsewhere
We thank Pam Schulte and Uwe Boshof from the Fraunhofer IME for their technical assistance especially for helping conduct the bioassay. The skilful technical assistance of Patrizia Reckwald (RKI) and of Dan Balkema (FLI) is gratefully acknowledged. A.T. and M.B. wish to thank Anja Schreiter and Corina Loeschner for their help in setting up the PMCA procedure used in this study at the RKI.