Scrapie Agent (Strain 263K) can transmit disease via the oral route after persistence in soil over years

Abstract

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 PrP(Sc) 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 PrP(Sc) 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.

Figure 1. Western blot analysis of short-time incubation experiments.

a) Western blot detection of PrP^C 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 PrP^Sc, extracted by using 1% SDS from soil contaminated with 263 K scrapie brain homogenate from hamsters after 1 h of incubation (dilution series). PrP^Sc 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.

Figure 2. Western blot analysis of long-time incubation experiments.

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 PrP^Sc 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.

Figure 3. Western blot detection of PrP27-30 extracted from prion-contaminated soil after 21 months (M21) and respective surrounding samples.

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.

Figure 4. Western blot analysis of hamsters orally challenged with contaminated soil and western blot typing of PrP27-30.

a) Western blot showing PrP^Sc 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).