Persistence of pathogenic prion protein during simulated wastewater treatment processes

Abstract

Transmissible spongiform encephalopathies (TSEs, prion diseases) are a class of fatal neurodegenerative diseases affecting a variety of mammalian species including humans. A misfolded form of the prion protein (PrP(TSE)) is the major, if not sole, component of the infectious agent. Prions are highly resistant to degradation and to many disinfection procedures suggesting that, if prions enter wastewater treatment systems through sewers and/or septic systems (e.g., from slaughterhouses, necropsy laboratories, rural meat processors, private game dressing) or through leachate from landfills that have received TSE-contaminated material, prions could survive conventional wastewater treatment. Here, we report the results of experiments examining the partitioning and persistence of PrPTSE during simulated wastewater treatment processes including activated and mesophilic anaerobic sludge digestion. Incubation with activated sludge did not result in significant PrPTSE degradation. PrPTSE and prion infectivity partitioned strongly to activated sludge solids and are expected to enter biosolids treatment processes. A large fraction of PrPTSE survived simulated mesophilic anaerobic sludge digestion. The small reduction in recoverable PrPTSE after 20-d anaerobic sludge digestion appeared attributable to a combination of declining extractability with time and microbial degradation. Our results suggest that if prions were to enter municipal wastewater treatment systems, most would partition to activated sludge solids, survive mesophilic anaerobic digestion, and be present in treated biosolids.

FIGURE 1

PrP^TSE extraction efficiency from aerobic and anaerobic sludge using 1% SDS. Brain homogenate (BH) or proteinase K (PK)-treated BH was incubated with each sludge and extracted using 1% SDS and vortexing. Extracted protein was compared to starting material by immunoblot analysis and densitometry. A minimum of three independent replicates were used for each experiment. (A) Extraction from aerobic sludge, (B) Extraction from anaerobic sludge, (C) Densitometric measurements of extraction of full-length and truncated PrP from each sludge relative to the highest densitometric value observed. Error bars represent the standard deviation of these densitometric measurements. A.U. = absorbance units

FIGURE 2

(A) Partitioning of PrP^TSE between supernatant and activated sludge solids. (B) Immunoblot sensitivity. PrP^TSE was semiquantitatively assessed by serial dilution to the limit of immunoblotting detection. The dilution at which no detectable immunoreactivity remained provided a basis for comparison with samples lacking immunoreactivity before dilution. Abbreviations: PK, proteinase K; Solids, settled activated sludge solids; Sup, supernatant (PrP^TSE not associated with settled activated sludge solids).

FIGURE 3

Extractability and partitioning of PrP^TSE in un-manipulated and manipulated anaerobic digester sludges. Abbreviations: SDS, extraction from settled anaerobic digester solids with 1% SDS; Sup, supernatant fraction from settled anaerobic digester solids.

FIGURE 4

(A) Flow chart of simulated mesophilic anaerobic sludge experiments. (B) PrP^TSE recovery after 0-20 day incubations with un-manipulated and manipulated anaerobic digester sludge. Day indicates the time infected brain homogenate was exposed to anaerobic sludge under mesophilic conditions (i.e., 37°C). BRIEF: The disease-associated form of the prion protein partitions to activated sludge solids and survives simulated mesophilic anaerobic sludge digestion.