Samuel E. Saunders

Prions in the environment Occurrence, fate and mitigation

Scrapie and CWD are horizontally transmissible, and the environment likely serves as a stable reservoir of infectious prions, facilitating a sustained incidence of CWD in free-ranging cervid populations and complicating efforts to eliminate disease in captive herds. Prions will enter the environment through mortalities and/or shedding from live hosts. Unfortunately, a sensitive detection method to identify prion contamination in environmental samples has not yet been developed. An environmentally-relevant prion model must be used in experimental studies. Changes in PrPSc structure upon environmental exposure may be as significant as changes in PrPSc quantity, since the structure can directly affect infectivity and disease pathology. Prions strongly bind to soil and remain infectious. Conformational changes upon adsorption, competitive sorption, and potential for desorption and transport all warrant further investigation. Mitigation of contaminated carcasses or soil might be accomplished with enzyme treatments or composting in lieu of incineration.

Replication Efficiency of Soil-Bound Prions Varies with Soil Type

ABSTRACT Prion sorption to soil is thought to play an important role in the transmission of scrapie and chronic wasting disease (CWD) via the environment. Sorption of PrP to soil and soil minerals is influenced by the strain and species of PrPSc and by soil characteristics. However, the ability of soil-bound prions to convert PrPc to PrPSc under these wide-ranging conditions remains poorly understood. We developed a semiquantitative protein misfolding cyclic amplification (PMCA) protocol to evaluate replication efficiency of soil-bound prions. Binding of the hyper (HY) strain of transmissible mink encephalopathy (TME) (hamster) prions to a silty clay loam soil yielded a greater-than-1-log decrease in PMCA replication efficiency with a corresponding 1.3-log reduction in titer. The increased binding of PrPSc to soil over time corresponded with a decrease in PMCA replication efficiency. The PMCA efficiency of bound prions varied with soil type, where prions bound to clay and organic surfaces exhibited significantly lower replication efficiencies while prions bound to sand exhibited no apparent difference in replication efficiency compared to unbound controls. PMCA results from hamster and CWD agent-infected elk prions yielded similar findings. Given that PrPSc adsorption affinity varies with soil type, the overall balance between prion adsorption affinity and replication efficiency for the dominant soil types of an area may be a significant determinant in the environmental transmission of prion diseases.

Detection, occurrence, and fate of emerging contaminants in agricultural environments

A total of 43 papers published in 2014 were reviewed ranging from detailed descriptions of analytical methods, to fate and occurrence studies, to measuring and predicting biological effects for a wide variety of emerging contaminants likely to occur in agricultural environments. New methods and studies on veterinary pharmaceuticals, natural and synthetics steroids, and antibiotic resistance genes in agricultural environments continue to expand our knowledge base on the occurrence and potential impacts of these compounds. This review is divided into the following sections: Introduction, Analytical Methods, Occurrence and Fate, Antibiotic Resistance Genes, and Risk Assessment.

Ethidium monoazide does not inhibit RT-PCR amplification of nonviable avian influenza RNA

A critical obstacle to using PCR to quantify viral titers is the distinguishment of viable and nonviable genomic material. Pretreatments of ethidium monoazide (EMA) have been effective in preventing PCR amplification of DNA from nonviable bacteria. To test whether an EMA pretreatment could be used with RT-PCR to quantify viable RNA virions, avian influenza virus (AIV) survival was measured in water through 28d using cell culture titration and real-time RT-PCR with or without EMA pretreatment. Cell culture titration yielded significantly lower titers than both RT-PCR procedures, and there was no significant difference between RT-PCR results with or without EMA. Ineffective binding of EMA to AIV RNA may have allowed nonviable AIV RNA to amplify. Furthermore, since AIV inactivation may take place by means other than membrane disruption, any pretreatment distinguishing viable and nonviable AIV virions by membrane integrity may not be practical.

Soil-mediated prion transmission: Is local soil-type a key determinant of prion disease incidence?

Prion diseases, including chronic wasting disease (CWD) and scrapie, can be transmitted via indirect environmental routes. Animals habitually ingest soil, and results from laboratory experiments demonstrate prions can bind to a wide range of soils and soil minerals, retain the ability to replicate, and remain infectious, indicating soil could serve as a reservoir for natural prion transmission and a potential prion exposure route for humans. Preliminary epidemiological modeling suggests soil texture may influence the incidence of prion disease. These results are supported by experimental work demonstrating variance in prion interactions with soil, including variance in prion soil adsorption and soil-bound prion replication with respect to soil type. Thus, local soil type may be a key determinant of prion incidence. Further experimental and epidemiological work is required to fully elucidate the dynamics of soil-mediated prion transmission, an effort that should lead to effective disease management and mitigation strategies.

Effects of solution chemistry and aging time on prion protein adsorption and replication of soil-bound prions

Prion interactions with soil may play an important role in the transmission of chronic wasting disease (CWD) and scrapie. Prions are known to bind to a wide range of soil surfaces, but the effects of adsorption solution chemistry and long-term soil binding on prion fate and transmission risk are unknown. We investigated HY TME prion protein (PrPSc) adsorption to soil minerals in aqueous solutions of phosphate buffered saline (PBS), sodium chloride, calcium chloride, and deionized water using western blotting. The replication efficiency of bound prions following adsorption in these solutions was also evaluated by protein misfolding cyclic amplification (PMCA). Aging studies investigated PrPSc desorption and replication efficiency up to one year following adsorption in PBS or DI water. Results indicate that adsorption solution chemistry can affect subsequent prion replication or desorption ability, especially after incubation periods of 30 d or longer. Observed effects were minor over the short-term (7 d or less). Results of long-term aging experiments demonstrate that unbound prions or prions bound to a diverse range of soil surfaces can readily replicate after one year. Our results suggest that while prion-soil interactions can vary with solution chemistry, prions bound to soil could remain a risk for transmitting prion diseases after months in the environment.

An Enzymatic Treatment of Soil-Bound Prions Effectively Inhibits Replication

ABSTRACT Chronic wasting disease (CWD) and scrapie can be transmitted through indirect environmental routes, possibly via soil, and a practical decontamination strategy for prion-contaminated soil is currently unavailable. In the laboratory, an enzymatic treatment under environmentally relevant conditions (22°C, pH 7.4) can degrade soil-bound PrPSc below the limits of Western blot detection. We developed and used a quantitative serial protein misfolding cyclic amplification (PMCA) protocol to characterize the amplification efficiency of treated soil samples relative to controls of known infectious titer. Our results suggest large (104- to >106-fold) decreases in soil-bound prion infectivity following enzyme treatment, demonstrating that a mild enzymatic treatment could effectively reduce the risk of prion disease transmission via soil or other environmental surfaces.

Resistance of Soil-Bound Prions to Rumen Digestion

Before prion uptake and infection can occur in the lower gastrointestinal system, ingested prions are subjected to anaerobic digestion in the rumen of cervids and bovids. The susceptibility of soil-bound prions to rumen digestion has not been evaluated previously. In this study, prions from infectious brain homogenates as well as prions bound to a range of soils and soil minerals were subjected to in vitro rumen digestion, and changes in PrP levels were measured via western blot. Binding to clay appeared to protect noninfectious hamster PrPc from complete digestion, while both unbound and soil-bound infectious PrPSc proved highly resistant to rumen digestion. In addition, no change in intracerebral incubation period was observed following active rumen digestion of unbound hamster HY TME prions and HY TME prions bound to a silty clay loam soil. These results demonstrate that both unbound and soil-bound prions readily survive rumen digestion without a reduction in infectivity, further supporting the potential for soil-mediated transmission of chronic wasting disease (CWD) and scrapie in the environment.

Prions in the Environment

Scrapie and CWD are horizontally transmissible, and the environment likely serves as a stable reservoir of infectious prions, facilitating a sustained incidence of CWD in free-ranging cervid populations and complicating efforts to eliminate disease in captive herds. Prions will enter the environment through mortalities and/or shedding from live hosts. Unfortunately, a sensitive detection method to identify prion contamination in environmental samples has not yet been developed. An environmentally-relevant prion model must be used in experimental studies. Changes in PrPSc structure upon environmental exposure may be as significant as changes in PrPSc quantity, since the structure can directly affect infectivity and disease pathology. Prions strongly bind to soil and remain infectious. Conformational changes upon adsorption, competitive sorption, and potential for desorption and transport all warrant further investigation. Mitigation of contaminated carcasses or soil might be accomplished with enzyme treatm...

Methane and carbon dioxide production from simulated anaerobic degradation of cattle carcasses.

Approximately 2.2million cattle carcasses require disposal annually in the United States. Land burial is a convenient disposal method that has been widely used in animal production for disposal of both daily mortalities as well as during catastrophic mortality events. To date, greenhouse gas production after mortality burial has not been quantified, and this study represents the first attempt to quantify greenhouse gas emissions from land burial of animal carcasses. In this study, anaerobic decomposition of both homogenized and unhomogenized cattle carcass material was investigated using bench-scale reactors. Maximum yields of methane and carbon dioxide were 0.33 and 0.09m(3)/kg dry material, respectively, a higher methane yield than that previously reported for municipal solid waste. Variability in methane production rates were observed over time and between reactors. Based on our laboratory data, annual methane emissions from burial of cattle mortalities in the United States could total 1.6Tg CO(2) equivalents. Although this represents less than 1% of total emissions produced by the agricultural sector in 2009, greenhouse gas emissions from animal carcass burial may be significant if disposal of swine and poultry carcasses is also considered.