Chronic wasting disease (CWD), an environmentally transmissible, fatal prion disease is endemic in North America, present in South Korea and has recently been confirmed in northern Europe. The expanding geographic range of this contagious disease of free-ranging deer, moose, elk and reindeer has resulted in increasing levels of prion infectivity in the environment. Soils are involved in CWD horizontal transmission, acting as an environmental reservoir, and soil mineral and organic compounds have the ability to bind prions. Upper horizons of soils are usually enriched with soil organic matter (SOM), however, the role of SOM in prion conservation and mobility remains unclear. In this study, we show that incubation of PrPCWD with humic acids (HA), a major SOM compound, affects both the molecular weight and recovery of PrPCWD. Detection of PrPCWD is reduced as HA concentration increases. Native HA extracted from pristine soils also reduces or entirely eliminates PrPCWD signal. Incubation of CWD prions with HA significantly increased incubation periods in tgElk mice demonstrating that HA can reduce CWD infectivity.

Author summary

Chronic wasting disease (CWD) is a contagious prion disease affecting several species of captive and wild cervids. Environmental prion contamination plays a major role in increasing incidence of CWD, with CWD infectivity being released into the environment by decaying carcasses, or shedding of biological fluids including urine, feces, and saliva. Horizontal transmission of CWD involves soils as an environmental reservoir of infectivity. Here, we tested the role of a soil organic matter compound, humic acid, for its ability to bind CWD prions and impact infectivity. A wide range of humic acid concentrations were examined representing the extensive spectrum of humic acid levels present in native soils. We found that incubation of CWD prions with high concentrations of humic acids (>2.5 g L-1) decreases the both CWD-prion signal and infectivity, whereas lower levels of humic acids did not significantly impact protein stability or infectivity. Our study provides new insights into soil-prion interactions, prions persistence in soil, and their bioavailability to grazing animals.


Chronic wasting disease (CWD), a fatal prion disease affecting free ranging white-tailed deer, mule deer, elk, moose and reindeer as well as farmed cervids, is the only spongiform encephalopathy (along with scrapie) that is environmentally transmitted. Currently, CWD-infected cervids are present in 3 provinces of Canada, 25 US states, South Korea, Norway and Finland, and its geographic range continues to expand among free-ranging cervids. Evidence suggests that horizontal transmission of CWD involves soils as an environmental reservoir of infectivity []. Soil-related CWD transmission in cervids may occur orally [], although intranasal and aerosol routes of transmission are also possible [,]. The CWD agent may enter the soil via alimentary secretions, blood, and decomposing infected carcases []. While prion infectivity persists in the soil for a few years in laboratory conditions [], infectivity of soils in CWD-endemic regions remains largely unknown. Different soil compounds, mineral and organic, can differentially bind prions and change their infective properties []. Montmorillonite (mte) mineral particles bind prions avidly and increase their infectivity []; kaolinite (kte) and quartz microparticles (common soil minerals) also may increase disease transmission []. Upper horizons of soils are usually enriched with soil organic matter (SOM) but the role of SOM in prion conservation and mobility remains unclear. SOM is defined as all biologically derived organic matter that resides within the soil matrix and is divided into living and non-living components []. Humus is the most important and specific part of SOM; it is a mixture of amorphous organic materials that contains identifiable biomolecules (e.g. polysaccharides, lipids, proteins etc.) and non-identifiable molecules (humic substance) []. Humus is an extraordinarily complex, molecularly flexible material that can be fractionated into specific humic substances: fulvic acids (FA), humic acids (HA), and humin. Humic acids are comprised of weak aliphatic (carbon chains) and aromatic (carbon rings) organic acids which are insoluble in water under acid conditions, but soluble in water under alkaline conditions. Humic acids have average molecular weights varying from 10kDa-103kDa for soil-derived material []. They are considered to be flexible linear polymers that exist as random coils with cross-linked bonds. In water solution, the HA are large, dynamic supramolecular associations, held together by hydrophobic interactions, which are easily disrupted and capable of exhibiting micellar properties [].

Due to the wide variety of soil types, composition and amount of SOM differs between locations. For example, in western Canada, there are at least 12 great groups of soils [] and each is comprised of different amounts and types of organic compounds. SOM content in the upper soil horizons vary from 0.5% in the surface horizon of Regosolic soils, to 30% in the LHF (plant litter) horizon of most soils in the northern part of Canada. The ratio of different humic compounds also varies across soil types. In soils where the vegetative (biologically productive) period is longer, the humus has more HA than FA (e.g. in Chernozems) compared to soils that are less biologically active (due to non-optimal temperatures and moisture regimes), where the humus contents are mostly FA (e.g. in Luvisols) [,]. For Chernozemic soils of Northern American CWD-endemic regions, HA abundance was estimated between 0.75%-1.4% []. In Luvisolic and Brunisolic soils, the upper horizon contains high amounts of SOM, but insignificant amounts of HA []; for boreal region soils, the HA content is estimated as 0.1–0.7% [].

Since most of SOM is concentrated in the surface soil horizon and HA is a major component of SOM, it likely plays an important role in the fate of shed prions. Although the absolute concentration of HA in soil is significantly less than the abundance of mineral particles, HA is more biologically and chemically active and can adsorb on mineral particles creating films on the surfaces and masking them. Analyses of HA-prion interactions, and their impact on CWD infectivity, are important factors in determining the fate of PrPCWD in soil environments. While a series of studies have investigated prion binding to soils varying in SOM content, the direct interaction of HA with PrPCWD has not been studied [,]. Studies using non-infectious recombinant PrP (recPrP) indicated that HA and other SOM compounds have a strong affinity to the recPrP []. However, due to difference in structure of infectious and non-infectious prions, they may interact differently with SOM compounds. It has previously been shown that hamster prions, when incubated with low concentrations of HA, remain infectious to hamsters with little biological impact on the incubation period []. The objective of this study was to determine, by examining a more complete range of HA concentrations, how interactions with HA affects PrPCWD and resulting infectivity.

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