These experiments address the critical question of whether soil particle–bound prions are infectious by an environmentally relevant exposure route, namely, oral ingestion. Oral infectivity of soil particle–bound prions is a conditio sine qua non
for soil to serve as an environmental reservoir for TSE agent. The maintenance of infectivity and enhanced transmissibility when TSE agent is bound to the common soil mineral Mte is remarkable given the avidity of the PrPTSE
–Mte interaction [22
]. One might expect the avid interaction of PrPTSE
with Mte to result in the mineral serving as a sink, rather than a reservoir, for TSE infectivity. Our results demonstrate this may not be the case. Furthermore, sorption of prions to complex whole soils did not diminish bioavailability, and in two of three cases promoted disease transmission by the oral route of exposure. While extrapolation of these results to environmental conditions must be made with care, prion sorption to soil particles clearly has the potential to increase disease transmission via the oral route and contribute to the maintenance of TSE epizootics.
Two of three tested soils potentiated oral prion disease transmission. The reason for increased oral transmissibility associated with some, but not all, of the soils remains to be elucidated. One possibility is that components responsible for enhancing oral transmissibility were present at higher levels in the Elliot and Bluestem soils than in the Dodge soil. The major difference between the Dodge soil and the other two soils was the extremely high natural organic matter content of the former (34%, [22
]). The Dodge and Elliot soils contained similar levels of mixed-layer illite/smectite, although the contribution of smectite layers was higher in the Dodge soil (14%–16%, [22
]). The organic matter present in the Dodge soil may have obstructed access of PrPTSE
to sorption sites on smectite (or other mineral) surfaces.
The mechanism by which Mte or other soil components enhances the oral transmissibility of particle-bound prions remains to be clarified. Aluminosilicate minerals such as Mte do not provoke inflammation of the intestinal lining [39
]. Although such an effect is conceivable for whole soils, soil ingestion is common in ruminants and other mammals [25
]. Prion binding to Mte or other soil components may partially protect PrPTSE
from denaturation or proteolysis in the digestive tract [22
] allowing more disease agent to be taken up from the gut than would otherwise be the case. Adsorption of PrPTSE
to soil or soil minerals may alter the aggregation state of the protein, shifting the size distribution toward more infectious prion protein particles, thereby increasing the specific titer (i.e., infectious units per mass of protein) [41
]. In the intestine, PrPTSE
complexed with soil particles may be more readily sampled, endocytosed (e.g., at Peyer's patches), or persorbed than unbound prions. Aluminosilicate (as well as titanium dioxide, starch, and silica) microparticles, similar in size to the Mte used in our experiments, readily undergo endocytotic and persorptive uptake in the small intestine [42
]. Enhanced translocation of the infectious agent from the gut lumen into the body may be responsible for the observed increase in transmission efficiency.
Survival analysis indicated that when bound to Mte, prions from both BH and purified PrPTSE
preparations were more orally infectious than unbound agent. Mte addition influenced the effective titer of infected BH to a lesser extent than purified PrPTSE
. Several nonmutually exclusive factors may explain this result: (1) other macromolecules present in BH (e.g., lipids, nucleic acids, other proteins) compete with PrPTSE
for Mte binding sites; (2) prion protein is more aggregated in the purified PrPTSE
preparation than in BH [45
], and sorption to Mte reduces PrPTSE
aggregate size, increasing specific titer [41
]; and (3) sorption of macromolecules present in BH to Mte influences mineral particle uptake in the gut by altering surface charge or size, whereas the approximately 1,000-fold lower total protein concentration in purified PrPTSE
preparations did not produce this effect.
We previously showed that other inorganic microparticles (kaolinite and silicon dioxide) also bind PrPTSE
]. All three types of microparticles are widely used food additives and are typically listed as bentonite (Mte), kaolin (kaolinite), and silica (silicon dioxide). Microparticles are increasingly included in Western diets. Dietary microparticles are typically inert and considered safe for consumption by themselves, do not cause inflammatory responses or other pathologies, even with chronic consumption, and are often sampled in the gut and transferred from the intestinal lumen to lymphoid tissue [39
]. Our data suggest that the binding of PrPTSE
to dietary microparticles has the potential to enhance oral prion disease transmission and warrants further investigation.
In conclusion, our results provide compelling support for the hypothesis that soil serves as a biologically relevant reservoir of TSE infectivity. Our data are intriguing in light of reports that naïve animals can contract TSEs following exposure to presumably low doses of agent in the environment [5
]. We find that Mte enhances the likelihood of TSE manifestation in cases that would otherwise remain subclinical (B and C), and that prions bound to soil are orally infectious (). Our results demonstrate that adsorption of TSE agent to inorganic microparticles and certain soils alter transmission efficiency via the oral route of exposure.