Alzheimer’s Disease Transmitted Through Environment, Food, Water

Alzheimer’s An Infectious Disease Fueled By Infectious Waste

Neurodegenerative disease, including Alzheimer’s disease and Parkinson’s disease, is the fastest-growing cause of death in the world. Misinformation and mismanagement are contributing to the surge. Alzheimer’s disease alone is killing 50-100 million people now. Experts suggest that the prevalence of brain disease will quadruple by 2050, if not sooner.

Death rates from heart disease and cancer are dropping globally due to advances in nutrition, medicine and disease management. Meanwhile, neurodegenerative disease is exploding. In the U.S., deaths from Alzheimer’s disease increased 71 percent from 2000 to 2013, while those attributed to heart disease decreased 14 percent. Similar trends are emerging around the world. The reasons are alarming.

To understand the threat, one must understand the dynamics and scope of the epidemic. Alzheimer’s, for example, is a member of an aggressive family of neurodegenerative diseases known as Transmissible Spongiform Encephalopathy (TSE). The operative word is “transmissible.”

Alzheimer's disease epidemic

TSEs also include Creutzfeldt-Jakob disease (CJD) Parkinson’s, Huntington’s, mad cow disease and chronic wasting disease in the deer family. Few, if any, mammals are immune. There is no cure. TSEs are always fatal.

  • Women are contracting neurodegenerative disease at twice the rate of men;
  • Spouses of those with Alzheimer’s disease are 600% more likely to contract the disease, which is further evidence that it is a transmissible disease. Caregivers, family members and others are in harm’s way because of disease mismanagement and misinformation; and
  • People in Finland, Iceland, Sweden and the United States have the highest prevalence of Alzheimer’s disease. Rates in North Dakota, South Dakota and Washington rival the highest rates in the world.

The epidemic is larger than anyone knows. Physicians are withholding millions of diagnoses from patients and their families. According to the Alzheimer’s Association, physicians in the U.S. only inform 45 percent of patients about their Alzheimer’s disease diagnosis. The same suppression is likely at work in most countries. Meanwhile, millions of people go undiagnosed and misdiagnosed.

prion disease epidemic

Dr. Stanley Prusiner, an American neuroscientist from the University of California at San Francisco, earned a Nobel Prize in 1997 for discovering and characterizing deadly prions and prion disease. He claims that all TSEs are caused by prions. President Obama awarded Prusiner the National Medal of Science in 2010 to recognize the importance of his research. According to Prusiner, TSEs all are on the same disease spectrum, which is more accurately described as prion (PREE-on) disease. CJD is at the extreme end of the spectrum.

Prions are unstoppable and the pathogen spreads through the bodily fluids and cell tissue of its victims. Prions shed from humans are the most deadly mutation. Prions shed from human victims demand more respect than radiation. Infected surgical instruments, for example, are impossible to sterilize and hospitals throw them away. Prions are in the blood, saliva, urine, feces, mucus, and bodily tissue of its victims. Many factors are contributing to the epidemic. Prions are now the X factor. Industry and government are not accounting for prions or regulating them. They are ignoring these deadly proteins completely, which violates the United States’ Bioterrorism Preparedness and Response Act of 2002. Other nations also are ignoring laws developed to protect food, air and water.

land application sewage sludge

“There is now real evidence of the potential transmissibility of Alzheimer’s,” says Thomas Wiesniewski M.D. a prion and Alzheimer’s researcher at New York University School of Medicine.

A new study published in the journal Nature renews concern about the transmissibility of Alzheimer’s disease between people. A second study by the same scientist in early 2016 adds to the stack of evidence.

Experts claim that at least 25 percent of Alzheimer’s diagnoses are not Alzheimer’s disease. These misdiagnoses are actually CJD, which is further up the prion disease spectrum. CJD, without dispute, is extremely infectious to caregivers and loved ones. Millions of cases of deadly CJD are being misdiagnosed as Alzheimer’s disease. Millions of patients and caregivers are being misinformed, misguided and exposed to an aggressive disease. Misdiagnosis and misinformation regarding prion disease is a matter of life and death. The mismanagement doesn’t end here.

Wastewater treatment plants, for example, are spreading this infectious waste far and wide because they are incapable of stopping prions. All by-products and discharges from wastewater treatment plants are infectious waste, which are contributing to the global epidemic of neurodegenerative disease among humans, wildlife and livestock. Wastewater treatment plants can’t detect or stop prions. Just ask the U.S. EPA and the industry trade organization—the Wastewater Effluent Federation. Sewage sludge (biosolids) and wastewater reclamation are causing widespread contamination.

biosolids land application and disease

Sewage Spreading Alzheimer’s Disease To Wildlife

Once unleashed on the environment, prions remain infectious. They migrate, mutate and multiply as they infect crops, water supplies, wildlife, livestock, sea mammals and humans. It’s a real world version of Pandora’s Lunchbox.

Deer, elk, moose and reindeer are now contracting prion disease from humans. To help cloak the epidemic, it’s called chronic wasting disease (CWD). Deer with CWD are proverbial canaries in a coal mine. They are being killed by government sharpshooters to help cover up the problem. It’s insane.

chronic wasting disease caused by prions

The risk assessments prepared by the U.S. EPA for wastewater treatment and sewage sludge (biosolids) are flawed and current practices of recycling this infectious waste is fueling a public health disaster. Many risks are not addressed, including prions and radioactive waste. They don’t mention prions or radionuclides because there is no answer. Most nations are making the same mistake. We’re dumping killer proteins on crops, parks, golf courses, gardens, ski areas, school grounds and beyond. Wind, rain and irrigation spread this infectious waste  throughout our communities and watersheds.

Failure to account for known risks is negligent. Crops for humans and livestock grown grown in sewage sludge absorb prions and become infectious. We’re all vulnerable to Alzheimer’s and other forms of prion disease right now due to widespread denial and mismanagement. It’s time to stop the land application of sewage sludge (LASS) in all nations. Safer alternatives exist.

Alzheimer's disease prevention

For example, the practice of dumping the sewage from billions of people on land and at sea adds fuel to the Alzheimer’s epidemic. Applying biosolids (sewage sludge) to cropland, or any watershed, demands reconsideration. The reuse of reclaimed wastewater for drinking water is reckless. Just think how many listeria outbreaks have happened from sewage being applied to our crops. The pathway between human sewage and food is proven. The pathogen is proven. The risk is very real. There is no proof to the contrary.

People and animals with prion disease represent an environmental nightmare. These killer proteins are unstoppable. Public and private protocols demand the truth and reform to help stop the Alzheimer’s epidemic.

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public relations firm and public affairs firm Denver and Phoenix

Crossbow Communications specializes in issue management and public affairs. Alzheimer’s disease, Creutzfeldt-Jakob disease, chronic wasting disease and the prion disease epidemic is an area special expertise. Please contact Gary Chandler to join our coalition for reform

Chronic Wasting Disease Fueled By Sewage Sludge

Biosolids Spreading Brain Disease

By Patrick Durkin

Those who think Wisconsin should just “learn to live” with chronic wasting disease are seeing their surrender take shape as “nature takes its course” on our deer herd.

In fact, folks near Spring Green are living the realities of such clichés. One farmer in the Wyoming valley of north-central Iowa County has shot 21 CWD-positive deer from his family’s 700 acres since 2008, with 11 falling since April 2012.

Those are just a few of the sick deer in a 144-square mile area where CWD (prion disease) is rising at “unprecedented” rates. That one-word assessment came from Bryan Richards, CWD project leader at the U.S. Geological Survey’s National Wildlife Health Center in Madison, after reviewing the latest CWD reports from Robert Rolley, a Department of Natural Resources researcher in the wildlife science bureau.

chronic wasting disease caused by prions

Rolley, Richards and about 50 other citizens, biologists and agency staff were at UW-Stevens Point on April 6 to help implement 62 recommendations from the “Deer Trustee Report,” Dr. James Kroll’s guide to revamping Wisconsin deer management.

Rolley, too, used one word — “frightening” — to assess CWD’s increase in the 12- by-12-mile area around the Wyoming valley. The eastern border of this diseased block abuts CWD’s core area (northeastern Iowa and northwestern Dane counties), where the disease was first discovered 11 years ago.

What constitutes “unprecedented” and “frightening?” First, realize the infection rate in the core area is increasing about 10 percent annually. That resembles annual infection rates of mule deer in southeastern Wyoming’s Converse area, where 40 to 50 percent of the herd is infected with CWD.

prion disease epidemic

Now consider north-central Iowa County:

• CWD’s annual growth rate for all deer (both sexes combined) 2½ years and older is 27 percent.

• Annual disease rates for adult bucks (18 months and older) are doubling every two to three years.

• Roughly every third buck 2½ years and older is infected, as is one in every six yearling bucks (18 months old). Infected deer live two years or less.

• Although the number of diseased females is lower, the infection rate for does 2½ years and older is growing 38 percent annually, faster than for males.

I’m not aware of data anywhere showing wild, free-range deer with similar infection rates,” Richards said. “The only thing worse was the Stan Hall farm (Buckhorn Flats, near Almond), whose penned herd of 76 deer went from one sick deer to 60 in five years.”

That might sound like mere statistics to some, but not to Matt Limmex, 49, an Iowa County dairy farmer who has spent his life on the family’s property. Of the 11 sick deer killed on Limmex’s lands the past year, six fell during 2012 gun seasons.

land application sewage sludge

The other five? Limmex shot them at the DNR’s request after noticing the “droolers and shakers” near his farmyard. In three cases, they were so sick they couldn’t flee when Limmex approached. The DNR retrieved the carcasses for testing and disposal.

“I hate to see this,” Limmex said. “It’s disheartening. I just want to get sick deer off the landscape.”

Limmex said his family deploys about 14 hunters each year during gun season. They’ve also used agricultural shooting permits since 1991 to control the herd. Even so, this is the first time he senses deer numbers decreasing.

“It seems like the disease might be affecting the herd now,” he said.

biosolids land application and disease

So, what’s causing CWD rates in the Wyoming valley to exceed those in the original disease zone? And will it shrink local herds, as experts have long predicted? No one knows, and our state and federal governments aren’t inclined to find out.

The only current DNR-funded deer research by the University of Wisconsin is studying whether predators are affecting North Woods whitetails. Meanwhile, Professor Michael Samuel at the University of Wisconsin is using federal funds to study if deer leave CWD-causing prions in feces, breeding scrapes and mineral licks. But that’s ending soon.

“We’re puzzled by what’s going on in the Wyoming valley,” Samuel said. “It’s very disturbing, but CWD research is on the way out. We could generate hypotheses and proposals to study what’s behind the increases, but I doubt we’d get the funding. There’s little interest in CWD these days, Wisconsin and nationwide.”

Imagine that. The world’s most “disturbing,” “frightening” and “unprecedented” CWD case is growing next door to our capital and flagship university, and our government won’t crack a window to sniff it.

Meanwhile, no group or coalition of hunters, doctors, veterinarians or environmentalists is holding politicians accountable, or funding the research themselves. There’ll be no shortage of shame as this stench spreads.

CWD News Via

public relations firm and public affairs firm Denver and Phoenix

Crossbow Communications specializes in issue management and public affairs. Alzheimer’s disease, Creutzfeldt-Jakob disease, chronic wasting disease and the prion disease epidemic is an area of special expertise. Please contact Gary Chandler to join our coalition for reform

Transmissibility Of Prion Disease In Soil

Sewage Sludge Expands Prion Pathways

Soil may serve as an environmental reservoir for prion infectivity and contribute to the horizontal transmission of prion diseases (transmissible spongiform encephalopathies [TSEs]) of sheep, deer, and elk. TSE infectivity can persist in soil for years, and we previously demonstrated that the disease-associated form of the prion protein binds to soil particles and prions adsorbed to the common soil mineral montmorillonite (Mte) retain infectivity following intracerebral inoculation. Here, we assess the oral infectivity of Mte- and soil-bound prions.

land application sewage sludge

We establish that prions bound to Mte are orally bioavailable, and that, unexpectedly, binding to Mte significantly enhances disease penetrance and reduces the incubation period relative to unbound agent. Cox proportional hazards modeling revealed that across the doses of TSE agent tested, Mte increased the effective infectious titer by a factor of 680 relative to unbound agent. Oral exposure to Mte-associated prions led to TSE development in experimental animals even at doses too low to produce clinical symptoms in the absence of the mineral.

We tested the oral infectivity of prions bound to three whole soils differing in texture, mineralogy, and organic carbon content and found soil-bound prions to be orally infectious. Two of the three soils increased oral transmission of disease, and the infectivity of agent bound to the third organic carbon-rich soil was equivalent to that of unbound agent. Enhanced transmissibility of soil-bound prions may explain the environmental spread of some TSEs despite the presumably low levels shed into the environment. Association of prions with inorganic microparticles represents a novel means by which their oral transmission is enhanced relative to unbound agent.

chronic wasting disease caused by prions

Transmissible spongiform encephalopathies (TSEs) are a group of incurable neurological diseases likely caused by a misfolded form of the prion protein. TSEs include scrapie in sheep, bovine spongiform encephalopathy (“mad cow” disease) in cattle, chronic wasting disease in deer and elk, and Creutzfeldt-Jakob disease in humans. Scrapie and chronic wasting disease are unique among TSEs because they can be transmitted between animals, and the disease agents appear to persist in environments previously inhabited by infected animals.

Soil has been hypothesized to act as a reservoir of infectivity and to bind the infectious agent. In the current study, we orally dosed experimental animals with a common clay mineral, montmorillonite, or whole soils laden with infectious prions, and compared the transmissibility to unbound agent. We found that prions bound to montmorillonite and whole soils remained orally infectious, and, in most cases, increased the oral transmission of disease compared to the unbound agent. The results presented in this study suggest that soil may contribute to environmental spread of TSEs by increasing the transmissibility of small amounts of infectious agent in the environment.

biosolids land application LASS

Citation: Johnson CJ, Pedersen JA, Chappell RJ, McKenzie D, Aiken JM (2007) Oral Transmissibility of Prion Disease Is Enhanced by Binding to Soil Particles. PLoS Pathog 3(7): e93. doi:10.1371/journal.ppat.0030093


Bovine spongiform encephalopathy, human Creutzfeldt-Jakob disease and kuru, sheep scrapie, and chronic wasting disease of deer, elk, and moose belong to the class of fatal, infectious neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs) or prion diseases. The precise nature of the etiological agent of these diseases remains controversial, but most evidence points to a misfolded isoform of the prion protein (PrPTSE) as the major, if not sole, component of the pathogen.

Sheep scrapie and cervid (deer, elk, and moose) chronic wasting disease are distinct among TSEs because epizootics can be maintained by horizontal transmission from infected to naïve animals, and transmission is mediated, at least in part, by an environmental reservoir of infectivity. The presence of an environmental TSE reservoir impacts several epidemiological factors including contact rate (the frequency animals come in contact with the disease agent), duration of exposure (time period over which animals come in contact with the pathogen), and the efficiency of transmission (the probability that an exposed individual contracts the disease).

The oral route of exposure appears responsible for environmental transmission of chronic wasting disease and scrapie; the propagation of bovine spongiform encephalopathy epizootics (feeding TSE-infected meat and bonemeal to cattle); the appearance of variant Creutzfeldt-Jacob disease in humans and feline spongiform encephalopathy in cats (presumably by consumption of bovine spongiform encephalopathy–infected beef); the spread of kuru among the Fore of Papua New Guinea (ritualistic endocannibalism); and outbreaks of transmissible mink encephalopathy (TME) in farm-reared mink. Following consumption, TSE agent is sampled by gut-associated lymphoid tissue, such as Peyer’s patches or isolated lymphoid follicles, and accumulates in lymphatic tissues before entering the central nervous system via the enteric nervous system. While ingestion is a biologically relevant TSE exposure route, oral dosing is a factor of ~105 less efficient than intracerebral inoculation in inducing disease in rodent models. The amounts of TSE agent shed into the environment are presumably small. The assumed low levels of TSE agent in the environment and the inefficiency of oral transmission have led to uncertainty about the contribution of environmental reservoirs of infectivity to prion disease transmission.

prion disease epidemic

We and others have hypothesized that soil may serve as a reservoir of TSE infectivity. Deliberate and incidental ingestion of soil by ruminants can amount to hundreds of grams daily. Prions enter soil environments via decomposition of infected carcasses, alimentary shedding, deliberate burial of diseased carcasses/material and possibly, urinary excretion. TSE agent persists for years when buried in soil. The disease-associated prion protein sorbs to soil particles, and the interaction of PrPTSE with the common aluminosilicate clay mineral montmorillonite (Mte) is remarkably avid. Despite this strong binding, PrPTSE–Mte complexes are infectious when inoculated into brains of recipient animals.

For TSEs to be transmitted via ingestion of prion-contaminated soil, prions bound to soil components must remain infectious by the oral route of exposure. We therefore investigated the oral infectivity of Mte- and soil-bound prions. We examined the effects of prion source (viz. infected brain homogenate [BH] and purified PrPTSE) and dose on disease penetrance (proportion of animals eventually exhibiting clinical TSE symptoms) and incubation period (time to onset of clinical symptoms) in experiments with Mte. We investigated the oral infectivity of soil particle–bound prions to Syrian hamsters using four dosing regimes: (1) infected BH mixed with Mte (BH–Mte mixtures), (2) isolated complexes of purified PrPTSE bound to Mte (PrPTSE–Mte complexes), (3) purified PrPTSE mixed with Mte (PrPTSE–Mte mixtures), and (4) PrPTSE mixed with each of three whole soils (PrPTSE–soil mixtures). The rationale for each dosing regime is described below. Survival analysis was used to assess risk of clinical disease manifestation and quantify differences in effective titer. Application of survival analysis to oral bioassays of TSE transmissibility is discussed in Figure S1 and Text S1.

biosolids land application and disease

Oral Infectivity of BH–Mte Mixtures

To examine the effect of Mte on the oral transmissibility of prions in BH, we incubated infected BH with clay particles for 2 h to allow sorption of the agent; controls lacking Mte were treated identically [22]. Three doses of 10% BH (30, 3, and 0.3 μL) were assayed. Diminished gastrointestinal bioavailability was expected to be evidenced by significant lengthening of incubation period, reduced disease penetrance, or both. Binding of either 30 or 3 μL of brain material to Mte yielded disease penetrance and incubation periods similar to BH alone (Figure 1A and 1B), a finding consistent with our previous report that a substantial fraction of PrPTSE in clarified BH binds to Mte and that Mte-bound prions remain infectious [22].

No Loss of Oral TSE Transmissibility Following Sorption of Prions from Infected BH to Mte (BH–Mte Mixtures)

The oral transmissibility of prions in 30 (A) and 3 (B) μL was not diminished by dosing with Mte. Indicates non-TSE intercurrent death. Animals dosed with Mte alone remained healthy throughout the course of the experiment (unpublished data).

Surprisingly, at the lowest BH dose (0.3 μL, Figure 2), sorption of TSE agent to Mte enhanced transmission, increasing disease penetrance and shortening incubation period. Adjusted for the amount of BH administered and combined across doses, Mte significantly enhanced oral transmissibility (p < 0.0001). Survival analysis indicated the risk of clinical disease manifestation relative to Mte-free controls was 3.03 (95% confidence interval [CI]: 1.68, 5.45), signifying an increase in the effective titer of TSE agent. While the influence of Mte was significant when tested across all BH doses, the effect was most readily observed at 0.3 μL. The dose-dependent difference in the influence of Mte on transmissibility may be attributable to competition between macromolecules in BH (e.g., lipids, other proteins, nucleic acids) with PrPTSE for sorption sites on the clay surface. Such competition was evidenced by detection of unbound PrPTSE and other proteins in incubations of Mte with 30 and 3 μL BH (unpublished data).

Ingestion of Mte mixed with a lower dose of TSE-infected BH (0.3 μL) markedly shortens incubation period and increases disease penetrance relative to an equal amount of unbound BH. * indicates non-TSE intercurrent death. Animals dosed with Mte alone remained healthy throughout the course of the experiment (unpublished data).

Oral Infectivity of PrPTSE–Mte Complexes

To examine the influence of Mte on oral transmissibility without the interference of other macromolecules from brain homogenate, we purified PrPTSE and inoculated hamsters using two different dosing regimes. The first dosing regime (PrPTSE–Mte complexes) was designed to directly assay the infectivity of PrPTSE sorbed to Mte surfaces (i.e., the amount of unbound PrPTSE was minimized in treatments containing Mte). Purified PrPTSE was clarified to remove large aggregates, and after 2-h incubation with Mte, PrPTSE–Mte complexes were separated from unbound protein by centrifugation through a sucrose cushion [22]. Hamsters were orally challenged with the isolated PrPTSE–Mte complexes [22] or an amount of unbound clarified PrPTSE (200 or 20 ng) equivalent to that introduced into the clay suspension (Table 1). Immunoblot analysis of the inocula (Figure S2A) demonstrated that the amount of PrP in the unbound samples was not less than that in PrPTSE–Mte complexes.

Prions Adsorbed to Mte Clay Are Infectious Perorally

Sorption of PrPTSE to Mte dramatically enhanced prion disease transmission (Table 1). Approximately 38% of animals receiving 200 ng of unbound clarified PrPTSE exhibited clinical symptoms with an incubation period for infected animals of 203 ± 33 (mean ± standard deviation) days post inoculation (dpi). In contrast, all animals orally dosed with an equivalent amount of Mte-bound PrPTSE manifested disease symptoms (incubation period = 195 ± 37 dpi), an enhancement of transmission comparable to that observed for the lowest BH dose (Figure 2). Animals inoculated with Mte alone or 10-fold less unbound clarified PrPTSE (20 ng) remained asymptomatic throughout the course of the experiment (>365 dpi), whereas 20 ng of clarified PrPTSE adsorbed to Mte produced TSE infection in 17% of animals. These data establish not only that the Mte-bound prions remain infectious via the oral route of exposure, but that agent binding to Mte increases disease penetrance, enhancing the efficiency of oral transmission.

Oral Infectivity of PrPTSE–Mte Mixtures

The second oral dosing regime using purified PrPTSE (PrPTSE–Mte mixtures) was designed to ensure that treatments with and without Mte contained equivalent PrPTSE doses. These experiments differed from those above in two important aspects. First, PrPTSE–Mte complexes were not separated from suspension prior to inoculation so that comparable amounts of infectious agent were administered to both treatment groups. In the first dosing regime, some PrPTSE may have been lost during sedimentation of PrPTSE–Mte complexes (Figure S2A). Second, the purified prion preparation was not clarified and therefore contained a range of PrPTSE aggregate sizes. The sizes of PrPTSE aggregates attached to Mte particles were expected to be more heterogeneous than those in the first dosing regime.

Compared to Mte-free controls, administration of purified PrPTSE mixed with Mte increased disease penetrance at all doses and shorted incubation times in the 1-μg PrPTSE treatment (Figure 3A). At the two lower doses (0.1 and 0.01 μg PrPTSE), binding of the agent to Mte dramatically increased disease penetrance (31%) at PrPTSE doses failing to yield clinical infection in 31 of 32 animals in the absence of the clay mineral (Figure 3B and 3C). Comparison of the survival curves in Figure 3A and 3C indicates that the 0.01-μg PrPTSE–Mte mixture was at least as infectious as 1-μg PrPTSE Mte-free samples, suggesting that sorption of purified PrPTSE to Mte enhanced transmission by a factor of ≥100.

Figure 3. Concurrent Peroral Administration of Mte and PrPTSE Dramatically Increases Disease Penetrance at Agent Doses That Typically Fail to Produce Clinical Symptoms (PrPTSE–Mte Mixture)

Mte increases disease penetrance and shortens incubation periods associated with ingestion of 1 μg of purified PrPTSE. Concurrent peroral dosage of lower, typically subclinical doses of purified PrPTSE (0.1 or 0.01 μg, [B and C]) with Mte increases disease incidence. Animals dosed with Mte alone remained healthy throughout the course of the experiment (unpublished data).

To quantify the contributions to changes in relative risk of prion dose and agent sorption to Mte, we constructed a multivariate Cox proportional hazards model with two covariates: log10 PrPTSE dose and Mte presence (Table 2). Each log10 increase in PrPTSE dose multiplies the relative risk by a factor of ~2 (i.e., a 10-fold increase in dose approximately doubles the risk of infection). Notably, sorption of purified PrPTSE to Mte multiplies the relative risk by a factor of ~8. These values allowed computation of a multiplicative equivalence factor between PrPTSE dose and Mte presence in the inoculum. Expressed in terms of PrPTSE dose, addition of Mte to the inoculum is equivalent to multiplying the PrPTSE dose by a factor of 680 (95% CI 16, ∞); that is, inclusion of Mte increases the effective titer of a given PrPTSE dose by 680-fold. Estimates of effective titer span a wide range (95% CI 16, ∞), and the present data do not allow us to place an upper bound on the increased risk associated with the presence of Mte in a sample. At a minimum, effective titer increased by 1.2 orders of magnitude, but the effect could be substantially larger. The best estimate of the Cox analysis represents a 2.8 order-of-magnitude increase in effective titer.

Estimated Hazard Ratios due to Prion Dose and Mte Addition

Strain PropertiesOral administration of Mte-bound PrPTSE did not appear to alter strain properties. Following limited proteinase K (PK) digestion, many PrPTSE strains can be discriminated by the size and glycoform pattern of PK-resistant core of PrPTSE (PrP-res) [3336]. Strain differences are also manifested in specific clinical symptoms. At the conclusion of the oral transmission experiments described above, the brains of clinically infected animals were assayed for PrP-res by immunoblotting (Figure S3). Differences in the molecular mass and glycoform distribution of PrP-res were not apparent between the treatment groups. Furthermore, clinical presentation of disease (symptoms or length of clinically positive period) did not differ between treatments.

The experiments described above were conducted using the Hyper (HY) strain of hamster-adapted TME agent (PrPHY). To further examine the strain stability of Mte-bound PrPTSE, we employed the Drowsy (DY) strain of hamster-passaged TME agent (PrPDY) to investigate the molecular mass of PrP desorbed from Mte and the effect of this clay mineral on oral transmissibility [35,36]. We previously reported the N-terminal cleavage of PrPHY extracted from Mte yielding a product similar in size to PK-digested PrPHY [22]. PK digestion of PrPHY and PrPDY results in products of characteristically different molecular masses [35,36]: the length of the PrPHY digestion product exceeds that of PrPDY by at least ten amino acids [35,36]. We found that extraction of bound PrPDY from Mte resulted in a product similar in molecular mass to PrPDY cleaved by PK (Figure 4). These data are consistent with the idea that strain properties are preserved when PrPTSE binds to Mte. DY agent is not orally transmissible [37], and we find that sorption of DY to Mte does not facilitate oral transmission (Text S1).

BH from hamsters clinically affected with either HY or DY agents were incubated with Mte to allow binding. Desorbed proteins were analyzed by SDS-PAGE and immunoblotting. Cleavage patterns of PrPHY and PrPDY extracted from Mte parallel PK cleavage patterns for the respective proteins: cleaved PrPDY migrates further (corresponding to a 1- to 2-kDa molecular mass difference) than cleaved PrPHY. Immunoblot used the PrP-specific antibody 3F4.

Natural soils are composed of a complex mixture of inorganic and organic components of various particle sizes. Smectitic clays such as Mte are important constituents of many natural soils and contribute significantly to their surface reactivity [38]. In natural soils, metal oxide and organic matter often coat smectite surfaces and may alter their propensity to bind PrPTSE. Furthermore, additional sorbent phases may be important in the binding of TSE agents to whole soils. We previously demonstrated that PrPTSE binds to whole soils of varying texture, mineralogy, and organic carbon content [22]. To examine the impact of agent binding to whole soil on oral TSE transmission, we incubated 1 μg of purified PrPTSE with each of three whole soil samples (Elliot, Dodge, and Bluestem soils) to allow sorption, and then orally dosed hamsters with the PrPTSE–soil mixtures. Soil-bound TSE agent remained infectious perorally, and two of the soils significantly enhanced oral disease transmission (Figure 5). Hazard ratios between Elliot (4.76 [95% CI: 1.38–16.4], p = 0.019) and Bluestem (6.04 [95% CI: 1.59–22.9], p = 0.013) soils and unbound PrPTSE indicate a significant increase in transmissibility, but no difference for the Dodge soil (1.66 [95% CI: 0.52–1.66], p = 0.578). The hazard ratios for the Elliot and Bluestem soils did not differ from one another (0.79 [95% CI: 0.19–3.25], p = 0.543) indicating statistical equivalence in transmissibility. The limited numbers of animals in the treatment groups precluded derivation of a multiplicative equivalence factor to equate the presence of Elliot or Bluestem soil with dose of infectious agent; however, substantially more animals in the Elliot and Bluestem treatment groups (14 of 16 animals, 87.5% penetrance) displayed clinical symptoms compared to the unbound PrPTSE treatment group (two of eight animals, 25% penetrance).

Prions Bound to Whole Soils Remain Orally Infectious and Some Soils Increase Transmission

Three soils (Dodge, Elliot, and Bluestem) were incubated in the presence of purified PrPTSE. The samples were orally dosed into hamsters and found to remain orally infectious. Agent association with Elliot and Bluestem soils increases disease incidence, whereas Dodge soil does not influence disease transmission. Animals dosed with soil alone remained healthy throughout the course of the experiment (unpublished data).

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 [22,40] 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 [4244]. 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 [22]. 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 [39,46,47]. 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 [5,79]. We find that Mte enhances the likelihood of TSE manifestation in cases that would otherwise remain subclinical (Figure 3B and 3C), and that prions bound to soil are orally infectious (Figure 5). Our results demonstrate that adsorption of TSE agent to inorganic microparticles and certain soils alter transmission efficiency via the oral route of exposure.

TSE agent source.

Syrian hamsters (cared for according to all institutional protocols) were experimentally infected with the HY or DY strain of hamster-adapted TME agent [48]. Brain homogenate, 10% w/v, was prepared in 10 mM NaCl. PrPTSE was purified to a P4 pellet from brains of hamsters infected with the HY strain using a modification of the procedure described by Bolton et al. [49,50]. The P4 pellet prepared from four brains was resuspended in 1 mL of 10 mM Tris (pH 7.4) with 130 mM NaCl. In the subset of experiments using PrPTSE–Mte complexes, larger prion aggregates were removed from the preparation by collecting supernatants from two sequential 5-min centrifugations at 800 g (clarification). Protein concentrations were determined using the Bio-Rad ( DC protein assay as directed by the manufacturer’s instructions.

Preparation of inocula and oral dosing.Four types of Mte- or soil-containing inocula were prepared: BH–Mte mixtures, PrPTSE–Mte mixtures, PrPTSE–soil mixtures, and PrPTSE–Mte complexes (see below). To prepare mixtures of BH or PrPTSE with Mte, the indicated amount of 10% brain homogenate (Figures 1 and 2) or PrPTSE (Figure 3) was added to 500 μL of 10 mM NaCl in the presence or absence of 500 μg of Na+-saturated Mte (particle hydrodynamic diameter = 0.5–2 μm) (prepared per [51]). Mixtures of PrPTSE and whole soils (Figure 5) were prepared by adding 1 μg of PrPTSE to 500 μL of 5 mM CaCl2 in the presence or absence of 1 mg of each soil type. Samples were rotated at ambient temperature for 2 h, like samples were pooled, and the equivalent of 500 μg of Mte or 1 mg of whole soil was orally inoculated into each hamster. We previously showed that absorption of purified PrPTSE to Mte was complete within 2 h [22].

Isolated PrPTSE–Mte complexes were prepared as previously described [22]. Briefly, the indicated amount of clarified PrPTSE (200 or 20 ng, Table 1) was added to 500 μg of Mte in 10 mM NaCl (500 μL final volume) per sample. Mixtures were rotated at ambient temperature for 2 h. Each PrPTSE–Mte suspension was placed over a 750-mM sucrose cushion prepared in 10 mM NaCl and centrifuged at 800 g for 7 min to sediment mineral particles and adsorbed PrPTSE. PrPTSE–Mte complexes were resuspended in 500 μL of 10 mM NaCl and pooled. The equivalent of 500 μg of Mte was orally inoculated into each hamster. To control for potential sedimentation of unbound PrPTSE, “mock” samples lacking Mte were processed identically, and any sedimented material was inoculated into hamsters. As a positive control, unbound PrPTSE (200 or 20 ng) was orally administered to hamsters. All oral inoculations were via pipette and voluntary consumption. Following oral dosing, hamsters were observed twice weekly for the onset of clinical symptoms [48] for at least 300 d, a period of time found sufficient to observe most or all clinical cases.

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