Badger Cull Or Mad Cow Disease

Badger Cull Covering Up Infected Soil

Despite a public outcry from the majority of people in the UK, the government and the agriculture industry have proceeded with a highly controversial badger cull. The official story is that it’s to help contain tuberculosis (TB), which can spread from badgers to cattle. In the livestock industry, TB is a death sentence for an infected animal and in some cases entire herds.

However, as ground zero in the war on Mad Cow disease, I’m curious if the UK has a hidden agenda that should be discussed. I propose that they test every dead badger for prion disease. As you probably know, prions are the deadly pathogen responsible for causing Mad Cow disease and other forms of prion diseases. Badgers are likely falling to prion disease because of infected soil. Killing them eliminates a symptom of a bigger health threat in the food and water supplies.

badger cull

Why not test the badgers for prion disease?

  • In the 1980s, the UK killed thousands upon thousands of cattle because of concerns over Mad Cow disease. It’s unknown how many of those cows actually had the disease, but several cases were verified and you don’t wipe out a nation’s entire herd over a frivolous concern.
  • Many of these infected carcasses were just buried, while others were incinerated and the smoke plumes filled the sky. This reckless disposal may have spread the disease further into the soil, groundwater, air and surface water. The world has a tendency to underestimate the prion threat. Buried carcasses may have attracted badger colonies.
  • Prions are deposited in the soil by infected animals’ urine, feces, blood, saliva and tissue.
  • Prions are known to persist in soil for a very long time (some mutated forms might be lethal forever).
  • Prions are known to mutate, migrate and multiply, which means prion-infected animals may have just been the tip of a growing iceberg.
  • Badgers live in the soil where the risk of prion exposure is high.
  • In Wisconsin, mink contracted prion disease by eating the carcasses (and protein meal) of downer cattle that likely had Mad Cow disease.
  • There is not an isolated case of Mad-cow, or any prion disease. The disease can’t be cured and infectious prions can’t be stopped. Cattle are still getting sick from prion disease across the UK and Europe despite the ban on feeding dead cattle back to other cattle. More than 170 cases have been confirmed since 1995.
  • The disease itself is contributing to deadly forms of environmental contamination that essentially recycle and redistribute the disease.
  • A responsible prion surveillance program will test the badger vector since they are determined to kill hundreds and possibly thousands of these tough little creatures. Such a test will help us understand the contagious nature of soil and water runoff.
  • Biosolids and reclaimed wastewater could be infecting badgers with prion disease from humans.

“Prions can survive for years in soil,” (Brown, et al 1991). “Animals can become infected from prions in soil,” (Miller, et al 2004).

biosolids land application and disease

Furthermore, prions can wash from the soil and migrate through irrigation and surface water runoff and settle in groundwater, streams, ponds, lakes, and oceans—where they proceed to multiply and mutate into even more abundant and lethal forms. Wildlife, livestock and humans (especially children) can ingest prions from soil exposure, water exposure, or food. We can’t afford to take the risk of further contaminating entire watersheds – increasing the pathway to humans, livestock, and wildlife downstream.

If the above chain of logic isn’t enough, let’s consider two other potential pathways.

  • Feedlot Waste. Farmers and feedlots will take cattle manure and urine from pens, corals and other holding facilities and spread it on their farmland and pastures where animals graze. My guess is that these are the same facilities that held thousands of at-risk animals back in the Mad Cow epidemic. Every infected animal in these facilities contaminated them permanently with their urine, feces, blood, and saliva. Those facilities are likely still in service today—exposing captive animals and spreading the risk further as fertilizer.
  • Biosolids and Reclaimed Sewage Water. Humans contract prion disease, too, which means our bodily fluids also can transport prions to new pathways (including reclaimed sewage water, sewage sludge—biosolids).

“Prions have been found in the blood and urine of CJD victims,” Gabizon, et al, 2001; Reichl, et al 2002. “Undertakers and medical facilities routinely discharge CJD infected blood and body fluids into public sewers,” Yale, UC Davis, Center for Disease Control.

EPA National Water Research Compendium 2009-2014 lists prions eight times as an emerging contaminant of concern in sewage sludge (biosolids), water and manure.

Renowned prion researcher, Joel Pedersen, University of Wisconsin, found that prions become 680 times more infective in certain soils.

Oral transmissibility of prion disease is enhanced by binding to soil particles. Dr. Pedersen and associates found that anaerobic digestion in sewage treatment did not inactivate prions in sludge. Persistence of pathogenic prion protein during simulated wastewater treatment processes.

biosolids land application LASS

In the July 3, 2010 issue of Veterinary Record, Dr. Pedersen stated, “the disposal of sludge was considered to represent the greatest risk of spreading (prion) infectivity to other premises.

“Given it is unlikely that the sewage treatment or pellet production processes can effectively deactivate prions, adopting measures to prevent the entry of prions into the sewer system is advisable,” Toronto (Canada) Department of Health, Nov. 2004.

“Pathogen free” is clearly not the case when the Class A sludge compost can contain infectious human and animal prions. Not only are livestock and wildlife at risk from ingesting prion infected soil and sludge, but humans, and particularly children, are especially at risk because their hand to mouth behavior results in the ingestion of dirt,
(Robischon, 1971; LaGoy, 1987; Binder, et al 1986; Gerba, et al 2002; CDC, Callahan, 2004).

In humans, we have conveniently given prion disease more sophisticated names, but they are equally as savage and deadly. After all, who wants to die from Mad Cow disease? Spin doctors call the human form Creutzfeldt-Jakob disease and often blur the lines by calling it Alzheimer’s disease. Pioneering research by Nobel Prize winners and others have now determined that Alzheimer’s disease is a prion disease, which explains why there is not a cure.

Alzheimer's disease epidemic

While the death rate from most major diseases are declining, the death rate from Alzheimer’s disease is rising (at least in the United States. My guess is that it’s a global trend.) The differing characteristics in the diseases are likely due to the fact that prions are known to mutate and the genetic makeup of different people and different species likely accounts for other variances in disease characteristics. Most coroners refuse to conduct an autopsy on the body of a person who had prion disease. They know how contagious the body is and how deadly prions are. Meanwhile, these people used public toilets from the point of infection until the day they died. Therefore, the sewage system is permanently contaminated with prions just from one sick patient. What if there have been thousands of infected people? Where has that sewage gone? Badger country. Cattle country. Watersheds that collect rainwater above the streams, ponds, lakes, rivers and oceans.

To make a long story a little longer, we have pissed in the pool. We owe it to ourselves and our grandchildren to understand the dangers of these foolish practices. Is the badger cull an attempt to rid the countryside of badgers before they are seen dying of prion disease and recycling it back to other animals and livestock? Or is it really just a TB containment precaution? I won’t get into the politics of the matter, but I will hammer on the logic of testing these innocent creatures. If there is a prion epidemic in our soil, we all deserve the truth while there is still a chance of partial containment. Once the prion problem reaches a tipping point, it will change life as we know it.

To learn more about prions and prion disease, please keep reading. We also have a library on this site archived for your review.

prion disease epidemic


Mad Cow disease, technically known as Bovine Spongiform Encephalopathy (BSE), is one of many deadly prion diseases (technically referred to as Transmissible Spongiform Encephalopathy (TSE).

We know prion disease as:

  • Mad-cow disease (or BSE in cattle)
  • Creutzfeldt-Jakob disease (CJD in humans)
  • Chronic wasting disease (CWD in wildlife such as deer, elk and moose)
  • Scrapie (sheep).

The common denominator in all of these diseases is the prion. In addition, about 10-25 percent of Alzheimer’s disease cases are misdiagnosed—they are actually CJD. In fact, according to Dr. Claudio Soto at the University of Texas, Alzheimer’s is a prion disease. When you sift through the smokescreen and lump these diseases together, it begs the question “do we have a deadly epidemic on our hands and is it being mismanaged.”

Prions are a form of protein that cannot be effectively stopped. They can’t be killed because they are not a virus or bacteria and they don’t contain DNA or RNA. These pathological proteins mutate, migrate, multiply, and intensify. Prion diseases kill everything in their path. In reality, there is no way to contain the disease. There is no cure—prion disease is always fatal.

Prions are a lethal threat to our food and water supplies. We also risk exposure and infection at hospitals, dental offices, restaurants, and through pet food. The buildup of prions in the environment will get worse with time. Mismanagement is accelerating the process. Various forms of prion disease are already spreading around the world, building up in soil and water, and building up in the bodies of virtually every living creature on the planet. The incubation period and the onset of clinical signs of the disease usually take years, which makes these diseases easier to ignore and more difficult to study.

Dr. Stanley Prusiner, an American neuroscientist from the University of California at San Francisco, earned a Nobel Prize in 1997 for discovering and studying deadly prions. President Obama awarded Prusiner the National Medal of Science in 2010 to recognize the growing significance of his discovery. Although his research is ongoing, we know enough about prions to sound the alarm on many levels.

U.S. Department of Homeland Security Sounds The Alarm

Prions are such a formidable threat that the U.S. government enacted the Bioterrorism Preparedness and Response Act of 2002, which included a provision to halt research on infectious prions in all but two laboratories. Now, infectious prions are classified as select agents that require special security clearance for lab research.

Thanks to Dr. Prusiner’s discovery and pioneering research, prion disease has been found in humans, livestock and a variety of wildlife species in several countries, including Austria, Canada, Czech Republic, Finland, Germany, Greece, Israel, Italy, Japan, Poland, Slovakia, Slovenia, Spain, United Kingdom, and the United States.

chronic wasting disease caused by prions

Not only are prion diseases a symptom of a much bigger problem, they are contributing to the buildup of prions in the environment.  A person or animal with prion disease is contaminating their immediate environment and exposing nearby humans and animals to deadly prions.

The Threat to Public Health

The prion pathogen spreads through urine, feces, saliva, blood, milk, soil, and the tissue of infected animals and humans—including muscle tissue. (Contrary to industry reports and a controversial statement from the World Health Organization, research suggests that milk is a pathway to prion exposure.

If a single person with prion disease discharges bodily fluids or feces into a public sewer system, that sewage system is permanently infected and the amount of contamination will multiply and intensify over time. Everything discharged from that sewage system—reclaimed water or biosolids—is at risk of spreading the contamination even further.

sewage treatment plant and disease

Humans with Creutzfeldt-Jakob Disease (CJD) also are shedding infectious prions into toilets, public sewers and elsewhere (including dental offices and eating utensils). Between 2 and 25 percent of the 4.5 million cases of Alzheimer’s disease and senile dementia victims in the U.S. alone are actually infected with sporadic CJD, creating the reality that many thousands of CJD victims are shedding infectious prions throughout their home and everywhere they visit, (Manuelidis, et al, 1989; Boller, et al, 1989, 1995; Harrison, 1991; Teixeira, 1995; Warren, et al, 2005).

Similar pathways exist from an infected cow, sheep, or deer. When infected animals use fields and feedlots, their urine, feces, saliva, and blood permanently contaminates those areas. That contamination becomes bio-available to every creature that follows the path of the infected animal.

mad cow disease and prions

Mad-Cow Precautions Are Inadequate and Short-Sighted

For example, in Canada, out of 55,415 cows tested for BSE in 2006, five head of cattle were identified with BSE. If the U.S. only tests 40,000 head of cattle and detects one animal with BSE out of that miniscule test group, we must assume that hundreds more infected animals are missed out of the millions milked and slaughtered each year. Based on those statistics, without comprehensive testing globally, we must assume that beef and dairy operations are producing hundreds of sick animals each year that are being milked, slaughtered and consumed by humans, pets, fish farms, and even other forms of livestock and poultry. In addition, the land at the feedlot or dairy is contaminated with manure and urine that often is scraped out and used as compost and fertilizer on farms and gardens, which expands the pathways for deadly prions to reach unsuspecting families.

With these characteristics and dynamics, there is not an isolated case of Mad-cow or any prion disease. The California dairy where the recent infected animal lived and produced milk is contaminated (two dairies have been quarantined since the discovery of that case of BSE). If the infected cow provided milk to a processor, that supply chain is now in question and those supply chains should be quarantined. In fact, all exposed milk should have been immediately recalled from that entire supply chain. The pathways of that milk still should be traced and condemned.

If the infected animal was rendered for pet food, that rendering plant is now permanently contaminated and will contaminate everything that is processed from now on—exposing our pets to the deadly disease and creating a new pathway in our homes—food bowls that also are permanently contaminated (in fact, an undisclosed rendering plant has been quarantined).

If livestock with BSE is actually processed at a slaughterhouse, that slaughterhouse also is permanently contaminated and will contaminate every carcass that follows the infected animal down the production line. Compounding the problem is the fact that liquid wastes from slaughterhouses are rinsed down the drainpipe and into the municipal sewage system, where they add to the risks associated with that waste stream.

 Deadly Prion Pathways

Milk and Meat: As stated earlier, highly infectious risk-material (brain and spine) is not the only pathway to prion exposure. Prions have been found in muscle tissue and milk.

Lessons From The United Kingdom: Initially, UK officials insisted the Mad-cow epidemic was not a risk to humans. After 150 human vCJD deaths, they admitted that they were wrong.

“Thousands of pages of grisly detail on meat-pie making and animal-feed milling might seem like a hard read. As bureaucrats digest the final report of Britain’s BSE inquiry, handed to ministers on October 2nd, 2000 stomachs at the Ministry of Agriculture, Fisheries and Food (MAFF) and the Department of Health must be churning. Not at the finer points of carcass-rendering, but at what is expected to be a thorough dissection of bureaucratic incompetence. Ministers will be considering the findings until the report is presented to Parliament on October 23rd. Three days later, the public will at last be allowed to read the report into Britain’s biggest public-health scandal for decades.

The independent inquiry was established by the UK government to work out the history of two epidemiological crises, bovine spongiform encephalopathy (mad-cow disease) and its human relative, new-variant Creutzfeldt-Jakob disease (VCJD). The inquiry’s three-person committee, headed by Lord Phillips, a high-court judge, was also asked to assess whether government and industry responded adequately to the situation as it evolved.

Roughly £27m ($39.4m) and 630 witnesses later, the Phillips report is widely expected to be the definitive word on what went wrong in Britain between the first documented cases of BSE in 1986 and the announcement in Parliament, ten years later, that the strange neurodegenerative condition appearing in a handful of young people, now called VCJD, was probably linked to mad-cow disease.” – The Economist, October 5, 2000.

The Full report from the UK’s BSE Inquiry is available here.

Furthermore, almost 4,000 Britons aged between 10 and 30 may be harboring the prion proteins that cause the human form of Mad-cow disease. The new estimate comes from direct analyses of human biopsies (tonsils), and is much higher than epidemiological projections of the likely number of deaths from variant Creutzfeldt-Jakob disease (vCJD).

treat Alzheimer's disease

For years, industry experts and government regulators insisted infectious prions could not be found in blood or muscle except for infected sheep and goats. Prions have since been found in blood and muscle of human vCJD and sCJD victims and in the leg muscle tissue of infected deer.

Even organic supplies are not immune from the prion problem. For example, if an organic farm is downstream from a traditional farm that has an animal with BSE, the water runoff from that farm will expose the organic operation downstream to deadly prions.

Let’s assume that everything that the beef and dairy industries, and the USDA, have said about the latest example of Mad-cow disease is true. The tested animal was sent to a rendering plant and was never destined for the food supply.

  1. How much milk did that dairy cow produce before it exhibited clinical signs of the deadly disease? Where did that milk go? On what date was this sick dairy cow withdrawn from the production line? We know that animals are contagious, and shed prions via bodily fluids, including milk, long before they exhibit clinical signs of the disease.
  2. How many other dairy cows have this fatal and contagious disease, but don’t exhibit the clinical signs, yet? How much milk are these animals producing every day?

Alzheimer's disease and contaminated dental surgical instruments

Surgical and Dental Procedures: We can’t sterilize surgical equipment used on people who have prion disease. Prions are so resistant to sterilization that surgical instruments used on a person with CJD must be disposed because they are permanently contaminated.  Hospitals have been sued successfully for exposing subsequent patients to deadly prions.

Dental and oral surgery settings have the same challenge, but those industries have ignored those risks for the most part.

Growth Hormones and Blood Transfusions: Most growth hormones are made from the pituitary brain of dead cattle or cloned from the DNA of that pituitary gland (bad idea). One infected gland in the production facility and all future products are permanently contaminated. Dairy and beef producers could be injecting BSE directly into live animals with this practice. Similar practices (taking the pituitary gland from cadavers) have killed people from prion disease, including this very recent case from May 2012:

It’s time to stop using growth hormones in beef and dairy cattle. Even if the hormone itself is free of prion disease, what does a growth hormone do to a prion? Has industry or regulators even conducted studies on this dynamic? People have contracted prion disease from infected hormones, infected blood and infected organ transplants. We must assume that the same risk is present for livestock.

Recent studies of variant Creutzfeldt-Jakob disease (vCJD) indicate that this disease is transmissible by blood. One case of probable transfusion-transmitted vCJD infection has been reported, and one case of subclinical infection has been detected. On February 9, 2006, a third case was announced by the UK Health Protection Agency

Each of the three patients had received a blood transfusion from a donor who subsequently developed clinical vCJD, which indicates that transfusion caused the infection.

Pet Food: How many infected animals are sent to a rendering plant, never tested for BSE, and are churned into food for dogs, cats, poultry, fish, and zoo animals? What is the likelihood that we are feeding deadly food to our pets? If and when contaminated, that food dish is another pathway for the prion pathogen to enter our homes and bodies, not to mention the risk to our pets. How often do you actually touch that pet food or the dish? How often do you wash that bowl in your kitchen sink?

Aquaculture: Many fish farms use specified risk material—SRM (brain and spinal cords) from slaughterhouses and rendering plants as protein meal. What is the likelihood that infected material from a slaughterhouse or a rendering plant was sent to a fish farm (either in a large lagoon or in the open ocean) and dumped into the water and consumed by farmed fish (and wild fish and mammals such as dolphins and whales). Since every microscopic prion can’t be consumed, how much water are we contaminating every year to extend this science experiment via new pathways.

This questionable practice puts the health of the fish at risk and those who eat the fish. Secondly, the water that the risk material is dumped becomes contaminated with prions, which threatens groundwater, surface water runoff, streams, rivers, and oceans with deadly prions. This factor could be contributing to the deaths of dolphins and whales and it could be contributing to prion disease in people. Many fish have contracted Whirling disease, which could be a form of prion disease (needs research that this author has not conducted, yet).

Animal Rendering & Anaerobic Digestion Of Carcasses: “It’s necessary to use additional heat at the end of the rendering process to fully inactivate pathogens.  However, even with this, prions are not inactivated,” APHIS/USDA, January 2005.

“While finished compost can be spread on farmland as fertilizer, if prions are present and the compost is used as fertilizer prions can re-enter the food chain through grazing plants, hay and straw obtained from those areas. Thus, composting should not be used to dispose of infected deer, elk, sheep, goats, or cattle. Composting is especially unsuitable for specified risk materials, especially neural tissues (skull and spinal cord) encased in bones. The indiscriminate use of composting and spreading its byproducts on agricultural land is inconsistent with the FDA feed rule, would dilute its integrity and invalidate all existing BSE/TSE risk assessment models. This is similar to what may have transpired with the CWD material, given the WIDNR (Wisconsin Department of Natural Resources) disposal policy was indeed implemented,” National Renderers’ Association response to USDA and APHIS, June 2005.

Sewage Sludge and Wastewater Reuse: Thousands of tons of sewage sludge (biosolids) are spread on farmland, parks, open spaces, and even lawns and gardens every year. In addition, millions of gallons of sewage water are being reclaimed for various uses.  Spreading sewage sludge and reclaimed sewage water on fields and in our watersheds is another foolish and risky practice. People with CJD (and many with Alzheimer’s disease) excrete prions in their urine, feces, saliva and blood and these recycling practices also are recycling, concentrating and expanding pathological pathways back to humans.

“Prions have been found in the blood and urine of CJD victims,” Gabizon, et al, 2001; Reichl, et al 2002. “Undertakers and medical facilities routinely discharge CJD infected blood and body fluids into public sewers,” Yale, UC Davis, Center for Disease Control.

Other prion-contaminated wastes dumped into sewers include waste from rendering plants (which process remains of up to two million potentially BSE infected downer cows each year), slaughterhouses, morticians, bio-cremation, taxidermists, butcher shops, veterinary clinics, necropsy labs, and hospitals.

Prions are not neutralized by sewage treatment. Therefore, prions become part of the sludge and create pathways to the disease on fields and water runoff. It’s time to quit spreading lies and pathogens on farmland and pastures where livestock graze and where surface water runs off into streams, rivers, lakes and ponds.

Sewage treatment does not inactivate prions. In fact, it concentrates the infectious prions in the sewage sludge being applied on home gardens, cropland, grazing fields and dairy pastures, putting humans, family pets, wildlife and livestock at risk.

“Prions are extremely resistant to inactivation by ultraviolet light, irradiation, boiling, dry heat, formaline, freezing, drying and changes in pH. Methods for inactivating prions in infected tissues or wastes include incineration at very high temperatures and alkaline hydrolysis,” U.S. EPA.

EPA National Water Research Compendium 2009-2014 lists prions eight times as an emerging contaminant of concern in sewage sludge (biosolids), water and manure.

Renowned prion researcher, Joel Pedersen, University of Wisconsin, found that prions become 680 times more infective in certain soils.

Oral transmissibility of prion disease is enhanced by binding to soil particles. Dr. Pedersen and associates found that anaerobic digestion sewage treatment did not inactivate prions in sludge. Persistence of pathogenic prion protein during simulated wastewater treatment processes,

In the July 3, 2010 issue of Veterinary Record, Dr. Pedersen stated, “the disposal of sludge was considered to represent the greatest risk of spreading (prion) infectivity to other premises.”

“Given it is unlikely that the sewage treatment or pellet production processes can effectively deactivate prions, adopting measures to prevent the entry of prions into the sewer system is advisable,” Toronto (Canada) Department of Health, Nov. 2004.

“Pathogen free” is clearly not the case when the Class A sludge compost can contain infectious human and animal prions. Not only are livestock and wildlife at risk from ingesting prion infected soil and sludge, but humans, and particularly children, are especially at risk because their hand to mouth behavior results in the ingestion of dirt,
(Robischon, 1971; LaGoy, 1987; Binder, et al 1986; Gerba, et al 2002; CDC, Callahan, 2004).

Given the volumes of research that clearly point to the risks associated with sewage sludge, how many cattle are being exposed to prions by grazing on land where sewage sludge (biosolids) has been applied? This exposure alone could spawn countless cases of Mad-cow disease around the globe every year. In addition, how many humans have ingested prions directly thanks to this foolish practice? How much water has been contaminated thanks to sewage sludge applications in our watersheds and directly injected into our rivers and oceans?

Sludge proponents claim that there aren’t enough prions in sludge to constitute an infectious dose. “Critics say that one example of outdated assumptions is the Harvard study’s assumption that a cow would have to eat one gram of infected material to come down with the disease. Most scientists now believe a cow would have to eat only 10 milligrams of infected material, a piece the size of a peppercorn, to catch the disease. That’s 100 times smaller than the assumption in the Harvard study. Recent British studies suggest the infectious dose could be 400 micrograms, which is 25 times smaller than 10 milligrams,” said Dr. Michael Hansen.

Above Sources on Sewage Risks Compliments of Helane Shields, Alton, NH.

Additional Documents & Research of Interest

  • “BSE has now been transmitted orally to 16 species,” (S. Dealler, 1995). Animals which have suffered fatal prion diseases include sheep, goats, cattle, pigs, bison, elk, mule and white-tailed deer, oxen, moose, domestic house cats, several species of macaques/monkeys, several species of lemurs, farmed mink, cougars, cheetahs, puma, ocelot, tiger, lion, kudu, oryx, eland, nyala, gemsbok and ankole.
  • Rendered sheep fed to cattle are believed to have initiated the Mad-cow epidemic. Intensive inbreeding of sheep for various genetic characteristics is thought to have spawned prion disease in sheep.
  • Contaminated meat and MBM feed are linked to zoo animal infections. A 1999 report documented three German zoo ostriches, which developed prion disease after eating feed made from downer cattle. An elephant at the Oakland Zoo died of prion disease.
  • Under ordinary circumstances, most sCJD cases go undiagnosed. Few autopsies are done on suspected sCJD victims because the families don’t want to incur the expense. What’s the point if their loved one is already dead? And the pathologist/medical examiner is reluctant to do an autopsy because he/she is concerned about their own risk of infection and the fact that expensive medical instruments may have to be discarded if the case is positive.
  • By binding to a common soil mineral, the misshapen proteins that cause chronic wasting disease in deer can be as much as 700 times more infectious than exposure to the proteins alone, according to researchers at UW-Madison. The finding, by UW-Madison animal health and biomedical science professor Judd Aiken, may help explain why CWD spreads orally among Wisconsin deer even though animals in the wild are exposed to relatively low levels of the infectious proteins, called prions. Herbivores, including deer and sheep, consume a fair amount of dirt each day as they graze. They also are known to consume soil as a source of minerals. Grazing cattle are known to ingest one kilogram of soil per day (2.2 pounds)

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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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