Airborne Prion Pathways
By James B. Woodward
February 19, 2004
Mr. Jonathan Akins
Environmental Engineer
Air Pollution Control Division – Stationary Sources Program
Colorado Department of Public Health & Environment
4300 Cherry Creek Drive South
Denver, Colorado 80246-1530
Subject: Draft Permit #03ME0664 issued to Colorado State University (CSU) for a bio-medical waste incinerator for disposal of pathological waste, some of which could potentially be infected with transmissible spongiform encephalopathies (TSEs), located at 425 29 Road, Grand Junction, Mesa County, Colorado.
Dear Mr. Akins:
The following are my comments concerning the air permit for the proposed Grand Junction incinerator. My comments are primarily concerned with the question of whether this incinerator will effectively destroy the agents thought to cause Transmissible Spongiform Encephalopathies (TSEs). Since I believe there is no assurance infective material will not be released into the environment, I request that the Air Pollution Control Division prohibit the disposal of waste contaminated or potentially contaminated with TSEs in this proposed incineration facility.
The proposed incinerator will be used to dispose of deer and elk heads (and perhaps carcasses) infected or potentially infected with Chronic Wasting Disease (CWD).
If the facility is permitted to burn TSE-infected tissues, the possibility exists that cattle with Bovine Spongiform Encephalopathy (BSE) or the newly discovered Bovine Amyloidotic Spongiform Encephalopathy (BASE)[1], and/or sheep with scrapie may be disposed of in this incinerator as well.
Surprisingly, the Colorado Department of Public Health & Environment has no departmental rules, regulations, policies, guidelines, and/or recommendations regarding the safe and effective disposal of TSE-contaminated animal carcasses and tissues. Over the past several months I have submitted Colorado Open Records Act requests asking for any such records or documents. None have been produced. Given the department’s lack of regulatory guidance on this matter and the scientific uncertainties surrounding cross-species transmission of TSEs, it would appear to be premature and irresponsible to allow burning of TSE-contaminated wastes in this proposed facility.
The draft permit requires an afterburner temperature of 1800° Fahrenheit (982° Celsius) with a retention time of two seconds. As you are aware, there is no scientific data suggesting that these time and temperature parameters will successfully eliminate detectable TSE infectivity. The parameters are drawn from a 1997 risk assessment prepared for the U.K. Environment Agency by the consulting firm Det Norske Veritas Limited (DNV). The report looked at the risks from disposing of BSE infected cattle in animal carcass incinerators, specifically the Vetspeed incinerator plant in Cambridge. The Vetspeed incinerator has approximately twice the capacity of the proposed Grand Junction unit. In addition, the Vetspeed incinerator uses a water-spray gas scrubbing unit for removal of particulates, which the study explains is one of the main protective measures against incomplete combustion.
The proposed incinerator would have no such pollution control equipment.
It is curious that the Colorado Department of Public Health & Environment would utilize the parameters from this study since the department apparently does not possess a copy of the actual study. The department seems to be relying on a brief excerpt of the study contained in a later report by the European Commission’s Scientific Steering Committee
(EC-SSC)[3]. A careful reading of the actual study reveals that the recommended conditions of operation, 850°C for at least two seconds, are not grounded in any direct scientific evidence. The authors state: “No experimental data are available on the effect of incineration on BSE infectivity as such.”
The typical method of testing for residual TSE infectivity is the bioassay. Suspect tissue or material is injected into the brains of test animals. The animals are monitored for clinical disease symptoms and are later autopsied and tested for evidence of a TSE. The DNV study did not use the bioassay to test incinerator ash or emissions. Rather, they used a surrogate measurement: the total protein content remaining in the ash. The theory is that, since the infective agent is thought to be a protein, the reduction in infectivity will be in proportion to the reduction in protein content.
In a 2003 report, the EC-SSC looked at this methodology and concluded that data to date suggest the assumption that TSE degradation is proportional to the degradation of other proteins is not necessarily justified. The heat resistance of the TSE agent may simply be greater than other proteins.
The department’s reliance on the DNV study for minimum time/temperature parameters for TSE waste incinerators may be misplaced. The study provides no direct evidence or experimental data supporting the parameters. And it is unclear why the department has disregarded the DNV recommendation calling for the use of pollution control equipment to remove potentially infective particulates.
Below is additional information describing the resistance of TSE agents to typical inactivation methods, problems associated with incineration, the alternative disposal method of alkaline tissue digestion, and cross species transmission of TSEs (particularly CWD).
TSE Inactivation Impossible
Writing in a study on prion transmission, Dr. C. Weissmann of the Medical Research Council Prion Unit of the Institute of Neurology in London stated:
“A striking feature of prions is their extraordinary resistance to conventional sterilization procedures, and their capacity to bind to surfaces of metal and plastic without losing infectivity.”
The Centers for Disease Control and Prevention reports that “Prions are characterized by extreme resistance to conventional inactivation procedures including irradiation, boiling, dry heat, and chemicals.”
The World Health Organization (WHO) writes, “TSE agents are unusually resistant to disinfection and sterilization by most of the physical and chemical methods in common use for decontamination of infectious pathogens.” The WHO goes on to note “infectivity is strongly stabilized by drying” and that “contaminated materials should be kept wet between the time of use and disinfection by immersion in chemical disinfectants.”[9]
In a scientific study on the heat resistance of the scrapie agent, Dr. Paul Brown observed that TSE agents “are notoriously resistant to most physical and chemical methods used for inactivating pathogens, including heat”.[10] Dr. Brown is a leading authority on prion diseases and prion inactivation. A 1998 article by Dr. Brown in the Lancet includes the following discussion of prion inactivation:
“The agents that cause TSE have been known almost since their discovery to have awesome resistance to methods that quickly and easily inactivate most other pathogens…TSE agents are very resistant to virtually every imaginable method of inactivation, and those methods found to be most effective may, in one test or another, fail to sterilise. It seems that even when most infectious particles succumb to an inactivating process, there may remain a small subpopulation of particles that exhibit an extraordinary capacity to withstand inactivation, and that, with appropriate testing, will be found to retain the ability to transmit disease.”
Dr. Brown’s discussion of heat resistant subpopulations refers to experiments by Dr. David Taylor, who is perhaps the world’s leading expert on prion inactivation. Dr. Taylor published a study in 1998 showing that during heat inactivation, small subpopulations of TSE agents can become rapidly heat-fixed, and that these thermostable
subpopulations may survive to resist further attempts at inactivation. The resistant subpopulations can be differentiated by their longer incubation periods in test animals.
In a study on the effect of dry heat on the scrapie agent, Dr. Taylor notes that prions “possess a number of properties which differentiate them from conventional microorganisms, including an exceptional resistance to inactivation by chemical and physical methods”.
The most relevant study on heat resistance of TSE agents was done by Dr. Paul Brown et al., published in 2000. Brown exposed one-gram samples of scrapie-infected hamster brains to various time and temperature parameters. The resulting samples were injected intracerebrally into healthy hamsters to test for residual infectivity. Samples exposed to 600°C and 1000°C for 5 minutes resulted in no detectable infectivity. A sample exposed to 600°C for 15 minutes, however, infected 5 of 18 hamsters.
The fact that 5 of the test animals became infected is remarkable, since it seems likely that exposure to 600°C for 15 minutes would decompose all organic compounds. This enigmatic result led Brown and colleagues to propose various explanations. They note “combustion is a series of pyrolysis and oxidation reactions that proceed rapidly but
incompletely,” and that 600°C is a “comparatively low combustion temperature”.
They theorize that incomplete combustion may have introduced elemental carbon into the combustion residues, and carbon has been reported to partially protect TSE infectivity. The researchers observed that only at 1000°C did it appear that most of the carbon residue had been oxidized. The most interesting theory proposed by Dr. Brown is that the heat created an inorganic replica of the prion’s molecular geometry. This inorganic “fossil template” could mimic the infectivity of the scrapie agent.[15]
Although the study’s authors write that the results suggest that such an inorganic template would have a decomposition point near 600°C, the fact is that this is a guess. Measurable infectivity was found at 600°C and none was detected at 1000°C. No samples were exposed to intermediate temperatures.
Incineration Of Deer With CWD
Regarding incineration, the European Commission’s Scientific Steering Committee (EC-SSC) concludes that:
“With respect to TSEs, it is generally assumed that incineration is a completely effective method for destroying TSE-like agents. However, there is no direct evidence for this. The dry heat experiments described in the literature may not be completely relevant to incineration because exposure to dry heat does not involve oxidative combustion, as occurs during incineration.”
“The possibility that incineration might not be completely effective is clearly being considered. For example, after incinerating materials that could be TSE-infected, the USDA soaks the resulting ash in sodium hydroxide for two weeks before disposal.”
The EC-SSC’s “Opinion on the use of small incinerators for BSE risk reduction,” dated January 16-17, 2003 notes the following:
1. Because of variability in incinerator design and performance characteristics, each incinerator facility needs to be the subject of a specific risk assessment.
2. There is no direct data on the TSE levels that may occur in airborne emissions and residual ash from incinerators.
3. Because of this lack of data it is not possible to assess the risks.
4. In the absence of reliable data on the possible residual infectivity of the ash, it should be disposed of in controlled landfills.
5. Unburned material is commonly noted in the ash from small
incinerators.
6. The level of expertise available for the management of small incinerators is highly variable because few such facilities can afford to employ specialists in incineration.
The EC-SSC’s report concludes:
“In view of the uncertainty regarding the risks due to BSE/TSE contamination of the fly and bottom ash and airborne emissions it is recommended that further research is conducted to identify the residual risks (along with attendant uncertainties) from the burial of ash (without further treatment,) in uncontained sites. It is essential that suitable monitoring methods are developed.”[17]
In a presentation to the Food and Drug Administration’s Transmissible Spongiform Encephalopathies Advisory Committee on July 17, 2003, Captain Edward Rau explained that “ash bed temperatures often may run 100°C lower than the actual air temperature” (in an incinerator’s burn chamber).[18] Captain Rau is an Environmental Health Officer with the National Institutes of Health and a co-researcher with Dr. Brown on the scrapie heat resistance study.
A 6,000 pound capacity incinerator burns nearly 3 million times the mass of the samples tested by Brown and Rau. Many factors can affect an incinerator’s ability to completely combust TSE contaminated waste material. As Captain Rau explains:
“Probability of survival in ash not only depends on a lot of factors, the load density, the turbulence, the type of equipment, other operational factors.”
“Particularly, as things are just inserted into the incinerator, you tend to get a boil off of some of the material, a flash burn. That can be carried over very quickly into the second chamber.”
“Reported temperatures for incinerators refer to the air. That is what is being monitored, and not the actual temperature in the ash. Under abnormal conditions, a lot of things can really go wrong, cold start up conditions, overloading, inadequate control of the under fire air flow.” [19]
Overloading, excessive load density, inadequate air turbulence, and insufficient control of under fire airflow can all negatively impact incinerator performance. Any of these conditions can result in incomplete heat penetration of the ash bed and insufficient residence time in the burn chamber.
Dr. David Taylor discusses this possibility in a paper on disposal issues related to TSE infected animals:
“Although incineration is generally regarded as the optimal method for the destruction of all microorganisms, there have been frequent reports of the discovery of organic material in the resulting ash. These findings indicate that the incineration process does not always perform according to the required standards.”
Equipment breakdowns during burn cycles are likely to occur at some point. These include electrical component failure, short circuits, and failure of fuel or air delivery systems. A common incinerator problem is refractory (firebrick) breakage. If the burn chamber is not inspected and repaired on a regular basis, a damaged refractory can expose the steel wall of the burn chamber to high temperatures resulting in a “burn-through” and emergency shutdown.
Malfunctions can force a shutdown in mid-cycle. The result is an unintended release of smoke from the stack since there is no way to effectively contain the combustion gases until the partially combusted waste material has cooled down. When an incinerator is in failure mode there is no way to close or contain the system to prevent release of infectious agents and other pollutants into the environment.
Operator training and supervision is crucial to incinerator performance. Operators of hazardous waste incinerators are required by law to take classroom training courses and annual refresher courses. The American Society of Mechanical Engineers has developed certification programs for hazardous and medical waste incinerator operators. The Colorado Department of Public Health & Environment, however, does not require classroom courses, refresher training, or operator certification.
Incinerator upsets and problems may occur often, even in new facilities. The Colorado Division of Wildlife (CDOW) operated two new pathological incinerators in the town of Craig, Colorado during the 2002-2003 hunting season. In daily logs obtained under the Colorado Open Records Act, the incinerator operator documented numerous problems and malfunctions. The occupants of another house about 1,000 feet from the incinerators told Mr. Switzer they experienced a “foul smell” from the incinerators. In a July 15, 2003 article in the Craig Daily Press, Ed Relaford confirmed that the stench of burning flesh permeated his neighborhood.
Alkaline Tissue Digestion and CWD
Alkaline tissue digestion is the only proven technology for prion inactivation that is capable of handling large volumes of animal carcasses. It is based on the process of alkaline hydrolysis, which is the breaking of chemical bonds by the insertion of water between atoms, catalyzed by alkali metal hydroxides.
The technology for hydrolyzing large quantities of animal carcasses was developed in 1992 by Dr. Gordon Kaye and Dr. Peter Weber, professors at Albany Medical College in New York. The equipment consists of an insulated, steam-jacketed, stainless steel pressure vessel with a lid. Carcasses (or heads) are loaded into a basket that is placed
inside the vessel. The load is automatically weighed, and the appropriate amounts of water and alkali are automatically added.
The vessel is sealed and the contents are heated by steam. The alkali solution is continuously recirculated and agitated. There are no moving parts inside the vessel. The tissues are dissolved and are hydrolyzed into smaller and smaller molecules.[26] The recommended digestion cycle time is six hours based on experiments done by Robert Somerville in 2002, funded by the UK Department of the Environment, Food, and Rural Affairs (DEFRA).[27]
The Somerville experiments involved digestion of sheep heads inoculated with mouse-passaged BSE agent. Different chemical and time parameters were tested. Samples of the resulting hydrolysate were diluted and injected into mice. Although infectivity remained after 3-hour digestion cycles, no infectivity was detectable after a 6-hour cycle.
Due to constraints specific to the Somerville experiments, the European Commission Scientific Steering Committee (EC-SSC) concluded in its April 2003 opinion that further studies are needed “before any final assurance could be given regarding the safety of the process with respect to TSE risks”.
It should be noted that the precautionary approach to digestion by the EC-SSC has never been applied to the process of incineration. This is perhaps because of the widely held, though not scientifically proven, assumption that incineration is an effective means of inactivating TSEs. Regardless, with respect to TSE inactivation, alkaline tissue digestion has been subjected to far greater scrutiny than incineration.
Responding to an early version of the EC-SSC’s opinion, Dr. David Taylor prepared a risk assessment of alkaline digestion assuming a digester with the capacity to process ten BSE-infected bovine carcasses. Dr. Taylor concluded that “a human would have to consume at least 126,000 litres (over 33,000 gallons) of effluent from the production process to have a 50% chance of developing variant CJD.”
Further reinforcing the effectiveness of alkaline hydrolysis are Dr. Taylor’s comments in his 2000 review of prion inactivation methods:
“The only methods that appear to be completely effective under worst-case conditions are strong sodium hypochlorite solutions or hot solutions of sodium hydroxide.”
Apparently convinced that alkaline tissue digestion is effective, the European Commission is in the process of adopting the final language of regulations approving alkaline hydrolysis for disposal of TSE-infected materials.
Compared to incinerators, alkaline digesters are safer particularly in the failure mode because the system is self-contained. In the event of loss of temperature and pressure, or mechanical breakdown, the contents remain inside the sealed vessel until repairs are completed and the process is restarted.
The effluent from a digester can either be dehydrated and the remaining solids landfilled, or an anaerobic digestion process can be utilized. Many facilities reduce the pH level of the effluent and release it into municipal sewer systems with no adverse effects.
Alkaline tissue digesters have already become the waste disposal technology of choice at a variety of installations. Health Canada’s Host Genetics and Prion Diseases Federal Laboratories in Winnipeg took delivery of a small digester in January 2000. In April of that year, the US Department of Agriculture’s (USDA) Animal Research Service lab in
Laramie, Wyoming installed a 1,500-pound capacity tissue digester. In February 2001, the USDA’s Animal and Plant Health Inspection Service in Ames, Iowa purchased a 7,000-pound digester to dispose of 350 sheep exposed to an unidentified prion disease.
Colorado State University’s Veterinary Diagnostic Lab (CSU-VDL) in Fort Collins installed a 2,000-pound digester in October 2001. The facility is a joint venture of CSU, the Colorado Department of Agriculture, and the USDA. During the 2002-2003 hunting season, the DOW contracted with the CSU-VDL to dispose of 140,189 pounds of deer and elk heads and carcasses, many infected with CWD.
The College of Veterinary Medicine at the University of Pennsylvania purchased a 7,000-pound unit last year specifically for prion destruction.[37] In a joint venture, the USDA, the Wisconsin Veterinary Diagnostic Laboratory (WVDL), and the Wisconsin Department of Agriculture, Trade and Consumer Protection are deploying a mobile tissue digester to be available for state use in dealing with the CWD “emergency” as well as other animal disease outbreaks. The USDA has provided $1 million for the purchase of the digester as part of a homeland security grant to the state and WVDL.
The Cornell College of Veterinary Medicine (CCVM) in Ithaca, New York is finalizing funding for an alkaline tissue digester to replace its 18-year-old pathological incinerator. In the Draft Environmental Impact Statement for the project, the CCVM concludes the following significant positive impacts on human health are to be expected:
“The significant reduction in air pollutants and greenhouse gases (e.g., NOx and CO2) would result in a positive impact on human health.”
“Moreover, the Proposed Action would provide more reliable treatment of animal remains infected with prions, the causative agents of Mad Cow Disease and other Transmissible Spongiform Encephalopathies (TSEs) compared with current treatment (i.e., incineration) based on research published to date, and would protect public health to the maximum extent in the event that prion-infected wastes were received by the CCVM.”
Compared to incineration, alkaline tissue digestion is a more reliable method of TSE inactivation, has undergone more scientific scrutiny, can be contained in failure mode, is simpler to operate, is a less complex technology, and costs less to operate per pound of waste.
Cross-Species Transmission of TSEs
The Raymond et al. 2000 in-vitro study found that the human molecular barrier to CWD is approximately as effective as the human molecular barrier to BSE.[40] While the transmission of BSE to humans is inefficient, it has certainly proven to be possible. U.K Department of Health statistics as of February 2, 2004 indicate that 139 people have died of definite or probable variant Creutzfeldt-Jakob Disease (vCJD). It is believed that variant CJD is caused by consumption of beef contaminated with BSE.
The study shows that, at the molecular level, humans are no less susceptible to CWD than to BSE. The study’s authors conclude “since humans have apparently been infected by BSE, it would seem prudent to take reasonable measures to limit exposure of humans (as well as sheep and cattle) to CWD infectivity.”
Many of the 692 cases of sporadic Creutzfeldt-Jakob Disease recorded in the U.K. since 1990 may also be linked to BSE. Sporadic CJD has been thought to occur spontaneously and not as a result of exposure to a pathogen. This theory was called into question in a 2002 study by Emmanuel Asante, John Collinge, and others showing that BSE could produce a disease indistinguishable from sporadic CJD. The authors conclude, “some patients with a phenotype consistent with sporadic CJD may have a disease arising from BSE exposure.”
Any natural or molecular barriers humans may have against CWD may be overcome by the agent’s potential for adaptation. In a study by scientists at the Rocky Mountain Laboratories in Hamilton, Montana, a strain of hamster scrapie gradually adapted to cause illness in mice that were previously not susceptible to the disease.
The research revealed that scrapie prions could persist in asymptomatic mice for years at levels too low for standard lab tests to detect. When the agent was transferred from the original group of mice to additional groups, the disease grew stronger making the newly infected mice sick. Researcher Richard Race explained: “The scrapie seemed to have learned how to deal with this new species, and it worked much better. It replicated faster in additional rounds of mice and even became more lethal to them.”
Race noted the study “confirmed that prion disease can adapt to new species”, and that “the process is slow and difficult to detect.” Applying his results to CWD, Race said: “If BSE were derived from sheep scrapie, then adaptation during passage in cattle may have increased its pathogenicity for humans. A similar situation could occur with CWD. CWD transmission to other cervids or livestock could change its characteristics, including its potential for transmission to people.”
In a similar study conducted in the U.K. on subclinical prion infection, results led the researchers to caution that “current definitions of the species barrier, which have been
based on clinical end-points, need to be fundamentally reassessed”.
Noted CWD researcher Dr. Elizabeth Williams from Laramie, Wyoming had the following to say about the risk to humans:
“I do think it is legitimate to be concerned about the potential for humans being susceptible to CWD. We don’t have evidence…but we can’t say it could never happen and we have to be prudent.”
CWD Transmission To Cattle
Regarding CWD transmission to cattle, the results are mixed. In an ongoing study in Wyoming, cattle fed brain tissue from CWD-infected mule deer have not gotten sick after 6 years. A study in Ames, Iowa has yielded different results. In 1997, 10 calves were inoculated intracerebrally with the same inoculum used in the Wyoming experiment. As of mid-2003, 5 calves had developed CWD.
Although the Wyoming study involves a more natural route, ingestion may simply result in a more prolonged incubation period. Alternatively, based on the work done by Race, et al. on TSE adaptation and species barriers, the test cattle in Wyoming may be asymptomatic carriers harboring a gradually adapting CWD agent.
Complicating matters, a recent study analyzing “glycoform” patterns of abnormal prion protein from CWD-affected deer and elk, scrapie-affected sheep and cattle, and BSE-affected cattle failed to identify patterns capable of reliably distinguishing these TSEs. Difficulty in identifying the source of a TSE following cross-species transmission adds to the uncertainty surrounding CWD and possible risks to cattle and humans. The authors write: “Sheep scrapie has been present in the United States since at least 1947, and in many geographical areas, sheep, deer, and elk share pastures and rangeland. If scrapie-affected sheep were present in these situations, then cross-species transmission might have occurred. Sheep scrapie is not thought to cause disease in humans, although passage through cattle appears to have changed this characteristic. It remains to be determined if the same will be true of CWD.”
The European Commission’s Scientific Steering Committee (EC-SSC) released a lengthy, comprehensive report and opinion on CWD in March 2003. Cross-species transmissibility experiments and risks are discussed in detail. The EC-SSC report reviews the successful transmission of CWD by intracerebral inoculation to ferrets, mice, a squirrel monkey, mink, a goat, and a sheep. The report concludes, “…it remains theoretically possible that the CWD-agent could infect humans.” — (More than theoretical!)
