Biological Safety Manual - Chapter 08: Agent Summary Statements (Section VIII: Prion Diseases)


Biological Safety Manual - Chapter 08: Agent Summary Statements (Section VIII: Prion Diseases)


Transmissible spongiform encephalopathies (TSE) or prion diseases are neurodegenerative diseases which affect humans and a variety of domestic and wild animal species (Tables 1 and 2).1,2 A central biochemical feature of prion diseases is the conversion of normal prion protein (PrP) to an abnormal, misfolded, pathogenic isoform designated PrPSc (named for "scrapie," the prototypic prion disease). The infectious agents that transmit prion diseases are resistant to inactivation by heat and chemicals and thus require special biosafety precautions. Prion diseases are transmissible by inoculation or ingestion of infected tissues or homogenates, and infectivity is present at high levels in brain or other central nervous system tissues, and at slightly lower levels in lymphoid tissues including spleen, lymph nodes, gut, bone marrow, and blood. Although the biochemical nature of the infectious TSE agent, or prion, is not yet proven, the infectivity is strongly associated with the presence of PrPSc, suggesting that this material may be a major component of the infectious agent.

A chromosomal gene encodes PrPC (the cellular isoform of PrP) and no PrP genes are found in purified preparations of prions. PrPSc is derived from PrPC by a posttranslational process whereby PrPSc acquires a high beta-sheet content and a resistance to inactivation by normal disinfection processes. The PrPSc is less soluble in aqueous buffers and, when incubated with protease (proteinase K), the PrPC is completely digested (sometimes indicated by the "sensitive" superscript, PrPsen) while PrPSc is resistant to protease (PrPres). Neither PrP-specific nucleic acids nor virus-like particles have been detected in purified, infectious preparations.

Table of Contents

  1. Occupational Infections
  2. Natural Modes of Infection
  3. Laboratory Safety
  4. Special Issues
  5. References

Occupational Infections

No occupational infections have been recorded from working with prions. No increased incidence of Creutzfeldt-Jakob disease (CJD) has been found amongst pathologists who encounter cases of the disease post-mortem.

Natural Modes of Infection

The recognized diseases caused by prions are listed under Table 1 (human diseases) and Table 2 (animal diseases). The only clear risk-factor for disease transmission is the consumption of infected tissues such as human brain in the case of kuru, and meat including nervous tissue in the case of bovine spongiform encephalopathy and related diseases such as feline spongiform encephalopathy. It is also possible to acquire certain diseases such as familial CJD by inheritance through the germline.

Table 1

The Human Prion Diseases
Disease Abbreviation Mechanism of Pathogenesis
Kuru   Infection through ritualistic cannibalism
Creutzfeldt-Jakob disease CJD Unknown mechanism
Sporadic CJD sCJD Unknown mechanism; possibly somatic mutation or spontaneous conversion of PrPC
to PrPSc
Variant CJD vCJD Infection presumably from consumption of BSE-contaminated cattle products and secondary bloodborne transmission
Familial CJD fCJD Germline mutations in PrP gene
Iatrogenic CJD iCJD Infection from contaminated corneal and dural grafts, pituitary hormone, or neurosurgical equipment
Gerstmann- träussler-Scheinker syndrome GSS Germline mutations in PrP gene
Fatal familial insomnia FFI Germline mutations in PrP gene

Table 2

The Animal Prion Diseases
Disease Abbreviation Natural Host Mechanism of Pathogenesis
Scrapie   Sheep, goats and mouflon Infection in genetically susceptible sheep
Bovine spongiform encephalopathy BSE Cattle Infection with prion-contaminated feedstuffs
Chronic wasting disease CWD Mule deer, white-tailed deer and Rocky Mountain elk Unknown mechanism; possibly from direct animal contact or indirectly from contaminated feed and water sources
Exotic ungulate encephalopathy EUE Nyala, greater kudu and oryx Infection with BSE-contaminated feedstuffs
Feline spongiform encephalopathy FSE Domestic and wild cats in captivity Infection with BSE-contaminated feedstuffs
Transmissible mink encephalopathy TME Mink (farm raised) Infection with prion-contaminated feedstuffs

Species-Specificity of Prions

Most TSE agents, or prions, have a preference for infection of the homologous species, but cross-species infection with a reduced efficiency is also possible. After cross-species infection there is often a gradual adaptation of specificity for the new host; however, infectivity for the original host may also be propagated for several passages over a time-span of years. The process of cross-species adaptation can also vary among individuals in the same species and the rate of adaptation and the final species specificity is difficult to predict with accuracy. Such considerations help to form the basis for the biosafety classification of different prions.

Laboratory Safety

Biosafety Level Classification

In the laboratory setting prions from human tissue and human prions propagated in animals should be manipulated at BSL-2. BSE prions can likewise be manipulated at BSL-2. Due to the high probability that BSE prions have been transmitted to humans, certain circumstances may require the use of BSL-3 facilities. All other animal prions are considered BSL-2 pathogens. However, when a prion from one species is inoculated into another the resultant infected animal should be treated according to the guidelines applying to the source of the inoculum. Contact APHIS National Center for Import and Export at (301) 734- 5960 for specific guidance. Although the exact mechanism of spread of scrapie among sheep and goats developing natural scrapie is unknown, there is considerable evidence that one of the primary sources is oral inoculation with placental membranes from infected ewes. There has been no evidence for transmission of scrapie to humans, even though the disease was recognized in sheep for over 200 years. The diseases TME, BSE, FSE, and EUE are all thought to occur after the consumption of prion-infected foods.1,2 The exact mechanism of CWD spread among mule deer, white-tailed deer and Rocky Mountain elk is unknown. There is strong evidence that CWD is laterally transmitted and environmental contamination may play an important role in local maintenance of the disease.2

Human Prion Diseases

In the care of patients diagnosed with human prion disease, Standard Precautions are adequate. However, the human prion diseases in this setting are not communicable or contagious.3 There is no evidence of contact or aerosol transmission of prions from one human to another. However, they are infectious under some circumstances, such as ritualistic cannibalism in New Guinea causing kuru, the administration of prion-contaminated growth hormone causing iatrogenic CJD, and the transplantation of prion-contaminated dura mater and corneal grafts. It is highly suspected that variant CJD can also be transmitted by blood transfusion.4 However, there is no evidence for bloodborne transmission of non-variant forms of CJD. Familial CJD, GSS, and FFI are all dominantly inherited prion diseases; many different mutations of the PrP gene have been shown to be genetically linked to the development of inherited prion disease. Prions from many cases of inherited prion disease have been transmitted to apes, monkeys, and mice, especially those carrying human PrP transgenes.

Special Issues

Inactivation of Prions

Prions are characterized by resistance to conventional inactivation procedures including irradiation, boiling, dry heat, and chemicals (formalin, betapropiolactone, alcohols). While prion infectivity in purified samples is diminished by prolonged digestion with proteases, results from boiling in sodium dodecyl sulfate and urea are variable. Likewise, denaturing organic solvents such as phenol or chaotropic reagents such as guanidine isothiocyanate have also resulted in greatly reduced but not complete inactivation. The use of conventional autoclaves as the sole treatment has not resulted in complete inactivation of prions.5 Formalin-fixed and paraffin-embedded tissues, especially of the brain, remain infectious. Some investigators recommend that formalin-fixed tissues from suspected cases of prion disease be immersed for 30 min in 96% formic acid or phenol before histopathologic processing (Table 3), but such treatment may severely distort the microscopic neuropathology.

The safest and most unambiguous method for ensuring that there is no risk of residual infectivity on contaminated instruments and other materials is to discard and destroy them by incineration.6 Current recommendations for inactivation of prions on instruments and other materials are based on the use of sodium hypochlorite, NaOH, Environ LpH and the moist heat of autoclaving with combinations of heat and chemical being most effective (See Table 4).5,6

Surgical Procedures

Precautions for surgical procedures on patients diagnosed with prion disease are outlined in an infection control guideline for transmissible spongiform encephalopathies developed by a consultation convened by the WHO in 1999.6 Sterilization of reusable surgical instruments and decontamination of surfaces should be performed in accordance with recommendations described by the CDC ( and the WHO infection control guidelines.6 Table 4 summarizes the key recommendations for decontamination of reusable instruments and surfaces. Contaminated disposable instruments or materials should be incinerated.


Routine autopsies and the processing of small amounts of formalin-fixed tissues containing human prions can safely be done using BSL-2 precautions.7 The absence of any known effective treatment for prion disease demands caution. The highest concentrations of prions are in the central nervous system and its coverings. Based on animal studies, it is likely that prions are also found in spleen, thymus, lymph nodes, and intestine. The main precaution to be taken by laboratorians working with prion-infected or contaminated material is to avoid accidental puncture of the skin.3 Persons handling contaminated specimens should wear cut resistant gloves if possible. If accidental contamination of unbroken skin occurs, the area should be washed with detergent and abundant quantities of warm water (avoid scrubbing); brief exposure (1 minute to 1N NaOH or a 1:10 dilution of bleach) can be considered for maximum safety.6 Additional guidance related to occupational injury are provided in the WHO infection control guidelines.6 Unfixed samples of brain, spinal cord, and other tissues containing human prions should be processed with extreme care at least in a BSL-2 facility.

Bovine Spongiform Encephalopathy

Although the eventual total number of variant CJD cases resulting from BSE transmission to humans is unknown, a review of the epidemiological data from the United Kingdom indicates that BSE transmission to humans is not efficient.8 The most prudent approach is to study BSE prions at a minimum in a BSL-2 facility. When performing necropsies on large animals where there is an opportunity that the worker may be accidentally splashed or have contact with high-risk materials (e.g., spinal column, brain, etc.) personnel should wear full body coverage personal protective equipment (e.g., gloves, rear closing gown and face shield). Disposable plasticware, which can be discarded as a dry regulated medical waste, is highly recommended. Because the paraformaldehyde vaporization procedure does not diminish prion titers, BSCs must be decontaminated with 1N NaOH and rinsed with water. HEPA filters should be bagged out and incinerated. Although there is no evidence to suggest that aerosol transmission occurs in the natural disease, it is prudent to avoid the generation of aerosols or droplets during the manipulation of tissues or fluids and during the necropsy of experimental animals. It is further strongly recommended that impervious gloves be worn for activities that provide the opportunity for skin contact with infectious tissues and fluids.

Animal carcasses and other tissue waste can be disposed by incineration with a minimum secondary temperature of 1000°C (1832°F).6 Pathological incinerators should maintain a primary chamber temperature in compliance with design and applicable state regulations, and employ good combustion practices. Medical waste incinerators should be in compliance with applicable state and federal regulations.

The alkaline hydrolysis process, using a pressurized vessel that exposes the carcass or tissues to 1 N NaOH or KOH heated to 150°C, can be used as an alternative to incineration for the disposal of carcasses and tissue.5,9 The process has been shown to completely inactive TSEs (301v agent used) when used for the recommended period of time.

Handling and Processing of Tissues From Patients With Suspected Prion Disease

The special characteristics of work with prions require particular attention to the facilities, equipment, policies, and procedures involved.9 The related considerations outlined in Table 3 should be incorporated into the laboratory's risk management for this work.

Table 3

Tissue Preparation for Human CJD and Related Diseases
1. Histology technicians wear gloves, apron, laboratory coat, and face protection.
2. Adequate fixation of small tissue samples (e.g., biopsies) from a patient with suspected prion disease can be followed by post-fixation in 96% absolute formic acid for 30 minutes, followed by 48 hours in fresh 10% formalin.
3. Liquid waste is collected in a 4L waste bottle initially containing 600 ml 6N NaOH.
4. Gloves, embedding molds, and all handling materials are disposed as regulated medical waste.
5. Tissue cassettes are processed manually to prevent contamination of tissue processors.
6. Tissues are embedded in a disposable embedding mold. If used, forceps are decontaminated as in Table 4.
7. In preparing sections, gloves are worn, section waste is collected and disposed in a regulated medical waste receptacle. The knife stage is wiped with 2N NaOH, and the knife used is discarded immediately in a "regulated medical waste sharps" receptacle. Slides are labeled with "CJD Precautions." The sectioned block is sealed with paraffin.
8. Routine staining:
  1. slides are processed by hand;
  2. reagents are prepared in 100 ml disposable specimen cups;
  3. after placing the coverslip on, slides are decontaminated by soaking them for 1 hour in 2N NaOH;
  4. slides are labeled as "Infectious-CJD."
9. Other suggestions:
  1. disposable specimen cups or slide mailers may be used for reagents;
  2. slides for immunocytochemistry may be processed in disposable petri dishes;
  3. equipment is decontaminated as described above or disposed as regulated medical waste.

Table 4

Prion Inactivation Methods for Reusable Instruments and Surfaces
1. Immerse in 1 N NaOH, and heat in a gravity displacement autoclave at 121oC for 30 minutes. Clean and sterilize by conventional means.
2. Immerse in 1 N NaOH or sodium hypochlorite (20,000 ppm) for 1 hour. Transfer into water and autoclave (gravity displacement) at 121oC for 1 hour. Clean and sterilize by conventional means.
3. Immerse in 1N NaOH or sodium hypochlorite (20,000 ppm) for 1 hour. Rinse instruments with water, transfer to open pan and autoclave at 121oC (gravity displacement) or 134oC (porous load) for 1 hour. Clean and sterilize by conventional means.
4. Surfaces or heat-sensitive instruments can be treated with 2N NaOH or sodium hypochlorite (20,000 ppm) for 1 hour. Ensure surfaces remain wet for entire time period, then rinse well with water. Before chemical treatment, it is strongly recommended that gross contamination of surfaces be reduced because the presence of excess organic material will reduce the strength of either NaOH or sodium hypochlorite solutions.
5. Environ LpH (EPA Reg. No. 1043-118) may be used on washable, hard, non-porous surfaces (such as floors, tables, equipment, and counters), items (such as non-disposable instruments, sharps, and sharp containers), and/or laboratory waste solutions (such as formalin or other liquids). This product is currently being used under FIFRA Section 18 exemptions in a number of States. Users should consult with the State environmental protection office prior to use.

(Adapted from, 10,11)

Working Solutions

1 N NaOH equals 40 grams of NaOH per liter of water. Solution should be prepared daily. A stock solution of 10 N NaOH can be prepared and fresh 1:10 dilutions (1 part 10 N NaOH plus 9 parts water) used daily.

20,000 ppm sodium hypochlorite equals a 2% solution. Most commercial household bleach contains 5.25% sodium hypochlorite, therefore make a 1:2.5 dilution (1 part 5.25% bleach plus 1.5 parts water) to produce a 20,000 ppm solution. This ratio can also be stated as two parts 5.25% bleach to three parts water. Working solutions should be prepared daily.

CAUTION: Above solutions are corrosive and require suitable personal protective equipment and proper secondary containment. These strong corrosive solutions require careful disposal in accordance with local regulations.

Precautions in Using NaOH or Sodium Hypochlorite Solutions in Autoclaves

NaOH spills or gas may damage the autoclave if proper containers are not used. The use of containers with a rim and lid designed for condensation to collect and drip back into the pan is recommended. Persons who use this procedure should be cautious in handling hot NaOH solution (post- autoclave) and in avoiding potential exposure to gaseous NaOH, exercise caution during all sterilization steps, and allow the autoclave, instruments, and solutions to cool down before removal. Immersion in sodium hypochlorite bleach can cause severe damage to some instruments.


  1. Prusiner SB. Prion diseases and the BSE crisis. Science. 1997;278:245-51.
  2. Williams ES, Miller MW. Transmissible spongiform encephalopathies in non-domestic animals: origin, transmission and risk factors. Rev Sci Tech Off Int Epiz. 2003;22:145-56.
  3. Ridley RM, Baker HF. Occupational risk of Creutzfeldt-Jakob disease. Lancet. 1993;341:641- 2.
  4. Llewelyn CA, Hewitt PE, Knight RS, et al. Possible transmission of variant Creutzfeldt-Jakob disease by blood transfusion. Lancet. 2004;7;363:417-21.
  5. Taylor DM, Woodgate SL.Rendering practices and inactivation of transmissible spongiform encephalopathy agents. Rev Sci Tech Off Int Epiz. 2003;22:297-310.
  6. World Health Organization. []. Geneva (Switzerland): The Organization; [updated 2006 Sept 21; cited 2006 Sept 21]. 2000. WHO Infection Control Guidelines for Transmissible Spongiform Encephalopathies. Report of a WHO Consultation, Geneva, Switzerland, 23-26 March 1999.
    Available from:
    Chapter 8: Section VIII7
  7. Ironside JW and JE Bell. The 'high-risk' neuropathological autopsy in AIDS and Creutzfeldt- Jakob disease: principles and practice. Neuropathol Appl Neurobiol. 1996;22:388-393.
  8. Hilton DA. Pathogenesis and prevalence of variant Creutzfeldt-Jakob disease. Pathol J. 2006;208:134-41.
  9. Richmond JY, Hill RH, Weyant RS, et al. What's hot in animal biosafety? ILAR J. 2003;44:20-7.
  10. Ernst DR, Race RE. Comparative analysis of scrapie agent inactivation methods. J. Virol. Methods. 1994; 41:193-202.
  11. Race RE, Raymond GJ. Inactivation of transmissible spongiform encephalopathy (prion) agents by Environ LpH. J Virol. 2004;78:2164-5.


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