Ebook: Radiation Inactivation of Bioterrorism Agents

The use of and problems associated with biological weapons have been of concern to NATO and non-NATO military organizations for many years. Until recently most of the readily available literature addressed the military issues associated with the possible use of biological weapons on the battlefield, the medical effects of the various agents, and what was known about medical prophylaxis and treatments. Information on other needed countermeasures, such as decontamination or public health issues associated with exposure of civilian populations were largely overlooked. This perspective changed dramatically after the events in the United States in the fall of 2001 in response to the contamination of the U.S. Mail system with powdered anthrax spores. Surprisingly, this use of a biological warfare agent, which gained widespread international attention, was not in the context of a military operation, but a terrorist action that involved civilians. The contaminated facilities were both private and government owned, including important mail-sorting and government offices within the Washington DC area. The exposed populations, including those killed by the attack, were exclusively civilians, and not those whose responsibilities would previously have been expected to put them at risk. Most likely, the intensity of the situation, sense of vulnerability of national security, and urgency to provide solutions were magnified by the context of the other unprecedented terrorist attacks that had occurred a few weeks earlier in the United States on 11 September.

Among the significant problems and defensive weaknesses that the anthrax attack revealed was the lack of established industrial-scale decontamination methods for large volumes of heterogeneous objects (e.g., the mail) or for complex physical environments, (e.g., the U.S. Postal Service sorting facilities). Ultimately, these two microbial decontamination problems were solved in very different ways. The contaminated mail was treated with ionizing radiation while the contaminated government buildings were treated with vapor/gasphase chemicals. The most urgent problems at the time, decontaminating the mail and establishing a process for prophylactic treatment of the mail, were solved relatively quickly. This was largely due to the robust radiation biology and technical base derived from the industrial use of ionizing radiation. For many years this industry has successfully used radiation to sterilize complex objects, and indeed most modern hospitals are wholly dependent on the wide variety of medical devices and supplies sterilized by such methods. Contributing to the speed of response was the fact that the attack occurred within the United States and in the "home town" of many of the technical experts and decision makers, allowing official response to be coordinated rapidly.

This volume presents the papers delivered at the NATO Advanced Research Workshop on Radiation Inactivation of Bioterrorism Agents, 7–9 March 2004 in Budapest, Hungary. The conference was graciously hosted by the Frédéric Joliot-Curie National Research Institute for Radiobiology and Radiohygiene, Budapest, Hungary and organized by Dr. L.G. Gazsó and Dr. C.C. Ponta. The conference was in part the outcome of the co-organizers' forward thinking in this area and previous recommendations on the use of ionizing radiation for biological weapon agent inactivation (VI. Int. Symposium on Protection Against Chemical and Biological Warfare Agents, Stockholm, 1998 and Symposium on Nuclear, Biological and Chemical Threats in the 21st Century, Helsinki, 2000). Wisely, the conference brought together experts from across a number of professional disciplines and geographic boundaries from the private sector, government, scientific research, and international regulatory agencies.

The conference papers within this volume cover many of the factors essential to the successful application of ionizing radiation to biological agent inactivation. Consideration of international law and treaty issues and defining what constitutes various kinds of attacks are reviewed, which are likely to be important if there is need for a multinational response. Because the most efficient application of radiation requires the total dose be well matched to the sensitivity of the microorganism(s) concerned, there were several valuable reports detailing progress on precise, accurate, rapid, and field-ready diagnostics to assay the type of microbial contamination. A strength of this conference was the inclusion of facility operators and experts on process control, safety, and dosimetry. Their operational knowledge, detailed information on the current state of the art, descriptions of facility capabilities, explanation of dosimetry standards, and presentation of available technology and emerging techniques provide a strong technical base. Only from such a technical base is it possible to consider what resources are available, determine those that could be used most effectively in any particular situation where there has been the illicit use of biological agents, and provide a high degree of assurance of the effectiveness of the decontamination effort. Also addressed was the radiation sensitivity of several types of agents of concern, including bacteria, bacterial spores, and viruses. Furthermore, factors that could alter an agent’s radiation sensitivity were discussed. Several conference participants presented information on the U.S. response to the mail contamination, the approach that was taken, and some of the lessons learned. This conference also provided a forum for radiation experts on a broad regional basis to meet one another or become reacquainted. Potentially, this may be one of the most important facets of the conference. An important aspect of the U.S. response was rapidly making the needed connections and coordination among the appropriate scientists, private sector facility operators, and regulatory officials.

The conference recommendations were encapsulated in a formal memo to the International Atomic Energy Agency. In brief, the memo made following recommendations: (a) there is a need for a comprehensive assessment of the potential use of ionizing radiation for the destruction of biologically hazardous materials, (b) a need to assemble a committee of experts to develop and maintain a database on the use of radiation technology for biological agent defeat and to identify critical areas that still need to be addressed, (c) consider organizing an experts’ meeting to advise the Coordinated Research Project on possible future Member States’ actions, and (d) compile a list of radiation sources and locations capable of contributing to biological agent inactivation.

This workshop is a valuable basic reference for the use of radiation decontamination technologies against bioterrorism agents. The conference and its proceedings also provide a template for future highly cooperative and productive meetings to facilitate international interactions among those concerned with preparing responses to biological agent attacks. Hopefully these proceedings will stimulate support and foster collegial efforts in research on these technologies, which will not only improve their use in biological agent defeat but also broaden their applicability for medical and industrial processing.

R. Joel LOWY, Thomas B. ELLIOTT, Michael O. SHOEMAKER, Gregory B. KNUDSON and Marc F. DESROSIERS

Developments concerning radiation technologies' applications are discussed in the paper. The industrial irradiators based on gamma isotope and electron or X rays, accelerator driven sources are reviewed.

Present applications: polymers and rubber processing, sterilization and food irradiation are reported. Future possible developments in the field of natural polymers' processing and nanomaterials/nanomachines engineering are presented as well. The technological breakthrough achieved in the field of applications of radiation processes for environment protection illustrates new opportunities of the process utilization. This refers to the inactivation of biological warfare agents by ionizing radiation to the same extent. The economical and social aspects are shortly underlined.

Finally, the role of the Agency's programmes in promoting the above and the progress achieved is well described by this panoramic view of the status of radiation technology presented in this paper.

Radiation safety of gamma‐ and electron irradiation facilities is of basic significance concerning the safety of personnel involved in construction, operation and maintenance, the safety of the products treated as well as the environment of the facility. The safety principles with respect to the design and construction of the facilities, requirements concerning the basic parts and the operation of the plants, the reliable control of running irradiation facilities, the role of emergency planning and the role of licences are discussed. Recent measures to increase safety are also mentioned.

Several types of dosimeters are used in radiation processing, including calorimeters, thin radiochromic plastic films or sheets, free radical dosimeters, luminescent dosimeters and liquid dosimeters. These dosimeters, their properties and applications are briefly described in this paper.

A wide spread industrial application of radiation treatment is sterilization. The control of any process is a responsible and documented action for safe release of goods to the end‐user. In radiation sterilization the control strategy has a validation step followed by routinely monitoring of validated parameters. Validation procedure establishes the sterilization conditions of a specific product treated in a specific irradiation facility. During validation the bioburden level before sterilization and radiation sensitivity of microorganisms found on the items (D₁₀ value) are measured. They contribute together with an agreed SAL (Sterility Assurance Level) to choose the irradiation time direct related to the sterilization dose. Parametric release to the market is accepted if routine process proceeded in validated conditions. For radiation inactivation of biological weapon, validation concept can not be applied in an orthodox way mainly because the bioburden level can not be measured. ALARA (As Low As Reasonable Achievable) concept is a possible approach to establish a strongly motivated treatment dose.

The manufacturer of the product has responsibility for the quality of the product including the selection of the appropriate sterilizing dose. Several approaches to select the dose can be used depending on the batch size and device bioburden level.

Ionising radiation interacting with matter produces the most different radiation‐physical, radiation‐chemical, and radiation‐biological effects. In this contribution an accent on industrial applications of radiation sources is given. The possibilities of their application in chemical technology, namely in technology of radiation synthetic reactions, other polymeric reactions and to production of new macromolecular substances are reviewed. There are meant also radiation wastewater and sewage technologies, irradiation techniques in air cleaning processes, radiation sterilization in medicine, preparation of radiopharmaceuticals, etc. Some information on current possibilities of Slovak accelerators usage for irradiation technologies is brought.

The sensitivity of microorganisms towards high energy radiation varies widely: different types, species and strains exhibit greatly different radiation sensitivities. Certain environmental factors are also able to influence the actual radiation response. The basic principles of radiation damage, radiosensitivity of microorganisms and physical, chemical and biological dose modifying factors are reviewed in this paper.

Egypt has already an advanced radiation processing programs in different areas such as medical sterilization, food irradiation and industrial applications. Such technologies already are existed in Egypt since 1972 at that time The National Center for Radiation Research and Technology (NCRRT) was established. It was aiming at promoting research and development using ionizing radiation. Since that time, Egypt has been engaged in collaboration with Mediterranian countries through IAEA, AFRA member states and Arab Atomic Energy Agency. Radiation processing of medical and food products in addition to industrial applications is the main concern and consideration at NCRRT. The center is divided into three divisions, including twelve departments, in addition to central laboratories and industrial radiation processing facilities. The main objectives of NCRRT are to facilitate the concrete application of radiation technology in environmental studies as well as the processing of selected materials for the benefits of peaceful applications in our daily life.

Main three categories of chemical, biological, radiological and nuclear (CBRN) terrorism (often depicted as ultra‐ or superterrorism) according to form and material source are suggested. Differences between terrorism using weapons of mass destruction (WMD) and CBRN terrorism are explained, the WMD terrorism being only one of the three categories of CBRN terrorism.

CBRN terrorism involves in the first line misuse of the WMD, in the second line use of non weaponised toxic,contagious and radioactive materials, or primitive nuclear explosive devices.The third category implies violent strikes against infrastructures of present civilised and industrialised societies causing accidents with release of toxic agents, highly infectious materials and radionuclides resembling the pushing mechanism of disastrous wartime strikes with conventional weapons rather than peace‐time accidents caused by personal, material or system failures.

Examples of already executed cases of CB‐terrorism support this approach and categorisation of these highest forms of terrorism.

The terrorist anthrax epidemic reported in October – December 2001 in the USA demonstrated that biological weapon is possible to be used nowadays.

Difficulties in controlling potential biological agents (mainly dual use pathogens or genetically modified organisms) raise important concerns on the possible use in bioterrorism.

The preventive counter‐bioterrorism strategy is based on:

• Promoting strong national legislation for preventing and combating bioterrorism;

• Adopting international regulations/recommendations against bioterrorism at national level

• Setting national plans and structures of biosecurity in human, animal and plant (bioprevention, biodefense)

• Controlling non‐proliferation of biological weapon; controlling circulation of natural highly pathogenic organisms of natural or genetically modified agents as well as the size and types of different equipments for research and bioproduction activities.

• Integrated activities in public health: epidemic surveillance, suitable stocks of elected or new vaccines, network of well trained and equipped laboratories for a rapid detection and identification of microorganisms.

Since October 2001, bio‐agent risk has become a high priority threat factor and one of the key issues of the national and international anti‐terrorist activities. The presentation is going to summarize known microbial agents in accordance with their potential or suitable for being used as biological weapons, and also the hazards or burdens attributed to the proliferation of such weapons or their bioterrorist use. Consisting mainly of natural pathogens, at this time various lists are known from different sources aiming to categorize microbial agents for this respect. Since 2001 WHO and CDC bio‐agent classifications have become the most acknowledged and widely used ones. Such systemic collections of pathogens combined with the specific risk estimation viewpoints are not only theoretically useful for preventive military medical purposes, but it can directly and successfully assist NBC defence practices, too. Although in the era of genomics the real value of any lists is rather limited, yet they can provide useful references and practical guidance to the medical aspects of force protection planning, as well as for the entire spectrum of medical defence activities and efforts, including the development of newer and more efficient decontamination procedures.

Microbiological agents as well as bio‐threats show some distinguishing and unique features if compared to other agents of mass destruction. For certain agents, lethality can be expected at concentrations far below the threshold of the conventional field detection capabilities. Their ability for multiplication instead of decomposition is very characteristic leading rather to the expansion than the attenuation of the consequences afterwards.

These specificities underline the importance of and need for a deployable bio‐laboratory equipped with very rapid and sensitive methods. Until recently, when the development of recent molecular biological sciences gave an opportunity to introduce fast and very sensitive laboratory techniques, we have had only immunochemical techniques for these purposes.

To meet the requirements and as a first step, Hungarian Defence Forces developed and introduced a fieldable (molecular) biological‐laboratory using up‐to‐date technology. The system is easily transportable and has all the parts needed for a complex molecular biological laboratory except housing. Readiness is reached 1.5 hours after deployment, and the first results are possible to get even in 3 to 6 hours depending on the number and characteristics (quality) of the samples. The whole staff consists of 4 persons. The mobile laboratory has been developed to detect hazardous agents from environmental and biological samples, and was successfully tested in several field exercises. In order to improve its bio‐sampling and identification capabilities, as well as to become entirely independent from the hosting environment, the development of a mobile and fully containerized version has been initiated.

In October 2001, first class letters, which were laced with Bacillus anthracis spores, were sent to political and media targets resulting in five deaths and 22 illnesses, significant mail service disruption, and economic loss. The White House Office of Science and Technology Policy established a technical task force on mail decontamination that included three key agencies: the National Institute of Standards and Technology (NIST); the Armed Forces Radiobiology Research Institute; and, the United States Postal Service. A cooperative effort between this task force and industry led to protocols for the processing of letter and parcel mail.

Currently, NIST is examining the technical issues and barriers to the use of ionizing radiation to mitigate bioterrorism agents in high‐risk passenger luggage. The purpose of this work is to develop irradiation specifications, procedures, and protocols that will ensure that broad classes of bioterrorism agents in passenger luggage will be neutralized without damaging luggage contents and inconveniencing passengers with long delays. This work focuses on three areas: the assembly of critical input data, the development of a coupled computational‐experimental verification approach for estimating the radiation dose that can be delivered to passenger luggage and the application of the computations to a larger variety of luggage configurations followed by the development of specifications, procedures, and protocols for the irradiation of passenger luggage.

An analysis of the expectations for growth in these and other homeland security areas where irradiation technology can be applied will be discussed.

State Research Center of Virology and Biotechnology VECTOR is one of Russia's largest scientific research and production facilities whose major activities are focused on carrying out basic and applied research in a wide area of natural sciences, development and manufacture of therapeutic, preventive, and diagnostic products for public health and agriculture.

Methods for rapid identification of orthopoxviruses pathogenic for humans using a RFLP, PCR, multiplex PCR, real‐time PCR, and microarray assays are described.

The Anthrax attacks in the Fall of 2001 resulted in a heightened awareness of the role radiation plays in the inactivation of BioTerrorism Agents. After a review of thousands of proposals to decontaminate mail, only X‐ray and Electron Beam solutions were implemented by the United States Postal Service. A brief overview of the current process used to sanitize the US mail is presented.

Often over time, radiation solutions for industrial, scientific, and government applications are displaced by less sophisticated alternatives. A comparison of the radiation solution to other technologies is presented to suggest why, in the case of BioTerrorism, the radiation solution is here to stay. Military, homeland defense, mail, and commercial applications for radiation inactivation of Bio‐Terrorism agents are growing. One of those applications is also presented here.

The Armed Forces Radiobiology Research Institute (AFRRI) in the U. S. Department of Defense conducts biomedical research on the effects of ionizing radiation. It has the largest radiobiology program in the United States and is a national resource in the response to nuclear and radiation accidents. Bacterial spores are potential biological weapons because they can be prepared and distributed by aerosol, they endure harsh environmental conditions, and they are infectious. Decontamination procedures for large concentrations of spores must be effective and practical. We determined the dose response of bacterial spores to three qualities, or types, of ionizing radiation.

Inactivation of dry and hydrated bacterial spores with gamma radiation has been more thoroughly studied than spore inactivation with neutron radiation. Decimal‐reduction curves were produced at AFRRI for Bacillus atrophaeus (B. subtilis var. niger, B. globigii, “BG”), B. pumilus, B. thuringiensis, and B. anthracis Sterne spores, both wet and dry, using doses of 0.3 to 7.2 kGy neutrons delivered at a dose rate of 44 to 49 Gy/min (D_n/D_T=0.95) in the AFRRI training, research, isotope‐producing General Atomic (TRIGA) Mark‐F nuclear reactor, and doses of 0.6 to 24.0 kGy gamma rays delivered at dose rates of 112 to 120 Gy/min in the AFRRI cobalt‐60 (⁶⁰Co) gamma‐photon irradiation facility. Decimal‐reduction curves were constructed by plotting the spore survival fraction in terms of colony‐forming units vs. radiation dose. All four species showed greater sensitivity to neutron radiation than to gamma radiation, regardless of the state of hydration. Dry spores of all four species were more sensitive to gamma radiation than were hydrated spores. In contrast, the state of hydration, whether dry or hydrated, of spores of B. subtilis and B. pumilus, which were embedded in filter paper strips, did not affect their sensitivity to neutron radiation. Wet B. thuringiensis spores were only slightly more sensitive to neutrons than were B. thuringiensis spores in dry powdered form. Furthermore, the species most resistant to neutron and gamma radiations was a concentrated B. anthracis Sterne spore suspension. When the starting spore concentration, the Bacillus species used, the radiation quality (neutron or gamma), and the state of hydration are known, the radiation decimal‐reduction curves generated in these studies can be used to predict bacterial spore survival.

Electron‐beam radiation (e‐beam) has been used to inactivate microorganisms in spices, fresh food, medical components, and hazardous waste. AFRRI assessed the efficacy of using an e‐beam for decontamination of bulk biological agents and of byproducts of the decontamination procedures such as wipes and aqueous runoff. Biological agent surrogates were tested under controlled conditions to determine the effectiveness of e‐beam for decontamination. Using the AFRRI linear accelerator (LINAC) to deliver doses of 2 to 20 kGy at a dose rate of 1 kGy/min, radiation decimal‐reduction curves were constructed for Bacillus atrophaeus spores in a dry powder and B. anthracis Sterne spores in a slurry. Doses of 0.25 to 1.0 kGy were delivered to vegetative Gram‐negative bacterial cells of Serratia marcescens. The LINAC produced 13‐MeV electrons at 30 pulses/sec with a 4‐µsec pulse width generated through a water scatterer. Spore samples were irradiated in an array of three screw‐capped polystyrene tubes. Dosimetry was performed at the beginning of each experimental run with LiF:Ti,Mg thermoluminescent dosimeters (TLDs), product type TLD‐100 (Bicron®). TLDs were processed with the Bicron®/Harshaw Model 5500 Automatic TLD Reader. The inactivation data for dry B. atrophaeus spores, B. anthracis Sterne spores, and S. marcescens were fitted to a mathematical formula. The e‐beam decimal‐reduction curves for the bacterial spores were similar to our previously generated gamma‐photon radiation curves. The vegetative bacterial cells of S. marcescens were more susceptible to high‐speed electrons than were the bacterial spores. These experimental findings support the concept of using a truck‐mounted transportable LINAC in the field for decontaminating bulk materials that are contaminated with pathogenic bacteria.

The terrorist attack in September 2001, with the dissemination of Bacillus anthracis spores in letters sent through the U.S. Postal Service, brought home the reality of bioterrorism. These attacks have heightened concerns about future large‐scale aerosol attacks with powders of B. anthracis spores and other pathogens that cause smallpox, pneumonic plague, tularemia, and viral hemorrhagic fevers, as well as toxins such as botulinum toxin, ricin or Staphylococcus enterotoxin B. Means to prevent the use of these agents, and to manage the consequences of their use, have become a high priority. One of the tools that can play a role in disease prevention and consequence management is nonionizing radiation in the form of germicidal short‐wavelength ultraviolet (UV) light. This article presents background information on the pathogen Bacillus anthracis, the causative agent for anthrax, and its susceptibility to killing by germicidal UV. The results of two experimental studies are also presented that examine UV inactivation of B. anthracis vegetative cells and spores, and spores of closely related Bacillus species, in suspension, dried on surfaces, and as free‐flowing powders.

Exposure of viral pathogens to ionizing radiation can be an effective method of neutralization. Potential applications include decontaminating materials and providing non‐infectious antigens for diagnostic and therapeutic applications. As radiation can completely penetrate through most biological and non‐biological materials, there can be high assurance that all the viruses have been completely exposed. This report will include recent experimental work investigating the radiation sensitivity of influenza A and vaccinia viruses, as well as a brief review of the published literature. Data to be presented includes the dose range for virus inactivation, comparisons of different types of ionizing radiation and putative molecular mechanisms. Biological and methodological parameters affecting radiation sensitivity and its measurement will also be discussed.

The Radiation Technology Group of ITN Portugal is developing two different projects that could contribute to the prevention and early detection of microbiological war: “Control of the environment in surgical rooms at Army hospitals and the impact on the incidence of cross‐infection” and “Sanitation of chicken eggs by ionising radiation”.

The first project focuses on the development and improvement of alternative techniques to control the environment in surgical rooms leading to the detection and identification of nosocomial microorganisms in a Portuguese Hospital. A database can then be constructed that could demonstrate the relation between the improvement of the airborne conditions and the hospital infection agents. This project includes a continuous monitorization of the surgery rooms' natural air bioburden and nosocomial microorganisms, and the consequent updating of the database. This study will be carried out based on the molecular type of isolated strains from infected patients during surgery and the correlation with surgical environment isolated strains. This procedure could lead to a model for the detection of emerging microorganisms that could become hospital infectious agents.

The second project uses the decontaminating capacity of ionising radiation to inactivate pathogenic microorganisms in eggs and will be presented in a different section.

These two studies, although in different microbiological areas, are examples of how we could deal with potential harmful public health agents: detection and prevention.

Bioterrorism is an event in a civil setting that is equivalent to an epidemic in a medical scenario. The collaboration between medicine and public health is also important in the clinical diagnostics and epidemiological surveillance in response to the emergence of a biological agent.

The malicious contamination of food for terrorist purposes is a real and current threat, and deliberate contamination of food at one location could have global health implications. The key to prevent food terrorism is to enhance existing food safety programmes in order to protect food production systems. Typical food safety management programmes within the food industry include good manufacturing practice and “hazard analysis and critical control point” – HACCP. Food irradiation could be one additional food safety tool that serves as a complement to other food safety technologies such as the HACCP.

With the purpose to apply the food irradiation as a food safety tool, the Radiation Technology group at the ITN is developing a project entitled “Sanitation of chicken egg by ionizing radiation”, which applies the decontaminating capacity of the ionising radiation to inactivate pathogenic microorganisms in eggs. Salmonella spp. and Campylobacter spp. are eggs natural contaminants and the leading causes of bacterial gastroenteritis in humans. In this study, the D_values of reference strains of Salmonella and Campylobacter were determined and sub‐lethal gamma radiation doses were applied to artificially contaminated eggs, in order to predict which irradiation dose could guarantee egg sanitation. Based on the results obtained and to guarantee organoleptic acceptable and safe eggs, a radicidation dose of 1.5 kGy is thus proposed.

Monitoring programmes with a rapid follow‐up are important if variation in product is seen that could indicate deliberate contamination. Since a reliable detection step to identify the hazard is crucial in any production process, we validated a PCR method to detect Salmonella and Campylobacter contamination, directly from eggs and egg‐products.