Preface

Pathogenic microorganisms and their toxins have always posed a significant threat to humans, animals, and plants that become exposed and infected naturally. But now there is a newly recognized threat – the deliberate use of pathogenic microorganisms and toxins as weapons in acts of bioterrorism or in the commission of biocrimes. Long before the germ theory of disease was understood, man knew that pathogenic microorganisms and toxins were useful as weapons. However, as the twentieth century came to a close, the perceived difficulties in production, weaponization, and deployment of these biological weapons as well as a belief that moral restraints would preclude the use of these weapons gave many a false sense of security. Recently, a number of events have served to focus attention on the threat of terrorism and the potential for the use of biological weapons against the military, civilian populations, or agriculture for the purpose of causing illness, death, or economic loss. This threat is a significant concern in the United States as well as internationally.

Bioterrorist attacks occur as one of two scenarios: overt and covert. In either scenario, biologic agents could be introduced into populations by several routes, including aerosol; contamination of food, water, or medical products; fomites; or the release of infected arthropod vectors. The deliberate nature of such dissemination will often be obvious, as in the case of multiple mailed letters containing anthrax spores (ie overt). Because we currently lack the ability to conduct extensive real-time monitoring for the release of a biologic agent, a covert release of a microorganism of toxin in a population would be likely to go unnoticed for some time, with individuals exposed leaving the attack area before the act of terrorism became evident. Because of the incubation period, the first signs that a microorganism or toxin has been released may not become apparent until hours or weeks later, when individuals become ill and seek medical care.

Bioterrorism presents many challenges, particularly when compared to chemical, radiological, or nuclear terrorism. These challenges reflect the dual-use nature of many technologies that can be used for either beneficial purposes or bioterrorism; intentional threats that can coexist with similar and naturally occurring threats; the complexity of the interaction of the threat with the environment, the human immune system, the society/social structure; and rapid advances in biotechnology. Preparing for and responding to bioterrorism in the twenty-first century has brought together diverse

#### VIII Preface

disciplines with different backgrounds and cultures, for example intelligence agencies, public health and medical professionals, law enforcement officers, scientists, and the commercial sector. It remains a learning process.

Many biological agents can cause illness in humans, but not all are capable of affecting public health and medical infrastructures on a large scale. As part of an effort to focus public health preparedness efforts, the United States Centers for Disease Control and Prevention (CDC) developed a prioritized list of biological agents taking into consideration criteria such as high morbidity and mortality; potential for person-toperson transmission, directly or by vector; low infectious dose and high infectivity by aerosol, with a commensurate ability to cause large outbreaks; ability to contaminate food or water supplies; lack of a specific diagnostic test and/or effective treatment; lack of a safe and effective vaccine; potential to cause anxiety in the general public and in healthcare workers; and potential for weaponization. The final category assignments (A, B, or C) of agents for public health preparedness were based on an overall evaluation of the ratings the agents received in the aforementioned criteria. Agents placed in Category A (eg Yersinia pestis, botulinum neurotoxins) were considered to have a moderate to high potential for large-scale dissemination, the greatest potential for adverse public health impact with mass casualties, and a requirement for broadbased public health preparedness efforts. Agents placed in Category B (eg rickettsia, ricin, staphylococcal enterotoxins) also have some potential for large-scale dissemination with resultant illness, but generally cause less illness and death and, therefore, would be expected to have lower public health and medical impact. Biological agents that were not believed to present a high bioterrorism risk to public health but which could emerge as future threats, as scientific understanding of these agents improved were placed in Category C.

This book was written to provide a resource to scientists, epidemiologists, clinicians, and others in the field of bioterrorism defense. Not all possible topics are described. Instead, representative topics, authored by international experts provide a unique and state-of-the-art perspective on this field. The book consists of this preface and nine other chapters that address the broad areas of (1) detection capabilities (ie spatiotemporal disease surveillance; diagnostic bioterrorism response strategies; methods for detecting the presence of botulinum neurotoxins in food and other biological samples; and detection of bacillus spores by surface-enhanced Raman spectroscopy) and (2) characteristics of specific pathogenic microorganisms and toxins (ie Rickettsia and Rickettsial diseases; recent advances in the development of vaccines against Yersinia pestis; botulinum neurotoxins; ricin; and staphylococcal enterotoxins).

#### **Dr. Stephen A. Morse**

Associate Director of Science, Division of Bioterrorism Preparedness and Response, and Director of the Environmental Microbiology Program, CDC, USA

VIII Preface

disciplines with different backgrounds and cultures, for example intelligence agencies, public health and medical professionals, law enforcement officers, scientists, and the

Many biological agents can cause illness in humans, but not all are capable of affecting public health and medical infrastructures on a large scale. As part of an effort to focus public health preparedness efforts, the United States Centers for Disease Control and Prevention (CDC) developed a prioritized list of biological agents taking into consideration criteria such as high morbidity and mortality; potential for person-toperson transmission, directly or by vector; low infectious dose and high infectivity by aerosol, with a commensurate ability to cause large outbreaks; ability to contaminate food or water supplies; lack of a specific diagnostic test and/or effective treatment; lack of a safe and effective vaccine; potential to cause anxiety in the general public and in healthcare workers; and potential for weaponization. The final category assignments (A, B, or C) of agents for public health preparedness were based on an overall evaluation of the ratings the agents received in the aforementioned criteria. Agents placed in Category A (eg Yersinia pestis, botulinum neurotoxins) were considered to have a moderate to high potential for large-scale dissemination, the greatest potential for adverse public health impact with mass casualties, and a requirement for broadbased public health preparedness efforts. Agents placed in Category B (eg rickettsia, ricin, staphylococcal enterotoxins) also have some potential for large-scale dissemination with resultant illness, but generally cause less illness and death and, therefore, would be expected to have lower public health and medical impact. Biological agents that were not believed to present a high bioterrorism risk to public health but which could emerge as future threats, as scientific understanding of these

This book was written to provide a resource to scientists, epidemiologists, clinicians, and others in the field of bioterrorism defense. Not all possible topics are described. Instead, representative topics, authored by international experts provide a unique and state-of-the-art perspective on this field. The book consists of this preface and nine other chapters that address the broad areas of (1) detection capabilities (ie spatiotemporal disease surveillance; diagnostic bioterrorism response strategies; methods for detecting the presence of botulinum neurotoxins in food and other biological samples; and detection of bacillus spores by surface-enhanced Raman spectroscopy) and (2) characteristics of specific pathogenic microorganisms and toxins (ie Rickettsia and Rickettsial diseases; recent advances in the development of vaccines against Yersinia

Associate Director of Science, Division of Bioterrorism Preparedness and Response,

and Director of the Environmental Microbiology Program, CDC,

**Dr. Stephen A. Morse** 

USA

pestis; botulinum neurotoxins; ricin; and staphylococcal enterotoxins).

commercial sector. It remains a learning process.

agents improved were placed in Category C.

**1** 

*USA* 

**Current Methods for Detecting the** 

**Presence of Botulinum Neurotoxins** 

**in Food and Other Biological Samples** 

Luisa W. Cheng1, Kirkwood M. Land2 and Larry H. Stanker1 *Foodborne Contaminants Research Unit, Western Regional Research Center, 1Agricultural Research Service, U.S. Department of Agriculture, Albany, CA, 2Department of Biological Sciences, University of the Pacific, Stockton, CA,* 

Botulinum neurotoxins (BoNTs) are some of the most lethal human bacterial toxins and the causative agent of botulism (Arnon et al., 2001; Simpson, 2004). The usual routes of intoxication for BoNTs are oral ingestion of clostridial spores or pre-formed toxin, manifested as infant, foodborne and adult onset botulism. An increasingly common route of intoxication is associated with intravenous drug use resulting in wound botulism. BoNTs are also classified as Select Agents and have been used as agents of bioterrorism (Arnon et al., 2001; Bigalke and Rummel, 2005). Potential methods for toxin exposure include intentional contamination of the food and drink supply, or by aerosol spread, leading to

Usually, an identification of botulism is made through clinical manifestations and diagnosis, with subsequent confirmation by laboratory identification of clostridial spores or toxin in foods, environmental or clinical samples (CDC, 1998; Lindström and Korkeala, 2006; Solomon and Lilly, 2001). The speed of recovery from botulism increases with the timely administration of antitoxin or medical interventions (Arnon et al., 2001; Simpson, 2004). Thus, sensitive and rapid toxin detection and diagnostic methods are critical for improved

Due to the potential for bioterrorism use, much effort and resources have been dedicated to the development of detection methods, treatment, and prevention of botulism. A multitude of assay formats have been developed over many years, with in some cases, reported sensitivities at the attomolar level (Grate et al., 2010). Many assays were designed for use in the validation of toxin production, for commercial purposes, or for high-throughput screening methods to identify therapeutics that inhibit toxin function. These highly sensitive methods usually detect highly purified BoNT samples and are used in research type applications. Many such assays are not usable for the detection of BoNT contamination in food or other complex samples. This chapter focuses on the diagnostic methods for toxin detection and the challenges encountered while adapting analytical methods for the

recovery time, as well as, facilitate the epidemiologic study of outbreaks.

detection of BoNTs in foods and other biological and environmental samples.

**1. Introduction** 

inhalational botulism.
