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Microbial Forensics is a rapidly evolving scientific discipline. In the last decade, and particularly due to the anthrax letter attacks in the United States, microbial forensics has become more formalized and has played an increasingly greater role in crime investigations. This has brought renewed interest, development and application of new technologies, and new rules of forensic and policy engagement. It has many applications ranging from biodefense, criminal investigations, providing intelligence information, ...
Microbial Forensics is a rapidly evolving scientific discipline. In the last decade, and particularly due to the anthrax letter attacks in the United States, microbial forensics has become more formalized and has played an increasingly greater role in crime investigations. This has brought renewed interest, development and application of new technologies, and new rules of forensic and policy engagement. It has many applications ranging from biodefense, criminal investigations, providing intelligence information, making society more secure, and helping protect precious resources, particularly human life. A combination of diverse areas is investigated, including the major disciplines of biology, microbiology, medicine, chemistry, physics, statistics, population genetics, and computer science.
Microbial Forensics 2nd Edition is fully revised and updated and serves as a complete reference of the discipline. It describes the advances, as well as the challenges and opportunities ahead, and will be integral in applying science to help solve future biocrimes.
Microbial Forensics describes the new and growing field of Microbial Forensics- the science that will help bring to justice criminals and terrorists who use biological material to cause harm. This book describes the foundation of the field of microbial forensics and will serve as a basic primer to initiate those scientists and officials that have an interest in the topic. It covers a variety of areas from forensic science, to microbiology, to epidemiology, to bioinformatics, and to legal issues. Provides the real science beyond that displayed on TV and in the movies
Arnold F. Kaufmann and Paul S. Keim
The Aum Shinrikyo, an apocalyptic religious sect based in Japan, first came to worldwide attention in 1995 as the result of the sect's deadly sarin gas attack on the Tokyo subway system. Subsequent investigations revealed that the Aum Shinrikyo had launched earlier attacks with both chemical and biological agents. The biological attacks utilizing Bacillus anthracis spores and botulinum neurotoxin were notably unsuccessful, with failure to produce any casualties in at least seven alleged attempts over several years beginning in 1990. This chapter discusses an attack in 1993 that was launched from the Aum Shinrikyo headquarters building, then located in Kameido, a Tokyo suburb, with emphasis on laboratory and epidemiological studies.
THE AUM SHINRIKYO: A BRIEF HISTORY
To put the Kameido incident into context, a brief history of the Aum Shinrikyo and its founder, adapted primarily from reviews by Hudson, Smithson, and Tu, is useful. The Aum Shinrikyo was founded by Shoko Asahara, whose birth name was Chizuo Matsumoto. Born into poverty in 1955 and suffering severe visual impairment due to infantile glaucoma, Matsumoto was sent at a young age to a government-subsidized boarding school for the blind. He purportedly felt abandoned by his family, which may have later led to an Aum Shinrikyo rule that followers were to cut off relationships with their parents to attain the supreme truth.
Having limited vision in one eye, Matsumoto developed influence over the other blind students, who paid him for various services. During his student years, he developed a reputation as a bully and con artist. After high school graduation in 1975, Matsumoto established a successful acupuncture clinic, but he had to move to Tokyo in 1977 due to his involvement in a fight that resulted in injury to several persons. About this time, his stated ambitions included becoming the leader of a robot kingdom or the Prime Minister of Japan.
In Tokyo, he found work as an acupuncturist and enrolled in a preparatory school for the Japanese college entrance examination, with a goal of qualifying for matriculation in Tokyo University. Failing the entrance examination, Matsumoto married and established an acupuncture clinic and a natural foods shop. As a sideline, he concocted an alcohol extract of tangerine skins that he marketed as a miracle drug for weight loss and a variety of other conditions. His success in sales of this product attracted the attention of the police and subsequent arrest and imprisonment for violating the Japanese Cosmetics and Medical Instruments Act. This experience may have contributed to his animosity toward established authority.
After his move to Tokyo, Matsumoto became interested in religion and, in 1981, joined Agon Shu, a new religion based on Buddhism and yoga. In 1984, Matsumoto quit the Agon Shu and established Aum Shinsen, a yoga club that grew rapidly from 15 to more than 1000 members. He also changed his name to Shoko Asahara or Bright Light in Japanese. Following a trip to India in 1986–1987, Asahara changed the name of the yoga club to Aum Shinrikyo. Aum is a Hindu syllable representing the spoken essence of the universe, and Shinrikyo is derived from the Japanese words for "supreme truth." In 1989, Aum Shinrikyo was officially recognized as a religious sect in Japan, giving the sect tax advantages as well as the ability to claim the members' work in the sect's various enterprises as voluntary. The sect's growth continued with spread to other countries, including the United States, Germany, and Russia.
Sect members lived a spartan life and were expected to cut off all associations from their past life, to take a chastity vow, and to turn over all their assets to the Aum Shinrikyo. They were subjected to a heavy diet of their master's "wisdom," often simultaneously undergoing food and sleep deprivation. Members were expected to labor voluntarily in the sect's various commercial enterprises, such as sales of herbal teas and natural medications, operation of noodle shops, health clubs and babysitting services, and computer-related services. Those who balked were driven ever harder, drugged, and confined. In some extreme cases, defectors were murdered.
To carry out its activities, the Aum Shinrikyo developed a complex organizational structure consisting of 22 ministries plus the Offices of Religious Members. The latter was charged with recruitment of persons having needed skills, such as members of the Japanese Self Defense Forces and scientists. In effect, the Aum Shinrikyo assumed the form of a shadow government, which could supplant the existent Japanese government if Asahara's ambitions were fulfilled.
By 1990, with membership in the tens of thousands spread over six countries and an estimated 300 million to a billion dollars available, the Aum Shinrikyo was well positioned to further Asahara's ambitions and delusions. The Aum Shinrikyo initially attempted a conventional approach to political power by fielding a slate of 25 candidates for the national elections that year. None of the candidates, including Asahara, was even modestly successful. Asahara believed that the Japanese government had cheated him rather than that the electorate was put off by the doomsday overtones of the candidate's speeches. His belief was reinforced by the fact that the number of votes received by all the candidates was far fewer than the number of Aum Shinrikyo members in Japan.
Based on a pastiche of apocalyptic scenarios drawn from various religions, Asahara preached that Japan was destined to suffer a number of overwhelming catastrophes, including a poison gas attack by the United States. Asahara and his followers would survive the looming Armageddon and evolve into a super-race dominating the world. He became more vocal in expressing this belief after the humiliating electoral defeat in 1990. Not content to allow the catastrophes to occur in their own time, Asahara initiated development of chemical and biological weapons to speed up the process.
Only the chemical weapons program had some success. After overcoming initial production problems, the Aum launched an attack with sarin gas in Matsumoto City in June 1994. The attack targeted judges presiding over a land use dispute between the Aum Shinrikyo and a local real estate agent. Suspecting that the judges would make a decision unfavorable to the cult, Asahara ordered their assassinations. This gave an opportunity to test the effectiveness of their sarin on humans. The sarin release utilized a spray device and resulted in 311 known casualties, with 58 hospitalized, including seven deaths. The judges were unharmed.
Investigation of the Matsumoto City attack proceeded slowly, without definitive evidence linking the crime to a specific individual or group. The Aum Shinrikyo was suspected, and a sarin degradation product was detected in soil near a building on an Aum Shinrikyo compound, the Seventh Satayan, in Kamakiuishiki. The police did not seek a warrant to search the facility because of a conservative interpretation of pertinent laws. In an unrelated kidnapping case, however, the police found fingerprint evidence that an Aum Shinrikyo member was involved. This gave justification for obtaining a warrant to investigate the facility. In March 1995, Asahara learned of the plans for a police raid from Aum members within the Japanese Self Defense Forces. In a ploy to distract the police and buy time, Asahara decided to release sarin in the Tokyo subway system. Two days later, the attack was carried out, resulting in several thousand casualties, including 12 fatalities.
The Aum Shinrikyo cult had been thought to be an odd group and even a nuisance on occasion. Investigations after the Tokyo sarin attack revealed a more sinister aspect of the sect and its leader. In addition to the chemical weapons development program, the Aum Shinrikyo was found to have been actively pursuing biological weapons, albeit without success due to incompetence. In particular, the ineffective release of B. anthracis spores in Kameido was discovered, leading to the investigations discussed in this chapter.
THE KAMEIDO ANTHRAX INCIDENT
On June 29–30, 1993, complaints about foul odors were registered with local environmental health authorities in Kameido in the Tokyo metropolitan area. The odors originated from the eight-story headquarters building of the Aum Shinrikyo. Some of the exposed persons reported appetite loss, nausea, and vomiting. Birds and pets were also reportedly ill, but the nature of these illnesses was not defined. The environmental health officials requested permission to inspect the headquarters building, but Aum Shinrikyo members at the scene refused. The officials checked the building's exterior, collected air samples, and began surveillance of activities at the building. Other than the nuisance posed by the odor, definitive human health risks could not be identified.
On the morning of July 1, neighbors began to complain about loud noises and an intermittent mist originating from one of two structures on the roof which were thought to be cooling towers (Figure 1.1). As the day progressed, 118 complaints about foul odors were received from nearby residents, primarily to the south of the building. Winds (2–4m/sec) that day blew from a northeasterly direction. Light rain (7mm total) fell in the early morning, with cloudy conditions thereafter. The temperature ranged from 16.9 to 19.9°C throughout the day.
A "gelatin-like, oily, gray-to-black" fluid from the mist coming off the "cooling towers" collected on the side of the building. Samples of the fluid were collected by the environmental health officials and stored in a refrigerator (4°C) for later testing.
The next day, July 2, Shoko Asahara agreed to stop using the rooftop devices and to clean and vacate the building. An environmental inspection found no equipment, including the rooftop devices, remaining in the building, and black stains on the walls were the only notable observation.
The problem, apparently being resolved, was largely forgotten until 1996. Police investigations of the sarin attack on the Tokyo subway system revealed that the Aum Shinrikyo was also involved in bioterrorism. Following the conservative Japanese policy of not revealing criminal evidence until the time of trial in court, the true nature of the Kameido incident was first disclosed to the public in May 1996 when Asahara was arraigned. Aum Shinrikyo members had confessed that the odors resulted from efforts to aerosolize a liquid suspension of B. anthracis spores. The motive was to trigger an inhalational anthrax epidemic and a subsequent world war. The war would culminate in the Aum Shinrikyo members becoming a super-race that would rule the world in accord with Asahara's preaching.
Many questions about the incident remained unanswered. For example, did the attack actually occur? If so, were B. anthracis spores utilized? Could the specific B. anthracis strain and its origin be identified? Was the strain virulent? Why did the attack apparently fail? Had illnesses occurred but gone undetected? Investigations were initiated to better characterize the alleged attack and its consequences.
MICROBIAL FORENSICS INVESTIGATION
Fluid that had been collected from the Aum Shinrikyo headquarters building in July 1993 and subsequently stored at 4°C was examined in January 2000 for bacterial content. Polymerase chain reaction (PCR) screening of the fluid was positive for B. anthracis. Microscopic examination of the fluid with malachite green and safranin staining revealed spores, nonspecific debris, and bacterial cells other than large bacilli. The fluid was cultured by spreading on sheep blood agar plates and incubating at 37°C under ambient CO2 concentration (Figure 1.2). Based on the number of bacterial colonies observed on the plates after incubation, the fluid contained approximately 4 X 104 bacterial colony forming units (CFU) per milliliter. Most colonies grew only weakly and were morphologically inconsistent with normal B. anthracis characteristics when grown under these conditions. The poorly growing bacteria were not further characterized.
About 10% of the colonies on the plates were typical of B. anthracis, being large and having a nonhemolytic, "gray ground glass" appearance (Figure 1.2). The number of observed colonies consistent with B. anthracis was consistent with a concentration of 4 X 103 CFU/ml of the fluid. Forty-eight of these colonies were purified by single-colony streaking and subjected to the eight-locus multiple-locus variable-number tandem repeat analysis (MLVA) (6). All 48 colonies were B. anthracis and had an identical MLVA genotype, although the VNTR marker on the pX02 plasmid failed to amplify. The genotype of all 48 strains was vrrA, 313bp; vrrB1, 229bp; vrrB2, 162bp; vrrC1, 583bp; vrrC2, 532bp; CG3, 158bp; pX01-aat, 129bp; pX02-at, no amplification. The lack of PCR amplification at the pX02 markers is consistent with strains that are missing the pX02 plasmid entirely. Amplification of these loci can occur in closely related Bacillus cereus strains, but actual amplicon sizes had been observed previously only in B. anthracis. The MLVA genotype observed was consistent with results obtained with the Sterne anthrax vaccine strain (Figure 1.3). The Sterne strain is a member of the A3.b diversity cluster, and in a study of 419 isolates, only four naturally occurring B. anthracis strains in the electronic database of worldwide isolates had the same seven-marker genotype, although these strains were pX02 positive.
The Sterne 34F2 anthrax vaccine strain is available commercially in Japan for veterinary use. It had been reported previously that the Aum Shinrikyo had obtained a veterinary vaccine strain of B. anthracis, which may have been used by them for bioterrorist attacks (1). Results are consistent with this previously unsubstantiated report. The Sterne strain has low virulence due to lack of the pX02 plasmid, which is the location of genes coding for the ability to produce capsule, a major virulence factor of B. anthracis.
Culture-confirmed anthrax is a nationally notifiable disease in Japan, with physicians being required to report all cases. Only four human anthrax cases were reported during the 1990s, with a single case being reported in Tokyo. The Tokyo case was diagnosed in a man who was in his eighties and resided in Sumida-ward, which is adjacent to Kameido-ward. The case occurred in August 1994 and had no obvious association with the 1993 Kameido incident.
Could additional anthrax cases from the 1993 attack have gone unrecognized or unreported? A retrospective case-detection survey was conducted in 1999 to address this question (4). Using the official registry of "foul odor" complaints, the residences of the 118 complainants were mapped to identify the area of presumed highest risk. The 0.33-km2 high-risk area determined by this approach contained approximately 3400 households and 7000 residents. A telephone survey was conducted of physicians at 39 medical facilities (15 internal medicine, 7 dermatology, and 15 other specialties) serving the area. None of these physicians had treated cases of anthrax, unexplained serious respiratory illnesses, or hemorrhagic meningitis, a common complication of systemic anthrax in residents of the high-risk area.
A number of lessons can be learned from the Kameido incident. The investigation suffered from a failure to detect the incident at the time. The Aum Shinrikyo did not attract much official attention until the sarin attack in Matsumoto City in 1994. The Japanese culture is very tolerant of varying religious beliefs, an attitude reflecting Japanese constitutional guarantees of religious freedom. The Aum Shinrikyo was but one of more than 180,000 minor religions active in Japan. The police policy of conservative interpretation of pertinent laws was another factor. Also, the policy of not revealing details about a criminal investigation until the time court procedures are initiated slowed communications between pertinent agencies, delaying investigation even after the Aum Shinrikyo's attempt to utilize biological weapons first became known during the investigation of the 1995 sarin attack on the Tokyo subway (3). Awareness of potential threats, a low threshold of suspicion, and active sharing of information between governmental agencies having pertinent expertise and/ or authority are some key components in early detection of terrorism incidents.
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Introduction - The Rapidly Evolving Discipline of Microbial Forensics;
PART 1 INVESTIGATIVE GENETICS - A. Criminal; The Kameido anthrax incident: A microbial forensics case study; Microbial Forensic Investigation of the Anthrax-Letter Attacks;
B. Civilian; Foodborne Outbreaks: What’s New, What’s Not, and Where Do We Go from Here?; Genotype and Geography; the global distribution of bovine tuberculosis; Tracing meat products through production and distribution chain from farm to consumer; Microbial Forensics of RNA Viruses: Foot-and-Mouth Disease Virus; Forensic Plant Pathology;
PART 2 EMERGING INFECTIONS Influenza Forensics; Keeping Track of Viruses; Microbial Forensic Analysis of Trace and Unculturable Specimens; Molecular Microbial Surveillance and Discovery in Bioforensics;
PART 3 BIOSECURITY Assessment of the Threat; The Select Agent Regulations; Biosurety in the Post-9/11 Era; Forensic public health: epidemiologic & microbiologic investigations for biosecurity
PART 4 SUBJECT AREAS Forensic Analysis in Bacterial Pathogens; Rickettsia and Coxiella; Forensics and Epidemiology of Fungal Pathogens; Ricin Forensics: Comparisons to Microbial Forensics; Forensic Aspects of Biologic Toxins;
PART 5 METHODS The Use of Host Factors in Microbial Forensics; Collection and Preservation of Microbial Forensic Samples; Sampling for Microbial Forensic Investigations; Toxin Analysis Using Mass Spectrometry; Electron Beam-Based Methods for Bioforensic Investigations; Proteomics Development and Application for Bioforensics; High-throughput Sequencing; Genomics; The Design of Genomic Signatures for Pathogen Identification and Characterization; Non-Biological Measurements on Biological Agents; Inferential Validation and Evidence Interpretation; Microbial Forensic Investigations in the Context of Bacterial Population Genetics; Population Genetic Considerations in Statistical Interpretation of Microbial Forensic Data; Biorepositories and their Foundations – Microbial Forensic Considerations;
PART 6 NATIONAL LEVEL CAPABILITIES A U.S. Research Strategy for Microbial Forensics: From Genesis to Implementation; The National Bioforensic Analysis Center; Microbial Forensics in Australia - The Australian Federal Police Perspective; Validation of Microbial Forensics in the Scientific, Legal and Policy Contexts;
PART 7 MICROBIAL FORENSICS CURRICULA AND TRAINING Microbial Forensics: Educating the Workforce and the Community; So You Really Want to be an Expert Witness? A Primer for the Occasional Expert Witness; Conclusion – Microbial Forensics, What Next?