Biological weapon identification

DEFINITION: Identification of weapons of mass destruction that are based on bacteria, viruses, fungi, and toxins produced by these microorganisms.

SIGNIFICANCE: Heightened concerns regarding the possibility of bioterrorist attacks have led to increased emphasis on microbial forensic science. Microbial forensic data may be presented in court as evidence in cases of terrorist attacks.

Virtually all disease-causing microorganisms are potentially useful as biological weapons. The most important candidates for biological weapons are microorganisms that cause diseases with high human mortality rates, such as anthrax, smallpox, plague, encephalitis, and hemorrhagic fever. In addition, biological weapons that are designed to wipe out crops or kill livestock could cause mass starvation and devastating economic losses.

In 2001, the public in the United States became aware of biological weapons as a result of a series of attacks involving mail containing Bacillus anthracis (the bacterium that causes anthrax). Since that time, the possibility that terrorists might employ biological weapons, many of which are easily produced and spread, has been a growing concern. In response to the threat of terrorist attacks, the US government has led efforts to develop quick and efficient methods of biological weapon identification, ultimately leading to the establishment of the new scientific discipline of microbial forensics. In general, the identification of microorganisms is based on techniques that rely on microscopic examination, analysis of the growth and metabolic functions of the microbes (growth-dependent and biochemical tests), and immunological and genetic tests.

Growth-Dependent and Biochemical Tests

Classic methods of microbial identification involve preliminary examination of stained specimens under a microscope, followed by growth-dependent tests. Growth-dependent tests are based on the growth patterns of microorganisms on artificial food sources (media). Particular media can be selected that will produce microbial populations—known as colonies—that have distinctive appearance and color. By comparing the reactions on these media with the known characteristics of different species of microorganisms, scientists can usually identify which microbe is present. However, most growth-dependent tests do not provide results that are extremely specific; that is, they may not distinguish among closely related microorganisms.

To aid in definitive microorganism identification, scientists have developed a series of biochemical tests that can be used to differentiate even the most closely related microbes. These tests are based on the identification of various metabolic reactions and products of different microbes. Microbial species can often be identified on the basis of fermentation patterns and the production of different chemical compounds, such as indole or hydrogen sulfide. Microorganisms are not easily identified by a single biochemical test, so it is usually necessary to perform several tests. A number of rapid identification systems are available that allow several (approximately twenty) biochemical tests to be performed quickly on a particular microorganism.

Immunological Tests

Immunological tests utilize antibodies that are produced in response to the presence of a specific microorganism; actually, they respond to the presence of specific molecules, called antigens, on the microorganism cell surfaces. Antibodies are proteins produced by the body that recognize and bind to those antigens. Specific antibodies for many known disease-causing microorganisms are commercially available. Immunological tests vary in the ways they make the antigen-antibody reaction visible; some show obvious clumps and precipitates, whereas others show color changes or the release of fluorescence.

An example of an immunological test is the agglutination test, which is performed routinely in hospitals to determine blood types. In an agglutination test, antibody-antigen complexes form visible clumps on a test glass slide. Extremely sensitive immunological tests called immunoassays permit rapid and accurate measurement of trace bioweapon agents. These methods are being used increasingly in criminology. A good example of such an immunoassay is the enzyme-linked immunosorbent assay (ELISA). A positive result in this immunoassay is the appearance of a colored product. ELISA is a common screening test for the antibodies to toxins and bacteria that may be used as bioweapons. In radioimmunoassays, antibodies are labeled with radioactive isotopes and traced. Immunological methods are especially important for the identification of viruses, as other identification methods are not suitable to them, and growth times are long.

Genetic Tests

Genetic tests of microorganisms are based on the detection of the unique (deoxyribonucleic acid) sequences of potential weapon microorganisms. Certain viruses maintain their genetic material in the form of RNA (ribonucleic acid), which can be converted into corresponding DNA for detection purposes. One particular technique has been widely used for identifying microorganisms based on their DNA sequences: polymerase chain reaction (PCR).

Two variations of the PCR technique have been adopted for identification: PCR and real-time PCR. Both utilize specific sets of primers (short DNA sequences) to amplify and detect DNA sequences unique to a particular microorganism. In PCR, amplified DNA sequences are subjected to separation by electrophoresis, where negatively charged DNA fragments move toward the positive pole. Separated DNA fragments can be classified by the distance they traveled, depending on their molecular size. Each microorganism exhibits a characteristic DNA moving pattern by which it can be identified. In real-time PCR, detection of a microorganism’s amplified DNA and confirmation of that microorganism’s presence are sensed by activation of a fluorescent dye. Officials of the United Nations used portable PCR detectors when they conducted their 2002-2003 inspections of Iraqi facilities for weapons of mass destruction. These detectors can identify a single B. anthracis bacterium in an average kitchen-sized room.

Ongoing Challenges

Although, in most cases, agents used as biological weapons could be identified easily within twenty-four hours, prosecutors may have difficulty proving that microorganisms identified in the homes or laboratories of suspects are in fact the same microorganisms used as weapons or intended for such use. One problem with making legal arguments based on weapon microbe identification is that some potentially dangerous microorganisms, such as B. anthracis, are found widely in soil. A thus must prove that the microbes submitted as in a given case are the same microbes used in the attack in question, and not simply microorganisms that have been transported into the suspect’s home or lab accidentally. In the 2020s, researchers were experimenting with electrochemical sensing, which isolates the biological agent, so it is easier to identify. This technique measures changes in the environment caused by the introduction of the biological weapon. Scientists can identify the weapon by the type of change it causes.

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