The US would retaliate to bio-attacks.

Several weapons of mass destruction have been developed in various countries, like nuclear bombs, chemical weapons, and biological agents. The concept of using infection as a weapon is one of the most dangerous things to cause terrorism [1]. Terrorists can get control of these weapons and can create havoc. A number of countries in the 20th century have been affected by various bio-attacks [2]. Bioterrorism is defined as the deliberate dissemination of biological agents to cause illness or death among humans, animals or plants [3]. These bio-agents include viruses, bacteria, fungi, and toxic chemicals [4] [Table 1). These agents can be found in nature but can also be intentionally mutated to increase their death or disease capability. Some agents (e.g., chickenpox) are communicable and can spread from one person to another via air, water, food, touch etc., while some (e.g., anthrax) are non-communicable [5]. In addition, many of these bioagents are modified to make them more deadly by combining them with other chemicals and are then disseminated in the population [6]. Bioterrorism is the most common choice for terrorists as they are easy and inexpensive to obtain; moreover, these agents are hard to detect [7]. The threats of bioterrorism are frequently developing due to fast-growing technological skills and increasingly rapid growth in molecular biological sciences and biotechnology. The rapid proliferation of transnational terrorist organizations and their access to funding, equipment and expertise to produce biological weapons is another important factor that adds to the complexity of the situation. Therefore, a bioterrorism threat must be identified and handled in order to reduce adverse health effects and avoid fatality [8,9]. Although bio-crimes are restricted relative to other crimes, these actions raise concerns about how forensic evidence can be used in criminal proceedings to ascertain the source of microorganisms used as weapons. Bob Stevens, an American Media employee, was diagnosed with systemic anthrax on October 4, 2001. The report of this diagnosis sparked concerns that the disease resulted from an act of clandestine bioterrorism [10]. This Anthrax letter attack has brought a new reality to the world and made people more aware of a global bioterrorism risk. Moreover, the incident illustrated that the U.S was inexperienced in describing the biological facts of that case. In turn, a strong forensic capacity was developed to investigate bioterrorism and bio-crime [11]. Microbial forensics can be considered a scientific discipline for examining evidence from bioterrorism, bio-crime or illegal release of microorganisms/toxins for purposes of attribution. Besides the traditional role to prosecute crimes, law enforcement is now enhancing it to address the growing threat in the area of bio-crime and bioterrorism [12].

History of bioterrorism

Biological weapons are not simply an issue for the 21st century, but people have been using infectious agents for centuries in conflicts [26]. The Romans used to practice biological warfare by poisoning the water source in the wells of their enemies. Such terror tactics of the bio-war continued far into the 20th century [27]. Various countries, especially Germany and France, developed secret bio-weapon programs like infecting cattle feed with B.anthracis and B. mallei to infect their enemies during world war I [28]. There is much evidence in history that various nations used bioweapons to kill their enemies (Table 2). In 1336, Mongolian invaders used catapults to impel cadavers of bubonic plague victims across the walls of the Caffa town in the current country of Ukraine to kill city dwellers [29].

In 1785, Tunisian forces took advantage of plague-contaminated clothes as a bioweapon under the occupation of La Calle to make their enemies sick. During Pontiac's Rebellion (1763), British officials deliberately introduced smallpox to Native Americans in order to spread deadly diseases among nearby tribes [30]. During the Chinese-Japanese war in the late 1930s −1940s, Japan used the plague as a biological weapon. The bombs were loaded with fleas contaminated with plagues and were dropped from the aircraft in two Chinese cities. About 580,000 Chinese people lost their lives as a result of the Japanese bio-weapons program [31]. In Oregan (1984), supporters of Bhagwan Shree Rajneesh attempted to direct the local elections by infecting local people with Salmonella Typhimurium Bacteria. About 751 people were affected by the attack by extreme food toxicity. In the past, cattle were used as a secondary agent as a carrier of infection. For example, during the first world war, Germans used Glanders (Burkholderia mallei) to infect horses and mules [29]. In 1916, Chevy Chase, Maryland, developed a laboratory for the Glander and anthrax culture, and cultured organisms were supplied to Germans to infect animals in the U.S. During the war in Afghanistan in the early 1980s, the former Soviet Union also used Glanders as a biological weapon [4]. Biological and Toxin Weapons Conservation (BTWC) came into existence in 1975. This International Agreement intends on ending the production development of bio-warfare weapons and was signed by more than 100 Nations. Despite the deal, bio-weapon threats from terrorists continue to worry global health authorities [32].

Categories of bio-agents

U.S. Centers for Disease Control and Prevention (CDC) outlines three main categories of possible biological agents to be used as weapons, based on ease of dissemination or transmission, rate of mortality, social disruption, public health readiness and public health planning requirements [31,41]. ‘Category A’ contains the agents which are considered as most vulnerable, and a significant amount of research into biodefense has been directed to those agents. Such agents pose a significant risk to public safety and national security and can be transmitted and disseminated rapidly, leading to high mortality. There could be potentially significant global health consequences, triggering public concerns or requiring specific public health disaster response actions. ‘Category B’ includes agents that potentially disseminate moderately. These agents lead to infection at moderate rates and cause low mortality and need significant improvements and developments in the diagnostic capability of the CDC . Finally, Pathogens that are regarded as threats of emerging infectious diseases that may be engineered for mass transmission are included in ‘Category C’ [[42], [43]]. To analyze the risks posed to public health by various bio-agents, the CDC considers the level of disease outbreak, method of transmission, the effectiveness, and efficacy of vaccination, prevention, diagnosis and treatment for disease caused by different bio-agent items [6,31,32]. The examples and perspectives of the three categories are mentioned in Table 3.

Detection of biological warfare agents

Various coupled molecular and microbiological sensory techniques are used to detect biowarfare agents. Currently, antibody-based immunoassays, biochemical spectrometry, microbiological culture and genomic analysis PCR (used in the USA Biowatch Programme) are used for the identification of biological agents. They are extremely effective, flexible and specific techniques. Some of these detection methods have drawbacks, including isolation difficulties, extraction, and purification of test samples, low detection ability for the identification of pathological and etiological variations, and physiological or structural variations [8]. In 2001, anthrax mailings were critical for the investigation to establish if the agents were of the same strain. Random Amplification of Polymorphic DNA (RAPD) can be employed to study DNA Profiles produced by different isolates. This technique can differentiate Bacillus and non-Bacillus Isolates and demonstrate genetic differences among the strains of Bacillus cereus closely linked to Bacillus anthracis. Therefore, this method can be employed to link isolates used in biological attacks to each other and determine if the bio-agents have a common origin [44]. Continuous environmental surveillance of infectious diseases and associates of bio-warfare includes the implementation of extremely sensitive and precise functional methodologies. Real-time polymerase chain reaction (PCR) analysis is one effective approach for clinical diagnostics. However, this approach is limited by the use of single-use consumables in commercial PCR instruments for environmental surveillance. The mechanical complexity is greatly increased, as complicated robotic systems have to refill the discarded components. The flow-through Polymerase Chain Reaction Instrument System is one of the developed systems used to continuously monitor infectious biological agents. A reusable, flow-through PCR module that acts as an integral part of the whole system was introduced. This system was rigorously tested using genomic DNA from B. anthracis and revealed a high repetitiveness, sensitivity, and performance. This system consists of four new pairs of oligonucleotide primers, each specific for the A, B, E or F gene of botulinum toxin [45]. A fiber-optic, micro-based, multiplexed array composed of DNA probe microsensors used for the rapid identification of various bio-warfare agents was developed by L. Song et al. The high density of such an array could handle additional types of probes while retaining high reliability on the array for every type of probe. Additional probe types can be introduced to the array without disrupting the efficiency of the microsphere. The capability of the array to expand the probe types allows for incorporating other possible bio-agents when the genome sequences are available [46]. E. Legally (2004) studied the real-time detection of bacteria that move through water with an optical sensor based on a starburst Dendrimer film with a lipophilic fluorophore [47]. The sensor was designed by means of a covalent association between the amine-terminated poly-amidoamine dendrimer with the silanized glass. The molecule is introduced through host-guest interaction into the dendrimer layer. Automated real-time detection and quantitation of bacteria are achieved with a charging detector coupled camera and customized imaging and analysis tools and software. The sensor responds within one minute after the bacteria is injected into a water flow system.

Biochemical systems for biological target identification are based on microbial metabolism-related enzyme activity. They are usually not as precise as approaches based on antibody or nucleic acid methods, as the target enzyme or product can be found in other species. The electronic nose system is one the example of technology that detects the metabolic products of organisms. This system uses a transducer (e.g., cantilever), a conducting polymer coated by a chemical that comes in contact with volatile substances or gasses to make a sensing response [48].

There are numerous collections of probes and primers that can be used for certain pathogens or bio-threat agents. Some commercial companies recently have started to supply PCR-based kits to identify bio-threatening agents. Such kits remove the need for a comprehensive design of the primer and probes and enable rapid detection and surveillance of biowarfare agents. Most of such kits come along with already prepared controls and DNA samples to be tested [49]. Immunoassay techniques for identifying infectious diseases, drugs, and toxins are being used widely in the various medical, pharmaceutical and food industries. Moreover, the immunological identification of bio-agents such as bacteria, viruses, spores, and toxins is successfully used, with the concept that any material that can trigger an immune response can be described as an antigen. The various kinds of immunoassays have already been investigated for bio-threat detection [49]. The BioPen helps frontline soldiers in assessing if they are exposed to biologically dangerous agents in less than 20 min. With a small LCD, the latest diagnostic system is user-friendly, requires the user no preliminary training and is self-sufficient. This may also be used to test drinking water, identify environmental contaminants, and diagnose various serious diseases, for example, hepatitis B and C and even some forms of cancer, more reliably and efficiently than is now being tested. A special device called BioPen developed by the Ben Gurion University team for antigen detection uses the application ofthe widely employed Enzyme-Linked Immunosorbent Assay technique [50]. Different countries have launched bio-threat surveillance systems (e.g., BioWatch Programme by the U.S.), laboratories and a range of monitoring instruments for early detection of bio-attack. BioWatch is a united states initiative for identifying pathogen release into the air as part of a biological attack on major cities in America. This system allows the investigator with a rough idea about the source and the timing of the attack. It also provides forensic scientists with a small bio-agent sample for analysis and maintains the custody chain [51,52]. Genome editing which enables the engineering of specific genes also poses a great security challenge for the agencies. There are multiple guidelines set by the different regulatory bodies around the globe to keep the advancement of genome editing technology under check and balance [53,54].

Bio-attack investigation

The identification process of bio-attack is split into three phases: identify the biological agent responsible for the unprecedented outbreak of disease, identify the outbreak as being naturally or intentionally at the origin, and if the attack is intentional, identification of the state, organization or individual responsible for the attack is made [55]. Various kinds of facts can be linked to biological attacks [56]. Microbial forensics may play a major role in this situation. The bio-crime detection process relies on whether bio-agent exposure is transparent/open or secret. As per the FBI investigation process, in case of an open attack, a place where the disease outbreaks are designated as a crime scene and entry to that place is restricted in order to collect and preserve evidence. Forensic experts then examine the genetic makeup of the agent on the basis of physical, biological, and genetic properties. The analysis is then correlated with the data of known bio-agents accessible from nature, preserved in research laboratories or collected from suspect parties. In such cases, medical professionals, law enforcement and policy communities work collaboratively to deal with the bio-attack [55]. Medical experts aim to identify the infectious agents, law enforcement officials collect evidence and identify the perpetrators and national policy communities assess the origins of the bio-attack to retaliate actions relatively in a short time [57].