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 Table of Contents  
Year : 2018  |  Volume : 4  |  Issue : 3  |  Page : 161-166

Bioterrorism threat: A review of microbial forensics source-tracing of some bioterrorism agents

1 Department of Histopathology and Cytology, Forensic Science Unit, Federal College of Veterinary and Medical Laboratory Technology, Vom, Plateau State, Nigeria
2 Viral Research Division, National Veterinary Research Institute, Vom, Plateau State, Nigeria

Date of Web Publication28-Sep-2018

Correspondence Address:
Mr. Bobmanuel Chimaroke Echeonwu
Department of Histopathology and Cytology, Forensic Science Unit, Federal College of Veterinary and Medical Laboratory Technology, Vom, Plateau State
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jfsm.jfsm_5_18

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Global perception and consciousness of the threat of bioterrorism seem to have diminished in recent past following achievements in decades of global fight against deadly infectious diseases such as plague and smallpox. However, with recent advancement in biotechnology and the arrival on the scene of amoral and rogue states as well as terrorist groups, there is a justifiably heightened global apprehension that bacteria, viruses, and toxicogenic fungi can be weaponized and used to cause great harm to humans and agricultural biodiversity. We now have on our hands the dilemma of dual-use of biotechnology. This review brings to the fore an aspect of microbial forensics – bioagent source-tracing (attribution) that is very key in mounting an appropriate response to the evident threat of bioterrorism. This article places a little more emphasis on the bioagent Yersinia pestis, and the technique of whole-genome sequence typing adjudged the most effective technique for building databases for bioterrorism-associated agents and public health important pathogens. The need for international sharing of data and databases of bio-agents is emphasized, as this would enable global applicability of bio-agent source-tracing in cases of bioterrorism.

Keywords: Bioagent, bioterrorism, microbial forensics, source-tracing, whole-genome sequence typing

How to cite this article:
Echeonwu BC, Nwankiti OO, Chollom SC, Olawuyi KA. Bioterrorism threat: A review of microbial forensics source-tracing of some bioterrorism agents. J Forensic Sci Med 2018;4:161-6

How to cite this URL:
Echeonwu BC, Nwankiti OO, Chollom SC, Olawuyi KA. Bioterrorism threat: A review of microbial forensics source-tracing of some bioterrorism agents. J Forensic Sci Med [serial online] 2018 [cited 2022 Oct 2];4:161-6. Available from: https://www.jfsmonline.com/text.asp?2018/4/3/161/242514

  Introduction Top

In the last couple of centuries, infectious diseases of viruses, bacteria, protozoans, fungi, and nematodes became lesser issues to human survival than they had previously been. Smallpox, for instance, in most years was responsible for about 10% of deaths in children in London, whereas the periodic episodes of  Yersinia More Details pestis outbreaks (plague) might wipe out about 30% of any infected population.[1] Smallpox, however, has been eliminated from the world since 1979, while the plague is today largely unknown in the developed world and is a very rare occurrence in the world entirely. The world had enjoyed these and other similar success stories in the fight against infectious diseases, until the emergence on the scene of amoral states or terrorist groups and individuals that have the propensity to deliberately use these hitherto severe infectious disease-causing microorganisms to inflict debilitation and death on targeted human populations in a bid to advance whatever political or socioreligious positions they advance.

  The Bioterrorism Threat – Brief History and Contemporary Assessment Top

Bioterrorism has been defined as the deliberate act of using pathogenic organisms or their by-products such as toxins, to cause harm to vulnerable humans, animals, or plants including the environment, by individuals or groups (e.g., lone offenders or terrorist groups) for the purpose of menacing societal safety or stability, or to cause panic.[1],[2] Bioterrorism is a little different from biological warfare, which is the use of biological agents or living organisms by the military in war circumstances.[3] There may be criminal, ideological, religious, or political reasons or motives behind acts of bioterrorism and/or biological warfare, but whichever in the case, the effects on humans, plants, and animals (the generality of biodiversity) are nonetheless devastating. Resources such as water or food [4] as well as manufactured materials [5] may also be targets of the bioterrorist.

The threat of bioterrorism to humans and agricultural biodiversity has been recognized a long time ago.[6] History offers several examples of the intentional use of disease-causing agents or in some cases in the past infected cadavers as weapons to cause harm to the enemy.

Historical evidence of bioterrorism threat

Some examples of historical biological warfare or examples of the intentional use of disease-causing agents in the past are summarised in [Table 1].
Table 1: Some bioterrorism events in history

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Assessment of contemporary bioterrorism threat

According to Powers and Ban,[16] the assessments of bioterrorism threat over the years have been either unfocused or narrowly focused on single factors. The failure of threat assessment methodologies to put into consideration all the factors that make up the threat has been responsible for the imbalance between the threat assessments and the preparedness measures.

In assessing the contemporary threat of bioterrorism, one needs to consider the fact that the Internet and other open literature contain information and knowledge required to develop biological weapons and the fact that many of these pathogens associated with bioterrorism exist naturally within host animals or the vector organisms.[2] In addition to this, the ease and low cost with which biological agents may be dispersed is also a cause for concern. As far back as in 1998,[17] it had been estimated that while for conventional weapons, it may cost up to $2000/km2 to inflict civilian casualty and about $800/km2 for nuclear weapon use, it would only cost about $1/km2 to cause civilian casualties using biological weapons.

With current biotechnological advancement, there is an almost palpable fear that bacteria and viruses might be modified in diverse ways to make them into more powerful biological weapons.[1] Modifications to the traditionally known biological agents (e.g., Bacillus anthracis,  Brucella More Details, Francisella tularensis, and Y. pestis) could be by way of making them antibiotic resistant, vaccine evading, decontaminant insensitive, or environmentally stable.[2] There have been speculations also that bioterrorists could take advantage of advanced biotechnology to engineer biological agents that target specific races, creating a stealth virus capable of incorporating itself into the DNA of such targeted populations. The creation of a different virus by recombining IL4 gene into mousepox virus [18] is a case in which advanced biotechnology has been demonstrated. There have also been cases where the Spanish flu virus of 1918 and the poliovirus were recreatedin vitro using advanced biotechnology.[19],[20] It is little wonder, therefore, that scientists in various fields who are following the trends in bioterrorism and biotechnology are getting more worried about the possible misuse of biotechnology.[21]

  The Scientific/investigative Response to Bioterrorism: Source-Tracing Top

In the event of a bioterrorism attack, several questions need to be asked and answered in the course of responding and investigating the incident. Such questions and issues would border on the safety of the public as well as first responders, investigation of the suspected site, sample collection and transfer, isolation and identification of the pathogen, and source-tracing of the identified bioagent.[2]

In the case of the anthrax letter attack in the United States for instance, a number of issues had to be addressed regarding the relatedness or otherwise and cause of the disease cases in the four states involved, the relatedness and origin of the bacterial isolates from the different states, as well as the identity of the perpetrators who released the agent.[22] Answers to these questions are within the jurisdiction of microbial forensics, which is expected to employ relevant traditional investigative methodologies, as well as molecular techniques that are established (and where necessary, newer techniques that are under development), in order to identify and trace the source of the pathogen.

Microbial forensic investigations generally consist of three inter-related stages; identification, characterization, and attribution (source-tracing).

  • Identification: This is the stage at which the bioagent used in the attack is identified. When an attack is fully a covert one, it may not be detected until animals and/or humans begin to manifest the signs of the disease. Therefore, identifying the pathogen will mainly be from an animal or public health response rather than from a microbial forensics response. However, if the perpetrator announces the attack, as in an overt scenario, then the appropriate response would be from the microbial forensics angle. A microbial forensics approach will also be appropriate in cases where authorities are acting on intelligence information to locate and take possession of the attack strain before the attack happens
  • Characterization: This next step relates to the process of determining if the event was an intentional one or an unintentional one. If an attacker has announced his/her intentions to release a pathogen initially, then this stage would not be necessary. However, in cases where an outbreak exhibits unusual characteristics and therefore arouses suspicion of a deliberate release of pathogen or where the perpetrator only claims responsibility after signs of the disease begin to manifest, then it becomes very important to determine the cause of the disease
  • Attribution: This third step which may indeed be referred to as source-tracing seeks to locate or identify the source of the “weapon strain” pathogen as well as the perpetrator of the attack. It would also yield useful evidence that would be necessary to prosecute the bioterrorist.[23]

Tracing of pathogen(s) used in an attack to a particular source is paramount and takes a central position in the bid to identify and criminally prosecute perpetrators of bioterrorism acts.[22] This onerous task of microbial forensic source-tracing has an expanded scope [24] and should not be limited only to cases of deliberate bioterror attacks (whether overt or covert) with the intention to criminally prosecute; it should also be applied to civil cases such as epidemiological investigations of serious diseases of public health importance for proper tracking of outbreaks.

Attribution in microbial forensics will be involved with tasks such as identifying and collecting samples, handling and preservation of the recovered samples, selection of appropriate analytical methods, casework analysis, result interpretation, as well as quality assurance and validation. Microbial forensics, therefore, needs to be a field that brings together a range of other well-established fields which would include phylogenetics, microbial genomics, bioinformatics, forensic information, computer science, in addition to the traditional microbiology, and epidemiology.[2]

This review essentially focuses on currently available methods and techniques for this third component (attribution) of microbial forensic investigation in the event of a bioterrorist attack.

A wide array of laboratory analytical techniques and methodologies to include molecular sequencing, biochemical analyses, microbial cultures, electron microscopy, mass spectrometry, and crystallography may be required for microbial forensics, and these go far beyond those employed in medical diagnoses or epidemiological investigations.[25]

For some years now, molecular techniques have been employed in what has been termed as molecular epidemiology, to trace sources of outbreaks of microbial diseases. According to Budowle et al.,[26] genetic markers are needed which can have a significant impact on statistical inferences relating to the evidence in microbial forensics. Some of these markers are based on single-nucleotide polymorphisms (SNPs), insertions and deletions, repetitive sequences, mobile elements, housekeeping genes, pathogenicity islands, structural genes, resistance and virulence genes, whole-genome sequences, sexual and asexual reproduction, conjugation, transduction, horizontal gene transfer, lysogeny, gene conversion, gene duplication, recombination, rearrangements, and mutational hotspots. Technologies based on nucleic acids include multilocus sequence typing (MLST) and an improved form of it, ribosomal multilocus sequence typing,[27] multilocus variable-number tandem-repeat (VNTR) analysis (MLVA), clustered regularly interspaced short palindromic repeats (CRISPRs), whole-genome sequencing, and microarray. In this regard, the advent of high-throughput methods such as the massively parallel sequencing technique otherwise known as next-generation sequencing in recent times greatly and positively impacts the capabilities of forensic genetics and by extension microbial forensics.[28],[29] Whichever choice of the above-listed techniques is adopted for use, the necessity of a reference database for the markers cannot be overemphasized.[2]

Source-tracing of prospective bioterrorism agents –  Yersinia pestis More Details in view

Y. pestis, which causes bubonic and pneumonic plagues, is recognized as one of the world's most dangerous bacterial pathogen. It has been credited with the deaths of close to 200 million people during the cause of history.[30] The pathogen, however, exists naturally in wild rodents and is transmitted between rodents by their flea ectoparasites, which move from one dead host to another for blood meals.

Y. pestis is believed to be the descendant of  Yersinia pseudotuberculosis More Details, and according to Morelli et al.,[31] their evolutionary divergence is believed to have occurred some 2600–28,000 years ago. There is currently a wide range of Y. pestis intraspecies groups classified as biovars, ectotypes, subspecies genotypes, plasmidovars, etc., which have resulted from intraspecific modifications over the years.

In recent years, a large number of sources for polymorphism as well as typing methods have emerged owing partly to the technology available for whole-genome sequencing which has enabled the generation of whole-genome sequence data for a lot of bacterial species and for strains within the same species. Some of the typing methods that have been applied to Y. pestis include (as mentioned above) SNPs, MLVA, MLST, different region (DFR) typing, and analysis.[2] These recent methods, including the CRISPR technique, are fast becoming preferred methods than previous methods such as insertion sequence typing using pulsed-field gel electrophoresis, Southern blotting, or ribotyping. These earlier techniques, though more expensive, are not suitably applicable to creation of international databases.[32]

Using single-nucleotide polymorphisms for source-tracing of Yersinia pestis

According to Achtman et al.,[33] the evolutionary closeness of Y. pestis to Y. pseudotuberculosis hinders their differentiation by MLST. However, in their work of 2004, Achtman et al. were able to identify 76 conservative SNPs from within 3250 orthologous coding sequences. This was based on the comparative analysis of three whole genomes of Y. pestis which represented different biovars of the pathogen. They proposed a genetic typing scheme based on a three-branched phylogenetic tree using SNP variations. Many more SNPs have been discovered using whole-genome sequencing analyses, which would be more discriminating when used for genetic typing.[31]

The etiology of the Black Death was successfully traced using SNP information during which time multiple importation instances of the pathogen into Europe was inferred and it was suggested that the modern biovars of Y. pestis – Orientalis and Medievalis – were descendants of the ancient plague pathogen.[34]

Multilocus variable-number tandem-repeat analysis in Yersinia pestis tracing

Tandem repeat genomic polymorphism in mammals has been very useful in genetic mapping and is still the basis of forensic DNA fingerprinting. These repeat sequences are usually classified depending on the varying ranges of base pair numbers, into satellite (megabases of DNA in association with chromatin), minisatellites (repeat units of about 6–100 bp across hundreds of base pairs), or microsatellites (with repeat units of between 1–5 bp across a few tens base pairs).[2]

Several studies [35],[36] have shown the importance of mini- and micro-satellite tandem repeat polymorphisms as markers in the identification of pathogenic organisms, even for highly monomorphic newly emerged species. There are numerous VNTR sequences in the Y. pestis genome which are frequently found within gene coding regions. According to Klevytska et al.,[37] VNTR sequences are seen averagely at 2.18 arrays/10 kbp, evenly distributed across the genome. The tetranucleotide repeat sequence (CAAA) n was identified by Adair et al.[38] in Y. pestis genome and shown to exist in nine allelic forms possessing high diversity.

MLVA has been shown to be able to classify distantly related strains as well as distinguish between closely related strains. Employing 42 and 25 VNTR loci, Klevytska et al.[37] and Pourcel et al.,[39] respectively, were able to observe vast differences among these loci from representative Y. pestis strains. Pourcel et al. succeeded in classifying 180 Y. pestis isolates into 61 distinct genotypes with correct distribution of the three biovars into the three major branches. They went ahead to propose that a subset of seven VNTR markers would be adequate for a quick comparison of a new strain with their collection which they say can be achieved easily, using a web-based facility. The technique of MLVA has been employed by scientists from both the United States and Europe to create databases for over 1000 strains of Y. pestis due to the fact that the technique can easily be standardized.

Sample application cases in history

B. anthracis spores used to carry out the 2001 “white powder letter” bioterrorist attack was source-traced using genome sequencing, VNTR, and MLVA techniques. It was eventually uncovered that the bioagent was an Ames strain, “homemade in the USA.” Later, with a combination of systematic microbiological analysis with comparative genomics and whole-genome sequencing of spores from the same case, uncommon morphological mutants were uncovered [Table 2].[40],[41]
Table 2: Historical cases of successful source-tracing of bioagents

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In 2001, Keim et al.[42] reported how the MLVA technique was used retrospectively to investigate and successfully trace the source of a 1993 aerosolization of B. anthracis spores over Kameido, Japan, by the Aum Shinrikyo cult group. The publication detailed how eight VNTR markers and a missing pXO2 gene plasmid were used to successfully identify the attack strain as the Sterne vaccine strain of B. anthracis that was locally available in Japan for veterinary use.

Clustered regularly interspaced short palindromic repeat analysis

CRISPRs are well-defined genetic structures present within the genome of many bacteria and most Archaea and sometimes are present in multiple copies per genome, being surrounded by CRISPR-associated genes (cas). CRISPRs consist of repeat sequences interspaced with nonrepetitive elements also called “spacers.”[43] CRISPR analysis has already been applied to the investigation of the epidemiology of Mycobacterium tuberculosis in a typing method referred to as “spoligotyping.” A similar approach would be useful for Y. pestis phylogenetic investigations, considering the fact that it (CRISPR analysis) seems to be a good tool for ancient DNA analyses.[2]

Whole-genome sequence analysis

Using whole-genome sequence analysis, it is possible to identify different types of genetic variations, ranging from SNPs, gene loss/gain, gene rearrangement, VNTRs, to CRISPRs. This would yield comprehensive amount of information about bacterial genome diversity, leading to a fine-tuned source-tracing analysis. This genome-wide variation analysis method is referred to as whole-genome sequence typing (WGST). Databases for source-tracing could be built which contain whole-genome sequences of even hundreds of bacterial strains using the capabilities of the next-generation sequencing technology that is rapidly advancing.[2]

Other methods applicable to source-tracing though not fully developed include DFR typing, bacterial mass fingerprinting, cellular fatty acid typing, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

Sample application cases in history

WGST was applied in 2009 to attribute the source of a fatal laboratory-acquired Y. pestis infection to a research laboratory in Chicago, USA [Table 2].[44] Furthermore, the Vibrio cholerae outbreak in Haiti following the Earthquake in 2010 was eventually traced using WGST to Nepalese strains. They were very closely related with just 1 or 2 base pair differences, providing a strong evidence in support of the theory that the outbreak was caused by Nepalese soldiers who were serving in Haiti as United Nations peacekeepers [Table 2].[45]

  Conclusion Top

The anthrax attacks of 2001 in the United States was a wake-up call to the world on the threat of bioterrorism, though the fear or apprehension of the possibility of such an event had always been there. That incident drew the attention of the United States and the world to the inadequate preparedness for bioterrorism attacks. A key component of the response preparedness effort would be the ability to quickly identify the bioagent used in an attack so as to know what sort of medical response would be appropriate for the situation. This is the point at which epidemiological surveillance and microbial forensics need to interplay. Microbial forensics would further be required to trace the source of the agent in order to gather necessary evidence to identify and prosecute perpetrators of a biocrime. Source-tracing, therefore, takes a central position in the bid to identify and criminally prosecute perpetrators of bioterrorism acts.

The task of source-tracing can be achieved using the various advanced biotechnological tools highlighted above. WGST is adjudged for now by this review, to be the most effective technique for building databases for bioterrorism-associated as well as public health important pathogens,[45] and a properly selected MLVA follows after, due to the relative ease with which it may be standardized. However, it is going to be effective only through the sharing of data and databases containing information about these pathogens.

Having highlighted these, it is important to note also that transfer of bioterrorism-associated materials including the pathogens and their DNAs or other by-products usually come under strict restrictions and in many instances is actually prohibited by law. This poses a serious challenge to data sharing which would then make it very difficult to establish international databases. While it is easier for scientists in a single country to put together genetic database for bioterror agents or indeed other pathogens of public health importance, such regionally limited databases would, in turn, limit the ability to attribute to, or in fact, exclude international offenders. Like Yang and Keim have said,[2] “wisdom is indeed needed on how to bridge this gap of data sharing, as it remains key to effective bioterrorism agents source-tracing.”

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Conflicts of interest

There are no conflicts of interest.

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  [Table 1], [Table 2]


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