The terms ‘microbe’ and ‘microorganism’ are used interchangeably, but there are varying ideas about which organisms should they include. There is common consensus that the prokaryotic bacteria and archaea are ‘microbes’, but some definitions also include eukaryotes such as unicellular fungi, (e.g. yeasts) and protozoa; and some even include the rickettsia and viruses. For the purposes of this chapter, we will consider ‘microbe’ to encompass the bacteria and archaea. The use of viruses as forensic indicators will also be covered, although the debate about whether these are even living entities has been going on for decades without any prospect of ever being resolved.
The involvement of microbes and viruses in legal cases is increasing as advances in technology, especially molecular biology, facilitate their identification, and courts of law become willing to accept non‐human DNA‐based evidence. In addition, there is enhanced awareness among the public of the dangers posed by pathogens spread deliberately or through reckless behaviour by naturally infected individuals, and this has led to changes to the law. Furthermore, there has been an increase in the numbers of individuals and groups threatening to release pathogens maliciously, or simply to cause distress and gain publicity. The majority of threats to release pathogens are hoaxes, but the need for rapid pathogen identification and tracking has become a priority, as has the need to prosecute people making the fraudulent claims.
There is currently a lot of research into how microbial communities, commonly referred to as ‘microbiomes’, vary between and interact with their environment. Microbiomes often consist of hundreds or even thousands of microbial species and strains. Therefore, to speed up the analysis, one approach is to use amplicon sequencing coupled with next generation sequencing (NGS). Amplicon sequencing involves using sets of universal PCR primers to mass‐amplify sequences that identify selected groups of microorganisms. The gene 16S rRNA is a universal primer for archaea and bacteria, 18S rRNA is a primer for unicellular eukaryotes such as protozoa, whilst the internal transcribed spacer (ITS) region is used for the identification of fungi. The PCR product is then subject to NGS, which enables millions of strands of DNA to be sequenced in parallel. An alternative approach is shotgun metagenomics followed by NGS, in which the whole genomes of all the microbes present in the sample are isolated, fragmented, and then sequenced (Jovel et al. 2016). Amplicon sequencing is widely used but only provides an indication of taxonomic diversity. This is because the microbes within a community are distinguished based on variation associated with a specific marker gene. By contrast, in shotgun sequencing, the whole genome of all the microbes present is compared and this enables both the taxonomic and functional composition of the microbial community to be identified. With both approaches, the sequences are compared with those on databases and the result is a list of organisms present and an estimate of their relative abundance. Viruses are even more abundant and diverse than microbes and all prokaryotic and eukaryotic cells are subject to viral infection. The virus community, or ‘virome’, associated with an organism consists of infective viruses, endogenous retroviral elements that have become inserted into the host genome (in the case of the human genome, this might be as much as 8%), bacteriophages that infect bacteria and archaeal viruses that infect archaea. The virome therefore directly and indirectly affects (and is affected by) the organism and the associated microbiome (Zou et al. 2016). Some workers include viruses within their definition of the ‘microbiome’, although most studies to date consider microbes and viruses separately.
Even when we are healthy, we are host to vast numbers of microbes, often referred to as the ‘human microbiome’. Indeed, it is often stated that there are 10 times more bacterial cells in our bodies than there are human cells. This, however, is an overestimate and the real ratio is approximately 1 : 1 (Sender et al. 2016). Furthermore, because bacteria are so much smaller than human cells, this would equate to only about 2 kg of weight in a typical adult human. Nevertheless, although estimates of microbial abundance in our bodies have been downgraded, they are extremely numerous and affect many aspects of our physiology.
The different regions of the body harbour distinct microbial communities (Knight et al. 2017). These communities consist of a normal resident flora, transient organisms, and in the case of disease, pathogens. For example, the colon microbiome differs from the microbiome of our hands and both differ from the microbiome that lives in our mouth. Variations in the constitution of microbiomes impacts upon our health and even upon our behaviour. For example, our microbiome influences our sleep patterns, our mental state (e.g. depression), and autism spectrum disorders (Logan et al. 2016). The US National Institute of Health operates ‘The Common Fund Human Microbiome Project’ and this is a good source of information on the human microbiome in health and disease (commonfund.nih.gov/hmp). Microbiome analysis is increasingly combined with metabolomics, in which the profile of low molecular weight metabolites associated with a biological system (e.g. gut fluid, skin surface, plant leaf, or soil) is studied. In the case of animals and plants, the metabolomic profile reflects the combined genomic activity of the animal or plant and the microbiome associated with it, and the effect of the environment and extrinsic factors – such as drugs – upon them both. This complex interaction of processes results in distinct metabolomic profiles and some workers consider these could provide identification features in forensic investigations (Castillo‐Peinado and de Castro 2016; Schmedes et al. 2017). As yet, there have been few attempts to use virome analysis as a forensic indicator, although it may prove useful in the future.
Whilst the extent to which personal identification from microbiome analysis is possible remains uncertain, there is no doubt that it yields a lot of information about us. For example, microbiome analysis indicates sex, health, diet, association with others (the longer, closer, and more intimate the association two people have, the more aspects of their microbiome that they share), and recent travel (e.g. presence of microbes with restricted distributions that have temporarily colonised the body). Therefore, the collection and storage of samples is undertaken with the same attention to regulations required for human DNA. Informed consent is necessary for the collection of any sample that can identify a specific individual and their characteristics (e.g. sex, sexuality, health, religion). Similarly, police authorities should request microbiome analysis as part of criminal proceedings on the same basis as for human DNA and fingerprints. The fact that one is sequencing microbial DNA rather than human DNA is immaterial. It is the ability to use this information to identify an individual and his/her characteristics that is important.
The bacteria, especially the streptococci naturally present in our mouth, are extremely diverse and distinct from those in our guts. This diversity differs between individuals and the composition is relatively stable over the times measured to date (Leake et al. 2016). However, there is a circadian rhythm in the abundance and gene activity of many of the microbes constituting the oral microbiome (Takayasu et al. 2017) and it is likely that similar rhythms occur in other microbiomes. The oral microbiome is also affected by oral health/disease, our lifestyle and diet, and there is even a suggestion that it indicates ethnicity (Mason et al. 2013). This could prove useful in the investigation of bite injuries – whether to the victim or the assailant. Human bites are nasty because our saliva contains bacteria capable of causing a serious wound infection. For example, if you punch someone in the face and make contact with his or her teeth, you are liable to suffer a clenched fist injury and this is liable to infection. Borgula et al. (2003) and Rahimi et al. (2005) asked volunteers to bite themselves, after which samples were taken over varying time periods. They demonstrated that live bacteria could be recovered from the bite site as well as from fabrics, for at least 24 hours afterwards, if it was relatively undisturbed. Furthermore, the genomic profiles of the bacteria recovered from the bite sites indicated the person responsible. In subsequent work, Kennedy et al. (2012) demonstrated that, in an experimental setting, streptococcal DNA profiling linked bite victim to the ‘assailant’ with a high degree of certainty. However, the bite site was swabbed after only 3 hours and, as in previous work, for ethical reasons the ‘assailant’ and ‘victim’ was the same person. To what extent microbiome analysis can provide a reliable identification in more ‘real world’ scenarios currently awaits study.
Saliva contains DNA (and is therefore the best means of identification) but it also contains nucleases. Therefore, in a saliva stain, the DNA may degrade and not yield a full STR profile. In addition, should an assailant's DNA not be on a DNA database, then the additional information provided by a microbial profile could help in the search for the suspect. In a similar manner, the microbiome associated with pubic hair might link two (or more) people together in cases of sexual assault (Williams and Gibson 2017). As with bite injuries, human DNA is the most appropriate indicator in this scenario, but it may be absent for a variety of reasons (e.g. failure to ejaculate). It is difficult to extract human DNA from faeces and therefore the faecal microbiome may be an alternative forensic indicator (Quaak et al. 2017). The microbes that live on our skin are transmitted to everything that we touch. Preliminary work suggests that the skin microbial profile can identify who last used a computer keyboard or mobile phone (Fierer et al. 2010; Lax et al. 2015). Although fingerprint and human DNA analysis are the preferred means of analysis, because they are well‐established methods, quicker, and cheaper, it is sometimes useful to have additional evidence; for example, if there are only partial prints or an incomplete STR profile.
The study of soil is known as pedology, although many workers refer to it as simply ‘soil science’. Because of the importance of soil characteristics for agriculture, the UK has a good, mapped soil database and this is a useful resource for linking a person, animal, or object to a geographical location. This is being expanded upon as part of the SoilFit project coordinated by the Macauley Institute in Aberdeen (McKinley 2013). In a forensic context, soil particles are often found on shoes, clothing, vehicles, animals, plants, and objects of all kinds. If identifiable characteristics are ascribable to the soil particles, it becomes feasible to link them to a location or to link people/animals/objects together.
Soil is a complex substance and, with the exception of a few organic soils (e.g. peat‐based soils), the bulk of it is composed of mineral material. In addition to its mineral component, soil contains varying amounts of decaying animal and vegetable matter and this supports a huge variety of microbes. Currently, most forensic analysis of soil samples is based upon their mineral and chemical composition. However, in most cases, all that can be said is that there is a match between two or more samples – and this does not necessarily mean that they are related.
Most soil microbes cannot be cultured in the laboratory, but DNA analysis indicates that the soil microbial community is highly diverse and differs considerably over short distances and with the season. There is therefore an interest in adapting ecological studies on the soil microbiome, so that they could be used as forensic indicators (Young et al. 2017). As with many other forms of forensic evidence, it is the presence of rare microbial species or genotypes with restricted distributions that provide the best evidence of a match between samples. In an experimental study, Demanèche et al. (2017) demonstrated that the soil microbiome could distinguish soil samples taken at a mock crime scene from those sampled 25 m away and identify soil samples that are ‘contaminated’ with soil from the crime scene. At the time of writing, there was a case report in which the soil microbial profile was used to match samples taken from the clothing of a rape victim and her alleged attacker (Uitdehaag et al. 2016), but this analysis is in the early stages of development. It remains to be seen whether soil microbial profiling provides a reliable unique identifier or whether, like mineral analysis, it is limited to stating that there is a ‘close similarity’ between two or more samples. In addition, although the cost of microbiome analysis is reducing all the time, it is unlikely to ever be cheap. It is therefore likely to face competition from use of FTIR analysis that is at a similar early stage of development for use on soil samples; if effective, the spectroscopic method would be both quicker and less expensive (Margenot et al. 2016).
Many of the bacteria present upon or within our bodies are also important in the decay process and anything that restricts their activity, such as low temperature, lack of oxygen, or the absence of water, prolongs decomposition. In a very dry environment, especially if combined with strong air currents and extreme heat or cold, a human body will mummify and can remain in this state for hundreds, or even thousands of years. Similarly, under constantly frozen conditions, a body, if undisturbed, will be preserved for many years. Conversely, under warm, moist conditions, such as in the tropics, a body will decompose extremely quickly, even in the absence of invertebrates and other detritivores.
Bacterial colonisation of a dead body usually begins from the intestine, which is naturally home to large numbers faecal bacteria, including coliforms (e.g. Escherichia coli), and enterococci (e.g. Enterococcus faecalis). After death, the bacteria present in the gut replicate into the surrounding tissues and fluids and a dead body therefore tends to decompose from the inside to the outside. By contrast, if the body is frozen immediately after death, then once it is returned to above 0 °C, decomposition tends to occur from the outside to the centre. This is because the outer surface will defrost a long time before the internal organs – although precisely how long will depend upon the ambient temperature. As the body starts to decay, the pH becomes more acidic owing to the release of acids during autolysis and the products of bacterial fermentation. It also becomes anaerobic, because the bacteria use up all the oxygen and the circulation system has ceased to operate. This favours the growth of obligate anaerobic bacteria such as Bacterioides spp. and Clostridium spp. Consequently, the majority of the bacteria found in a dead body tend to be anaerobic and spore forming species, such as Clostridium perfringens. C. perfringens is a common soil organism that can also be found in the human intestine, the female genital tract, and surrounding skin. There are numerous strains of C. perfringens and it is implicated in a range of pathogenic infections, ranging from food poisoning to cellulitis and gas gangrene. It is therefore essential to take care when handling dead bodies and taking tissue samples, because there are serious risks of contamination of forensic evidence and of personal infection. Wounds whether formed before or after death, are another source of entry and the bacteria spread rapidly via the blood vessels. Decay is therefore more rapid in a person who suffered from septicaemia or other bacterial infections at the time of death. By contrast, decay is delayed where there is excessive blood loss, because the lack of fluid in the blood vessels slows the spread of bacteria through the body.
There are an increasing number of studies on changes in the bacterial flora in and around a dead body – sometimes referred to as the ‘thanatomicrobiome’. Some workers consider that the composition of the microbiome associated with a corpse and the soil surrounding it could indicate the post‐mortem interval (PMI) with equivalent accuracy to forensic entomology (Finley et al. 2015; Javan et al. 2016). In addition, microbiome analysis has an advantage over entomology, because microbes are invariably present from the moment of death. Consequently, thanatobiome analysis should not be (at least theoretically) affected by seasonality, locality, and body accessibility. In a highly detailed analysis of microbial decomposition in both dead humans and mice, Metcalf et al. (2016) demonstrated that there is a clear microbial succession, with certain groups appearing at predictable time intervals. This work was undertaken with corpses lying on the soil surface and whilst soil type was not a factor in the development of the microbial communities, the soil was the source of most of the microbes involved in decomposition. As one would expect, the human microbiome and the mouse microbiome are different and the development of the microbiome in the two groups of corpses was not the same. It therefore follows that animal models can be used to research the processes that affect the development of the microbiome after death, but not to identify specific bacteria as indicators of the human PMI. Belk et al. (2018) discuss the best analytical approaches to using the corpse microbiome and that of the surrounding soil for determining the PMI.
Although the use of microbiome analysis to determine the PMI clearly has potential, it should be emphasised that this work is still in its developmental stages. For example, the work of Metcalf et al. (2016) utilised only four human corpses. This is not a criticism, because obtaining any human bodies for this sort of research is incredibly difficult. However, a great deal more needs to be known about how taphonomic factors affect the development of microbiomes after death before they can be considered as offering reliable PMI indicators. For example, Pechal et al. (2017) demonstrated that as a frozen body thaws, there are marked changes with time in the composition of its associated microbial flora. Largely, this reflects the extent to which the original flora survives the period of freezing and begins to resume activity as the temperature rises. Similarly, the presence of a corpse lying on the soil surface causes different changes to the underlying soil microbiome in the summer and the winter (Carter et al. 2015). It might therefore be expected that the microbiome will develop differently if a body is indoors lying on a bed compared to one lying on the soil outside or hanging by the neck from a rope. Similarly, it is uncertain whether the microbiome would develop differently after death if the person had been suffering from an infection or ill health, or he/she had been taking antibiotics. Furthermore, to what extent would detritivores affect the development of the microbiome? Maggots have complicated interactions with microbes: on the negative side, they feed upon microbes and secrete antibiotic substances, but they also stimulate microbial growth through liquefying tissues, facilitate aerobic microbial decomposition and, in the case of maggot feeding masses, cause a localised increase in temperature. Maggots feeding upon a dead body develop their own associated microbiome (Weatherbee et al. 2017) and this can be expected to influence that developing upon the corpse. Similarly, large detritivores, such as dogs and birds that dismember a corpse, will influence decomposition by allowing oxygen access, thereby facilitating the growth of aerobic microbes and increasing the speed with which the remaining parts dry out – and both these factors will influence the composition of the corpse microbiome.
The analysis of microbiomes promises a wealth of forensic information, but there remain formidable challenges to overcome before it is employed in routine analysis (Table 13.1). At the timing of writing, there were insufficient studies to know whether there are characteristics within a person's microbiome that are truly unique and that remain so throughout their life. Each microbiome region appears to contain a relatively stable microbial flora and a transient flora that are both affected by external factors such as diet and the environment. As might be expected, the flora is rapidly altered by chemicals that are bactericidal or bacteriostatic. For example, the consumption of antibiotics affects all microbiomes, soaps and detergents affects the skin microbiome, whilst toothpaste and mouthwashes affect the oral microbiome. After ‘challenge’, the stable microbiome returns within a shorter or longer period. However, this variability would undoubtedly be exploited in court to cast doubt on the reliability of the identification. Whilst 90% certainty might sound good, in a court of law the jury needs to be convinced that the identification is accurate ‘beyond reasonable doubt’ and a 10% doubt is too large to send someone to prison for life.
Table 13.1 The benefits and limitations of microbiomes as forensic indicators.
| Benefits of microbiome analysis
Microbes are always present. Wide variety of applications: can provide information about living organisms, dead organisms, and environmental samples. Wide range of information provided: Individual identity, gender, health, association with humans/animals/ecosystem/country Limitations of microbiome analysis All aspects of study from sample collection to statistical analysis and interpretation require validation and standardisation. Cost |
We need more experiments employing ‘real‐life’ scenarios and using sufficient individuals to determine whether characteristic microbiome profiles are detectable in forensic samples after a range of times under a variety of environmental conditions. There also needs to be agreement on accepted protocols for the analysis of microbiomes and whether a microbiome database is feasible or, if microbiomes change with age and environmental exposure, even worthwhile. There needs to be agreed reference samples for calibration and quality control, agreed means of statistical analysis, and what constitutes sufficient ‘similarity’ to be a genuine match. This will be particularly important in a court of law when presenting evidence to a jury. A microbiome is not a constant, like a fingerprint or a DNA STR profile, but it is a dynamic entity. A microbiome is influenced by numerous factors such as health, disease, diet, drugs, antibiotics, and circadian rhythms. Therefore, in the absence of robust supporting evidence, it is easy to argue that any similarities between a suspect's microbiome profile and that found at the crime scene are the consequences of chance or that anything less than a 100% perfect match (and it would be highly unlikely to obtain this, even under perfect conditions) would be a mis‐identification. There are also the cost implications to consider. Although sequencing costs have reduced enormously over recent years, microbiome analysis is far more complicated than that required for standard forensic DNA analysis and funding remains a serious issue for all police forces. It is therefore not surprising that, as of 2017, there had been no criminal cases in which human microbiome analysis had been presented in court. For at least the near future, microbiome analysis is therefore likely to remain a feature of rare ‘special cases’, in which the accepted conventional techniques (i.e. fingerprinting, DNA, and trace evidence analysis) failed to yield sufficient information.
Many microbes and viruses exhibit regional variations that might help identify where a person grew up or their recent travel. For example, in Japan it is possible to trace the origins of unidentified bodies by genotyping the JC virus (JCV) (Ikegaya and Iwase 2004; Ikegaya et al. 2007). JCV is a DNA virus belonging to the Family Polyomaviridae that infects most of us during childhood and is usually asymptomatic, except in immunocompromised people. Once infected, the same viral genotype is found in our kidneys for the rest of our life. In addition, the virus is excreted in urine – this may have forensic importance where urine splashes are found at a crime scene. One can extract JCV DNA from bodies that are up to 10 days old, thus facilitating the identification of decayed corpses (Ikegaya et al. 2002). A related polyomavirus, BK virus (BKV), can also be isolated from kidneys and used in a similar manner. Furthermore, because the distributions of the genotypes of the two viruses are not the same, one can narrow down the likely origin of an individual. Both JCV and BKV are detectable in semen (Khalili and Jeang 2010; Rotondo et al. 2017) and therefore may help identify offenders in cases of sexual assault.
JCV and BKV genotyping works a bit like stable isotope ratio analysis, in that it does not indicate identity or ethnicity but suggests a country or region of origin. Furthermore, like stable isotope ratio analysis, its effectiveness depends upon the extent and reliability of databanks. Hepatitis B virus (Inoue et al. 2014) and the bacterium Helicobacter pylori (Nagasawa et al. 2013) both exhibit geographical variation and are potential forensic indicators. Although many pathogens express regional differences, almost all studies on them are related to the epidemiology of disease and not to their forensic potential. Nevertheless, this approach could be useful when an unidentified body is found, whose details do not match those of any ‘missing person’ and for whom there are no police records; for example, if it is suspected that an unidentified body might be that of an illegal immigrant/someone who was trafficked illegally. Since the 1990s, many women from Asia and Eastern Europe have been sold into prostitution in the UK and elsewhere and it remains an ongoing problem. Women who have been trafficked are at serious risk of being killed by their clients or their pimps and it is then a difficult job to determine who they were or where they came from. However, you need to exercise caution if using pathogens as geographical indicators. For example, genotype 4 of the Hepatitis C virus is often referred to as the ‘African genotype’, but it also has a high prevalence in North East Poland. This appears to have nothing to do with immigration but to intravenous drug use. Once a pathogen genotype is introduced into an area, it sometimes spreads rapidly among the local population and appears as an anomalous ‘island’ among the resident pathogen genotype(s).
Changes in the abundance, diversity, and distribution of microbes within the body can be of importance in determining the cause of death. For example, there may be a dispute about whether a person died of an ongoing disease or a fatal infection resulting from a medical procedure, diagnosing a case of fatal food poisoning, determining whether a person died of a drug overdose or an infection acquired through illicit drug use, or whether a person died of natural causes or a malicious act.
Samples for microbial culture or DNA analysis should be taken from dead bodies with great care, to avoid the possibility of contamination. If numerous species of bacteria and other microbes are isolated from a sample, there is a strong possibility that contamination occurred through either the process of decay or the sampling and handling techniques employed. There is no consensus about how samples should be taken, nor on the most appropriate tissues and fluids to use. However, the choice will be at least partly affected by the organism/diagnosis targeted (Fernandez‐Rodriguez et al. 2015). For example, because of the ‘open access’ between the lungs and the outside world, cultures isolated from post‐mortem lung tissue often yield false positives because of contamination. Even when we are alive and healthy, our airways are home to a natural microbiome – although this is altered in conditions such as asthma and lung disease. In the absence of other indicators, it is therefore difficult to determine whether microbes grown from post‐mortem tissues were an important factor in the cause of death, the result of non‐pathogenic infections already present before death, or microbes that colonised the body after death. There is uncertainty about how quickly and by what route microbes move into the blood and closed organs, such as the liver, after death. An early suggestion that microbes gained access to the blood through ‘agonal spread’ is now considered unlikely (Riedel 2014). This is the theory that microbes enter the bloodstream during the agonal period; there is no consensus on how this period should be defined, but one suggestion is that it is time between when the cardio‐respiratory system stops and brain death occurs. The most likely route is through ‘transmigration’ of bacteria from the gut and other mucosal surfaces after death into the blood and lymph vessels. How quickly this transmigration takes place is hard to determine, owing to the difficulty of avoiding contamination. Nevertheless, it is probably strongly affected by taphonomic conditions and in particular temperature. In living people, the bacterium Staphylococcus aureus, which is not motile, gains access to deep body tissues by attaching to mycelia of the opportunistic fungus Candida albicans, as these grow into breaks in the epidermis. Possibly, similar associations occur between other bacteria and fungi in dead bodies. If a body is transferred to a morgue and stored at low temperatures immediately after death, samples can be taken with confidence up to 48 hours later. The presence of a single species of bacteria, especially a pathogenic species, points towards an infection, but the presence of several species of bacteria, and in particular species normally associated with the gut flora, suggests either transmigration or contamination. Raised levels of C‐reactive protein in the blood provides further evidence of bacterial infection before death (Christoffersen 2015).
Some microbial infections result in the formation of skin lesions that might be mistaken for signs of physical assault (e.g. Prahlow and Linch 2000). This is especially the case in vulnerable persons, such as the very young, the mentally disturbed and those suffering from senility, who are unable to relate what has happened to them. For example, Nields et al. (1998) describe a case in which a 4‐year‐old child who died of streptococcal toxic shock syndrome was initially thought to been a victim of child abuse. Fortunately, the bacteria were identified from samples taken at autopsy and no one was charged with assault. Streptococcal toxic shock syndrome is caused by infection with toxigenic strains of Streptococcus pyogenes (also known as group A Streptococcus, (GAS). It may result in organ failure and shock – the consequences of which can be rapidly fatal. In many cases, it is not known how the bacteria enter the body, but a common feature of the syndrome is the development of necrotizing fasciitis in which there is destruction of the fascia (fibrous tissues that cover and separate the muscles) and fat. Following this, the overlying skin may die, split, and break. Consequently, the damaged region may, at first sight, be thought to have resulted from an infected wound.
Food poisoning is extremely common and many people automatically assume that any bout of vomiting or diarrhoea is a consequence of something they ate. However, some experts believe that up to 50% of such cases result from infections acquired from other sources, such as pets, contact with faeces, contact with someone who was already infected, or failure to follow simple hygiene rules such as washing hands. The most common microbes causing food poisoning in the UK are the bacteria Campylobacter spp. (which causes the majority of cases), Salmonella spp., E. coli O157, C. perfringens and Listeria monocytogenes. A number of viruses, including Norovirus, can be transmitted through contaminated food and their symptoms can be confused with bacterial food poisoning. Most cases of food poisoning, although unpleasant, are not life threatening and occur as individual or sporadic events. An outbreak is defined as occurring when two or more people fall ill having consumed a common batch of food. Occasionally food poisoning may prove fatal, especially in the very young, the elderly, and the infirm. Food poisoning usually results unintentionally from the incorrect storage or cooking of food that allows the bacteria within it to survive and replicate to dangerously high levels or from poor hygiene practices that result in the transfer of bacteria from contaminated food or surfaces to previously uncontaminated food. Identifying the source of an outbreak of food poisoning requires the offending organism(s) to be cultured from patients' faeces and questioning those affected to determine their common food source. This may prove difficult because not everyone who consumes contaminated food need fall ill and if the food has all been eaten or thrown away, it may be impossible to prove that it was the source of the infection.
The intentional contamination of food with foreign bodies (e.g. razor blades, glass) and poisons (e.g. mercury) is common practice, usually by disaffected individuals with a grudge against the manufacturer or society or with a view to blackmail. There are few reports of the deliberate use of microbes, either through introducing them or through poor cooking or storage practices in order to cause food poisoning. However, it is a possibility where someone wishes to cause distress but not death. It would be difficult to prove, though it would carry a real risk of unintentionally causing fatalities. There are also many false claims of food poisoning, either through maliciousness or to gain insurance money. For example, in 2016, Spanish hoteliers suggested that false claims of food poisoning by tourists on all‐inclusive holidays cost the industry £52 million.
In addition to identifying the species of organism responsible for causing food poisoning, it is also necessary to identify the strain involved. Distinguishing between strains of bacteria that have been isolated in culture using tests based on differences in cell surface proteins or metabolic pathways is time‐consuming and often not specific enough. Therefore, a variety of DNA and mass spectrometric‐based methods such as MALDI‐TOF (matrix‐assisted laser desorption ionising time‐of‐flight mass spectrometry) are increasingly being used (Pavlovic et al. 2013).
Most of us have blamed a family member or friend for catching a cold, but proving it is difficult. This is because cold viruses are usually transmitted in aerosols and therefore we could acquire them from any infectious person in our vicinity. Where the disease is sexually transmitted, or blood‐borne, the list of potential sources of infection is reduced. In sexually transmitted infections (STIs), it can be difficult to ascertain whether person A infected person B or B infected A, or whether both A and B were already infected before they had sexual contact with one another. However, if person A and person B are infected with different strains/genotypes of the STI, then it is certain that their infections are not linked. Sex offenders often have multiple victims and even if caught, they are at a high risk of re‐offending once released from jail. As a group, sex offenders are more likely to be infected with STIs than the general population (Giotakos et al. 2003) and it is common for their victims to become infected (Jauréguy et al. 2016; Sathirareuangchai et al. 2015). Therefore, it is potentially feasible to link victims and assailants together through STI transmission. This could be useful if it is impossible to obtain human DNA from semen because of late presentation or lack of ejaculation. However, apart from the transmission of HIV, there are few published instances in which this was done.
To link the donor and recipient of an infectious disease, one needs to identify a genetic marker that meets three criteria. First, it exhibits sufficient variation to exclude unlinked individuals. Second, the variation is easily identifiable, and third, genetic variation arises neither so slowly nor so fast that it compromises one's ability to link infected people together. In cases of child abuse, there is no doubt about the direction of disease transfer. Therefore, genetic typing of STIs provides corroborating evidence of who abused the child. This is useful when the abuse takes place within families, where it would not be suspicious to find the suspect adult's DNA on the child's clothes or bed linen. Although perinatal transmission of Neisseria gonorrhoeae can take place if the mother is infected, this usually results in the baby suffering from eye infections or meningitis rather than a genital infection. There is a possibility of contaminative transmission through poor personal hygiene and the sharing of communal towels (Goodyear‐Smith 2007) and the bacteria are known to remain viable on clothing for several hours or even days. This is not thought to be a common occurrence but appropriate sampling would be advisable. Chlamydia trachomatis infections are extremely common – in the UK, the incidence is approximately 200 000 new diagnoses per year. Although most cases of infection are acquired through sexual transmission, perinatal transmission is possible. In the latter case, the disease is most often manifested in the eyes. However, it can also cause pneumonia and has been implicated in the development of sudden infant death syndrome (SIDS). In rare instances, perinatal transmission of C. trachomatis results in long‐lasting infection of the child's vagina and anus (de Barbeyrac et al. 2010), but it is much more likely that infection of these sites indicates sexual abuse.
The difficulty of proving the direction of transmission is probably the main reason that there are few records of criminal prosecutions for transmitting STIs. In addition, the wording and interpretation of the law is also important. In England, reference is often made to the case of Regina v Clarence (1888) 22 QBD 23 (1886–90). Clarence knew that he was infected with gonorrhoea, but did not tell his wife. Subsequently, she contracted the infection when they had consensual sexual intercourse. She was understandably upset and complained to the police who charged him under s.20 and s.47 of the ‘Offences Against the Person Act 1861’. Section 20 makes it an offence to ‘unlawfully and maliciously inflict any grievous bodily harm upon any person’, whilst section 47 makes it an offence to carry out ‘an assault occasioning bodily harm to any person’. Clarence was initially convicted, but on his subsequent appeal he was acquitted after an 11 : 4 verdict in his favour. This was because his wife had consented to sexual intercourse and therefore assault and battery had not occurred: the transmission of gonorrhoea was a consequence of sexual intercourse and did not ‘constitute a negation of consent’. It is probable that he might have been successfully prosecuted under s.23 of the same act that prohibits ‘maliciously administering any poison or other destructive or noxious thing’ (Orr 1989). However, this case is mentioned to emphasise that for a successful prosecution it is important to have a clear idea of which law has been broken as well as reliable evidence. Currently, the majority of cases of prosecutions for transmitting an STI take place in America under tort law – that is, in civil courts – rather than as criminal prosecutions. These often involve rich and famous individuals with a view to obtaining an out‐of‐court settlement before the case comes to trial (Eidsmoe and Edwards 1999).
HIV is a human retrovirus that affects the immune system and in time can cause the condition ‘Acquired Immunodeficiency Syndrome’, or AIDS. There are two species of the virus: HIV‐1 and HIV‐2. HIV‐1 is the more widespread of the two and the major cause of AIDS around the world, whilst HIV‐2 transmission is largely restricted to West Africa. Although HIV is primarily a sexually transmitted disease, the virus is also transmitted via blood transfusions, shared needles by intravenous drug users, accidental or malicious wounding (e.g. needlestick injuries), and an unfortunate number of infections in children arise through perinatal transmission at the time of birth.
The virus infects and destroys cells bearing the CD4 molecule, which includes T‐helper cells and a subset of blood and tissue macrophages. Following replication, the pro‐viral genome becomes integrated into the host chromosome, where it may remain quiescent or enter a cycle of production of progeny virus that results in the cell's destruction. The clinical manifestations of HIV infection are a result of depletion of the T‐helper cell population and thereby an impairment of the body's ability to respond to other infectious agents. Although AIDS remains a major cause of mortality in developing countries, provided an HIV positive individual receive suitable antiretroviral therapy, they can now expect to live a more‐or‐less normal lifespan.
HIV/AIDS is a problem throughout the world and health workers have to treat all patient samples as though they might be infected. In the UK it is illegal to test a person for HIV without their consent, because they must have counselling on being told the result – so unless a patient or person in police custody already knows that they are HIV positive and volunteers the information, or agrees to a test, there is no way of finding out. Forensic pathologists are well aware of the risks of contracting HIV and other diseases, such as Hepatitis B (HBV), Hepatitis C (HCV), Mycobacterium tuberculosis, and Neisseria meningitidis, whilst performing an autopsy and that this risk does not disappear, even if the body is many days old. Consequently, anyone handling a dead body or body parts should wear cut‐resistant undergloves and appropriate protective clothing and work with extreme caution.
HIV is a single‐stranded RNA virus, the RNA genome being enclosed within a protein envelope (or capsid). It belongs to the Family Retroviridae, genus Lentivirus. The term ‘lenti’ is derived from the Latin ‘lentus’ that means ‘slow’ or ‘inactive’, because many viruses belonging to this genus exhibit long incubation periods. However, like other RNA viruses, HIV is also capable of rapid multiplication under the right conditions. Once the HIV virus enters a cell, the enzyme reverse transcriptase, which is part of the virus particle, converts the viral RNA into single‐stranded DNA and then constructs a complementary strand of DNA for it. This DNA is then integrated into the host cell genome by the virally encoded enzyme integrase. Once integrated, the viral genome is replicated along with the host DNA by normal host cell mechanisms. The process by which the viral genome is incorporated into the host genome is the reverse of what normally happens within the cell, i.e. DNA is synthesised from an RNA template rather than RNA being synthesised from a DNA template. Hence the name of the enzyme: reverse transcriptase. Viral reverse transcriptase exhibits a high error rate, because unlike DNA polymerase, it has no proofreading ability. Consequently, the combination of speedy replication and poor proofreading leads to a rapid rate of mutation. An infected individual therefore contains a genetically diverse HIV population – albeit one in which most viruses will share common sequences. The HIV population will be even more diverse if the host is repeatedly infected on several occasions and if infected by different donors.
Because HIV rapidly evolves all the time, it is impossible to obtain an exact match between the virus profile from an alleged donor and recipient. Indeed, the profile in each of them changes constantly and the longer the time gap between infection and sampling, the more variation evolves. Consequently, matching virus profiles between donor and recipient depends upon calculating the evolutionary relationship of their virus populations – these are known as phylogenetic tree reconstructions. Those working on HIV transmission chains use neighbour joining (NJ) and maximum likelihood methods (ML) most frequently. These determine the likelihood (probability) that a proposed phylogenetic tree and hypothetical evolutionary history would explain the observed virus profile. By generating many phylogenetic trees using computer simulations, the one with the highest likelihood (probability) can be identified: Wilson et al. (2013), provide a detailed consideration of the methodology. It is essential that the phylogenetic tree includes control samples from persons who carry the same HIV subtype as the donor and recipient and live in the same locality at the same time. This ensures that the phylogenetic tree includes a reliable estimate of the evolutionary profile of the local virus populations. The inclusion of sufficient and appropriate control samples is crucial. If there are not enough controls or they include inappropriate examples, then it can result in a false impression of similarity or difference between the alleged donor and recipient viral profiles. Furthermore, the samples being compared should be, ideally, processed separately, both temporally and spatially, to minimise the risk of contamination.
The profile of the virus in donor and recipient starts to diverge from the moment of transfer. The rate at which base substitutions arise varies between regions of the genome and stage of disease; for example, whether it is quiescent or rapidly progressing. Evolutionary models are needed because base change mutations do not take place in a uniform manner along the whole genome. In addition, transitions and transversions take place at different rates and HIV exhibits a skewed base composition. Base transitions are where one purine or pyrimidine is substituted for another (e.g. adenine for guanine or cytosine for thymine), whilst transversions are where a purine is substituted for a pyrimidine (e.g. adenine for cytosine) or a pyrimidine is substituted for a purine (e.g. thymine for adenine). Transitions are far more common than transversions. HIV has high adenine, low cytosine levels, and because different bases exhibit different transition/transversion rates, this needs to be taken account of when modelling evolutionary profiles. Despite this, overall the substitution rate is relatively constant and can be factored into the construction of the phylogenetic tree. Furthermore, most forensic cases involve samples taken within one or a few years of the alleged transfer of infection, thereby reducing the amount of divergence that could be expected. Nevertheless, phylogenetic trees cannot inform on the direction of transmission and even if two individuals are found to contain similar viral profiles, it does not mean that other people were not involved in the transmission chain. If only two or three sequences were compared, it might be argued that differences would be found if other areas of the viral genome were compared. Consequently, phylogenetic trees cannot by themselves provide conclusive proof of transmission from one person to another and they must be considered alongside other evidence (Abecasis et al. 2011). Learn and Mullins (2003) have suggested that rather than making categorical judgements of association, statements along the lines of ‘the viral sequences from person A and person B display a high level of similarity’ or ‘the viral sequences are compatible with the possibility that person A infected person B’ should be used.
In the UK and several other countries, persons who know that they are HIV positive and do not inform their partner(s) before indulging in unprotected sex or biting, or otherwise intentionally attempt to infect someone, are liable to prosecution and can be sent to jail. Criminalising the sexual transmission of HIV has proved extremely controversial, as is anything connected to HIV, its treatment, and transmission. One of the arguments against is that it might prevent someone from seeking an HIV test, because if you do not know that you are infected with a disease, you cannot be accused of knowingly spreading it. From a strictly legal point of view, proof of malicious intent is difficult because, where sex was consensual, it relies on one person's word against another's that he/she intended to commit harm. Successful prosecutions have, however, been brought. In a landmark UK case in March 2001, a man who had known of his HIV status for almost a year was prosecuted for allowing his girlfriend to become infected. His girlfriend claimed that she was not aware of his HIV status when they began their sexual relationship and he only admitted it to her after she had discovered from a blood test that she too had become HIV positive. The man denied this version of the events, but the jury did not believe him and he was jailed for 5 years. The chances of transmission of HIV (and some other STIs) are much higher during rape or sexual assault than in consensual sex. This is owing to the violence resulting in genital or anal abrasions through which the virus can enter the bloodstream. Victims of assault are therefore always offered an HIV test and counselling when they report the attack. During the early stages of the AIDS epidemic, a rumour spread in some countries that sex with a virgin is a cure for the disease and this has led to a terrible increase in the number of rapes of young girls. The magical power of virgins is a longstanding and widespread belief and Howe (1950) relates a case in England (presumably – since the report lacks dates and place names) in which a 7‐year‐old girl was raped by a man suffering from gonorrhoea in the belief that this would cure of him of the disease. Despite the huge advances in forensic science, it is a sad reflection on justice in England and Wales that although the number of reported cases of rape has risen, less than 6% of these result in a conviction. This emphasises that the successful resolution of a crime depends on more than the development of ever more sophisticated laboratory techniques.
A remarkably heartless case of deliberate HIV transmission occurred in St Charles, Missouri, USA in 1998. A father, who worked as a phlebotomist, stole infected blood from his place of work and injected it into his 11‐month‐old son, who was lying sick in hospital suffering from an asthma attack. Doctors treating the child became concerned when a blood test revealed that he had somehow contracted HIV. They contacted the police who became suspicious of the father when he reportedly told his ex‐girlfriend that she would never claim any child support off him because the boy would not live that long. The father was convicted based on largely circumstantial evidence and received a life sentence; the child subsequently developed AIDS.
Any infectious agent that affects our nervous system can bring about changes in our ability to control our bodies. For example, poliovirus infection leads to the destruction of motor neurons in the spinal cord, resulting in paralysis that can be fatal. When the infection causes damage to those areas of the brain that control our behaviour, then the result can be a dramatic change in the way in which we speak to and interact with other people. This can lead previously well‐behaved persons to commit crimes, place themselves in a situation in which they could be harmed, or commit suicide. Syphilis, for example, causes a wide range of pathological symptoms, among which is damage to the nervous system – this is neurosyphilis. Syphilis is a sexually or congenitally transmitted disease caused by the bacterium Treponema pallidum and probably introduced into Europe by sailors returning from South America in the 1400s. The name syphilis derives from a character in a poem, ‘Syphilis siva morbus Gallicus’ (Syphilis or the French pox) by the Italian physician Girolamo Fracastoro. In the poem, Syphilis is a swineherd (or goatherd – translators differ as to his occupation) who is afflicted with the disease by the god Apollo for lack of respect. Although syphilis was once a common cause of both morbidity and mortality, with the advent of modern antibiotics, it became a rare disease. However, in recent years there has been a marked rise in the number of infections in the UK and many other countries. For example, the reported incidence of syphilis in England and Wales increased by over 1000% between 1997 and 2008. Subsequently, the number of newly‐diagnosed cases in England rose from 2650 to 5288 per year between 2010 and 2015. Most of the new cases have arisen through transmission between men who have sex with men and in particular those aged over 25. Neurosyphilis can occur at any stage during infection, although it is normally associated with the later ‘tertiary’ stage. In addition to paralysis, neurosyphilis can manifest itself in a variety of behavioural ways such as mood changes, irritability, pretentious, and overbearing behaviour, and dementia.
Cat scratch fever is caused by the bacterium Bartonella henselae, which infects cat fleas. As its name suggests, is transmitted to humans by scratch wounds inflicted by cats that have previously been scratching their fleas and so carry the bacteria on their claws. It is a relatively common disease but is normally benign and self‐limiting. However, sometimes it can cause a high fever, enlarged lymph nodes, and changes in behaviour that may include extreme aggression. Harvey et al. (1991) describe a case in which a 27‐year‐old truck driver underwent such marked changes in behaviour that he was thought to be exhibiting signs of drug abuse. Therefore, he was sacked from his job and was eventually forcibly taken to the local hospital by the police following complaints from his wife. In a similar fashion, viral encephalitis caused by a variety of different viruses (e.g. Herpes simplex, Borna Disease Virus (BDV), Epstein–Barr Virus, and a range of arboviruses), has been linked to odd, confused, and occasionally antisocial behaviour. AIDS may also result in behavioural changes, including loss of memory, irritability, depression, and dementia.
The protozoan parasite, Toxoplasma gondii, is an extremely common infection – up to one‐third of us are infected. If we acquire the parasite whilst still in our mother's womb, the consequences can lead to hydrocephaly, mental retardation, and death. However, most of us are infected after birth and the parasite encysts within our tissues, including the brain, without causing any obvious pathology. Nevertheless, infection has been associated with schizophrenia and testing sero‐positive for T. gondii links with epilepsy, aggression, propensity to commit suicide, and some personality disorders (Sugden et al. 2016).
There is a complex series of interactions between human behaviour, Hepatitis C virus (HCV), and both legal and illegal drugs. To begin with, people who are naturally impulsive and given to risk taking are particularly at risk of developing drug addiction that may include intravenous drug use. This leads to an increased risk of contracting HCV and about half of those infected suffer neuropsychiatric symptoms – including impulsiveness (Fábregas et al. 2014) – although the mechanism is currently uncertain. Interferon‐α is often prescribed as part of the treatment of HCV, but it can have side effects of causing depression, aggression, and suicidal behaviour.
Infections that cause anti‐social behavioural changes create medico‐legal problems because for many pathogens, only a small proportion of those suffering the disease exhibit mental problems, and of those an even smaller percentage commit offences as a result. In addition, the relationship between behaviour and disease can be difficult to disentangle. It is therefore a difficult clinical judgement to determine whether a person committed a crime because of being infected with the disease and therefore not responsible for their actions.
The interactions between humans, pathogens, and both therapeutic and illegal drugs, can be important in a variety of forensic contexts:
Any drug that is injected exposes the user to the risk of blood‐borne infections unless care is taken. The sharing of needles is well known to increase the risk of contracting HIV and HCV. What is less well known is that sharing snorting straws when taking cocaine or other opiates also poses a high risk of contracting HCV and therefore, presumably, other blood‐borne infections as well (Fernandez et al. 2016). This needs to be borne in mind when analysing transmission chains.
Between 2009 and 2017, there were over 70 reports of anthrax infection linked to heroin consumption in Europe (Berger et al. 2014). Many of these cases were fatal and acquired when injecting the drug rather than smoking it. Most of the heroin trafficked into Europe originates from Afghanistan, where anthrax is endemic, but it is not certain how the bacteria become associated with the drug. The most likely scenario is that contamination occurs when heroin is transported concealed among legal shipments of hides – which could include those that are naturally infected with anthrax. Another possibility is that anthrax infection occurs if heroin is cut (i.e. diluted) with ground‐up bones derived from infected animals by dealers seeking to maximise profitability (this could be checked by DNA analysis). It is also possible that contamination of the equipment used to process the heroin with anthrax spores occurs, although how that might happen is uncertain. Single nucleotide polymorphism and Multiple Loci Variable number of repeats tandem Analysis (MLV) assays of anthrax bacteria isolated from infected users has indicated that they are all closely related to strains naturally occurring in Turkey, but not Afghanistan (Grunow et al. 2013; Price et al. 2012). This lends credence to the suggestion that the drug becomes contaminated at some point during its shipment. Unfortunately, the anthrax genotype found is too widely distributed within Turkey to be able to narrow down the region through which the drug is being shipped. In addition to anthrax, a number of other potentially fatal diseases associated with spore‐forming bacteria can be transmitted by intravenous drug abuse, including botulism (Clostridium botulinum), tetanus (Clostridium tetani), and Clostridium novyi infection. Botulism and tetanus infections occur sporadically and therefore considered ‘chance events’. By contrast, C. novyi infections resemble those of anthrax and occur in temporal and spatial clusters that suggest batch contamination (Hope et al. 2012).
After death, many drugs present in the body's tissues and fluids are metabolised and degraded. The speed and extent to which breakdown occurs depends upon the drug and the environmental circumstances. Microbes may even cause an increase in some drug concentrations after death. For example, alcohol levels rise following death because of microbial fermentation. Similarly, gamma‐hydroxybutyric acid (GHB) is naturally present at a low level in our bodies, but this level increases enormously when it is used as either a recreational or a therapeutic drug – or for criminal purposes. GHB is a class C drug that is taken at low doses as an ‘upper’ by clubbers who know it as ‘liquid ecstasy’. However, at higher doses, it causes confusion and even coma and is notorious for being used to spike drinks in ‘date rape’. In high doses, GHB can be lethal. For example, between August 2014 and September 2015, Stephen Port murdered three men at his flat in London, UK, by giving them an overdose of GHB. He dumped two bodies in the graveyard of the nearby St Margaret's church, Barking and the other close to it. On the last victim, Daniel Whitworth, he planted a bottle of GHB and a fake suicide note suggesting that he had killed one of the other victims and then committed suicide out of guilt. Surprisingly, in the initial investigation, the bottle was not tested for fingerprints or DNA and neither was the blanket wrapped around Daniel Whitworth's body examined forensically. However, suspicions were not aroused by the fact that the same woman walking her dog discovered two of the bodies in almost the same place. This further indicates that basic police work is vital for any case to succeed. For another example of a case report involving GHB, see Mehling et al. (2016). After GHB is ingested, there is an initial heightened concentration in the blood, after which the levels decline again within a few hours. There is also a rise in the levels of GHB following death, although not as high as those seen immediately after taking the chemical as a drug. It is therefore of forensic interest to know the extent to which levels of GHB found in a body might be ascribed to natural causes as opposed to misuse. Pseudomonas aeruginosa, a common bacterium associated with decomposing tissues, produces GHB, although not in sufficient quantities to account for all of the natural increase. Therefore, other bacteria and autolytic processes must contribute to the rise. After death, lower levels of GHB are found in the brain than in the peripheral circulation and this may be a more suitable tissue to assay – although once advanced decay sets in, the levels in the brain rise considerably (Thomsen et al. 2017).
Biological warfare has been practised in a minor and infrequent way since antiquity (Mayor 2003), but its impact has been small compared to conventional weaponry. The development of biological warfare agents was banned under the Geneva Convention many years ago – not that that this put a stop to it! During the Cold War era, both Western democratic governments and Eastern communist regimes devoted considerable effort to the development of agents causing human and animal diseases as a means of causing huge casualties to the enemy (Wheelis et al. 2006). Following the physical and economic collapse of the Soviet Empire, there has been international concern that pathogens developed by the Soviet scientists and their knowledge of how to produce and deliver them might find their way into the hands of terrorists and ‘rogue governments’ who bear grudges against the Western world. The USA is so convinced of the threats that it is currently spending billions of dollars in developing strategies to combat them. With so much money readily available, it is not surprising that some scientists talk up the threat in their quest for research funds and advancement. This is not to deny that real risks exist where state sponsored production of biological weapons is concerned, but there is also a great deal of hype, which is further fuelled by the media in constant search of disaster stories. In the UK, the Advisory Committee on Dangerous Pathogens, grades organisms 1–4, based on the risks they pose; those in Hazard group 4 being considered the most dangerous. The US Centres for Disease Control and Prevention (CDC) lists pathogens A–C according to their potential for use in bioterrorism and those on the A‐list are considered the most dangerous.
A wide range of viruses and bacteria has been suggested for possible use in biological warfare (Table 13.2), and a few examples are considered here to illustrate how they are spread, identified, and traced.
Table 13.2 Some human pathogens investigated as biowarfare agents in the past or currently considered likely to be spread maliciously.
| Micro‐organism | Transmission |
| Viruses | |
| Smallpox | Inhalation |
| Hantavirus Pulmonary Syndrome | Inhalation |
| (HPC) | |
| West Nile Virus | Mosquito bite |
| Ebola Virus | Contact |
| Marburg Virus | Contact, sexual transmission possible |
| Lassa Fever Virus | Contact, sexual transmission possible |
| Bacteria | |
| Anthrax, Bacillus anthracis | Inhalation, ingestion, breaks in skin |
| Plague, Yersinia pestis | Flea bite, inhalation (pneumonic plague) |
| Salmonella food poisoning, | Ingestion |
| Salmonella typhimurium | |
| Typhoid, Salmonella typhi | Ingestion |
| Cholera, Vibrio cholerae | Ingestion |
| Tularaemia, Francisella tularensis | Tick bites, ingestion, contact |
| Brucellosis, Brucella spp. | Contact, ingestion |
| Botulism, Clostridium botulinum | Ingestion |
| Rocky Mountain spotted fever, | Tick bite |
| Rickettsia rickettsii | |
| Epidemic typhus, | Louse bite/ faeces |
| Rickettsia prowazekii |
Biological warfare agents are sometimes said to be ‘cheap and easy’ to produce, as if all microbes are the same and that they are ‘simple to deliver’ to their target. Growing highly infectious, rapidly lethal bacteria or viruses usually requires Category 4 containment facilities and highly trained scientists, otherwise the production staff would soon become infected and die. Even with access to the facilities of a modern biodefence laboratory, working with dangerous pathogens is a risky occupation. For example, in 2004, three researchers at Boston University's medical campus working on the bacterium which causes tularaemia became infected (Dalton 2005). Fortunately, the infections were not fatal and it is not normal for tularaemia to be spread by person‐to‐person contact. However, it was some time before it was realised that these infections had occurred, and with a different pathogen the disease could have unintentionally spread into the nearby community. Terrorists would also face the problems of establishing a suitable dose to incapacitate or kill the intended target(s) and a means of delivering that dose. Biological agents cannot usually be delivered in a conventional bomb, because they would be destroyed in the heat of the explosion. Although several governments have developed bombs and missile warheads to deliver biological agents, this represents a level of resources and skill beyond most terrorist organisations. A more low‐tech approach is to release the agent in the form of an aerosol, although this is not as simple as it sounds. For a start, the aerosolizer must generate droplets of around 0.5–5 μm. If they are smaller than this, the droplets may not be retained in the lungs and if they are larger, they tend to fall to the ground before reaching their intended target. Furthermore, the effectiveness of the aerosol will be strongly affected by environmental conditions such as humidity and air currents. For example, at low humidity the droplets will rapidly evaporate, whilst the turbulent air currents associated with the high‐rise buildings typical of many cities often pull air upwards and disperse it away from street level. Theoretically, a disease might be spread by a suicidal ‘biobomber’ becoming intentionally infected and then infecting the target population by travelling around a city on crowded public transport. However, dying in a sudden explosive ‘blaze of glory’ is one thing, but intentionally dying over a period of days from a painful incapacitating disease, in a public place and without drawing attention to oneself, calls for a different and rare type of suicidal individual. The use of pathogens by disaffected individuals or organisations is therefore most likely to be aimed at causing terror and disruption of public services rather than widespread mortality. The release of the agent would probably be publicised by the terrorists at the time of release, just as is the case with many of their conventional bombs. Alternatively, there would be the threat of a release in order to blackmail the government. This requires small amounts of agent and the delivery device need not even be very effective, but would still result widespread publicity and panic. These issues are, however, beyond the scope of this chapter.
The majority of infectious diseases that spread through the population result from natural outbreaks or human negligence. For example, in August 2002, almost 200 people at the Barrow Arts Centre (Leicestershire, UK) contracted Legionnaires' disease (caused by the bacterium Legionella pneumophila) of whom 7 died. This occurred because bacteria replicated within an air conditioning unit that then sprayed them into the surrounding air. This led to charges of unlawful killing against the technical and design services manager responsible for the site and the local council.
Many microbes produce toxins that could be employed to kill or injure an individual or community. For example, the bacterium C. botulinum produces botulinum toxin – which is one of the most poisonous substances known – whilst a number of fungi produce chemicals known as mycotoxins, some of which are potentially fatal. These toxins have a range of chemical structures and consequently their presence cannot be detected or characterised by standard molecular techniques. However, most are identifiable by antibody based assays and mass spectrometric methods (Dupré et al. 2015; Zhu et al. 2014). Unfortunately, these techniques usually require preliminary sample purification and this adds to the time and cost of the analyses. The microbes producing these toxins live naturally in the environment and many have widespread distribution. Consequently, for some of them, such as botulism, accidental contamination or poor food preparation are the most likely causes of the poisoning.
In 1990, the Aum Shrinrikyo cult in Japan made several attempts to disseminate botulinum toxin via sprays, balloons, and contaminating the water supply, but they were singularly unsuccessful (Wheelis et al. 2006). There are many logistical difficulties associated with using C. botulinum as a biological weapon. First, you need a strain that produces the toxin (not all of them do), then it must be reared under anaerobic conditions, and the toxin then has to be recovered and finally disseminated. Although state organisations have developed C. botulinum for use in warfare, the information to overcome these problems is not generally available.
The first task in any disease outbreak is to identify the organism involved. This involves standard medical/veterinary/laboratory techniques. Most disease outbreaks are a consequence of natural circumstances and unless an individual or terrorist group claims responsibility, it is only through epidemiological studies that criminal activities would be suspected (Table 13.3).
Table 13.3 Clues that would provide an early indication of the malicious spreading of a microbial pathogen.
Source: Adapted from Morse and Khan (2005).
|
Forensic science becomes involved in a case of suspected bioterrorism after the identification of a pathogen and the consequent need to determine where it originated. In the case of pathogens known for their biological warfare potential, this might be done from a comparison of its genetic profile or stable isotope ratio (Kreuzer‐Martin et al. 2004) with that of cultures held legitimately by laboratories throughout the world. Ideally, there should be a list of all persons who have access to the cultures in each of these laboratories. This requires a reliable database and a level of cooperation between countries that is steadily improving but far from perfect. For example, from the molecular characteristics of an isolate of Bacillus anthracis, it would be possible to determine whether a person was suffering from a naturally acquired anthrax infection or from a variety modified in the laboratory. However, for several biological agents, there are no tests to identify to strain or sub‐strain level, and/or the tests are not yet fully validated.
The bacterium B. anthracis causes anthrax and owes much of its pathogenicity to the secretion of a toxin containing three proteins (Liu et al., 2014). One of these, protective antigen, facilitates the entry of two toxic enzymes, lethal factor and oedema factor, into the host cell. The genes coding for the toxin are found on plasmids and these have now been sequenced. In addition, the full genomic sequence of several strains of B. anthracis is known – this facilitates diagnosis and improves our understanding of how it causes disease. The bacteria are Gram‐positive, relatively large, and rod‐shaped. In clinical specimens, the bacteria have a capsule and are usually seen singly or in twos. Spores are produced when growth conditions become sub‐optimal and are therefore not found in living tissues. They are, however, formed when the host organism dies or when the bacteria are deposited on the soil. By contrast, in culture, anthrax bacilli form long chains, they do not have a capsule, and spores are produced. Anthrax spores are notoriously difficult to destroy and survive in soil for decades, particularly if it contains high calcium levels. For example, in recent years in Siberia, there have been several cases of anthrax in humans and thousands in reindeer. These are thought to result from global warming releasing spores produced during anthrax outbreaks in the late nineteenth and early twentieth centuries. In 2004, a batch of anthrax, shipped to researchers at the Children's Hospital and Research Centre at Oakland USA for vaccine production, was found to contain live and still lethal bacteria, despite being heat inactivated and tests done to check that they no longer grew in culture. The ability of anthrax spores to survive for years, coupled with the possibility of engineering the bacteria to express enhanced pathogenicity and resistance to antibiotics, has led to them becoming prime candidates as biological warfare agents.
Clinically, there are three principal forms of anthrax: cutaneous, gastrointestinal, and inhalation. Cutaneous anthrax results from the bacteria gaining access to the body via wounds and breaks in the skin’s surface. It initially manifests itself as a painless papule that subsequently develops into a black necrotic ulcer. The name anthrax is derived from the Greek α῍νθραξ meaning coal. The lesion results in an inflammatory response that is surrounded by an extensive red oedema. Many cases of cutaneous anthrax resolve themselves, but infections can be fatal if there are complications such as invasion of the blood supply leading the bacteria to being disseminated around the body. Cutaneous anthrax is the most common form of human disease and was often acquired by persons who worked with infected animals or animal products such as leather. It used to be a common disease in Europe and known as ‘wool sorters’ disease’ in the UK, from its association with the trade. Surprisingly, the fingers are seldom affected, but lesions are common on the hands, arms, and neck (shaving often results in skin nicks through which the bacteria can invade). Following improvements in animal care, hygiene, and the availability of an effective animal vaccine, the disease is seldom seen in humans in developed countries. In the UK, there were only 19 cases between 1981 and 2009, but then a spike of 5 infections in 2010 associated with contaminated heroin. Gastrointestinal anthrax is acquired through eating food contaminated with anthrax spores and causes lesions in the gastrointestinal tract, through which the bacteria invade the rest of the body, causing septicaemia and death. Naturally acquired cases of inhalation anthrax are rare, but they do occur. It results from breathing in the spores and is therefore the form most likely to result from a bioterrorist attack. Anthrax spores are 1–2 μm in size and therefore of a size likely to be retained in the lungs. They are ingested by macrophages present in the alveoli, in which they germinate, and are then transported to the nearby lymph nodes and via the blood stream around the body. Initial symptoms resemble those of a common cold but once the lungs are infected, severe breathing difficulties ensue and the disease is rapidly fatal. Human‐to‐human transmission of anthrax is not thought to occur naturally.
There are several reports in the literature of people contacting anthrax when they unknowingly used infected animal hides to make traditional drums (Stratidis et al. 2008). Some of those infected died from inhalation anthrax, which, as already mentioned, is seldom a feature of natural infections, but would be an expected consequence of anthrax being used as a biological weapon. This can lead to false alarms in which accidental infections are mistaken for evidence of terrorist activity. Marston et al. (2011) describe how they used DNA sequencing to identify the source of anthrax spores derived from animal hides that caused five human infections in the USA. Three of the infections were linked to anthrax strains circulating in West Africa, whilst one was linked to a strain found in Pakistan. It was not possible to identify the sources of the anthrax infections with greater precision, because of a lack of information on the geographical distribution of anthrax genotypes. Indeed, one of the human infections was identified as a novel MLVA‐8 genotype (GT 149) and in the absence of import documentation for the animal hides, it was impossible to determine where it had originated. This study therefore demonstrated the effectiveness of genotyping for identifying likely geographical origins of anthrax infections, but that it is essential to have a reliable and comprehensive database.
The ‘gold standard’ for the identification of anthrax is to culture it on sheep blood agar at 37 °C under aerobic conditions. This, however, will only provide species level identification and for forensic purposes, molecular and chemical tests are required to determine the strain of the infection and its likely source. A variety of tests is employed including immunoassays (Seo et al. 2015), mass spectrometry (MALDI‐TOF‐MS) (Duriez et al. 2016), and PCR (Seiner et al. 2013). However, ideally, one requires a lab‐on‐a‐chip device, such as the one described by Gao et al. (2015), which could be used to enable rapid identification under field conditions. This is because it is important to know as rapidly as possible, first, whether a suspect powder (or other sample) contains viable anthrax spores and if it does, whether or not these belong to a pathogenic strain.
The various strains of anthrax differ markedly in their pathogenicity. This trait is used in both the development of vaccines, where a lack of pathogenicity is a virtue, and the development of biological warfare agents, where high pathogenicity is required. Followers of the Aum Shrinikyo cult in Japan were probably not aware of this when they released anthrax spores in Kameido (a region near Tokyo) in 1993 (Wheelis et al. 2006). Cult members sprayed crude liquid culture medium containing a suspension of spores from the eighth floor of a building. Nobody fell ill, although several people complained of the smell. To allay the locals' complaints, a cult representative called a press conference in which he claimed that the smell resulted from the cult's religious practices. The locals were unimpressed, their complaints continued, the police became involved, and the cult ceased spraying and vacated the site. Subsequent analysis indicated that the cult's apparatus was crudely made, leaky, and produced large droplets rather than an effective aerosol. Furthermore, the spore concentration was probably too low to be effective, even if a more virulent strain had been used. Their true intentions only became known later following the cult's more successful release of Sarin gas on a Tokyo underground station. This resulted in several deaths and a thorough investigation of the cult's activities. Retrospective analysis of fluid samples collected and stored by the authorities, because of the complaints about the smell, resulted in the discovery of anthrax spores from which it was possible to grow colonies of bacteria. DNA isolation and MLVA (Multiple Locus Variable number tandem repeats Analysis) genotyping of these colonies demonstrated that the anthrax belonged to the Sterne vaccine strain (Keim et al. 2001). MLVA typing is useful for strain identification of a number of bacteria (e.g. Yersinia pestis (plague) and Mycobacterium tuberculosus (TB)), although it is not suitable for all species. The technique involves identifying and analysing suitable loci (markers) on the bacterial genome. For example, Lista et al. (2006) have identified 25 markers for typing strains of anthrax. The loci are amplified and the PCR products separated based on their length (size) – the length being determined by the number of tandem repeats they contain. Strains are distinguishable based on the number of loci at which they differ. The Sterne34F2 vaccine strain is widely available in Japan, where it is used in the preparation of animal vaccines and it is probable that a cult member obtained a vial of animal vaccine from which a large number of bacteria were subsequently cultured and released. Thankfully, being non‐pathogenic, it posed little risk to humans.
Between September and October 2001, letters containing anthrax spores were sent via the mail to media outlets and two US senators in Washington DC and New York City, USA. The letters were posted on two separate occasions. They resulted in 18 confirmed cases of anthrax from which 5 people died. Of these cases, 7 people contracted cutaneous anthrax, all of whom survived, and 11 contracted inhalation anthrax, of whom only 6 survived. This indicates the high incidence of inhalation anthrax when contracted via a bioterrorism attack and the difficulty in treating this form of the disease.
The subsequent investigation became known as the ‘Amerithrax Investigation’. This was conducted over several years and some of the conclusions remain controversial. The letters were addressed by hand and contained photocopies of brief handwritten notes indicating the nature of their contents, threats to America and Israel, and ended with ‘Allah is great’. The letters were crudely written and included spelling mistakes and appeared to suggest that they were sent by a Muslim extremist organisation. Subsequent investigations indicated that this was highly unlikely, although some people remain desperately keen to implicate the usual suspects of Iraq and Syria. The postmarks on the letters indicated that they were sent from Trenton, New Jersey, and although this area included several hundred post‐boxes, only one of them, close to Princeton University, tested positive for anthrax bacilli.
The letters were tightly sealed, but it is impossible to place dried anthrax spores inside a standard envelope without contaminating their outer surface (Beecher 2006) and so people involved in handling them became infected as well as those who opened them. This would suggest that whoever sent the letters had access to facilities that reduced personal risk when filling them, had probably been immunised against infection and did not lick the seal! When opened, the letters released a powdery substance. In some of the letters, the powder was of a coarse brown consistency, whilst in others it had a fine white appearance. Subsequent analysis indicated that the powders represented different grades of the same strain of anthrax and probably derived from two production batches. Somewhat embarrassingly, molecular analysis demonstrated that the anthrax belonged to the ‘Ames’ strain that was developed in America from a sick cow that died in 1980. Furthermore, whilst a number of laboratories in the US and elsewhere held this strain at the time, all could trace their stocks back to the US Army Medical Research Institute of Infectious Disease (USAMRIID). Anthrax colonies grown from the spores contained in the letters exhibited four morphotypes (growth characteristics) and whole genome analysis identified four variant genotypes (Rasko et al. 2011). These variants were screened against all the available Ames control strains. The only complete match was with spore stock RMR‐1029 that was maintained by the prime suspect at USAMRIID. The interpretation of this evidence is still disputed, because some commentators believe that a single person working alone could not have carried out the Amerithrax incident. They allege that it was equally possible for a sub‐culture derived from the RMR‐1029 stock held at USAMRIID to be the source of the spores used in the letters (Velsko 2015). They are less forthcoming about who might have made the sub‐culture or where it was maintained.
Although the Ames strain is highly virulent, it is mainly used in the development of vaccines. There is confusion in the literature as to whether the anthrax spores were ‘weaponized’. Examples of ‘weaponization’ are, if the spores are milled into a size that is small enough to remain in suspension in the air or coated with an additive such as silica to reduce clumping and aid aerosolization. Once aerosolized, spores can travel long distances on air currents and contaminate surfaces over a wide area. Initial reports indicated that at least some of the anthrax letters contained ‘weaponized’ spores (Inglesby et al. 2002). However, according to Beecher (2006), a ‘widely circulated misconception is that the spores were produced using additives and sophisticated engineering supposedly akin to military weapon production’. This is significant, because it increases the number of individuals capable of sending the letters. Scanning electron microscope photographs of the spores indicate exceptionally pure samples with no evidence of silica particles or ‘milling’. According to Ember (2006), there is a report of silica being detected using X‐ray mass spectrometry among spore samples. However, others suggest that this is owing to confusing the peak for compound silica (SiO2) with that for the element silicon (Si). Although silicon has been reported in the spore coat of the related Bacillus cereus, other workers have failed to confirm its presence in significant amounts and its presence in anthrax spores needs verifying. The amount of silicon associated with the anthrax spores recovered from the letters (1.5%) is also a source of controversy, with some claiming that this level could only arise from weaponisation. In addition, there are conflicting reports about how easy it is to produce anthrax powders with the characteristics and silicon composition of those found in the letters.
Bomb pulse dating demonstrated that the spores were produced sometime in the previous two years before they were despatched in the letters (Tuniz et al. 2004). Once anthrax spores form, their metabolism effectively ceases. Therefore, they have a 14C signature that indicates their year of production. Stable isotope analysis indicated that the water used in the culture of the anthrax spores originated from the northeastern states, thereby providing further proof that the attack was fully organised within the USA (Kreuzer‐Martin and Jarman 2007).
Therefore, 17 years after the posting of the letters, we can be certain that the anthrax belonged to the Ames strain, but not whether the spores were weaponized. We can be confident that the anthrax was produced shortly before the letters were sent and therefore it did not originate from an old stockpile that had somehow made its way onto the black market. There is also evidence that the anthrax was grown in the USA. In 2008, the US Department of Justice identified Dr Bruce Edwards Ivens as the principal suspect, but he committed suicide before he was brought to trial. Officially, he is considered solely responsible for the attacks, but this view remains controversial. For example, there is uncertainty of whether Ivens was in the locality of the post‐box the anthrax letters were mailed from on the day they were sent. Ivens had worked at USAMRIID for many years, where he undertaken research on a number of pathogens, including anthrax. Ironically, he was also employed to analyse the Washington letters during the early stages of the investigation. However, some estimates suggest it would have taken him several months to culture sufficient spores to use in the letters and therefore it is uncertain how he could have done this without being detected. There remains a suspicion that he may have had help from persons who remain unknown or he might have supplied the spores to a ‘third party’. However, unlike some terrorist bomb outrages, it is inconceivable that the attack was perpetrated from the kitchen of the average citizen. What is certain is that although there have been numerous hoaxes and false alarms concerning anthrax since the letters were sent, there have been no repeats anywhere in the world. This suggests that this style of terrorism is not easy, simple, or cheap.
Smallpox is caused by a Variola virus, and is related to Cowpox virus and Monkeypox virus; Chickenpox is caused by the Varicella zoster virus, which is a Herpesvirus, so is not related to smallpox. The term ‘pox’ is an old word and refers to a disease that causes the formation of pustules. Smallpox gained its common name, not in reference to the size of the pustules but to distinguish it from syphilis that was referred to as the ‘great pox’. It is spread by droplet infection, by direct contact with an infectious person, or from contact with clothing or bedding previously used by a person suffering from smallpox. The incubation time is approximately three weeks, after which flu‐like symptoms are expressed; a person becomes infectious two to three days before these symptoms appear. A few days after the onset of the disease, the patient's temperature returns to normal and they develop a vesicular rash on the face and limbs that spreads to cover the whole body. Lesions in the mouth and throat release large amounts of virus that are then aspirated in the saliva and droplets in the breath. Over the course of 7–14 days, these vesicles enlarge, rupture, and start to heal over – at which point the patient ceases to be infectious. However, in about half of all cases, the fever then returns and widespread internal haemorrhage leads to death within a few days. The disfiguration and high mortality makes smallpox a frightening disease. However, it is also a rare example of how a pathogen can be eradicated – the world was officially declared free of smallpox in 1980. Eradication was possible because an effective vaccine was available, everyone who is infected with the virus develops the symptoms (therefore, they could be identified and treated before the disease was transmitted further), and all countries in the world were willing to cooperate with the WHO eradication campaign. The last serious smallpox outbreak in Europe occurred in 1972 in the former Yugoslavia and resulted in 175 people becoming infected of whom 35 died. At the time, it was compulsory for children to be vaccinated against smallpox, but as part of the containment process against the 1972 outbreak, the entire Yugoslav population was re‐vaccinated. The smallpox vaccine is given using a bifurcated needle and causes the formation of a characteristic scar. In Yugoslavia, the vaccine was injected into the upper right arm of the recipient. Therefore, any Caucasian with two smallpox vaccination scars on their upper right arm is almost certainly someone who was born and living in Yugoslavia until at least 1972 (Nikolić et al. 2014).
Although the disease is eradicated, stocks of the virus are maintained in the USA at CDC Atlanta and in Russia at the Vektor Institute, Novoskibirsk, ostensibly to facilitate vaccine development should the disease re‐emerge. There are continuous rumours that the security of these stocks has been breached and that other sources are held elsewhere in the world. The countries and organisations accused of holding illicit stocks all deny this. Unfortunately, it is impossible to prove a negative and the consequences if the disease was released are dire. For example, in the UK, vaccination against smallpox ceased in the 1960s and therefore by 2019 no one under 54 would have been vaccinated, whilst the immunity of older people has declined. Therefore, the majority of the UK population is susceptible. Computer models of how the disease would spread indicate that within 3 weeks, 10 people deliberately infected from an initial ‘index case’ would each have inadvertently infected a further 10 people (i.e. 100 people). However, the index person would have a short ‘window of opportunity’ during which they would be infectious, but their diseased condition not patently obvious from the facial rash – and they would probably be feeling extremely ill. Regardless of how the virus is introduced into the population, the diagnosis of smallpox anywhere in the world would indicate the release of an illicitly held virus. The UK government has contingency plans for dealing with smallpox, as it has for all biological and chemical weapons (www.dh.gov.uk, www.phe.org.uk). It is expected that smallpox would spread rapidly owing to the low immunity in the population, the speed and frequency with which people travel during their daily lives, and the unfamiliarity of people, including doctors and nurses, with the symptoms of the disease. Alert levels (1–6) are to be declared by the Chief Medical Officer and if there is ‘an overt release’ (i.e. the terrorists gave a specific warning), the police would lead the contingency plan. If the release is ‘covert’ (i.e. there was no warning, but cases of disease were positively identified), the plan would be led by the Department of Health. Regional smallpox diagnosis and response groups are established, which include a smallpox diagnostic expert and smallpox management and response teams. Confirmation of the disease would require electron microscopy and PCR analysis of the virus. At Alert Level 3 (Outbreak occurring in the UK), the contingency plan is to control the spread of smallpox and minimise disruption and inconvenience by identifying positive cases and isolating them in ‘smallpox centres’, then identifying all their possible contacts and vaccinating them. Mass vaccination of the whole population would only be considered at Alert Level 4, when there would be large multiple outbreaks across the country, in which the cases could not be linked. The value of vaccinating the general population has to be balanced against the one in a million chance of death resulting from reactions against the vaccine itself (in a population of greater than 60 million, this is a serious consideration) and the problems of supplying sufficient doses of vaccine.
Although most of the focus on bioterrorism concerns diseases and toxins that cause human mortality, agricultural bioterrorism has the potential to cripple a country's economy. This was exemplified by the UK Foot and Mouth Disease (FMD) outbreak of 2001, in which the direct and indirect costs to the country were put at over £12 billion. There is no suggestion that this outbreak was a consequence of a malicious act, but it indicates how expensive and disruptive such incidents can be.
During the twentieth century, a wide variety of pathogens were investigated by governments for their potential to affect agriculture (Wheelis et al. 2006). These have included viruses, bacteria, fungi, and insects, either to destroy crops or to kill or incapacitate farm animals (Table 13.4). The prime focus of this research was to disrupt the enemy's economy and reduce their ability to feed themselves. Few of these agents were used in anger, and even fewer had any significant impact. However, the use of herbicides has been more widespread – such as the use of Agent Orange (this contained n‐butyl 2,4‐dichlorophenoxyacetate (2,4‐D) and n‐butyl 2,4,5‐trichlorophenoxyacetate (2,4,5‐T) by the Americans in Viet Nam during the 1960s – and devastating. Agriculture is particularly vulnerable to terrorist attack, because it takes place over large unsecured stretches of land that are, for the most part, impossible to police. In the case of crops, many of them are grown as monocultures over large areas and their genetic similarity makes them vulnerable to pathogen attack. Farm animals are vulnerable to transmissible diseases when brought together and mingled when taken to market or for slaughter. In addition, they are routinely transported long distances within and between countries, thereby facilitating the spread of disease. In some cases, farm animals are kept indoors at high densities and although the buildings can be secured to some extent, the animals' proximity to one another makes them vulnerable to infectious pathogens. Furthermore, the presence of plant or animal disease within a country may make it impossible to export produce and home consumption may drop – for example, bovine spongiform encephalopathy (BSE) in UK cattle prevented the export of meat to the European Union for many years.
Table 13.4 Some domestic animal pathogens investigated as biowarfare agents in the past or currently considered likely to be spread maliciously.
| Micro‐organism | Host | Transmission |
| Viruses Foot and mouth disease Rinderpest African swine fever Avian influenza (Fowl plague) Newcastle disease Bacteria Brucellosis, Brucella spp. Glanders, Burkholderia mallei Melioidosis, Burkhoderia pseudomallei Psittacosis, Chlamydia psittaci |
Cattle, sheep, pigs Cattle, (sheep and pigs can be infected) Pigs Birds, can be transmitted to man Birds, can be transmitted to man Cattle, can be transmitted to man Horses + other equids, can be transmitted to man Cattle, sheep, goats, pigs, cats + many others, can be transmitted to man Birds, can be transmitted to man |
Contact, contamination Contact, ingestion Contact, ingestion, tick vectors Contact, contamination Contact, contamination Contact, ingestion Contact, inhalation Contamination, ingestion Inhalation |
Despite the inherent vulnerability of the agricultural system to malicious spreading of pests and diseases, there are few instances in which it has been targeted by terrorist groups or other disaffected individuals (Keremidis et al. 2013). This is because it lacks the obvious impact of human casualties or damage to the infrastructure (e.g. an explosion). However, should such cases occur, the pathogens could be identified and traced using similar techniques to those described previously for human pathogens.
FMD virus (FMDV) is an RNA virus, containing a single strand of positive sense RNA within a capsid. Taxonomically, FMDV is a picornavirus belonging to the genus Aphthovirus. It is highly contagious and infects cattle, sheep, goats, pigs, and other cloven hooved mammals. Humans are rarely infected. (The human disease of Hand Foot and Mouth (HFMD) is a quite separate infection caused by a picornavirus in a completely different genus – Enterovirus – that includes species associated with common childhood infections). FMD spreads by contamination with virus‐infected saliva and faeces. It is rarely fatal in adult animals, although the young suffer high levels of mortality. Although not fatal to adults, it is debilitating and causes lost productivity (e.g. growth, milk yield). Blisters and sores form on the feet leading to lameness and they are also found around the nose and tongue. No cases of FMD had been diagnosed in the UK for 20 years, until it was identified in a pig sent to slaughter in February 2001. However, by this time the disease was established in several parts of the country. The large‐scale movement of farm livestock that is common practice nowadays aided its spread. The official policy to control FMD was to slaughter all animals likely to have had contact with an infected animal, regardless of whether they themselves exhibited symptoms, and to restrict the movement of animals and humans over large areas of the country. After about 11 months, the outbreak was over but caused the slaughter of over 6 million animals, huge financial loss, and widespread disruption.
Like other RNA viruses, FMDV exhibits rapid multiplication and mutation rates and this can make identifying the source of an infection complicated. It is currently possible to identify the strain of an FMDV, but not its origin. The strain that caused the 2001 outbreak in the UK belonged to the type O pandemic strain (PanAsia) – one that has been spreading and causing outbreaks in many parts of the world for over 20 years. Isolates of this strain from different countries and regions within countries indicates that they are all closely related. However, a close match was found to a South African isolate, but whether this means that this was the source of the UK outbreak or that the UK and South African isolates shared a common source is not known (Mason et al. 2003). There is no evidence to explain when or how the virus entered the UK, although some people suspect that bushmeat or illegal meat imports may have been to blame.
A second FMD outbreak of FMD occurred in Surrey in August to September 2007. It was brought under control more quickly than the 2001 outbreak, although it too resulted in the slaughter of many animals and a lot of disruption. After the first case was diagnosed, the origins of the disease were quickly established – and proved to be highly embarrassing. The virus almost certainly came from a research and development site at Pirbright, at which three organisations, the government Institute of Animal Health, Merial Animal Health Ltd., and Stabilitech Ltd. all worked with FMDV to varying extents. The virus probably escaped via a leaky drainage system connecting the site to sodium hydroxide treatment tanks (that would have killed the virus). Phylogenetic analysis indicated that the FMDV responsible for the outbreak belonged to the strain O1 BFS and extremely closely related to that used at Pirbright. However, all three organisations at Pirbright used virus with slight differences from one another and therefore it was not possible to state categorically which organisation the virus originated from (www.hse.gov.uk/press/2007/e07032.htm). Phylogenetic analysis indicated that the viruses found at all infected farms were closely related and it was possible to predict the sequence in which nearby farms acquired their infections. How the virus transferred from Pirbright to the farms is uncertain, but probably involved virus infected soil and water contaminating footwear and vehicles. The first farm to be infected was only 3 miles from Pirbright, with a road that directly linked it to the site. The infection then probably transmitted between farms, although at least one of them may have been directly infected from Pirbright. This case illustrates how difficult it can be for even highly regulated officially sanctioned institutions to maintain effective biosecurity. Consequently, bioterrorists are likely to find it even harder to keep their activities secret and to prevent their microbes from escaping too early.
Microbial profiling offers enormous potential as a means of linking people, animals, plants, and objects to one another and/or to a geographical region. Changes in microbial profiles could potentially be useful in estimating the PMI and/or the length of time a body (or any other organic or inorganic object) has rested upon the soil surface. However, before this is possible, the techniques will need to be refined, standardised, and validated.
Molecular analysis of STIs offers the potential for linking assailant and victim in cases of sexual assault in which human DNA evidence is lacking or unreliable. Despite decades of sex education and health awareness campaigns, STIs remain remarkably common in the UK. Consequently, they are involved in many cases of sexual assault. As already mentioned, the appropriate standards and validations would be required and to be truly effective, a national database is needed.
Bioterrorism always captures the headlines but it will, hopefully, remain a rare event. Despite this, it is inevitable that large amounts of resources will continue to be spent on methods for detecting potential pathogens under ‘field’ conditions. These will probably involve refinements of the ‘lab‐on‐a‐chip’ techniques. (There is always a market for paranoia and one can already purchase one's own anthrax detection kits on the internet – not that they are particularly reliable.) These devices need to identify quickly the many hoaxes and suspicious powders, packages, etc. reported every year.