ABSTRACT Biological weapons are a thorny issue, for their destructive capabilities and for the potential to generate panic and terror among the affected people. Used since pre-Christian times, biological weapons have resulted in the decimation of whole populations and have changed the geopolitics of several places. In this work, a summary of the main war and terrorist activities carried out by biological weapons over the time is presented. Moreover, main biological warfare agents and related pathology are considered, according to American Centers for Disease Control and Prevention (CDC) priority classification. At last, the emerging more destructive potential of biological agents, due to the widespread introduction of biotechnology, is analyzed. 1. INTRODUCTION DaSilva, in his work published in 1999, defines the Biological warfare as the intentional use of microorganisms, and toxins, generally of microbial, plant or animal origin to produce disease and death in humans, livestock and crops (DaSilva, 1999). Biological warfare and bioterrorism are very complex subjects, mainly due to the many agents that can be used as a weapon and for the wide range ways for the dissemination in the environment and population. A biological event provides for the presence of at least two actors: one or more pathogens (bacteria, viruses, or toxins) and a vehicle for their dissemination. What makes biological threat so troubling? In addition to high spread capacity and lethality of biological agents, the invisibility and the extremely difficult short-term revelation makes impossible an immediate contrast with the subsequent increase of the affected. In fact, most of biological weapons (unless of toxins) have a peculiar quality that other non-conventional weapons (chemical and radiological ones) do not show: biological agents are able to multiply in the host organism and be transmitted in turn to new hosts, generating in this way not predictable effects on the population, both in terms of number of victims and in terms of spread geography. Among the reasons which make biological weapons attractive, there is their very low cost when compared to both conventional and unconventional ones. NATO sources, according to data processed in 1969 by U.S. experts, reported the following costs for an attack on an area of 1 km2 to civilian populations with different weapons: a) $1/Km2 for biological weapons, b) $600/Km2 for chemical, c) $800/Km2 for nuclear, d) $2,000/Km2 for conventional armaments (NATO, 1996). Therefore, it seems clear therefore that, in financial terms, an attack with biological weapons would be strongly cheaper when compared to any other one. In the second decade of the 21st century, to make the situation more troubling, there is the simplicity with which it is possible to produce large quantities of biological agents with facilities and expertise available to everyone, even to terrorist and paramilitary groups. The aim of this work is to present an overview on biological warfare and bioweapons, as well as to assess the state of the art on the actual offensive capability through this unconventional weapon. 2. HISTORYCAL 2.1 Pre Word Wars age The use of biological agents as war weapons is not a modern era novelty. Although it isn’t easy to identify a definite time when the use of bioweapons began, ancient information reported that in pre-Christian era, around 300 B.C., Greeks were using animal cadavers to contaminate water wells of the opponent. This strategy was also used the by Romans and the Persians (SIPRI, 1971). In a later period, during the battle of Tortona (Italy) in 1155, bodies of dead soldiers and animals were used to contaminate water wells by Barbarossa (Clarke, 1968). In the 14th century, during the siege of Kaffa by Tartar (now Feidisuja, Ukraine, a city on the Black Sea at that time under the control of the Genoese), a biological attack allowed Tartars to win the siege. During the siege, among the Tatar army, an epidemic of plague was spread: the besiegers thought to shoot the cadavers of their dead comrades within the walls of the city of Caffa (Wheelis, 2002). This resulted in a turning point in the war: the Genoese fled from Caffa, carrying with them the sick. On the return trip to Genoa, they landed several ports in the Mediterranean sea; although some sources believe a possible correlation between the epidemic of plague in Caffa and the pandemic that decimated the population of Europe in the following decades (Black Death), most of the authors share the view of two events independent (Wheelis, 2002). In 1422, during the siege of Carolstein, Lithuanian soldiers catapulted within the city cadavers of death soldiers and 2,000 chariots of excrement (Newark, 1988), frightening the population affected and spreading lethal fevers in many cases. We will have to wait for more than three centuries to find a new documented use of biological agents as a war weapon: during the French-Indian War (1754-1767), the British commander Sir. Jeffrey Amherst ordered the distribution of blankets infected with smallpox to decimate the population of Indian tribes hostile to the British (Bhalla and Warheit, 2004). The distribution of infected blankets occurred in the summer of 1763, and the resurgence of the virus among the indigenous lasted for over 200 years (Riedel, 2004). 2.2 World War I and II Several biological warfare actions carried out during the World War are not sufficiently confirmed in the literature. However is frequently reported that Germans inoculated cattle with Bacillus anthracis and Pseudomonas mallei, responsible to cause severe diseases such as anthrax and glanders, before sending them in enemy states (SIPRI, 1971; Poupard and Miller, 1992; Hugh-Jones, 1992). It should be considered that the First World War saw the large-scale use of non-conventional chemical weapons, therefore it must be expected until the Second World War to find a more extensive use of biological weapons. During World War II many countries conducted research programs on the development of bioweapons; the Japanese program, conducted under the direction of Captain Shiro Ishii, was certainly most ambitious (1892-1959). The research in this direction started in 1928; during this year Captain Ishii visited many European and American countries in order to learn useful techniques and information about the possible uses of biological weapons. Returned to homeland, Captain Ishii was substantial granted in order to constitute a massive bioweapons research center, called the 731 units, located at Beiyinhe, in Manchuria. The research center could count on a staff of over 3,000 scientists, mainly microbiologists. The experiments were conducted on pow, principally Koreans, Chinese and Russian soldiers. Prisoners were used to test numerous bioweapons, including Yersinia pestis, Vibrio cholera, Neisseria meningitidis and Bacillus anthracis (Leitenberg, 2001). Christopher and colleagues report that during this research, several thousand prisoners died as a result of the experiments conducted on them (Christopher et al., 1997). However, the mortality rate around the area 731 remains very high for several years. If we consider for the total count these deaths also, we reach the considerable sum of 200,000 deaths as a result of the activities carried out by Captain Ishii (Harris, 2002). In 1942, the wrong control of the infection spread resulted in the death of 1,700 Japanese soldiers (Sokolski and Ludes, 2001). Many other nations carried out experiments on potential biological agents, but information reported in the literature are rather skinny. It is also important to remember the experiments conducted in 1942 by the British army on the Island of Gruinard, off the Scotland coast, where anthrax dirty bombs were tested (Manchee et al., 1981). The island was contaminated and uninhabitable until 1990, when an extensive land decontamination was carried out (Aldhous, 1990). 2.3 Post World Was age Until World War II, United States remained considerably behind respect the other nations in the research fields on biological weapons. In the United States, the golden age for both the test and development of bioweapons in the United States was immediately after the conclusion of World War II, when U.S. received the results of the experiments performed by the Japanese unit 731. The U.S. could also rely on working directly with Captain Ishii, the former director of the unit 731 (Christopher et al., 1997). In September of 1950, the U.S. Navy conducted an experiment on civilians in order to assess the vulnerability of a large American coastal town to a biological attack: in the San Francisco Bay a cloud of Serratia marcescens (a low pathogenic bacterium mainly responsible for infections of skin and respiratory tract) was spread by boat. The infection struck, as a result of subsequent checks, almost the entire population (1 million people). Even though the bacterium had to be almost harmless, several individuals showed the effects of respiratory diseases and some of them died (Christopher et al., 1997). Few years later (1956-1958), in Georgia and Florida, swarms of mosquitoes, probably carriers of yellow fever, were released in order to verify the vulnerability to an air attack. Even though the documents are still top secret, several sources report that some individuals died for the bite of insects. A last large scale experiment which is documented, consists in the dissemination of Bacillus subtilis in the New York subway in the summer 1966. The experiment resulted in the infection, although without consequences, of more than one million people. It demonstrated that the spread of a pathogen in the whole subway network from a single station, due to the displacement of air in the tunnels, was possible (Zygmunt, 2006). In the seventies, USSR conducted an ambitious research program on biological weapons; however, unlike the United States programs, on which the secrecy has been partially removed, an aura of mystery on Russian research programs still remains. Some information about the research carried out in the USSR are provided in the paper published by Davis in 1999 on Emerging Infectious Disease. The USSR, between 1973 and 1974, formed an organization called "Chief Directorate for Biological Preparation" (Biopreparat), with the purpose to develop and produce biological weapons. Although there are no unambiguous data about the number of individuals employed by the Biopreparat, it is believed that more than 50,000 people were working in the whole system connected to the structure, including scientists and technicians, who could count on 52 research and producing factories. In these structures, high amounts of etiologic agents of plague, tularemia, anthrax, glanders, smallpox and Venezuelan equine encephalomyelitis were studied and produced. Not only biological agents from natural sources were studied in the Soviet Union. The Soviets studied and applied technologies of genetic engineering in order to increase the aggressiveness of biological agents through biotechnology The aim of this work was the production of a new more dangerous, more easily spread and more difficult to identify and combat generation of biological weapons (Davis, 1999). Among the countries that developed a massive program on bioweapons research, in the post-war era, we should quote Iraq. Iraq started its research and development program in the field of biological warfare in 1974, contextualizing it in an organization called "State Organization for Trade and Industry" (Davis, 1999). The program consisted on the study and production of botulinum toxin, anthrax, aflatoxin and ricin, as well as antiplants and viral agents (Rotavirus, Infectious Hemorrhagic Conjunctivitis, Camel Pox) (Leitenberg, 2001). The program involved about 300 scientists, who completed their training in Western Europe countries (Leitenberg, 2001). 2.4 International treaties The first measures against the use of biological weapons were taken in the 19th century during the Hague Conference in 1899, and then confirmed in the same place in 1907: with the document "Laws and Customs of War on Land", signed by 24 countries, the prohibition against the use of poisoned arms was ratified (Leitenberg, 2001). In 1925, with the awareness of the horrors of World War I, especially as regards the use of chemical weapons, the Geneva Protocol was signed. It "Prohibited the use in war of Asphyxiating, Poisonous or Other Gases, and of Bacteriological methods of Warfare". Although this treaty was signed by a considerable number of nations (even though it was ratified by the U.S. not until the mid-seventies), it prohibited the use of biological agents as weapons only, but not their development or their stocking (Christopher et al., 1997). In view of the limited effectiveness of the Protocol of Geneva in the control of the bioweapons development and proliferation, in 1972 the "Conventions on the Prohibition of the Development, Production and Stockpiling of Bacteriological (Biological) and Toxin Weapons and on Their Desctruction"was initialed. Initially signed by over 100 nations, the Convention became effective in 1975; however this convention, such as that Geneve one, has several loopholes: first of all, it does not provide guidelines for the protocol compliance verification; moreover it prohibits the use and development of bioweapons in “quantities that have no justification for prophylactic, protective or other peaceful purposes” (Riedel, 2004). However, it is evident how this assertion is open to interpretation; de facto it does not define threshold quantities or substantial limitations to the development and production of bioweapons (SIPRI 1971 b, 1973). The events consequent to the ratification of the Biological Weapons Convention of 1972, up to the most recent, have confirmed this observation. 2.5 Last decades: bioterrorism and biotechnology advent Even after the entry into force of the "Conventions on the Prohibition of the Development, Production and Stockpiling of Bacteriological (Biological) and Toxin Weapons and on Their Destruction", a large number of countries went on to develop, produce and test biological agents for military purposes. Since the 80s, increasingly terrorist groups have begun to consider the bioweapon as an economic and highly destabilizing way for civil society. It must be considered that the large scale advent of biotechnology, and the reduced difficulty in genetically modified organisms producing, has made the potential creation of multi-drug resistant pathogens, or with enhanced virulence factors possible. The use of biological agents in the last decades is mainly attributable to terrorist groups, more or less isolated, who used the bioweapons as a strategy to defend extremist religious ideas striking the civilian population or sensible government targets. In 1984 in The Dalles, Oregon, United States, a group of extremist followers of Bhagwan Shree Rajneesh (also known as Osho), contaminated the salad in 10 different salad bar with the pathogen of salmonellosis, Salmonella thyphimurium, in order to disable the population. 751 people contracted the disease and several of them were hospitalized; although there were no fatalities, this terrorist act is considered the largest bioterrorist attack in the history of the United States for involved people (Török et al., 1997). In the nineties the Japanese cult of Aum Shinrikyo tested different biological weapons, including botulin toxin, anthrax, cholera, and Q fever; in 1993, during a humanitarian mission in Africa, it tried to obtain samples of the Ebola virus. Between 1990 and 1995, the cult attempted to carry out several bioterrorist actions in Tokyo using vaporized biological agents including botulinum toxin and anthrax spores. Fortunately, the attacks were unsuccessful (Olson, 1999). A significant bioterrorist event happened in the United States contextually to the dramatic attacks to the World Trade Center in New York in September 2001. The release of Bacillus anthracis spores through the U.S. postal system was carried out with letters addressed to the press and to government officials. There were 22 confirmed cases of anthrax contamination, 12 cutaneous and 10 inhalational cases. The 12 cutaneous responded positively to the antibiotic treatment, while of the 10 inhalational cases, 4 were fatal (McCarthy, 2001). In 2002, in Manchester, UK, six terrorists were arrested for having been found in possession of ricin and in 2004, traces of the same toxin were found at the Dirksen Senate Office Building in Washington DC. So, it appears evident then that the use of biological agents has moved, in recent times, to terrorist groups. This creates very strong concerns: the use of biological weapons by terrorists can generate unexpected scenarios characterized by massive destructive potential. 3. BIOTERRORISM AND BIOWEAPONS The American Center for Disease Control and Prevention (CDC) defines a bioterrorism attack as “the deliberate release of viruses, bacteria or other germs (agents) used to cause illness or death in people, animals, or plants” (CDC, 2013). CDC classifies the most important bioterrorism agents into three categories (tab. 1): a) Category A: Agents that can be easily disseminated or transmitted from person to person. They result in high mortality rates and have the potential for major public health impact. They might cause public panic and social disruption, and require special action for public health preparedness. b) Category B: Agents that are moderately easy to disseminate. They result in moderate morbidity rates and low mortality, and require specific enhanced diagnostic capacity and disease surveillance. c) Category C: Emerging agents that could be engineered for mass dissemination in the future because of their availability. They are easy to produce and disseminate. They are potentially linked to high morbidity and mortality rates, and major health impact. Table 1: Major biological agents possible used as bioweapons (CDC, 2013). Groups Diseases Agents A Anthrax Bacillus anthracis Botulism Clostridium botulinum toxin Plague Yersinia pestis Smallpox Variola major Tularemia Francisella tularensis Viral hemorrhagic fevers Filoviruses and Arenaviruses B Brucellosis Brucella spp. Epsilon toxin Clostridium perfringens Food safety threats Salmonella spp., E.coli O157:H7, Shigella Glanders Burkholderia mallei Melioidosis Burkholderia pseudomallei Psittacosis Chlamydia psittaci Q fever Coxiella burnetii Ricin toxin Ricinus communis Staphylococcal enterotoxin B Staphylococcus spp. Typhus fever Rickettsia prowazekii Viral encephalitis Alphaviruses Water safety threats Vibrio cholerae, Cryptosporidium parvum C Emerging infectious diseases Nipahvirus and Hantavirus Generally, biological agents (included those used as weapons) can be further classified according to certain characteristics that define the hazard to health (NATO, 1996): a) Infectivity: The aptitude of an agent to penetrate and multiply in the host; b) Pathogenicity: The ability of the agent to cause a disease after penetrating into the body; c) Transmissibility: The ability of the agent to be transmitted from an infected individual to a healthy one; d) Ability to neutralize: It means to have preventive tools and/or therapeutic purposes. Biological agents can be transmitted through one or more ways. The transmission modes are the following (La Placa, 2010): a) Parenteral: Agents that are transmitted through body fluids or blood; b) Airway (by droplets): Agents that are emitted by infected people, which can then be inhaled by surrounding people; c) Contact: Through which the agents present on the surface of the infected organism can infect another organism; d) Oral-fecal route: Through objects, foods or other items contaminated with the feces of infected patients, or through sexual contact. 3.1 Bioweapons There are numerous pathogens (bacteria, viruses and toxins) that cause diseases in humans, animals and plants; however, only very few possess the characteristics to be a bioweapon. In literature, in 1997 Eitzen describes the characteristics that make a biological agent a potential bioweapon. Ideally, a biological weapon should be easy to find or to produce. In order to develop a biological attack towards sensitive targets or the population, large amounts of biological agents are in fact required; it must be considered that it is necessary quite a number of biological agents (or a certain amount of toxin) to generate a disease in an target. The ideal bioweapon also must hold a high capacity to incapacitate the affected or, alternatively, be highly lethal. It is appropriate to choose an agent with an incubation period depending on whether immediate or delayed effects are required. Other important characteristics for a biological weapon are the route of transmission, and hence, the ease of dissemination with an appropriate method of delivery. Finally, the stability of the agent must be assessed, especially when large quantities must be stored for indefinite periods (Eitzen, 1997). Below some of the key features of the most relevant biological agents (included in A category by CDC) are reported, categorized according to the biological origin. 3.2 Bacteria Bacillus anthracis. Bacillus anthracis is a Gram-positive, non-motile, facultative anaerobic endospore forming bacteria, usually surrounded by a capsule; it is the etiological agent of anthrax. The disease occurs most frequently when an epizootic or enzootic of herbivores becomes infected after acquiring spores from direct contact with contaminated soil. In humans, the disease can occur when exposed to infected animals, tissue from infected animals or high concentrations of anthrax spores. Antrhax endospores have no measurable metabolism, do not divide, and are resistant to drying, heat, ultraviolet and ionizing radiation, chemical disinfectant and other forms of stress, remaining not a word in the environment for years (Bhalla and Warheit, 2004): survival in soil for up 200 years has been reported (Yuen, 2001). The disease is caused by the action of a toxin produced by the vegetative bacillus; the toxin consists of three components: protective antigen (PA), edema factor (EF) and lethal factor (LF). PA binds to cell receptors, mediating the entry of EF and LF into the cell. Another anthrax virulence factor is the D-glutamic acid polypeptide capsule of the vegetative form (WHO, 2004). Three types of anthrax infection can occur: cutaneous, inhalation and gastro intestinal; cutaneous form is the most common and is characterized by dermal ulcers, painless, non-scarring, pruritic papule progressing to a black depressed eschar with swelling of adjacent lymph glands and oedema (WHO, 2004). Local lymphadenitis and fever can occur, but septicaemia is rare (Moquin and Moquin, 2002); untreated cutaneous anthrax can become systemic and it is fatal in 5-20% of cases. Gastro-intestinal and inhalation form are less common; inhalation starts with influenza-like symptoms that include fever, fatigue, chills, non-productive cough, vomiting, sweats, myalgia, dyspnoea, confusion, headache and chest and/or abdominal pain, followed by the development of cyanosis, shock, coma and death. Gastro-intestinal form is characterized by fever, nausea, vomiting, abdominal pain and bloody stools. Oropharyngeal infection, on the other hand, is accompanied by oedematous swelling of the neck, often followed by fever and lymphoid involvement (WHO, 2004). There is no evidence of direct person-to-person spread (Yuen, 2001). After exposure, the incubation period is reported to range from 1 to 7 days, possibly extending up to several weeks. Some vaccines are administered to prevent the disease, such as live spore vaccines based on attenuated strains, and cell-free vaccines based on anthrax protective antigen (PA) (WHO, 2004). Regarding therapy, there are three types of antibiotics that are effective against B. anthracis: ciprofloxacin, tetracyclines and penicillins (Bhalla and Warheit, 2004). For laboratory diagnosis and research, manipulations involving clinical specimens Biosafety Level 2 practices are recommended. However, for manipulations involving activities with a significant aerosol production, Biosafety Level 3 practices are advised (WHO, 2004). Clostridium botulinum. Clostridium botulinum is a spore forming and obligate anaerobe, etiological agent of botulism; it can be isolated from the soil, its natural habitat. Four species of C. botulinum are known, characterized by different genomes and their common botulinum toxin. In addition, 7 distinct antigenic types of botulinum toxin (A-G) are defined by the absence of cross-neutralization. The toxin is responsible for the disease and it is a dichain polypeptide: a heavy chain of 100 KDa is joined by a single disulfide bond to a 50 KDa light chain that is a Zn- containing endopeptidase that blocks acetylcholine-containing vesicles from fusing with the terminal membrane of the motor neuron, resulting in flaccid muscle paralysis (Arnon et al., 2001). Botulinum toxins are the most lethal toxins known and all seven types act in similar ways. Death often occurs as a result of paralysis of pharyngeal and diaphragmatic muscles, followed by respiratory arrest (Bhalla and Warheit, 2004). Three forms of human botulism exist: food-borne, wound, and intestinal. All forms of botulism are caused by absorption of botulinum toxin into the circulation from a wound or mucosal surface; after infection, the incubation period depends on the rate and amount of toxin absorption: from 2 hours to 8 days. Patients affected by botulism are a febrile and present symmetric, descending flaccid paralysis with prominent bulbar palsies. Therapy consists of passive immunization with equin antitoxin, accompanied by supportive care. Botulism can be prevented by administration of a pentavalent (ABCDE) botulinum toxoid; a recombinant vaccine is in development. Biosafety Level 2 practices are recommended for manipulations in laboratory and Biosafety Level 3 practices are suggested for activities with high potential for aerosol or droplet production (Arnon et al., 2001). Yersinia pestis. Yersinia pestis is a Gram-negative non-motile, non-spore-forming coccobacillus, that grows both in aerobic and anaerobic conditions. It can remain viable for days in moist soil or water, but it is killed by direct exposure to sunlight (WHO, 2004). Bacterium is the etiological agent of plague, a disease that can affect humans and animals (La Placa, 2010); wild rodents are the pathogen reservoirs and transmission to other animals occurs through fleas, infected animal tissues, contaminated soil or respiratory droplet exposures. In endemic rural areas, persons who come in contact with wild rodent hostes of Y. pestis can be affected by the plague, which exists in two forms: bubonic plague and pneumonic plague (WHO, 2004). The first would result if fleas were used as carriers of disease; in this case, the incubation period is 2-6 days after exposure. Swelling of the Lymph nodes occurs (bubones), associated with onset of fever, chills, headache, followed by nausea and vomiting. Untreated bubonic plague causes septicemia. Pneumonic plague can occur from inhaling organisms or from exposure to infected blood; productive cough with blood-tinged sputum is a typical symptom of pneumonic plague, that can spread from person to person by coughing (La Placa, 2010). If started soon after infection, antimicrobial therapy is effective. It consists of administration of streptomycin or gentamicin. Alternative antimicrobial substances are: tetracyclines, doxycyclines, chloramphenicol, fluoroquinolones, ciprofloxacin, sulfonamides. Plague vaccine is advised only for high-risk groups (laboratory personnel): vaccination with killed or live attenuated Y. pestis is effective against bubonic but not against pneumonic plague. Biosafety Level 2 practices are recommended for activities involving infective materials and cultures. Biosafety Level 3 may be used in the case of high production of infectious aerosol or direct contact with infected fleas (WHO, 2004). Francisella tularensis. Francisella tularensis is a small, Gram-negative, non-motile, facultative intracellular, aerobic coccobacillus. It is responsible of tularemia, a zoonotic disease. Two bacterium sub-species exist: F. tulariensis tulariensis (Type A) and F. tularensis palaearctica (Type B). Type A is more virulent than Type B (WHO, 2004). The organism can survive for up to several weeks in soil, water, straw and soil (Bhalla and Warheit, 2004). Many wild animals (rabbits, beavers, muskrats, hares, voles) are bacterium reservoirs. Humans can be infected when bitten by arthropods, by ingestion of contaminated food and water, inhalation of contaminated aerosols; direct contact with infected animals is also dangerous for humans, but person-to-person transmission has not been observed. After infection, the incubation period is generally 3-5 days, but it can extend up to 14 days. Symptoms of the disease depend on the virulence of the infectious agent; furthermore two different clinical manifestations exist: ulceroglandular (75% of cases) and typhoidal (25% of cases) tularemia. The first is characterized by indolent ulcer at the site of entry and painful swelling of local lymph glands; the expression “typhoidal tularemia” indicates systemic illness without apparent site of primary infection. Painful pharyngitis and cervical lymphadenitis result from infection through ingestion of contaminated food or water (Bhalla and Warheit, 2004). It consists of administration of intramuscular streptomycin; parenteral gentamicin can be used as an alternative drug; for pre-exposure prophylaxis a live, attenuated vaccine is available. However, for antimicrobial prophylaxis, oral administration of doxocycline or ciprofloxacin is advised for a 14-day period following the last day of exposure. Biosafety Level 2 practices are recommended for routine manipulations of clinical specimens from human and animals. Biosafety Level 3 practices are recommended for manipulations including risk of infectious aerosol production (Bhalla and Warheit, 2004). 3.3 Virus Variola major and Poxviridae. Poxviridae comprise a family of genetically related, large, enveloped, DNA viruses that replicate exclusively within the cytoplasm of vertebrate or invertebrate cells (Moss, 2007). Only member of the genus Orthopoxvirus, which includes smallpox, monkeypox, vaccinia, and cowpox can infect humans. Of these, only smallpox is readily transmitted from person to person via saliva or nasal secretion droplets and contaminated objects. The most common clinicopathologic presentation of smallpox was a systemically virulent form of the disease known as variola major with a case mortality rate of up to 30 to 40%. Saliva or nasal secretion droplets from infected individual are responsible of inter-human transmission. After oropharyngeal or respiratory mucosa infection, and the asymptomatic, non-infectiuous period of incubation (7- 17 days), many patients present the high fever and the malaise of prodromal illness. A maculopapular rash then appears on the mucosa of the mouth and pharynx, face, and forearms, and spreads to the trunk and legs. This is the most contagious stage because of the high viral titers present in the oropharyngeal tissues. Within 1–2 days, that rash becomes vesicular and later pustular. Scabs subsequently develop that, if the person survives, leave pitted scars called pocks from which the word pox has been derived. More severe but much less common manifestations of variola major, known as malignant or hemorrhagic smallpox, are associated with a near 100% case fatality rate (Fenner et al., 1988). Humans are the only known hosts of the virus, facilitating the global Variola eradication, by the World Health Organization (WHO) in 1980 after a successful global vaccination campaign, which was subsequently discontinued (Fenner et al., 1988). The cessation of vaccination not only expose populations to the risk of a bioterrorist attack but also is increasing prevalence of zoonotic poxvirus such as monkeypox (Rimoin et al., 2010). Currently there are no available treatments for smallpox infection and the therapy involves supportive care as antipyretic and anti-inflammatory treatments to relieve pain and fever. Antibiotics are prescribed for eventual bacterial super-infections. Filoviridae. Filoviridae family (from latin filum, referring to shape of virion), consists of enveloped, negative-stranded, RNA viruses that cause severe zoonotic hemorrhagic fever in humans and nonhuman primates. The family includes two distinct genera: Marburgvirus and Ebolavirus. The genus Marburgvirus includes a single species, Marburg marburgvirus, which has two members, Marburg virus (MARV) and Ravn virus (RAVV). The genus Ebolavirus includes five species, each of which has a single member: Zaire ebolavirus (Ebola virus, EBOV), Sudan ebolavirus (Sudan virus, SUDV), Taï Forest ebolavirus (Taï Forest virus, TAFV), Bundibugyo ebolavirus (Bundibugyo virus, BDBV) and Reston ebolavirus (Reston virus, RESTV) (Adams & Carstens, 2012). The natural reservoir hosts of these viruses have not yet been identified. However, Ebola virus RNA has been detected in terrestrial mammals in the Central African. Evidence is emerging that African, Asian and possibly also European bats are natural reservoirs of filoviruses and these animals could transmit the virus directly to humans or via intermediate hosts, including gorillas and swine. Following transmission to humans, spread of the virus between individuals is the result of direct contact with blood or other body fluids from infected patients. Filoviruses exhibit different virulence in humans: EBOV and MARV infection is associated with case-fatality rates of up to 90% while RESTV seems to be apathogenic (Sanchez et al., 2006; Kuhn et al., 2011). In infected individuals, after the incubation period, ranging from 2 to 21 days, the onset of illness begins with generic flu-like symptoms characterized by high fever, severe headache and malaise followed by gastrointestinal symptoms including abdominal pain, severe nausea, vomiting, and watery diarrhea. The majority of patients also presents clear hemorrhagic manifestations such as ecchymoses, mucosal bleeding, and hematemesis. Fatalities typically occur 8–16 days following the onset of symptoms, with death usually the resulting of severe diffuse coagulopathy, multiorgan failure, shock and coma (Brauburger et al., 2012). There is no a specific therapy against filoviral infections and supportive care are provided to limit symptoms. (Clark et al., 2012). Due to the lack of approved therapeutics or vaccines along with the high lethality and infectivity, work with Filoviridae is restricted to high-containment Biosafety Level 4 (BSL-4) laboratories (U.S. Department of Health and Human Services, 2009). Arenaviridae. Arenaviridae family consists of enveloped, negative-stranded, bi-partite RNA viruses that cause chronic infections in rodents (animals) and zoonotically acquired disease in humans (Salvato et al., 2011). The genus Arenavirus includes 22 viral species which, based on genetic and geographical data are divided into 2 groups: the Old World (OW) and the New World (NW) complex. OW complex includes the world-wide distributed Lymphocytic choriomeningitis virus (LCMV), causing acute aseptic meningocephalitis in human and other viruses endemic to the African continent including Lassa virus (LASV) and Lujo virus (LUJV) causing hemorrhagic fever (HF). The larger group of NW arenavirus is further divided into 3 clades (A, B and C). Clade B is the more relevant in term of human pathology, since it contains most of HF-causing arenaviruses in South America. (Charrel & de Lamballerie, 2003). Virus transmission occurs usually through human contact with excretions or materials contaminated with the excretions of an infected rodent though secondary person-to-person transmission can occur with some arenaviruses, such as Lassa, Machupo, and Lujo viruses (Weber & Rutala, 2001). After 1-2 weeks incubation period, HFV infection produces a wide range of symptoms and pathology including headache, cough and sore throat, nausea, vomiting and diarrhea. Several complications can arise: pleural effusions, neurological complications, facial edema and bleeding from mucosal surface. Advanced stages of disease is often associated with shock and death (Schattner et al., 2013). No licensed vaccines as well as prophylactic or therapeutic treatments are available against arenavirus infection. Currently therapy consists of ribavirin administration, accompanied by supportive care (Vela, 2012). BSL-4 containment is required for all pathogenic hemorrhagic fever-causing arenaviruses while BSL 2/3 laboratory environment is advised for handling of other arenaviruses (U.S Department of Health and Human Services, 2009). Table 1: Fatality rate. Biological Agent Fatality rate (%) Reference Bacteria Bacillus anthracis Cutaneous: <1% * Respiratory: 75% Gastrointestinal: 25%-60% Clostridium botulinum Foodborne: 3-5% Wound and intestinal: 15% Yersinia pestis 8-10% ** Francisella tularensis Subspecies tularensis: 2% ***; † Subspecies holarctica: fatal cases are rare *** Virus Variola major 30% * Filoviridae 90% †† Arenaviridae 15-30% ††† *CDC, 2013; ** WHO, 2004; *** WHO, 2007; † Dennis et al., 2001; †† Warfield et al., 2005; ††† Briese et al., 2009. 4. DISCUSSION AND CONCLUSIONS The use of biological agents as weapons has its roots in ancient times, when the concepts of bacteria, toxin or virus were not known yet. Over 2000 years ago, rudimentary techniques of biological warfare resolved the first disputes among people; hand by hand with the evolution of modern science (especially in the XX century), the possibility of using biological agents as weapons has been refined. In the last decades the development of innovative biotechnology techniques has provided the knowledge to create more aggressive bioweapons. These new organisms give rise to great concern, because they can produce devastating and completely unexpected effects, of the same level or even higher than the most dangerous wild type biological agents. Although international conventions prohibit the use of biological agents with offensive purposes, it is known that many terrorist groups continue their research about the possible use of biological agents as weapons. The concerns related to biological agents are aroused, as well as the effects in terms of victims, both from the objective difficulties in the detection of a potential attack. A release of biological agents is difficult to detect with current technology, especially when it comes to a stand-off revelation compared to a point detection. 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Cenciarelli, O., Rea, S., Carestia, M., D'Amico, F., Malizia, A., Bellecci, C., et al. (2013). Biological Weapons and Bio-Terrorism : a review of History and Biological Agents. INTERNATIONAL JOURNAL OF INTELLIGENT DEFENCE SUPPORT SYSTEMS, VI(2), 111-129.

Biological Weapons and Bio-Terrorism : a review of History and Biological Agents.

BELLECCI, CARLO;GAUDIO, PASQUALINO;FIORITO, ROBERTO
2013-11-29

Abstract

ABSTRACT Biological weapons are a thorny issue, for their destructive capabilities and for the potential to generate panic and terror among the affected people. Used since pre-Christian times, biological weapons have resulted in the decimation of whole populations and have changed the geopolitics of several places. In this work, a summary of the main war and terrorist activities carried out by biological weapons over the time is presented. Moreover, main biological warfare agents and related pathology are considered, according to American Centers for Disease Control and Prevention (CDC) priority classification. At last, the emerging more destructive potential of biological agents, due to the widespread introduction of biotechnology, is analyzed. 1. INTRODUCTION DaSilva, in his work published in 1999, defines the Biological warfare as the intentional use of microorganisms, and toxins, generally of microbial, plant or animal origin to produce disease and death in humans, livestock and crops (DaSilva, 1999). Biological warfare and bioterrorism are very complex subjects, mainly due to the many agents that can be used as a weapon and for the wide range ways for the dissemination in the environment and population. A biological event provides for the presence of at least two actors: one or more pathogens (bacteria, viruses, or toxins) and a vehicle for their dissemination. What makes biological threat so troubling? In addition to high spread capacity and lethality of biological agents, the invisibility and the extremely difficult short-term revelation makes impossible an immediate contrast with the subsequent increase of the affected. In fact, most of biological weapons (unless of toxins) have a peculiar quality that other non-conventional weapons (chemical and radiological ones) do not show: biological agents are able to multiply in the host organism and be transmitted in turn to new hosts, generating in this way not predictable effects on the population, both in terms of number of victims and in terms of spread geography. Among the reasons which make biological weapons attractive, there is their very low cost when compared to both conventional and unconventional ones. NATO sources, according to data processed in 1969 by U.S. experts, reported the following costs for an attack on an area of 1 km2 to civilian populations with different weapons: a) $1/Km2 for biological weapons, b) $600/Km2 for chemical, c) $800/Km2 for nuclear, d) $2,000/Km2 for conventional armaments (NATO, 1996). Therefore, it seems clear therefore that, in financial terms, an attack with biological weapons would be strongly cheaper when compared to any other one. In the second decade of the 21st century, to make the situation more troubling, there is the simplicity with which it is possible to produce large quantities of biological agents with facilities and expertise available to everyone, even to terrorist and paramilitary groups. The aim of this work is to present an overview on biological warfare and bioweapons, as well as to assess the state of the art on the actual offensive capability through this unconventional weapon. 2. HISTORYCAL 2.1 Pre Word Wars age The use of biological agents as war weapons is not a modern era novelty. Although it isn’t easy to identify a definite time when the use of bioweapons began, ancient information reported that in pre-Christian era, around 300 B.C., Greeks were using animal cadavers to contaminate water wells of the opponent. This strategy was also used the by Romans and the Persians (SIPRI, 1971). In a later period, during the battle of Tortona (Italy) in 1155, bodies of dead soldiers and animals were used to contaminate water wells by Barbarossa (Clarke, 1968). In the 14th century, during the siege of Kaffa by Tartar (now Feidisuja, Ukraine, a city on the Black Sea at that time under the control of the Genoese), a biological attack allowed Tartars to win the siege. During the siege, among the Tatar army, an epidemic of plague was spread: the besiegers thought to shoot the cadavers of their dead comrades within the walls of the city of Caffa (Wheelis, 2002). This resulted in a turning point in the war: the Genoese fled from Caffa, carrying with them the sick. On the return trip to Genoa, they landed several ports in the Mediterranean sea; although some sources believe a possible correlation between the epidemic of plague in Caffa and the pandemic that decimated the population of Europe in the following decades (Black Death), most of the authors share the view of two events independent (Wheelis, 2002). In 1422, during the siege of Carolstein, Lithuanian soldiers catapulted within the city cadavers of death soldiers and 2,000 chariots of excrement (Newark, 1988), frightening the population affected and spreading lethal fevers in many cases. We will have to wait for more than three centuries to find a new documented use of biological agents as a war weapon: during the French-Indian War (1754-1767), the British commander Sir. Jeffrey Amherst ordered the distribution of blankets infected with smallpox to decimate the population of Indian tribes hostile to the British (Bhalla and Warheit, 2004). The distribution of infected blankets occurred in the summer of 1763, and the resurgence of the virus among the indigenous lasted for over 200 years (Riedel, 2004). 2.2 World War I and II Several biological warfare actions carried out during the World War are not sufficiently confirmed in the literature. However is frequently reported that Germans inoculated cattle with Bacillus anthracis and Pseudomonas mallei, responsible to cause severe diseases such as anthrax and glanders, before sending them in enemy states (SIPRI, 1971; Poupard and Miller, 1992; Hugh-Jones, 1992). It should be considered that the First World War saw the large-scale use of non-conventional chemical weapons, therefore it must be expected until the Second World War to find a more extensive use of biological weapons. During World War II many countries conducted research programs on the development of bioweapons; the Japanese program, conducted under the direction of Captain Shiro Ishii, was certainly most ambitious (1892-1959). The research in this direction started in 1928; during this year Captain Ishii visited many European and American countries in order to learn useful techniques and information about the possible uses of biological weapons. Returned to homeland, Captain Ishii was substantial granted in order to constitute a massive bioweapons research center, called the 731 units, located at Beiyinhe, in Manchuria. The research center could count on a staff of over 3,000 scientists, mainly microbiologists. The experiments were conducted on pow, principally Koreans, Chinese and Russian soldiers. Prisoners were used to test numerous bioweapons, including Yersinia pestis, Vibrio cholera, Neisseria meningitidis and Bacillus anthracis (Leitenberg, 2001). Christopher and colleagues report that during this research, several thousand prisoners died as a result of the experiments conducted on them (Christopher et al., 1997). However, the mortality rate around the area 731 remains very high for several years. If we consider for the total count these deaths also, we reach the considerable sum of 200,000 deaths as a result of the activities carried out by Captain Ishii (Harris, 2002). In 1942, the wrong control of the infection spread resulted in the death of 1,700 Japanese soldiers (Sokolski and Ludes, 2001). Many other nations carried out experiments on potential biological agents, but information reported in the literature are rather skinny. It is also important to remember the experiments conducted in 1942 by the British army on the Island of Gruinard, off the Scotland coast, where anthrax dirty bombs were tested (Manchee et al., 1981). The island was contaminated and uninhabitable until 1990, when an extensive land decontamination was carried out (Aldhous, 1990). 2.3 Post World Was age Until World War II, United States remained considerably behind respect the other nations in the research fields on biological weapons. In the United States, the golden age for both the test and development of bioweapons in the United States was immediately after the conclusion of World War II, when U.S. received the results of the experiments performed by the Japanese unit 731. The U.S. could also rely on working directly with Captain Ishii, the former director of the unit 731 (Christopher et al., 1997). In September of 1950, the U.S. Navy conducted an experiment on civilians in order to assess the vulnerability of a large American coastal town to a biological attack: in the San Francisco Bay a cloud of Serratia marcescens (a low pathogenic bacterium mainly responsible for infections of skin and respiratory tract) was spread by boat. The infection struck, as a result of subsequent checks, almost the entire population (1 million people). Even though the bacterium had to be almost harmless, several individuals showed the effects of respiratory diseases and some of them died (Christopher et al., 1997). Few years later (1956-1958), in Georgia and Florida, swarms of mosquitoes, probably carriers of yellow fever, were released in order to verify the vulnerability to an air attack. Even though the documents are still top secret, several sources report that some individuals died for the bite of insects. A last large scale experiment which is documented, consists in the dissemination of Bacillus subtilis in the New York subway in the summer 1966. The experiment resulted in the infection, although without consequences, of more than one million people. It demonstrated that the spread of a pathogen in the whole subway network from a single station, due to the displacement of air in the tunnels, was possible (Zygmunt, 2006). In the seventies, USSR conducted an ambitious research program on biological weapons; however, unlike the United States programs, on which the secrecy has been partially removed, an aura of mystery on Russian research programs still remains. Some information about the research carried out in the USSR are provided in the paper published by Davis in 1999 on Emerging Infectious Disease. The USSR, between 1973 and 1974, formed an organization called "Chief Directorate for Biological Preparation" (Biopreparat), with the purpose to develop and produce biological weapons. Although there are no unambiguous data about the number of individuals employed by the Biopreparat, it is believed that more than 50,000 people were working in the whole system connected to the structure, including scientists and technicians, who could count on 52 research and producing factories. In these structures, high amounts of etiologic agents of plague, tularemia, anthrax, glanders, smallpox and Venezuelan equine encephalomyelitis were studied and produced. Not only biological agents from natural sources were studied in the Soviet Union. The Soviets studied and applied technologies of genetic engineering in order to increase the aggressiveness of biological agents through biotechnology The aim of this work was the production of a new more dangerous, more easily spread and more difficult to identify and combat generation of biological weapons (Davis, 1999). Among the countries that developed a massive program on bioweapons research, in the post-war era, we should quote Iraq. Iraq started its research and development program in the field of biological warfare in 1974, contextualizing it in an organization called "State Organization for Trade and Industry" (Davis, 1999). The program consisted on the study and production of botulinum toxin, anthrax, aflatoxin and ricin, as well as antiplants and viral agents (Rotavirus, Infectious Hemorrhagic Conjunctivitis, Camel Pox) (Leitenberg, 2001). The program involved about 300 scientists, who completed their training in Western Europe countries (Leitenberg, 2001). 2.4 International treaties The first measures against the use of biological weapons were taken in the 19th century during the Hague Conference in 1899, and then confirmed in the same place in 1907: with the document "Laws and Customs of War on Land", signed by 24 countries, the prohibition against the use of poisoned arms was ratified (Leitenberg, 2001). In 1925, with the awareness of the horrors of World War I, especially as regards the use of chemical weapons, the Geneva Protocol was signed. It "Prohibited the use in war of Asphyxiating, Poisonous or Other Gases, and of Bacteriological methods of Warfare". Although this treaty was signed by a considerable number of nations (even though it was ratified by the U.S. not until the mid-seventies), it prohibited the use of biological agents as weapons only, but not their development or their stocking (Christopher et al., 1997). In view of the limited effectiveness of the Protocol of Geneva in the control of the bioweapons development and proliferation, in 1972 the "Conventions on the Prohibition of the Development, Production and Stockpiling of Bacteriological (Biological) and Toxin Weapons and on Their Desctruction"was initialed. Initially signed by over 100 nations, the Convention became effective in 1975; however this convention, such as that Geneve one, has several loopholes: first of all, it does not provide guidelines for the protocol compliance verification; moreover it prohibits the use and development of bioweapons in “quantities that have no justification for prophylactic, protective or other peaceful purposes” (Riedel, 2004). However, it is evident how this assertion is open to interpretation; de facto it does not define threshold quantities or substantial limitations to the development and production of bioweapons (SIPRI 1971 b, 1973). The events consequent to the ratification of the Biological Weapons Convention of 1972, up to the most recent, have confirmed this observation. 2.5 Last decades: bioterrorism and biotechnology advent Even after the entry into force of the "Conventions on the Prohibition of the Development, Production and Stockpiling of Bacteriological (Biological) and Toxin Weapons and on Their Destruction", a large number of countries went on to develop, produce and test biological agents for military purposes. Since the 80s, increasingly terrorist groups have begun to consider the bioweapon as an economic and highly destabilizing way for civil society. It must be considered that the large scale advent of biotechnology, and the reduced difficulty in genetically modified organisms producing, has made the potential creation of multi-drug resistant pathogens, or with enhanced virulence factors possible. The use of biological agents in the last decades is mainly attributable to terrorist groups, more or less isolated, who used the bioweapons as a strategy to defend extremist religious ideas striking the civilian population or sensible government targets. In 1984 in The Dalles, Oregon, United States, a group of extremist followers of Bhagwan Shree Rajneesh (also known as Osho), contaminated the salad in 10 different salad bar with the pathogen of salmonellosis, Salmonella thyphimurium, in order to disable the population. 751 people contracted the disease and several of them were hospitalized; although there were no fatalities, this terrorist act is considered the largest bioterrorist attack in the history of the United States for involved people (Török et al., 1997). In the nineties the Japanese cult of Aum Shinrikyo tested different biological weapons, including botulin toxin, anthrax, cholera, and Q fever; in 1993, during a humanitarian mission in Africa, it tried to obtain samples of the Ebola virus. Between 1990 and 1995, the cult attempted to carry out several bioterrorist actions in Tokyo using vaporized biological agents including botulinum toxin and anthrax spores. Fortunately, the attacks were unsuccessful (Olson, 1999). A significant bioterrorist event happened in the United States contextually to the dramatic attacks to the World Trade Center in New York in September 2001. The release of Bacillus anthracis spores through the U.S. postal system was carried out with letters addressed to the press and to government officials. There were 22 confirmed cases of anthrax contamination, 12 cutaneous and 10 inhalational cases. The 12 cutaneous responded positively to the antibiotic treatment, while of the 10 inhalational cases, 4 were fatal (McCarthy, 2001). In 2002, in Manchester, UK, six terrorists were arrested for having been found in possession of ricin and in 2004, traces of the same toxin were found at the Dirksen Senate Office Building in Washington DC. So, it appears evident then that the use of biological agents has moved, in recent times, to terrorist groups. This creates very strong concerns: the use of biological weapons by terrorists can generate unexpected scenarios characterized by massive destructive potential. 3. BIOTERRORISM AND BIOWEAPONS The American Center for Disease Control and Prevention (CDC) defines a bioterrorism attack as “the deliberate release of viruses, bacteria or other germs (agents) used to cause illness or death in people, animals, or plants” (CDC, 2013). CDC classifies the most important bioterrorism agents into three categories (tab. 1): a) Category A: Agents that can be easily disseminated or transmitted from person to person. They result in high mortality rates and have the potential for major public health impact. They might cause public panic and social disruption, and require special action for public health preparedness. b) Category B: Agents that are moderately easy to disseminate. They result in moderate morbidity rates and low mortality, and require specific enhanced diagnostic capacity and disease surveillance. c) Category C: Emerging agents that could be engineered for mass dissemination in the future because of their availability. They are easy to produce and disseminate. They are potentially linked to high morbidity and mortality rates, and major health impact. Table 1: Major biological agents possible used as bioweapons (CDC, 2013). Groups Diseases Agents A Anthrax Bacillus anthracis Botulism Clostridium botulinum toxin Plague Yersinia pestis Smallpox Variola major Tularemia Francisella tularensis Viral hemorrhagic fevers Filoviruses and Arenaviruses B Brucellosis Brucella spp. Epsilon toxin Clostridium perfringens Food safety threats Salmonella spp., E.coli O157:H7, Shigella Glanders Burkholderia mallei Melioidosis Burkholderia pseudomallei Psittacosis Chlamydia psittaci Q fever Coxiella burnetii Ricin toxin Ricinus communis Staphylococcal enterotoxin B Staphylococcus spp. Typhus fever Rickettsia prowazekii Viral encephalitis Alphaviruses Water safety threats Vibrio cholerae, Cryptosporidium parvum C Emerging infectious diseases Nipahvirus and Hantavirus Generally, biological agents (included those used as weapons) can be further classified according to certain characteristics that define the hazard to health (NATO, 1996): a) Infectivity: The aptitude of an agent to penetrate and multiply in the host; b) Pathogenicity: The ability of the agent to cause a disease after penetrating into the body; c) Transmissibility: The ability of the agent to be transmitted from an infected individual to a healthy one; d) Ability to neutralize: It means to have preventive tools and/or therapeutic purposes. Biological agents can be transmitted through one or more ways. The transmission modes are the following (La Placa, 2010): a) Parenteral: Agents that are transmitted through body fluids or blood; b) Airway (by droplets): Agents that are emitted by infected people, which can then be inhaled by surrounding people; c) Contact: Through which the agents present on the surface of the infected organism can infect another organism; d) Oral-fecal route: Through objects, foods or other items contaminated with the feces of infected patients, or through sexual contact. 3.1 Bioweapons There are numerous pathogens (bacteria, viruses and toxins) that cause diseases in humans, animals and plants; however, only very few possess the characteristics to be a bioweapon. In literature, in 1997 Eitzen describes the characteristics that make a biological agent a potential bioweapon. Ideally, a biological weapon should be easy to find or to produce. In order to develop a biological attack towards sensitive targets or the population, large amounts of biological agents are in fact required; it must be considered that it is necessary quite a number of biological agents (or a certain amount of toxin) to generate a disease in an target. The ideal bioweapon also must hold a high capacity to incapacitate the affected or, alternatively, be highly lethal. It is appropriate to choose an agent with an incubation period depending on whether immediate or delayed effects are required. Other important characteristics for a biological weapon are the route of transmission, and hence, the ease of dissemination with an appropriate method of delivery. Finally, the stability of the agent must be assessed, especially when large quantities must be stored for indefinite periods (Eitzen, 1997). Below some of the key features of the most relevant biological agents (included in A category by CDC) are reported, categorized according to the biological origin. 3.2 Bacteria Bacillus anthracis. Bacillus anthracis is a Gram-positive, non-motile, facultative anaerobic endospore forming bacteria, usually surrounded by a capsule; it is the etiological agent of anthrax. The disease occurs most frequently when an epizootic or enzootic of herbivores becomes infected after acquiring spores from direct contact with contaminated soil. In humans, the disease can occur when exposed to infected animals, tissue from infected animals or high concentrations of anthrax spores. Antrhax endospores have no measurable metabolism, do not divide, and are resistant to drying, heat, ultraviolet and ionizing radiation, chemical disinfectant and other forms of stress, remaining not a word in the environment for years (Bhalla and Warheit, 2004): survival in soil for up 200 years has been reported (Yuen, 2001). The disease is caused by the action of a toxin produced by the vegetative bacillus; the toxin consists of three components: protective antigen (PA), edema factor (EF) and lethal factor (LF). PA binds to cell receptors, mediating the entry of EF and LF into the cell. Another anthrax virulence factor is the D-glutamic acid polypeptide capsule of the vegetative form (WHO, 2004). Three types of anthrax infection can occur: cutaneous, inhalation and gastro intestinal; cutaneous form is the most common and is characterized by dermal ulcers, painless, non-scarring, pruritic papule progressing to a black depressed eschar with swelling of adjacent lymph glands and oedema (WHO, 2004). Local lymphadenitis and fever can occur, but septicaemia is rare (Moquin and Moquin, 2002); untreated cutaneous anthrax can become systemic and it is fatal in 5-20% of cases. Gastro-intestinal and inhalation form are less common; inhalation starts with influenza-like symptoms that include fever, fatigue, chills, non-productive cough, vomiting, sweats, myalgia, dyspnoea, confusion, headache and chest and/or abdominal pain, followed by the development of cyanosis, shock, coma and death. Gastro-intestinal form is characterized by fever, nausea, vomiting, abdominal pain and bloody stools. Oropharyngeal infection, on the other hand, is accompanied by oedematous swelling of the neck, often followed by fever and lymphoid involvement (WHO, 2004). There is no evidence of direct person-to-person spread (Yuen, 2001). After exposure, the incubation period is reported to range from 1 to 7 days, possibly extending up to several weeks. Some vaccines are administered to prevent the disease, such as live spore vaccines based on attenuated strains, and cell-free vaccines based on anthrax protective antigen (PA) (WHO, 2004). Regarding therapy, there are three types of antibiotics that are effective against B. anthracis: ciprofloxacin, tetracyclines and penicillins (Bhalla and Warheit, 2004). For laboratory diagnosis and research, manipulations involving clinical specimens Biosafety Level 2 practices are recommended. However, for manipulations involving activities with a significant aerosol production, Biosafety Level 3 practices are advised (WHO, 2004). Clostridium botulinum. Clostridium botulinum is a spore forming and obligate anaerobe, etiological agent of botulism; it can be isolated from the soil, its natural habitat. Four species of C. botulinum are known, characterized by different genomes and their common botulinum toxin. In addition, 7 distinct antigenic types of botulinum toxin (A-G) are defined by the absence of cross-neutralization. The toxin is responsible for the disease and it is a dichain polypeptide: a heavy chain of 100 KDa is joined by a single disulfide bond to a 50 KDa light chain that is a Zn- containing endopeptidase that blocks acetylcholine-containing vesicles from fusing with the terminal membrane of the motor neuron, resulting in flaccid muscle paralysis (Arnon et al., 2001). Botulinum toxins are the most lethal toxins known and all seven types act in similar ways. Death often occurs as a result of paralysis of pharyngeal and diaphragmatic muscles, followed by respiratory arrest (Bhalla and Warheit, 2004). Three forms of human botulism exist: food-borne, wound, and intestinal. All forms of botulism are caused by absorption of botulinum toxin into the circulation from a wound or mucosal surface; after infection, the incubation period depends on the rate and amount of toxin absorption: from 2 hours to 8 days. Patients affected by botulism are a febrile and present symmetric, descending flaccid paralysis with prominent bulbar palsies. Therapy consists of passive immunization with equin antitoxin, accompanied by supportive care. Botulism can be prevented by administration of a pentavalent (ABCDE) botulinum toxoid; a recombinant vaccine is in development. Biosafety Level 2 practices are recommended for manipulations in laboratory and Biosafety Level 3 practices are suggested for activities with high potential for aerosol or droplet production (Arnon et al., 2001). Yersinia pestis. Yersinia pestis is a Gram-negative non-motile, non-spore-forming coccobacillus, that grows both in aerobic and anaerobic conditions. It can remain viable for days in moist soil or water, but it is killed by direct exposure to sunlight (WHO, 2004). Bacterium is the etiological agent of plague, a disease that can affect humans and animals (La Placa, 2010); wild rodents are the pathogen reservoirs and transmission to other animals occurs through fleas, infected animal tissues, contaminated soil or respiratory droplet exposures. In endemic rural areas, persons who come in contact with wild rodent hostes of Y. pestis can be affected by the plague, which exists in two forms: bubonic plague and pneumonic plague (WHO, 2004). The first would result if fleas were used as carriers of disease; in this case, the incubation period is 2-6 days after exposure. Swelling of the Lymph nodes occurs (bubones), associated with onset of fever, chills, headache, followed by nausea and vomiting. Untreated bubonic plague causes septicemia. Pneumonic plague can occur from inhaling organisms or from exposure to infected blood; productive cough with blood-tinged sputum is a typical symptom of pneumonic plague, that can spread from person to person by coughing (La Placa, 2010). If started soon after infection, antimicrobial therapy is effective. It consists of administration of streptomycin or gentamicin. Alternative antimicrobial substances are: tetracyclines, doxycyclines, chloramphenicol, fluoroquinolones, ciprofloxacin, sulfonamides. Plague vaccine is advised only for high-risk groups (laboratory personnel): vaccination with killed or live attenuated Y. pestis is effective against bubonic but not against pneumonic plague. Biosafety Level 2 practices are recommended for activities involving infective materials and cultures. Biosafety Level 3 may be used in the case of high production of infectious aerosol or direct contact with infected fleas (WHO, 2004). Francisella tularensis. Francisella tularensis is a small, Gram-negative, non-motile, facultative intracellular, aerobic coccobacillus. It is responsible of tularemia, a zoonotic disease. Two bacterium sub-species exist: F. tulariensis tulariensis (Type A) and F. tularensis palaearctica (Type B). Type A is more virulent than Type B (WHO, 2004). The organism can survive for up to several weeks in soil, water, straw and soil (Bhalla and Warheit, 2004). Many wild animals (rabbits, beavers, muskrats, hares, voles) are bacterium reservoirs. Humans can be infected when bitten by arthropods, by ingestion of contaminated food and water, inhalation of contaminated aerosols; direct contact with infected animals is also dangerous for humans, but person-to-person transmission has not been observed. After infection, the incubation period is generally 3-5 days, but it can extend up to 14 days. Symptoms of the disease depend on the virulence of the infectious agent; furthermore two different clinical manifestations exist: ulceroglandular (75% of cases) and typhoidal (25% of cases) tularemia. The first is characterized by indolent ulcer at the site of entry and painful swelling of local lymph glands; the expression “typhoidal tularemia” indicates systemic illness without apparent site of primary infection. Painful pharyngitis and cervical lymphadenitis result from infection through ingestion of contaminated food or water (Bhalla and Warheit, 2004). It consists of administration of intramuscular streptomycin; parenteral gentamicin can be used as an alternative drug; for pre-exposure prophylaxis a live, attenuated vaccine is available. However, for antimicrobial prophylaxis, oral administration of doxocycline or ciprofloxacin is advised for a 14-day period following the last day of exposure. Biosafety Level 2 practices are recommended for routine manipulations of clinical specimens from human and animals. Biosafety Level 3 practices are recommended for manipulations including risk of infectious aerosol production (Bhalla and Warheit, 2004). 3.3 Virus Variola major and Poxviridae. Poxviridae comprise a family of genetically related, large, enveloped, DNA viruses that replicate exclusively within the cytoplasm of vertebrate or invertebrate cells (Moss, 2007). Only member of the genus Orthopoxvirus, which includes smallpox, monkeypox, vaccinia, and cowpox can infect humans. Of these, only smallpox is readily transmitted from person to person via saliva or nasal secretion droplets and contaminated objects. The most common clinicopathologic presentation of smallpox was a systemically virulent form of the disease known as variola major with a case mortality rate of up to 30 to 40%. Saliva or nasal secretion droplets from infected individual are responsible of inter-human transmission. After oropharyngeal or respiratory mucosa infection, and the asymptomatic, non-infectiuous period of incubation (7- 17 days), many patients present the high fever and the malaise of prodromal illness. A maculopapular rash then appears on the mucosa of the mouth and pharynx, face, and forearms, and spreads to the trunk and legs. This is the most contagious stage because of the high viral titers present in the oropharyngeal tissues. Within 1–2 days, that rash becomes vesicular and later pustular. Scabs subsequently develop that, if the person survives, leave pitted scars called pocks from which the word pox has been derived. More severe but much less common manifestations of variola major, known as malignant or hemorrhagic smallpox, are associated with a near 100% case fatality rate (Fenner et al., 1988). Humans are the only known hosts of the virus, facilitating the global Variola eradication, by the World Health Organization (WHO) in 1980 after a successful global vaccination campaign, which was subsequently discontinued (Fenner et al., 1988). The cessation of vaccination not only expose populations to the risk of a bioterrorist attack but also is increasing prevalence of zoonotic poxvirus such as monkeypox (Rimoin et al., 2010). Currently there are no available treatments for smallpox infection and the therapy involves supportive care as antipyretic and anti-inflammatory treatments to relieve pain and fever. Antibiotics are prescribed for eventual bacterial super-infections. Filoviridae. Filoviridae family (from latin filum, referring to shape of virion), consists of enveloped, negative-stranded, RNA viruses that cause severe zoonotic hemorrhagic fever in humans and nonhuman primates. The family includes two distinct genera: Marburgvirus and Ebolavirus. The genus Marburgvirus includes a single species, Marburg marburgvirus, which has two members, Marburg virus (MARV) and Ravn virus (RAVV). The genus Ebolavirus includes five species, each of which has a single member: Zaire ebolavirus (Ebola virus, EBOV), Sudan ebolavirus (Sudan virus, SUDV), Taï Forest ebolavirus (Taï Forest virus, TAFV), Bundibugyo ebolavirus (Bundibugyo virus, BDBV) and Reston ebolavirus (Reston virus, RESTV) (Adams & Carstens, 2012). The natural reservoir hosts of these viruses have not yet been identified. However, Ebola virus RNA has been detected in terrestrial mammals in the Central African. Evidence is emerging that African, Asian and possibly also European bats are natural reservoirs of filoviruses and these animals could transmit the virus directly to humans or via intermediate hosts, including gorillas and swine. Following transmission to humans, spread of the virus between individuals is the result of direct contact with blood or other body fluids from infected patients. Filoviruses exhibit different virulence in humans: EBOV and MARV infection is associated with case-fatality rates of up to 90% while RESTV seems to be apathogenic (Sanchez et al., 2006; Kuhn et al., 2011). In infected individuals, after the incubation period, ranging from 2 to 21 days, the onset of illness begins with generic flu-like symptoms characterized by high fever, severe headache and malaise followed by gastrointestinal symptoms including abdominal pain, severe nausea, vomiting, and watery diarrhea. The majority of patients also presents clear hemorrhagic manifestations such as ecchymoses, mucosal bleeding, and hematemesis. Fatalities typically occur 8–16 days following the onset of symptoms, with death usually the resulting of severe diffuse coagulopathy, multiorgan failure, shock and coma (Brauburger et al., 2012). There is no a specific therapy against filoviral infections and supportive care are provided to limit symptoms. (Clark et al., 2012). Due to the lack of approved therapeutics or vaccines along with the high lethality and infectivity, work with Filoviridae is restricted to high-containment Biosafety Level 4 (BSL-4) laboratories (U.S. Department of Health and Human Services, 2009). Arenaviridae. Arenaviridae family consists of enveloped, negative-stranded, bi-partite RNA viruses that cause chronic infections in rodents (animals) and zoonotically acquired disease in humans (Salvato et al., 2011). The genus Arenavirus includes 22 viral species which, based on genetic and geographical data are divided into 2 groups: the Old World (OW) and the New World (NW) complex. OW complex includes the world-wide distributed Lymphocytic choriomeningitis virus (LCMV), causing acute aseptic meningocephalitis in human and other viruses endemic to the African continent including Lassa virus (LASV) and Lujo virus (LUJV) causing hemorrhagic fever (HF). The larger group of NW arenavirus is further divided into 3 clades (A, B and C). Clade B is the more relevant in term of human pathology, since it contains most of HF-causing arenaviruses in South America. (Charrel & de Lamballerie, 2003). Virus transmission occurs usually through human contact with excretions or materials contaminated with the excretions of an infected rodent though secondary person-to-person transmission can occur with some arenaviruses, such as Lassa, Machupo, and Lujo viruses (Weber & Rutala, 2001). After 1-2 weeks incubation period, HFV infection produces a wide range of symptoms and pathology including headache, cough and sore throat, nausea, vomiting and diarrhea. Several complications can arise: pleural effusions, neurological complications, facial edema and bleeding from mucosal surface. Advanced stages of disease is often associated with shock and death (Schattner et al., 2013). No licensed vaccines as well as prophylactic or therapeutic treatments are available against arenavirus infection. Currently therapy consists of ribavirin administration, accompanied by supportive care (Vela, 2012). BSL-4 containment is required for all pathogenic hemorrhagic fever-causing arenaviruses while BSL 2/3 laboratory environment is advised for handling of other arenaviruses (U.S Department of Health and Human Services, 2009). Table 1: Fatality rate. Biological Agent Fatality rate (%) Reference Bacteria Bacillus anthracis Cutaneous: <1% * Respiratory: 75% Gastrointestinal: 25%-60% Clostridium botulinum Foodborne: 3-5% Wound and intestinal: 15% Yersinia pestis 8-10% ** Francisella tularensis Subspecies tularensis: 2% ***; † Subspecies holarctica: fatal cases are rare *** Virus Variola major 30% * Filoviridae 90% †† Arenaviridae 15-30% ††† *CDC, 2013; ** WHO, 2004; *** WHO, 2007; † Dennis et al., 2001; †† Warfield et al., 2005; ††† Briese et al., 2009. 4. DISCUSSION AND CONCLUSIONS The use of biological agents as weapons has its roots in ancient times, when the concepts of bacteria, toxin or virus were not known yet. Over 2000 years ago, rudimentary techniques of biological warfare resolved the first disputes among people; hand by hand with the evolution of modern science (especially in the XX century), the possibility of using biological agents as weapons has been refined. In the last decades the development of innovative biotechnology techniques has provided the knowledge to create more aggressive bioweapons. These new organisms give rise to great concern, because they can produce devastating and completely unexpected effects, of the same level or even higher than the most dangerous wild type biological agents. Although international conventions prohibit the use of biological agents with offensive purposes, it is known that many terrorist groups continue their research about the possible use of biological agents as weapons. The concerns related to biological agents are aroused, as well as the effects in terms of victims, both from the objective difficulties in the detection of a potential attack. A release of biological agents is difficult to detect with current technology, especially when it comes to a stand-off revelation compared to a point detection. 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Rilevanza internazionale
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Esperti anonimi
Settore MED/18 - Chirurgia Generale
English
Biological Warfare (BW); biohazards; chemical, biological, radiological and nuclear (CBRN); biowarfare; bioweapons.
www.mastercbrn.com
Cenciarelli, O., Rea, S., Carestia, M., D'Amico, F., Malizia, A., Bellecci, C., et al. (2013). Biological Weapons and Bio-Terrorism : a review of History and Biological Agents. INTERNATIONAL JOURNAL OF INTELLIGENT DEFENCE SUPPORT SYSTEMS, VI(2), 111-129.
Cenciarelli, O; Rea, S; Carestia, M; D'Amico, F; Malizia, A; Bellecci, C; Gaudio, P; Gucciardino, A; Fiorito, R
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