Responding to the threat of bioterrorism: a microbial ecology perspective – the case of anthrax

Anthrax is a disease of herbivores caused by the gram-positive bacterium Bacillus anthracis. It can affect cattle, sheep, swine, horses and various species of wildlife. The routes for the spread among wildlife are reviewed. There are three kinds of human anthrax – inhalation, cutaneous, and intestinal anthrax – which differ in their routes of infection and outcomes. In the United States, confirmation of cases is made by the isolation of B. anthracis and by biochemical tests. Vaccination is not recommended for the general public; civilians who should be vaccinated include those who, in their work places, come in contact with products potentially contaminated with B. anthracis spores, and people engaged in research or diagnostic activities. After September 11, 2001, there were bioterrorism anthrax attacks in the United States: anthrax-laced letters sent to multiple locations were the source of infectious B. anthracis. The US Postal Service issued recommendations to prevent the danger of hazardous exposure to the bacterium. B. anthracis spores can spread easily and persist for very long times, which makes decontamination of buildings very difficult. Early detection, rapid diagnosis, and well-coordinated public health response are the key to minimizing casualties. The US Government is seeking new ways to deter bioterrorism, including a tighter control of research on infectious agents, even though pathogens such as B. anthracis are widely spread in nature and easy to grow. It is necessary to define the boundary between defensive and offensive biological weapons research. Deterring bioterrorism should not restrict critical scientific research. Electronic Publication     

城市小区环境生物剂气溶胶的扩散模拟研究

目的:研究城市小区中生物剂气溶胶的扩散模拟和污染区域的划分,为反生物恐怖危害评估及应急响应提供决策依据。方法:以典型生物剂炭疽为例,利用计算流体力学中的离散相模型对小区环境中生物剂气溶胶的扩散规律进行研究;对扩散后生物剂气溶胶的数目分布进行量化分析,结合吸入式炭疽的剂量-反应模型进行污染区域的划分。结果:通过计算机模拟,得到了生物剂气溶胶在小区环境中的扩散规律及数目分布,并依据人员感染炭疽概率的不同划分出小区内的污染区域。结论:利用离散相模型和剂量-反应模型,可以对城市小区中生物剂气溶胶的扩散规律进行模拟并划分污染区域,为反生物恐怖危害评估及应急响应提供决策依据。     

Bacillus anthracis contamination and inhalational anthrax in a mail processing and distribution center

AIMS: Four inhalational anthrax cases occurred in a large mail processing and distribution center in Washington, DC, after envelopes containing Bacillus anthracis spores were processed. This report describes the results of sampling for B. anthracis spores during investigations conducted in October and December 2001. METHODS AND RESULTS: Wet swabs, wet wipes, vacuum sock, and air-filter samples were collected throughout the facility to characterize the extent of building contamination. The results showed widespread contamination of B. anthracis spores, particularly associated with one delivery bar code sorter (DBCS) machine that had sorted the spore-containing envelopes and an area where the envelopes were handled by postal workers. Spore concentrations decreased as distance from the DBCS machine increased, but spores were widely dispersed into surrounding areas. CONCLUSION: The spatial distribution of culture positive samples was closely related to the work areas of the inhalational anthrax cases and supported epidemiological evidence that the workers became ill from exposure to B. anthracis spores in areas where the contaminated envelopes had travelled. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this investigation were used to guide decontamination efforts and provided baseline spore concentrations for follow-up measurements after the building had been cleaned. Implementing methods to reduce aerosolization and dispersion of dust within the facility would reduce postal workers' potential exposures to bioterrorism agents.     

Metabolic Network Analysis-Based Identification of Antimicrobial Drug Targets in Category A Bioterrorism Agents

The 2001 anthrax mail attacks in the United States demonstrated the potential threat of bioterrorism, hence driving the need to develop sophisticated treatment and diagnostic protocols to counter biological warfare. Here, by performing flux balance analyses on the fully-annotated metabolic networks of multiple, whole genome-sequenced bacterial strains, we have identified a large number of metabolic enzymes as potential drug targets for each of the three Category A-designated bioterrorism agents including Bacillus anthracis, Francisella tularensis and Yersinia pestis. Nine metabolic enzymes- belonging to the coenzyme A, folate, phosphatidyl-ethanolamine and nucleic acid pathways common to all strains across the three distinct genera were identified as targets. Antimicrobial agents against some of these enzymes are available. Thus, a combination of cross species-specific antibiotics and common antimicrobials against shared targets may represent a useful combinatorial therapeutic approach against all Category A bioterrorism agents.     

Lung dendritic cells rapidly mediate anthrax spore entry through the pulmonary route

Inhalational anthrax is a life-threatening infectious disease of considerable concern, especially because anthrax is an emerging bioterrorism agent. The exact mechanisms leading to a severe clinical form through the inhalational route are still unclear, particularly how immobile spores are captured in the alveoli and transported to the lymph nodes in the early steps of infection. We investigated the roles of alveolar macrophages and lung dendritic cells (LDC) in spore migration. We demonstrate that alveolar macrophages are the first cells to phagocytose alveolar spores, and do so within 10 min. However, interstitial LDCs capture spores present in the alveoli within 30 min without crossing the epithelial barrier suggesting a specific mechanism for rapid alveolus sampling by transepithelial extension. We show that interstitial LDCs constitute the cell population that transports spores into the thoracic lymph nodes from within 30 min to 72 h after intranasal infection. Our results demonstrate that LDCs are central to spore transport immediately after infection. The rapid kinetics of pathogen transport may contribute to the clinical features of inhalational anthrax.     

Risk of occupationally acquired illnesses from biological threat agents in unvaccinated laboratory workers

Many vaccines for bioterrorism agents are investigational and therefore not available (outside of research protocol use) to all at-risk laboratory workers who have begun working with these agents as a result of increased interest in biodefense research. Illness surveillance data archived from the U.S. offensive biological warfare program (from 1943 to 1969) were reviewed to assess the impact of safety measures on disease prevention (including biosafety cabinets [BSCs]) before and after vaccine availability. Most laboratory-acquired infections from agents with higher infective doses (e.g., anthrax, glanders, and plague) were prevented with personal protective measures and safety training alone. Safety measures (including BSCs) without vaccination failed to sufficiently prevent illness from agents with lower infective doses in this high-risk research setting. Infections continued with tularemia (average 15/year), Venezuelan equine encephalitis (1.9/year), and Q fever (3.4/year) but decreased dramatically once vaccinations became available (average of 1, 0.6, and 0 infections per year, respectively). While laboratory-acquired infections are not expected to occur frequently in the current lower-risk biodefense research setting because of further improvements in biosafety equipment and changes in biosafety policies, the data help to define the inherent risks of working with the specific agents of bioterrorism. The data support the idea that research with these agents should be restricted to laboratories with experience in handling highly hazardous agents and where appropriate safety training and precautions can be implemented.     

Diagnosing smallpox: would you know it if you saw it?

The intentional release of anthrax in the United States in 2001 and other recent acts of terrorism have highlighted the possibility of intentional release of smallpox by terrorists. Little is known about physicians' ability to diagnose smallpox, especially in the critical first days, when the potential for rapid control of transmission is greatest. During December 2002 and January 2003, primary care and emergency physicians at a large urban academic medical center were surveyed regarding the diagnosis and management of patients who present with vesicular rash illness. In addition to demographic and training-related questions, the questionnaire included items about perceived comfort in diagnosing and evaluating rashes, knowledge of the key differential diagnostic characteristics of chickenpox and smallpox, and the diagnostic interpretation of color photographs of patients with smallpox or chickenpox. Responses were summarized as a perceived comfort score, a differential diagnosis score, and a picture score. Of 266 eligible physicians, 178 (67%) responded. Of these, 95% thought clinicians need more education about bioterrorism; only 17% reported comfort in diagnosing smallpox. Although most physicians recognized pictures of smallpox and chickenpox, only 36% correctly answered 3 of 4 questions regarding differential diagnosis, an important aspect of identifying cases early. Those who were comfortable diagnosing rash illnesses had higher differential diagnosis scores. Strategies for bioterrorism-related training could take advantage of physicians' awareness of their own knowledge deficits.     

Medical control of bioterrorism

Against the recently accrued risk of bioterrorism, the Biotox plan has been set up in order to take in charge any person that could be facing a potential terrorist contamination. It has to take into consideration the components of bioterrorism, i.e. the incubation period, the function of bacterial or viral agents, the variable number of concerned people, the difficulty of alert launching, and the diagnosis of the responsible agent. This plan relies on hospitals comprising departments of infectious diseases. Those are specifically in charge of informing, organising, and coordinating the reception of people having been in contact or infected by infectious agents, in order to isolate and treat them properly. The recent experience has allowed to test this plan, to precise and correct some of its features.     

Anthrax lethal toxin blocks MAPK kinase-dependent IL-2 production in CD4+ T cells

Anthrax lethal toxin (LT) is a critical virulence factor that cleaves and inactivates MAPK kinases (MAPKKs) in host cells and has been proposed as a therapeutic target in the treatment of human anthrax infections. Despite the potential use of anti-toxin agents in humans, the standard activity assays for anthrax LT are currently based on cytotoxic actions of anthrax LT that are cell-, strain-, and species-specific, which have not been demonstrated to occur in human cells. We now report that T cell proliferation and IL-2 production inversely correlate with anthrax LT levels in human cell assays. The model CD4+ T cell tumor line, Jurkat, is a susceptible target for the specific protease action of anthrax LT. Anthrax LT cleaves and inactivates MAPKKs in Jurkat cells, whereas not affecting proximal or parallel TCR signal transduction pathways. Moreover, anthrax LT specifically inhibits PMA/ionomycin- and anti-CD3-induced IL-2 production in Jurkat cells. An inhibitor of the protease activity of anthrax LT completely restores IL-2 production by anthrax LT-treated Jurkat cells. Anthrax LT acts on primary CD4+ T cells as well, cleaving MAPKKs and leading to a 95% reduction in anti-CD3-induced proliferation and IL-2 production. These findings not only will be useful in the development of new human cell-based bioassays for the activity of anthrax LT, but they also suggest new mechanisms that facilitate immune evasion by Bacillus anthracis. Specifically, anthrax LT inhibits IL-2 production and proliferative responses in CD4+ T cells, thereby blocking functions that are pivotal in the regulation of immune responses.     

Innate immunity and biodefence vaccines

Host defence in vertebrates is achieved by the integration of two distinct arms of the immune system: the innate and adaptive responses. The innate response acts early after infection (within minutes), detecting and responding to broad cues from invading pathogens. The adaptive response takes time (days to weeks) to become effective, but provides the fine antigenic specificity required for complete elimination of the pathogen and the generation of immunologic memory. Antigen-independent recognition of pathogens by the innate immune system leads to the rapid mobilization of immune effector and regulatory mechanisms that provide the host with three critical advantages: (i) initiating the immune response (both innate and adaptive) and providing the inflammatory and co-stimulatory context for antigen recognition; (ii) mounting a first line of defence, thereby holding the pathogen in check during the maturation of the adaptive response; and (iii) steering the adaptive immune system towards the cellular or humoral responses most effective against the particular infectious agent. The quest for safer and more effective vaccines and immune-based therapies has taken on a sudden urgency with the increased threat of bioterrorism. Only a handful of vaccines covering a small proportion of potential biowarfare agents are available for human use (e.g. anthrax and small pox) and these suffer from poor safety profiles. Therefore, next generation biodefence-related vaccines and therapies with improved safety and the capacity to induce more rapid, more potent and broader protection are needed. To this end, strategies to target both the innate and adaptive immune systems will be required.     

The use of polymerase chain reaction for detection of the agents of glanders and melioidosis using experimental infection

Glanders and melioidosis are severe infectious diseases of people and animals. The causative agents of these infections refer to the potential agents of bioterrorism of group B. In this work the possibility of use of flagellin-based primers for the identification of B. mallei and B. pseudomallei and for diagnosis of experimental glanders and melioidosis was studied. The obtained results permit to make a conclusion that PCR using the developed primers may be recommended for the incorporation in the scheme of laboratory diagnosis of glanders and melioidosis both for the identification of clean cultures and in experimental clinical material.     

Proteomics for biodefense applications: progress and opportunities

The increasing threat of bioterrorism and continued emergence of new infectious diseases has driven a major resurgence in biomedical research efforts to develop improved treatments, diagnostics and vaccines, as well as increase the fundamental understanding of the host immune response to infectious agents. The availability of multiple mass spectrometry platforms combined with multidimensional separation technologies and microbial genomic databases provides an unprecedented opportunity to develop these much needed resources. An overview of current proteomic strategies applied to microbes and viruses considered potential bioterrorism agents is presented. The emerging area of immunoproteomics as applied to the development of new vaccine targets is also summarized. These powerful research approaches can generate a multitude of potential new protein targets; however, translating these proteomic discoveries to useful counter-bioterrorism products will require large collaborative research efforts across multiple basic science and clinical disciplines. A translational proteomic research paradigm illustrating this approach using influenza virus as an example is discussed.     

Problem of bioterrorism under modern conditions

It is practically impossible to discuss the problem of bioterrorism (BT) and to develop effective programs of decreasing the losses and expenses suffered by the society from the BT acts without evaluation of the threat and prognosis of consequences based on research and empiric data. Stained international situation following the act of terrorism (attack on the USA) on September 11, 2001, makes the scenarios of the bacterial weapon use (the causative agents of plague, smallpox, anthrax, etc.) by international terrorists most probable. In this connection studies on the analysis and prognostication of the consequences of BT, including mathematical and computer modelling, are necessary. The authors present the results of initiative studies on the analysis and prognostication of the consequences of the hypothetical act of BT with the use of the smallpox causative agent in a city with the population of about 1,000,000 inhabitants. The analytical prognostic studies on the operative analysis and prognostication of the consequences of the BT act with the use of the smallpox causative agent has demonstrated that the mathematical (computer) model of the epidemic outbreak of smallpox is an effective instrument of calculation studies. Prognostic evaluations of the consequences of the act of BT under the conditions of different reaction of public health services (time of detection, interventions) have been obtained with the use of modelling. In addition, the computer model is necessary for training health specialists to react adequately to the acts of BT with the use of different kinds of bacteriological weapons.     

What does the future hold for clinical microbiology?

In the past decade, clinical microbiology laboratories have undergone important changes with the introduction of molecular biology techniques and laboratory automation. In the future, there will be a need for more rapid diagnoses, increased standardization of testing and greater adaptability to cope with new threats from infectious microorganisms, such as agents of bioterrorism and emerging pathogens. The combination of the new tools that are now being developed in research laboratories, the general reorganization of clinical laboratories and improved communication between physicians and clinical microbiologists should lead to profound changes in the way that clinical microbiologists work.     

Reducing mortality from anthrax bioterrorism: strategies for stockpiling and dispensing medical and pharmaceutical supplies

A critical question in planning a response to bioterrorism is how antibiotics and medical supplies should be stockpiled and dispensed. The objective of this work was to evaluate the costs and benefits of alternative strategies for maintaining and dispensing local and regional inventories of antibiotics and medical supplies for responses to anthrax bioterrorism. We modeled the regional and local supply chain for antibiotics and medical supplies as well as local dispensing capacity. We found that mortality was highly dependent on the local dispensing capacity, the number of individuals requiring prophylaxis, adherence to prophylactic antibiotics, and delays in attack detection. For an attack exposing 250,000 people and requiring the prophylaxis of 5 million people, expected mortality fell from 243,000 to 145,000 as the dispensing capacity increased from 14,000 to 420,000 individuals per day. At low dispensing capacities (<14,000 individuals per day), nearly all exposed individuals died, regardless of the rate of adherence to prophylaxis, delays in attack detection, or availability of local inventories. No benefit was achieved by doubling local inventories at low dispensing capacities; however, at higher dispensing capacities, the cost-effectiveness of doubling local inventories fell from 100,000 US dollars to 20,000 US dollars/life year gained as the annual probability of an attack increased from 0.0002 to 0.001. We conclude that because of the reportedly rapid availability of regional inventories, the critical determinant of mortality following anthrax bioterrorism is local dispensing capacity. Bioterrorism preparedness efforts directed at improving local dispensing capacity are required before benefits can be reaped from enhancing local inventories.     

In vivo murine and in vitro M-like cell models of gastrointestinal anthrax

Bacillus anthracis is the causative agent of anthrax and is acquired by three routes of infection: inhalational, gastrointestinal and cutaneous. Gastrointestinal (GI) anthrax is rare, but can rapidly result in severe, systemic disease that is fatal in 25%–60% of cases. Disease mechanisms of GI anthrax remain unclear due to limited numbers of clinical cases and the lack of experimental animal models. Here, we developed an in vivo murine model of GI anthrax where spore survival was maximized through the neutralization of stomach acid followed by an intragastric administration of a thiabendazole paste spore formulation. Infected mice showed a dose-dependent mortality rate and pathological features closely mimicking human GI anthrax. Since Peyer's patches in the murine intestine are the primary sites of B. anthracis growth, we developed a human M (microfold)-like-cell model using a Caco-2/Raji B-cell co-culturing system to study invasive mechanisms of GI anthrax across the intestinal epithelium. Translocation of B. anthracis spores was higher in M-like cells than Caco-2 monolayers, suggesting that M-like cells may serve as an initial entry site for spores. Here, we developed an in vivo murine model of GI anthrax and an in vitro M-like cell model that could be used to further our knowledge of GI anthrax pathogenesis.     

Proteomics for biodefense applications: progress and opportunities

The increasing threat of bioterrorism and continued emergence of new infectious diseases has driven a major resurgence in biomedical research efforts to develop improved treatments, diagnostics and vaccines, as well as increase the fundamental understanding of the host immune response to infectious agents. The availability of multiple mass spectrometry platforms combined with multidimensional separation technologies and microbial genomic databases provides an unprecedented opportunity to develop these much needed resources. An overview of current proteomic strategies applied to microbes and viruses considered potential bioterrorism agents is presented. The emerging area of immunoproteomics as applied to the development of new vaccine targets is also summarized. These powerful research approaches can generate a multitude of potential new protein targets; however, translating these proteomic discoveries to useful counter-bioterrorism products will require large collaborative research efforts across multiple basic science and clinical disciplines. A translational proteomic research paradigm illustrating this approach using influenza virus as an example is discussed.     

The role of anthrolysin O in gut epithelial barrier disruption during Bacillus anthracis infection

Gastrointestinal (GI) anthrax, caused by the bacterial infection of Bacillus anthracis, posts a significant bioterrorism threat by its relatively high mortality rate in humans. Different from inhalational anthrax by the route of infection, accumulating evidence indicates the bypass of vegetative bacteria across GI epithelium is required to initiate GI anthrax. Previously, we reported that purified anthrolysin O (ALO), instead of tripartite anthrax edema and lethal toxins, is capable of disrupting gut epithelial tight junctions and barrier function in cultured cells. Here, we show that ALO can disrupt intestinal tissue barrier function in an ex vivo mouse model. To explore the effects of ALO in a cell culture model of B. anthracis infection, we showed that anthrax bacteria can effectively reduce the monolayer integrity of human Caco-2 brush-border expressor (C2BBE) cells based on the reduced transepithelial resistance and the increased leakage of fluorescent dye. This disruption is likely caused by tight junction dysfunction observed by the reorganization of the tight junction protein occludin. Consequently, we observe significant passage of vegetative anthrax bacteria across C2BBE cells. This barrier disruption and bacterial crossover requires ALO since ALO-deficient B. anthracis strains fail to induce monolayer dysfunction and allow the passage of anthrax bacteria. Together these findings point to a pivotal role for ALO within the establishment of GI anthrax infection and the initial bypass of the epithelial barrier.     

Recent outbreak of cutaneous anthrax in Bangladesh: clinico-demographic profile and treatment outcome of cases attended at Rajshahi Medical College Hospital

ABSTRACT: BACKGROUND: Human cutaneous anthrax results from skin exposure to B. anthracis, primarily due to occupational exposure. Bangladesh has experienced a number of outbreaks of cutaneous anthrax in recent years. The last episode occurred from April to August, 2011 and created mass havoc due to its dreadful clinical outcome and socio-cultural consequences. We report here the clinico-demographic profile and treatment outcome of 15 cutaneous anthrax cases attended at the Dermatology Outpatient Department of Rajshahi Medical College Hospital, Bangladesh between April and August, 2011 with an aim to create awareness for early case detection and management. FINDINGS: Anthrax was suspected primarily based on cutaneous manifestations of typical non-tender ulcer with black eschar, with or without oedema, and a history of butchering, or dressing/washing of cattle/goat or their meat. Diagnosis was established by demonstration of large gram-positive rods, typically resembling B. anthracis under light microscope where possible and also by ascertaining therapeutic success. The mean age of cases was 21.4 years (ranging from 3 to 46 years), 7 (46.7%) being males and 8 (53.3%) females. The majority of cases were from lower middle socioeconomic status. Types of exposures included butchering (20%), contact with raw meat (46.7%), and live animals (33.3%). Malignant pustule was present in upper extremity, both extremities, face, and trunk at frequencies of 11 (73.3%), 2 (13.3%), 1 (6.7%) and 1 (6.7%) respectively. Eight (53.3%) patients presented with fever, 7 (46.7%) had localized oedema and 5 (33.3%) had regional lymphadenopathy. Anthrax was confirmed in 13 (86.7%) cases by demonstration of gram-positive rods. All cases were cured with 2 months oral ciprofloxacin combined with flucoxacillin for 2 weeks. CONCLUSIONS: We present the findings from this series of cases to reinforce the criteria for clinical diagnosis and to urge prompt therapeutic measures to treat cutaneous anthrax successfully to eliminate the unnecessary panic of anthrax.     

Rabbit and nonhuman primate models of toxin-targeting human anthrax vaccines.

The intentional use of Bacillus anthracis, the etiological agent of anthrax, as a bioterrorist weapon in late 2001 made our society acutely aware of the importance of developing, testing, and stockpiling adequate countermeasures against biological attacks. Biodefense vaccines are an important component of our arsenal to be used during a biological attack. However, most of the agents considered significant threats either have been eradicated or rarely infect humans alive today. As such, vaccine efficacy cannot be determined in human clinical trials but must be extrapolated from experimental animal models. This article reviews the efficacy and immunogenicity of human anthrax vaccines in well-defined animal models and the progress toward developing a rugged immunologic correlate of protection. The ongoing evaluation of human anthrax vaccines will be dependent on animal efficacy data in the absence of human efficacy data for licensure by the U.S. Food and Drug Administration.