Evaluation of sponge wipe surface sampling for collection of potential surrogates for non-spore-forming bioterrorism agents.

AIM: Evaluate the efficacy of sponge wipe sampling at recovering potential bacterial surrogates for Category A and B non-spore-forming bacterial bioterrorism agents from hard, nonporous surfaces. METHODS: A literature survey identified seven nonpathogenic bacteria as potential surrogates for selected Category A and B non-spore-forming bacterial agents. Small (2 × 4 cm) and large (35.6 × 35.6 cm) coupons made from either stainless steel, plastic, or glass, were inoculated and utilized to assess persistence and surface sampling efficiency, respectively. Three commercially available premoistened sponge wipes (3M™, Sani-Stick®, and Solar-Cult®) were evaluated. RESULTS: Mean recoveries from persistence testing indicated that three microorganisms (Yersinia ruckeri, Escherichia coli, and Serratia marcescens) demonstrated sufficient persistence across all tested material types. Sampling of large inoculated (≥107 CFU per sample) coupons resulted in mean recoveries ranging from 6.6 to 3.4 Log10 CFU per sample. Mean recoveries for the Solar-Cult®, 3M™ sponge wipes, and Sani-Sticks® across all test organisms and all material types were ≥5.7, ≥3.7, and ≥3.4 Log10 CFU per sample, respectively. Mean recoveries for glass, stainless steel, and ABS plastic across all test organisms and all sponge types were ≥3.8, ≥3.7, and ≥3.4 Log10 CFU per sample, respectively. CONCLUSIONS: Recovery results suggest that sponge wipe sampling can effectively be used to recover non-spore-forming bacterial cells from hard, nonporous surfaces such as stainless steel, ABS plastic, and glass.

Poxviruses as Agents of Biological Warfare: The Importance of Ensuring Ethical Standards for Research with Viruses.

Historically, biological agents have been used to target various populations. One of the earliest examples could be the catastrophic effect of smallpox in Australia in the eighteenth century (as alleged by some historians). Modern biological techniques can be used to both create or provide protection against various agents of biological warfare. Any microorganism (viruses, bacteria, and fungi) or its toxins can be used as biological agents. Minnesota Department of Health has listed Smallpox (variola major) as a category A bioterrorism agent, even though it has been eradicated in 1980 through an extensive vaccination campaign. Category A agents are considered the highest risk to public health. Laboratory-associated outbreaks of poxviruses could cause unprecedented occupational hazards. Only two WHO-approved BSL-4 facilities in the United States and Russia are allowed to perform research on the variola virus. So, poxviruses present themselves as a classical case of a dual-use dilemma, since research with them can be used for both beneficial and harmful purposes. Although the importance of ethics in scientific research requires no further elaboration, ethical norms assume greater significance during experimentation with poxviruses. In this chapter, we will update the readers on the sensitive nature of conducting research with poxviruses, and how these viruses can be a source of potential biological weapons. Finally, specified ethical guidelines are explored to ensure safe research practices in virology.

Epidemiology of Pathogens Listed as Potential Bioterrorism Agents, the Netherlands, 2009‒2019.

We provide incidences (cases/10 million persons) in the Netherlands during 2009-2019 for pathogens listed as potential bioterrorism agents. We included pathogens from the highest categories of the European Medicines Agency or the US Centers for Disease Control and Prevention. Notifiable diseases and recently published data were used to calculate the average annual incidence. Coxiella burnetii had the highest incidence because of a Q fever epidemic during 2007-2010. Incidence then decreased to 10.8 cases/. Pathogens with an incidence >1 were Brucella spp. (2.5 cases), Francisella tularensis (1.3 cases), and Burkholderia pseudomallei (1.1 cases). Pathogens with an incidence <1 were hemorrhagic fever viruses (0.3 cases), Clostridium botulinum (0.2 cases), and Bacillus anthracis (0.1 cases). Variola major and Yersinia pestis were absent. The generally low incidences make it unlikely that ill-meaning persons can isolate these pathogens from natural sources in the Netherlands. However, the pathogens are stored in laboratories, underscoring the need for biosecurity measures.

Gain of Function and Loss of Control: Genetic Modification of Microbial Agents - Viruses "On Steroids".

Gain of Function refers to genetic modification to enhance certain properties of a biological agent. "Dual use research" refers to experiments which have a primary goal of benefitting humanity, but which could produce harm if misapplied. So, for example, a virus which was being genetically modified (GM) for altruistic reasons might become more transmissible or resistant to vaccines or antimicrobial medications. Such a GM virus has bioterrorism potential. The UN Biological Weapons Convention has not been universally approved and 10 States are not signatories to the Convention. The control of such experiments is variously controlled in certain jurisdictions but in Australia these experiments are well regulated through the Gene Technology Act 2000 (Cth), the National Health Security Act 2007 (Cth) and the Crimes (Biological Weapons) Act 1976 (Cth). The controls on such experiments in Europe and the United States are less precise. There are examples in the United States and Europe where the security provisions to contain microorganisms undergoing research including genetic modification have been breached. This threatens the health and safety of laboratory workers and the wider community.

Tularemia seroprevalence in humans in the region of the Hittite-Arzawa War (Inner Aegean Region), where the first biological weapon was used 3300 years ago.

BACKGROUND: : According to Egyptian records, tularemia emerged in the Canaan region, where it was first identified and spread to Anatolia over the Euphrates. It was used as an active biological weapon for the first time in the Hittite-Arzawa War in 1320-1318 BC. This study aimed to investigate the seroprevalence of tularemia in the Inner Aegean Region, which is thought to be the region where this war was fought 3300 years ago. METHODS: Tularemia seropositivity in humans was investigated in 27 villages/neighborhoods in 3 districts in each of Manisa, Kütahya, and Usak provinces. Before the study, the participants were informed about the disease via posters, and their blood samples were taken following filling out the questionnaire. Microagglutination tests were performed using in-house tularemia antigen and V plate for serological experiments. Rose-Bengal test was also performed on seropositive sera. RESULTS: Of the total of 410 people, 226 (55.12%) were male. The mean age of the volunteers was 43.72 years. The highest participation was from Kütahya Province. According to the results of the tularemia microagglutination test, seropositivity was detected in 6 cases. It was determined that all of the seropositive volunteers were in Kütahya. When the tularemia antibody titers were examined, seropositivity was determined at 1/20-1/160 titers. No positivity was detected in the Rose-Bengal test for cross-reaction. DISCUSSION: Kütahya has been identified as a risky region in terms of tularemia in the Inner Aegean Region. In order to use the resources in the country economically, first of all, the risk areas in terms of tularemia should be determined by serological studies in all regions. In order to increase awareness about the disease, physicians and filiation teams should be trained in risky areas. Surveillance studies should be conducted to identify and monitor possible sources in areas identified as risky.

ASSESSMENT OF THE CONSEQUENCES OF THE DETERIORATION OF THE EPIDEMIOLOGICAL SITUATION DURING HOSTILITIES.

OBJECTIVE: The aim: Scientific substantiation of the methodology for predicting the consequences of the worsening of the epidemic situation on the territory of Ukraine during military operations for the timely adoption of measures for the medical protection of military personnel in conditions of biological contamination. PATIENTS AND METHODS: Materials and methods: Determination and generalization of the impact of biological contamination due to the use of biological weapons were carried out considering the main determinants of the epidemic process using the index and coefficient of medical protection. Applied methods of scientific research: epidemiological, system, and information approach. RESULTS: Results: The authors proposed indicators that consider the pathogenicity of the infectious agent, contagiousness, the degree of non-specific protection of servicemen, specific protection of servicemen, and the sanitary-epidemiological state of the area of operations of troops (forces). Relevant epidemic situations were simulated, and the index and coefficient of medical protection were calculated to predict the consequences of the worsening of the epidemic situation to make timely decisions regarding the implementation of medical protection measures for military personnel in conditions of biological contamination during the repulsion of armed aggression. CONCLUSION: Conclusions: In the conditions of biological contamination, when biological weapons and biological terrorism are used, the epidemic process in the army is intensified, which requires timely decisions regarding the implementation of medical protection measures for military personnel in conditions of biological contamination.

Clinical Features of Patients Hospitalized for All Routes of Anthrax, 1880-2018: A Systematic Review.

BACKGROUND: Anthrax is a toxin-mediated zoonotic disease caused by Bacillus anthracis, with a worldwide distribution recognized for millennia. Bacillus anthracis is considered a potential biowarfare agent. METHODS: We completed a systematic review for clinical and demographic characteristics of adults and children hospitalized with anthrax (cutaneous, inhalation, ingestion, injection [from contaminated heroin], primary meningitis) abstracted from published case reports, case series, and line lists in English from 1880 through 2018, assessing treatment impact by type and severity of disease. We analyzed geographic distribution, route of infection, exposure to anthrax, and incubation period. RESULTS: Data on 764 adults and 167 children were reviewed. Most cases reported for 1880 through 1915 were from Europe; those for 1916 through 1950 were from North America; and from 1951 on, cases were from Asia. Cutaneous was the most common form of anthrax for all populations. Since 1960, adult anthrax mortality has ranged from 31% for cutaneous to 90% for primary meningitis. Median incubation periods ranged from 1 day (interquartile range [IQR], 0-4) for injection to 7 days (IQR, 4-9) for inhalation anthrax. Most patients with inhalation anthrax developed pleural effusions and more than half with ingestion anthrax developed ascites. Treatment and critical care advances have improved survival for those with systemic symptoms, from approximately 30% in those untreated to approximately 70% in those receiving antimicrobials or antiserum/antitoxin. CONCLUSIONS: This review provides an improved evidence base for both clinical care of individual anthrax patients and public health planning for wide-area aerosol releases of B. anthracis spores.

Systematic Review of Hospital Treatment Outcomes for Naturally Acquired and Bioterrorism-Related Anthrax, 1880-2018.

BACKGROUND: Bacillus anthracis can cause anthrax and is a potential bioterrorism agent. The 2014 Centers for Disease Control and Prevention recommendations for medical countermeasures against anthrax were based on in vitro data and expert opinion. However, a century of previously uncompiled observational human data that often includes treatment and outcomes is available in the literature for analysis. METHODS: We reviewed treatment outcomes for patients hospitalized with anthrax. We stratified patients by meningitis status, route of infection, and systemic criteria, then analyzed survival by treatment type, including antimicrobials, antitoxin/antiserum, and steroids. Using logistic regression, we calculated odds ratios and 95% confidence intervals to compare survival between treatments. We also calculated hospital length of stay. Finally, we evaluated antimicrobial postexposure prophylaxis (PEPAbx) using data from a 1970 Russian-language article. RESULTS: We identified 965 anthrax patients reported from 1880 through 2018. After exclusions, 605 remained: 430 adults, 145 children, and 30 missing age. Survival was low for untreated patients and meningitis patients, regardless of treatment. Most patients with localized cutaneous or nonmeningitis systemic anthrax survived with 1 or more antimicrobials; patients with inhalation anthrax without meningitis fared better with at least 2. Bactericidal antimicrobials were effective for systemic anthrax; addition of a protein synthesis inhibitor(s) (PSI) to a bactericidal antimicrobial(s) did not improve survival. Likewise, addition of antitoxin/antiserum to antimicrobials did not improve survival. Mannitol improved survival for meningitis patients, but steroids did not. PEPAbx reduced risk of anthrax following exposure to B. anthracis. CONCLUSIONS: Combination therapy appeared to be superior to monotherapy for inhalation anthrax without meningitis. For anthrax meningitis, neither monotherapy nor combination therapy were particularly effective; however, numbers were small. For localized cutaneous anthrax, monotherapy was sufficient. For B. anthracis exposures, PEPAbx was effective.

A multipathogen DNA vaccine elicits protective immune responses against two class A bioterrorism agents, anthrax and botulism.

The potential use of biological agents has become a major public health concern worldwide. According to the CDC classification, Bacillus anthracis and Clostridium botulinum, the bacterial pathogens that cause anthrax and botulism, respectively, are considered to be the most dangerous potential biological agents. Currently, there is no licensed vaccine that is well suited for mass immunization in the event of an anthrax or botulism epidemic. In the present study, we developed a dual-expression system-based multipathogen DNA vaccine that encodes the PA-D4 gene of B. anthracis and the HCt gene of C. botulinum. When the multipathogen DNA vaccine was administered to mice and guinea pigs, high level antibody responses were elicited against both PA-D4 and HCt. Analysis of the serum IgG subtype implied a combined Th1/Th2 response to both antigens, but one that was Th2 skewed. In addition, immunization with the multipathogen DNA vaccine induced effective neutralizing antibody activity against both PA-D4 and HCt. Finally, the protection efficiency of the multipathogen DNA vaccine was determined by sequential challenge with 10 LD50 of B. anthracis spores and 10 LD50 of botulinum toxin, or vice versa, and the multipathogen DNA vaccine provided higher than 50% protection against lethal challenge with both high-risk biothreat agents. Our studies suggest the strategy used for this anthrax-botulinum multipathogen DNA vaccine as a prospective approach for developing emergency vaccines that can be immediately distributed on a massive scale in response to a biothreat emergency or infectious disease outbreak. Key points • A novel multipathogen DNA vaccine was constructed against anthrax and botulism. • Robust immune responses were induced following vaccination. • Suggests a potential vaccine development strategy against biothreat agents.

A Collection of Molecular Fingerprints of Single Aerosol Particles in Air for Potential Identification and Detection Using Optical Trapping-Raman Spectroscopy.

Characterization, identification, and detection of aerosol particles in their native atmospheric states remain a challenge. Recently, optical trapping-Raman spectroscopy (OT-RS) has been developed and demonstrated for characterization of single, airborne particles. Such particles in different chemical groups have been characterized by OT-RS in recent years and many more are being studied. In this work, we collected single-particle Raman spectra measured using the OT-RS technique and began construction of a library of OT-RS fingerprints that may be used as a reference for potential detection and identification of aerosol particles in the atmosphere. We collected OT-RS fingerprints of aerosol particles from eight different categories including carbons, bioaerosols (pollens, fungi, vitamins, spores), dusts, biological warfare agent surrogates, etc. Among the eight categories, spectral fingerprints of six groups of aerosol particles have been published previously and two other groups are new. We also discussed challenges, limitations, and advantages of using single-particle optical trapping-Raman spectroscopy for aerosol-particle characterization, identification, and detection.

Botulinum Toxin in WW2 German and Allied Armies: Failures and Myths of Weaponization.

Botulinum toxin is nowadays approved as an effective medication for various neurological disorders. The extreme toxicity of this toxin-inducing botulism, a severe lethal muscle-paralyzing illness, has been well known since the seminal works of the end of the 19th century. Because of this toxicity, botulinum toxin was one of the first agents to be considered for use as a biological weapon. The Second World War was a crucial period for the first attempts to weaponize this toxin even if many unknown factors about botulinum toxin still existed at the outbreak of the war. Using documents from the British National Archives and other published sources, we discuss the main points of the attempts to weaponize this toxin in German and Allied armies. During WW2, Allied intelligence services regularly reported a major German threat related to the potential use of botulinum toxin as a biological weapon, especially during the preparation of Operation Overlord, the Allied invasion to liberate Europe. All these reports would ultimately prove to be inaccurate: botulinum toxin was not part of the German military arsenal even if some German scientists tried to use the results of the French pre-war military research. Misinformation spread by intelligence services stimulated military research at Porton Down facilities in England and at Camp Detrick in the USA. These studies led to a succession of failures and myths about the weaponization of botulinum toxin. Nevertheless, major progress (purification, toxoid) arose from the military research, providing useful data for the first steps of the therapeutic use of botulinum toxin in the post-war years.

Costs and benefits of provocation in bacterial warfare.

Competition in animals involves a wide variety of aggressive behaviors. One of the most sophisticated strategies for a focal actor is to provoke a competitor into uncontrolled aggression toward other competitors. Like animals, bacteria rely on a broad spectrum of molecular weapons, some of which provoke potential rivals by triggering retaliation. While bacterial provocation is well documented, its potential adaptive value has received little attention. Here, we examine the costs and benefits of provocation using mathematical modeling and experiments with Escherichia coli strains encoding colicin toxins. We show that provocation is typically costly in one-to-one encounters because a provoking strain receives a strong reciprocal attack compared with nonprovoking strains. By contrast, provocation can be strongly beneficial in communities including more than two toxin-producing strains, especially when the provoker is shielded from, or resistant to, its opponents' toxins. In these scenarios, we demonstrate that the benefit of provocation derives from a "divide-and-conquer" effect by which aggression-provoking toxin producers force their competitors into increased reciprocal aggression, leading to their cross-elimination. Furthermore, we show that this effect can be mimicked by using antibiotics that promote warfare among strains in a bacterial community, highlighting the potential of provocation as an antimicrobial approach.

Bioterrorism.

Bioterrorism is the deliberate release of viruses, bacteria, toxins, or fungi with the goal of causing panic, mass casualties, or severe economic disruption. From 1981 to 2018, there were 37 bioterrorist attacks worldwide. The Centers for Disease Control and Prevention (CDC) lists anthrax, botulism, plague, smallpox, tularemia, and viral hemorrhagic fevers as category A agents that are the greatest risk to national security. An emerging infectious disease (e.g., novel respiratory virus) may also be used as a biological agent. Clinicians may be the first to recognize a bioterrorism-related illness by noting an unusual presentation, location, timing, or severity of disease. Public health authorities should be notified when a biological agent is recognized or suspected. Treatment includes proper isolation and administration of antimicrobial or antitoxin agents in consultation with regional medical authorities and the CDC. Vaccinations for biological agents are not routinely administered except for smallpox, anthrax, and Ebola disease for people at high risk of exposure. The American Academy of Family Physicians, the CDC, and other organizations provide bioterrorism training and response resources for clinicians and communities. Clinicians should be aware of bioterrorism resources.

Protein biomarker elucidation for the verification of biological agents in the taxonomic group of Gammaproteobacteria using tandem mass spectrometry.

Some pathogenic microbes can be used for nefarious applications and instigate population-based fear. In a bio-threat scenario, rapid and accurate methods to detect biological agents in a wide range of complex environmental and clinical matrices, is of paramount importance for the implementation of mitigation protocols and medical countermeasures. This study describes targeted and shot-gun tandem MS based approaches for the verification of biological agents from the environmental samples. The marker proteins and peptides were elucidated by an exhaustive literature mining, in silico analysis of prioritized proteins, and MS/MS analysis of abundant proteins from selected bacterial species. For the shot-gun methodology, tandem MS analysis of abundant peptides was carried from spiked samples. The validation experiments employing a combination of shot-gun tandem MS analysis and a targeted search reported here is a proof of concept to show the applicability of the methodology for the unambiguous verification of biological agents at sub-species level, even with limited fractionation of crude protein extracts from environmental samples.

Drug Development against Smallpox: Present and Future.

Forty years after the last endemic smallpox case, variola virus (VARV) is still considered a major threat to humans due to its possible use as a bioterrorism agent. For many years, the risk of disease reemergence was thought to solely be through deliberate misuse of VARV strains kept in clandestine laboratories. However, recent experiments using synthetic biology have proven the feasibility of recreating a poxvirus de novo, implying that VARV could, in theory, be resurrected. Because of this new perspective, the WHO Advisory Committee on VARV Research released new recommendations concerning research on poxviruses that strongly encourages pursuing the development of new antiviral drugs against orthopoxviruses. In 2018, the U.S. FDA advised in favor of two molecules for smallpox treatment, tecovirimat and brincidofovir. This review highlights the difficulties to develop new drugs targeting an eradicated disease, especially as it requires working under the FDA "animal efficacy rule" with the few, and imperfect, animal models available.

Physicochemical Evidence that Francisella FupA and FupB Proteins Are Porins.

Responsible for tularemia, Francisella tularensis bacteria are highly infectious Gram-negative, category A bioterrorism agents. The molecular mechanisms for their virulence and resistance to antibiotics remain largely unknown. FupA (Fer Utilization Protein), a protein mediating high-affinity transport of ferrous iron across the outer membrane, is associated with both. Recent studies demonstrated that fupA deletion contributed to lower F. tularensis susceptibility towards fluoroquinolones, by increasing the production of outer membrane vesicles. Although the paralogous FupB protein lacks such activity, iron transport capacity and a role in membrane stability were reported for the FupA/B chimera, a protein found in some F. tularensis strains, including the live vaccine strain (LVS). To investigate the mode of action of these proteins, we purified recombinant FupA, FupB and FupA/B proteins expressed in Escherichia coli and incorporated them into mixed lipid bilayers. We examined the porin-forming activity of the FupA/B proteoliposomes using a fluorescent 8-aminonaphthalene-1,3,6-trisulfonic acid, disodium salt (ANTS) probe. Using electrophysiology on tethered bilayer lipid membranes, we confirmed that the FupA/B fusion protein exhibits pore-forming activity with large ionic conductance, a property shared with both FupA and FupB. This demonstration opens up new avenues for identifying functional genes, and novel therapeutic strategies against F. tularensis infections.

Bacillus anthracis as a biological warfare agent: infection, diagnosis and countermeasures.

AIM: Bacillus anthracis is a causative agent of zoonotic anthrax disease. In the last years, significant progress in therapy and diagnosis of anthrax was made. Concurrently, knowledge about anthrax progression, molecular pathology and release of anthrax toxin during the disease has improved. This review covers the recent progress in this field. METHODS: In this review, specifications of B. anthracis, anthrax disease, medical and biomedical countermeasures and diagnostic tools were surveyed. The actual literature was summarized and relevance of the microorganism as a biological warfare agent and the ways how to reduce its impact including therapeutic protocols were written and discussed. RESULTS: Currently, the microorganism is considered one of the top biological warfare agents due to lethality, long term stability of spores, easy dissemination and production. The recent research is focused on countermeasures suitable for reduction of consequences by a misuse of the microorganism in form of biological weapon (Tab. 3, Fig. 1, Ref. 101). Text in PDF www.elis.sk.

Next-Generation Sequencing for Biodefense: Biothreat Detection, Forensics, and the Clinic.

BACKGROUND: Next-generation sequencing (NGS) is revolutionizing a variety of molecular biology fields including bioforensics, biosurveillance, and infectious disease diagnostics. For pathogen detection, the ability to sequence all nucleic acids in a sample allows near limitless multiplexability, free from a priori knowledge regarding an etiologic agent as is typically required for targeted molecular assays such as real-time PCR. Furthermore, sequencing capabilities can generate in depth genomic information, allowing detailed molecular epidemiological studies and bioforensics analysis, which is critical for source agent identification in a biothreat outbreak. However, lack of analytical specificity, inherent to NGS, presents challenges for regulated applications such as clinical diagnostics and molecular attribution. CONTENT: Here, we discuss NGS applications in the context of preparedness and biothreat readiness. Specifically, we investigate current and future applications of NGS technologies to affect the fields of biosurveillance, bioforensics, and clinical diagnostics with specific focus on biodefense. SUMMARY: Overall, there are many advantages to the implementation of NGS for preparedness and readiness against biowarfare agents, from forensics to diagnostics. However, appropriate caveats must be associated with any technology. This includes NGS. While NGS is not the panacea replacing all molecular techniques, it will greatly enhance the ability to detect, characterize, and diagnose biowarfare agents, thus providing an excellent addition to the biodefense toolbox of biosurveillance, bioforensics, and biothreat diagnosis.

Who Should Be Driving US Science Policy?

Transparency was not always a desired aspect of medicine or STEM (science, technology, engineering, and math) research. In the late 1940s, the Nuremberg Code heralded a new era of informed patient consent, research subject protection, and the view that the public had a stake in emerging technology and should have some knowledge and input into the directions of scientific research. This understanding intensified in the United States with the very public discussions leading to the promulgation of the NIH Guidelines for Recombinant DNA Research in the 1970s. The way in which oversight of recombinant DNA research was handled is still the exception rather than the rule. Starting in the 1990s, various terror incidents led to the enactment of statutes and issuance of regulations that undermined the ability of scientists and research institutions to self-regulate and in some cases to disseminate information freely. This essay explores how the scientific community got to this status quo, and how it could regain some measure of control despite competing needs for transparency and security, so that research critical to biosecurity is supported rather than impeded.