Potential Bioterrorism Agent Research Articles

Development of a non-living vaccine against Burkholderia mallei (129.2)

Abstract Burkholderia mallei is a gram-negative highly pathogenic bacterium, responsible for glanders. B. mallei is considered potential bioterrorism agents, classified as such in list B by the Centers for Disease Control and Prevention. Currently there is no vaccine available against B. mallei. In this study, we aimed to identify the protective proteins of B. mallei. The candidate proteins were selected based on the previous data of proteomics and linear expression library (LEE) whole-genome screens coupled with known structures or functions of the proteins in other bacteria. The selected proteins were BMA-4, BMA-8, BMA-9, BMA-10, BMA-11 and BMA-12. The genes encoding each protein were cloned into pCDNA3.1 and pET28a. The His-tag proteins were then purified. BALB/c mice were vaccinated with DNA encoding each protein and then boosted 3 weeks later with the recombinant proteins. Two weeks after boosting, mice were infected with B. mallei ATCC23344. The result showed that, 19 days after infection, there was 100% survival in the BMA2821 and BMAA0768 groups, however only 78% survival in the control group. The study needs to be repeated and the result will be discussed.

EST‐based gene silencing for identification of macrophage resistance mechanisms against Francisella tularensis

Francisella tularensis (FT) is a gram‐negative, highly infectious, facultative intracellular pathogen that causes tularemia, and is a potential bioterrorism agent. Macrophages represent the main site of initial FT replication but the mechanism of survival and bacteriostasis upon activation remains poorly characterized. We used genome‐wide genetic screen to identify host genes responsible for survival of infection. We used macrophages (RAW 264.7) transduced with an EST‐lentiviral "knock‐down" library targeting 40,000 sequences. The macrophages were infected with attenuated Live Vaccine Strain (LVS) FT expressing GFP. We screened for EST interference resulting in a phenotype that allows survival of infection and intracellular bacterial proliferation. Flow cytometric sorting of cells with highest FT load (i.e. top 1% by green fluorescence) was performed. The EST inserts from sorted cells were amplified from genomic DNA by PCR, and corresponding EST inserts were identified by cloning and DNA sequencing. Initial screening has analyzed 1400 clones from 6 different sorts and has identified 7 genes of interest based on repeated detection. Validation of potential targets is underway to confirm gene expression and to assess effects of directed lentiviral knockdown of candidate genes. This approach may prove useful to identify mechanisms in macrophage innate immunity against intracellular pathogens.

Ricin poisoning and forensic toxicology

Ricin is one of the most fascinating poisons due to its high toxicity: as little as 500 microg can kill an adult. It gained fame by its use in the so-called 'umbrella murder' to kill the Bulgarian dissident Georgi Markov in 1978. Ricin also became known as a potential bio-terror agent to which people could be exposed through the air, food, or water. The origin, biochemistry, toxicity, and analytical procedures for the determination of ricin are summarized. The homicide of Markov is described as well as recent cases of criminal ricin use.

Identification of Thieno[3,2‐b]Pyrrole Derivatives as Novel Small Molecule Inhibitors of Neurotropic Alphaviruses

Neurotropic alphaviruses such as western, eastern, and Venezuelan equine encephalitis viruses cause serious and potentially fatal central nervous system infections in humans and are high-priority potential bioterrorism agents. There are currently no widely available vaccines or licensed therapies for these virulent pathogens. To identify potential novel antiviral drugs, we developed a cell-based assay with a western equine encephalitis virus replicon that expresses a luciferase reporter gene and screened a small molecule diversity library of 51,028 compounds. We identified and validated a thieno[3,2-b]pyrrole compound with a half maximal inhibitory concentration of <10 micromol/L, a selectivity index>20, and potent activity against live virus in cultured neuronal cells. Furthermore, a structure-activity relationship analysis with 20 related compounds identified several with enhanced activity profiles, including 6 with submicromolar half maximal inhibitory concentrations. In conclusion, we have identified a novel class of promising inhibitors with potent activity against virulent neurotropic alphaviruses.

Development and Characterization of Mouse Models of Infection with Aerosolized Brucella melitensis and Brucella suis

There is a need to identify vaccines that can protect against Brucella, a potential bioterrorism agent. We have developed mouse models of infection with aerosolized Brucella melitensis and Brucella suis and demonstrated their utility for the evaluation of vaccines using the model live B. melitensis vaccine strain Rev.1.

Characterization of rationally attenuated Francisella tularensis vaccine strains that harbor deletions in the guaA and guaB genes

Francisella tularensis, the etiologic agent of tularemia, can cause severe and fatal infection after inhalation of as few as 10–100 CFU. F. tularensis is a potential bioterrorism agent and, therefore, a priority for countermeasure development. Vaccination with the live vaccine strain (LVS), developed from a Type B strain, confers partial protection against aerosal exposure to the more virulent Type A strains and provides proof of principle that a live attenuated vaccine strain may be efficacious. However LVS suffers from several notable drawbacks that have prevented its licensure and widespread use. To address the specific deficiencies that render LVS a sub-optimal tularemia vaccine, we engineered F. tularensis LVS strains with targeted deletions in the guaA or guaB genes that encode critical enzymes in the guanine nucleotide biosynthetic pathway. F. tularensis LVSΔ guaA and LVSΔ guaB mutants were guanine auxotrophs and were highly attenuated in a mouse model of infection. While the mutants failed to replicate in macrophages, a robust proinflammatory cytokine response, equivalent to that of the parental LVS, was elicited. Mice vaccinated with a single dose of the F. tularensis LVSΔ guaA or LVSΔ guaB mutant were fully protected against subsequent lethal challenge with the LVS parental strain. These findings suggest the specific deletion of these target genes could generate a safe and efficacious live attenuated vaccine.

A mouse model for ricin poisoning and for evaluating protective effects of antiricin antibodies

Background. Ricin is a potential bioterrorism agent and no specific antidote or treatment exists for ricin poisoning. For this reason, we developed ricin-specific antibodies that were tested in a murine model of ricin poisoning for use as antidotes against symptoms of ricin poisoning. Methods. Mice were poisoned with a lethal dose of ricin (5 μg) and their temperature and general condition were monitored for determination of a surrogate and humane end point. Mice were then treated with injections of ricin and different combinations of polyclonal anti-ricin antibodies. Antibody effect was evaluated for various doses and using various time points from ricin to antibody injection. Also, the effect of adjuvant symptomatic treatment was examined. Brain, heart, intestines, kidney, liver, lung, pancreas, spleen, and stomach tissues were sampled for histopathological analysis. Results. The mouse model was reproducible and easy to use. A clear protective effect of both anti-ricin A-chain and anti-ricin B-chain antibodies–but not of irrelevant antibodies–was demonstrated with no added effect of symptomatic treatment. Conclusions. These data suggest that specific polyclonal antibodies against ricin A- and B-chain may reproducibly protect mice against ricin poisoning, even when the antibodies are administered up to 1.5 h after poisoning.

Recombinant attenuated Listeria monocytogenes vaccine expressing Francisella tularensis IglC induces protection in mice against aerosolized Type A F. tularensis

Fransicella tularensis, the causative agent of tularemia, is in the top category (Category A) of potential agents of bioterrorism. To develop a safer vaccine against aerosolized F. tularensis, we have employed an attenuated Listeria monocytogenes, which shares with F. tularensis an intracellular and extraphagosomal lifestyle, as a delivery vehicle for F. tularensis antigens. We constructed recombinant L. monocytogenes (rLm) vaccines stably expressing seven F. tularensis proteins including IglC (rLm/iglC), and tested their immunogenicity and protective efficacy against lethal F. tularensis challenge in mice. Mice immunized intradermally with rLm/iglC developed significant cellular immune responses to F. tularensis IglC as evidenced by lymphocyte proliferation and CD4+ and CD8+ T-cell intracellular expression of interferon gamma. Moreover, mice immunized with rLm/iglC were protected against lethal challenge with F. tularensis LVS administered by the intranasal route, a route chosen to mimic airborne infection, and, most importantly, against aerosol challenge with the highly virulent Type A F. tularensis SchuS4 strain.

Detection of Virulence-Associated Genes in Clinical Isolates of Bacillus anthracis by Multiplex PCR and DNA Probes

Anthrax is a zoonotic disease caused by Bacillus anthracis, and well recognized as a potential agent for bioterrorism. B. anthracis can be identified by detecting the virulence factors genes located on two plasmids, pXO1 and pXO2. The aim of the present study was to determine the presence of virulence genes in 27 isolates of B. anthracis isolated from clinical and environmental samples. For this purpose, multiplex PCR and DNA probes were designed to detect protective antigen ( pag), edema factor (cya), lethal factor (lef ), and capsule (cap) genes. Our results indicated that all the isolates contained all the above virulence genes, suggesting that the isolates were virulent. To the best our knowledge, this is the first study about the determination of virulence marker genes in clinical and environmental isolates of B. anthracis using multiplex PCR and DNA probes in India. We suggest that the above methods can be useful in specific identification of virulent B. anthracis in clinical and environmental samples.

Bioterrorism for the respiratory physician

Terrorist attacks by definition are designed to cause fear and panic. There is no question that a terrorist attack using biological agents would present a grave threat to stability of the society in which they were released. Early recognition of such a bioterrorist attack is crucial to containing the damage they could cause. As many of the most likely bioterrorism agents present with pulmonary disease, respiratory physicians may be crucial in the initial recognition and diagnosis phase, and certainly would be drawn into treatment of affected individuals. This review focuses on the biological agents thought most likely to be used by terrorists that have predominantly respiratory presentations. The primary focus of this review is on anthrax, plague, tularaemia, ricin, and Staphylococcal enterotoxin B. The pathogenesis, clinical manifestations and treatment of these agents will be discussed as well as historical examples of their use. Other potential bioterrorism agents with respiratory manifestations will also be discussed briefly.

Molecular identification and typing of Burkholderia pseudomallei and Burkholderia mallei: when is enough enough?

Burkholderia mallei and B. pseudomallei are highly pathogenic microorganisms for both humans and animals. Moreover, they are regarded as potential agents of bioterrorism. Thus, rapid and unequivocal detection and identification of these dangerous pathogens is critical. In the present study, we describe the use of an optimized protocol for the early diagnosis of experimental glanders and melioidosis and for the rapid differentiation and typing of Burkholderia strains. This experience with PCR-based identification methods indicates that single PCR targets (23S and 16S rRNA genes, 16S-23S intergenic region, fliC and type III secretion gene cluster) should be used with caution for identification of B. mallei and B. pseudomallei, and need to be used alongside molecular methods such as gene sequencing. Several molecular typing procedures have been used to identify genetically related B. pseudomallei and B. mallei isolates, including ribotyping, pulsed-field gel electrophoresis and multilocus sequence typing. However, these methods are time consuming and technically challenging for many laboratories. RAPD, variable amplicon typing scheme, Rep-PCR, BOX-PCR and multiple-locus variable-number tandem repeat analysis have been recommended by us for the rapid differentiation of B. mallei and B. pseudomallei strains.

Targeting inside-out phosphatidylserine as a therapeutic strategy for viral diseases

SummaryTargeting exposed anionic phospholipids on a spectrum of virus-infected cells can protect against lethal virus infections in vivo.

&lt;I&gt;Francisella&lt;/I&gt; Genes Required for Replication in Mosquito Cells

Francisella tularensis, a potential bioterrorism agent, is transmitted by arthropod vectors and causes tularemia in many mammals, including humans. Francisella novicida causes disease with similar pathology in mice. We show that F. novicida invades hemocyte-like cells of the SualB cell line derived from Anopheles gambiae and replicates vigorously within these cells. We used transposon knockouts of single genes of F. novicida to show that bacterial growth within these insect cells is dependent on virulence factors encoded in a bacterial pathogenicity island that has been linked to replication in mammalian macrophages. The virulence factors MglA, IglA, IglB, IglC, and IglD as well as PdpA and PdpB were necessary for efficient growth in insect cells, but PdpC and PdpD were not required. The SualB cell line presents a valuable model to study the interactions between this important pathogen and insect vectors.

In Vivo Himar1 Transposon Mutagenesis of Burkholderia pseudomallei

Burkholderia psedudomallei is the etiologic agent of melioidosis, and the bacterium is listed as a potential agent of bioterrorism because of its low infectious dose, multiple infectious routes, and intrinsic antibiotic resistance. To further accelerate research with this understudied bacterium, we developed a Himar1-based random mutagenesis system for B. pseudomallei (HimarBP). The transposons contain a Flp recombinase-excisable, approved kanamycin resistance selection marker and an R6K origin of replication for transposon rescue. In vivo mutagenesis of virulent B. pseudomallei strain 1026b was highly efficient, with up to 44% of cells transformed with the delivery plasmid harboring chromosomal HimarBP insertions. Southern analyses revealed single insertions with no evidence of delivery plasmid maintenance. Sequence analysis of rescued HimarBP insertions revealed random insertions on both chromosomes within open reading frames and intergenic regions and that the orientation of insertions was largely unbiased. Auxotrophic mutants were obtained at a frequency of 0.72%, and nutritional supplementation experiments supported the functional assignment of genes within the respective biosynthetic pathways. HimarBP insertions were stable in the absence of selection and could be readily transferred between naturally transformable strains. Experiments with B. thailandensis suggest that the newly developed HimarBP transposons can also be used for random mutagenesis of other Burkholderia spp., especially the closely related species B. mallei. Our results demonstrate that comprehensive transposon libraries of B. pseudomallei can be generated, providing additional tools for the study of the biology, pathogenesis, and antibiotic resistance of this pathogen.

Tularemia in a Park, Philadelphia, Pennsylvania

Tularemia in a Park, Philadelphia, Pennsylvania

A multicopy suppressor screening approach as a means to identify antibiotic resistance determinant candidates in Yersinia pestis

BackgroundYersinia pestis is the causative agent of plague and a potential agent of bioterrorism and biowarfare. The plague biothreat and the emergence of multidrug-resistant plague underscore the need to increase our understanding of the intrinsic potential of Y. pestis for developing antimicrobial resistance and to anticipate the mechanisms of resistance that may emerge in Y. pestis. Identification of Y. pestis genes that, when overexpressed, are capable of reducing antibiotic susceptibility is a useful strategy to expose genes that this pathogen may rely upon to evolve antibiotic resistance via a vertical modality. In this study, we explored the use of a multicopy suppressor, Escherichia coli host-based screening approach as a means to expose antibiotic resistance determinant candidates in Y. pestis.ResultsWe constructed a multicopy plasmid-based, Y. pestis genome-wide expression library of nearly 16,000 clones in E. coli and screened the library for suppressors of the antimicrobial activity of ofloxacin, a fluoroquinolone antibiotic. The screen permitted the identification of a transcriptional regulator-encoding gene (robAYp) that increased the MIC99 of ofloxacin by 23-fold when overexpressed from a multicopy plasmid in Y. pestis. Additionally, we found that robAYp overexpression in Y. pestis conferred low-level resistance to many other antibiotics and increased organic solvent tolerance. Overexpression of robAYp also upregulated the expression of several efflux pumps in Y. pestis.ConclusionOur study provides proof of principle for the use of multicopy suppressor screening based on the tractable and easy-to-manipulate E. coli host as a means to identify antibiotic resistance determinant candidates of Y. pestis.

Optimizing the Recovery of Surrogates for Bacterial Bioterrorism Agents from Ventilation Filters

AbstractHeating, ventilating, and air‐conditioning (HVAC) filters have the potential to serve as long‐term integrated samplers for biological particles that may be released intentionally or unintentionally into a public building. Methods for eluting bacteria that are surrogates for potential bioterrorism agents from HVAC filters were tested for their ability to recover as high a percentage of culturable bacteria as possible. A solution of 0.02% Tween‐80 in water was an effective eluent for Bacillus globigii (BG) spores whereas phosphate‐buffered saline was the most effective eluent for vegetative bacteria. Hand shaking was a more effective agitation method for high recovery rates than vortexing or mechanical shaking. The results indicate that BG spores can be eluted successfully while maintaining essentially 100% culturability. The recovery of the vegetative bacteria Mannheimia haemolytica and Yersinia ruckeri was 80% or higher under optimal conditions. The culturability of the vegetative bacteria eluted from HVAC filters declined substantially within as little as 2 h after the bacteria were loaded onto the filter. The culturability of the BG spores eluted from filters exhibited an exponential decay subsequent to loading with a half‐life of 8–9 days.

Coxiella burnetiiin Wild-caught Filth Flies

To the Editor: Coxiella burnetii, the agent of Q fever, is a bacterium and a potential agent of bioterrorism. The most frequent signs of infection in domestic animals are abortion and reduced fertility (1). Clinical signs of Q fever in humans vary from mild fevers to pneumonia, hepatitis, or death; atypical cases occur as other disorders, such as cholecystitis (1,2). Aerosols are the most common route of exposure, but oral transmission occurs (1). Some flies feed on the feces, milk, carcasses, or blood of domestic animals that can be infected with C. burnetii. These flies regurgitate and defecate when feeding and are mechanical vectors of bacteria (3,4). Flies have been shown to harbor, mechanically transport, and even support the growth of C. burnetii (4–6). It is known that house flies (Musca domestica) are possible mechanical vectors of C. burnetii because this organism survived 32 days in house flies and viable bacteria were shed by flies for 15 days (4). There are no studies of C. burnetii in field-collected flies. To examine the prevalence of C. burnetii in field-collected flies, we tested flies from farms, forests, ranches, and zoos. Flies that develop on animal dung, carcasses, feces, blood, or garbage are often called filth flies. Adult Calliphoridae, Hippoboscidae, Muscidae, and Sarcophagidae were collected from forests, zoos, ranches, and farms (Table). Flies were killed in 95% ethanol or by freezing. DNA was extracted from individual flies as described (7,8). A distilled water negative control was used for each extraction. Table Flies from the United States and Dominica assayed for Coxiella burnetii, 2004–2007 Individual DNA samples were tested, in duplicate, with a previously described TaqMan assay with a lower limit of detection of 1 C. burnetii organism (8). Positive and negative controls were used for all assays. Positive flies were verified by PCR and sequencing of 16S rRNA gene as described (9). Vouchers for each insect species were deposited in the Clemson University Arthropod Collection (Clemson, SC, USA), the University of Georgia Museum of Natural History (Athens, GA, USA), or the University of Wyoming Insect Collection (Laramie, WY, USA). Five of 363 flies were positive for C. burnetii DNA (Table). These flies included Stomoxys calcitrans, in which the adults feed on animal and human blood, and the blowflies Lucilia coeruleiviridis and L. sericata. C. burnetii–positive flies were obtained from carrion (1/12, 8.3%), a garbage bin of elephant feces (3/18, 16.7%), and a barn at a ranch (1/55, 1.8%). We sequenced 1,100 bp of the 16S rRNA gene from select DNA extracts, which were 99% identical with that of C. burnetii strain NC 002971. We detected DNA from C. burnetii in flies from a zoo, a ranch, and carrion in a forest. Laboratory data on house flies, which shed live C. burnetii for 15 days after exposure, suggest that related flies (e.g., S. calcitrans and Lucilia spp.) might also harbor viable C. burnetii. On the basis of our field data, S. calcitrans and Lucilia spp. should be studied as mechanical vectors of C. burnetii. Unlike many enteric bacteria, which require large inocula to cause disease, C. burnetii can be infectious at the level of 1 bacterium (10). If flies transmit C. burnetii, they pose an additional threat to human and animal health. The role of the sheep ked (Melophagus ovinus) in maintenance or transmission of C. burnetii is unknown. This fly is an obligate ectoparasite of sheep. It feeds on sheep blood, and feces from sheep keds can accumulate in the wool of sheep. Testing of sheep keds from infected sheep would help understand whether keds play a role in the epidemiology of C. burnetii.

Native Outer Membrane Proteins Protect Mice against Pulmonary Challenge with Virulent Type AFrancisella tularensis

Francisella tularensis is a gram-negative intracellular bacterium and the causative agent of the zoonotic disease tularemia. F. tularensis is a category A select agent and thus a potential agent of bioterrorism. Whereas an F. tularensis live, attenuated vaccine strain (LVS) is the basis of an investigational vaccine, this vaccine is not licensed for human use because of efficacy and safety concerns. In the present study, we immunized mice with isolated native outer membrane proteins (OMPs), ethanol-inactivated LVS (iLVS), or purified LVS lipopolysaccharide (LPS) and assessed the ability of each vaccine preparation to protect mice against pulmonary challenge with the virulent type A F. tularensis strain SchuS4. Antibody isotyping indicated that both Th1 and Th2 antibody responses were generated in mice after immunization with OMPs or iLVS, whereas LPS immunization resulted in only immunoglobulin A production. In survival studies, OMP immunization provided the greatest level of protection (50% survival at 20 days after infection with SchuS4), and there were associated 3-log reductions in the spleen and liver bacterial burdens (compared to nonvaccinated mice). Cytokine quantitation for the sera of SchuS4-challenged mice indicated that OMP and iLVS immunizations induced high levels of tumor necrosis factor alpha and interleukin-2 (IL-2) production, whereas only OMP immunization induced high levels of IL-10 production. By comparison, high levels of proinflammatory cytokines, including RANTES, granulocyte colony-stimulating factor, IL-6, IL-1alpha, IL-12p40, and KC, in nonvaccinated mice indicated that these cytokines may facilitate disease progression. Taken together, the results of this study demonstrate the potential utility of an OMP subunit (acellular) vaccine for protecting mammals against type A F. tularensis.

Genetic tools for allelic replacement in Burkholderia species.

Allelic replacement in the Burkholderia genus has been problematic due to the lack of appropriate counter-selectable and selectable markers. The counter-selectable marker sacB, commonly used in gram-negative bacteria, is nonselective on sucrose in many Burkholderia species. In addition, the use of antibiotic resistance markers of clinical importance for the selection of desirable genetic traits is prohibited in the United States for two potential bioterrorism agents, Burkholderia mallei and Burkholderia pseudomallei. Here, we engineered a mutated counter-selectable marker based on the B. pseudomallei PheS (the alpha-subunit of phenylalanyl tRNA synthase) protein and tested its effectiveness in three different Burkholderia species. The mutant PheS protein effectively killed 100% of the bacteria in the presence of 0.1% p-chlorophenylalanine. We assembled the mutant pheS on several allelic replacement vectors, in addition to constructing selectable markers based on tellurite (Tel(r)) and trimethoprim (Tp(r)) resistance that are excisable by flanking unique FLP recombination target (FRT) sequences. As a proof of concept, we utilized one of these gene replacement vectors (pBAKA) and the Tel(r)-FRT cassette to produce a chromosomal mutation in the Burkholderia thailandensis betBA operon, which codes for betaine aldehyde dehydrogenase and choline dehydrogenase. Chromosomal resistance markers could be excised by the introduction of pFLP-AB5 (Tp(r)), which is one of two constructed flp-containing plasmids, pFLP-AB4 (Tel(r)) and pFLP-AB5 (Tp(r)). These flp-containing plasmids harbor the mutant pheS gene and allow self curing on media that contain p-chlorophenylalanine after Flp-FRT excision. The characterization of the Delta betBA::Tel(r)-FRT and Delta betBA::FRT mutants indicated a defect in growth with choline as a sole carbon source, while these mutants grew as well as the wild type with succinate and glucose as alternative carbon sources.