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What Is Anthrax?

Anthrax is an acute infectious disease caused by the bacteria Bacillus anthracis and is highly lethal in its most virulent form. Anthrax most commonly occurs in wild and domestic herbivores, but it can also occur in humans when they are exposed to infected animals, tissue from infected animals, or high concentrations of anthrax spores. Still there are no case of people who got sick through contact with a diseased person. Anthrax means "coal" in Greek, and is used because victims develop black skin lesions.

Bacillus anthracis is a rod-shaped Gram-positive bacterium of size about 1 by 6 micrometres, and is the cause of the disease known as anthrax. B. anthracis was the first bacterium ever to be shown to cause disease, by Robert Koch in 1877. The specific name anthracis originates from the Greek word anthrax (ἄνθραξ), meaning coal, referring to the black skin lesions on the victims. The bacteria normally rest in spore form in the soil, and can survive for decades in this state. Once taken in by a herbivore, the bacteria start multiplying inside the animal and eventually kill it, then continue to reproduce in the carcass. Once they run out of nutrients there, they revert back to the dormant spore state.

Anthrax infection is rare but not remarkable in herbivores such as cattle, sheep, goats, camels, and antelopes. Anthrax can be found globally. It is more common in developing countries or countries without veterinary public health programs. Certain regions of the world (South and Central America, Southern and Eastern Europe, Asia, Africa, the Caribbean, and the Middle East) report more anthrax in animals than others.

When anthrax affects humans, it is usually due to an occupational exposure to infected animals or their products (such as skin and meat). Workers who are exposed to dead animals and animal products from countries where anthrax is more common may become infected with B. anthracis, and anthrax in wild livestock has occurred in the United States. Although many such workers are routinely exposed to significant levels of anthrax spores, most are not sufficiently exposed to develop symptoms.

Anthrax can enter the human body through the intestines, lungs (inhalation), or skin (cutaneous). Anthrax is non-contagious, and is unlikely to spread from person to person.

Pulmonary (pneumonic, respiratory, inhalation) anthrax infection initially presents with cold or flu-like symptoms for several days, followed by severe (and often fatal) respiratory problems. If not treated soon after exposure, before symptoms appear, inhalation infection is the most deadly, with a nearly 100% mortality rate. A lethal case of anthrax is reported to result from inhaling 10,000-20,000 spores. This form of the disease has also been known as Woolsorters' disease. Other routes have included the slicing up of animal horns for the manufacture of buttons, and handling bristles used for the manufacturing of brushes.

Gastrointestinal (gastroenteric) anthrax infection often presents with serious gastrointestinal difficulty, vomiting of blood, and severe diarrhea. Untreated, intestinal infection results in a 25-60% death rate.

Cutaneous (skin) anthrax infection presents with a large, painless necrotic ulcer (beginning as an irritating and itchy skin lesion or blister which is dark in color, usually concentrated as a black dot, somewhat resembling bread mold) at the site of infection, forming about a week or two after exposure. Unlike bruises or most other lesions, cutaneous anthrax does not cause pain. Cutaneous infection is the least deadly; without treatment, approximately 20% of all skin infection cases are fatal. Treated cutaneous anthrax is rarely fatal.

Treatment for anthrax infections includes large doses of intravenous and oral antibiotics, such as penicillin, ciprofloxacin, erythromycin, and vancomycin. For inhalation cases, antibiotic treatment is not very effective unless initiated within a day of exposure, before any symptoms appear. Antibiotic prophylaxis is crucial in cases of pulmonary anthrax to save lives. Some antibiotic-resistant strains are known.

A vaccine, produced from one component of the toxin of a non-virulent strand, is also available. The vaccine must be given at least four weeks before exposure to anthrax; annual booster injections are required to maintain immunity.

The spores can be trapped with a simple HEPA or P100 filter. Anthrax as an airborne threat can be prevented with a full-face mask. Unbroken skin is decontaminated simply with soap and water.

Bacillus anthracis is a rod-shaped gram-positive bacterium of size about 1 by 6 micrometres. It was the first bacterium ever to be shown to cause disease, by Robert Koch in 1877. The bacteria normally rest in spore form in the soil, and can survive for decades in this state. Once taken in by an herbivore, the bacteria start multiplying inside the animal and eventually kill it, then continue to reproduce in the carcass. Once they run out of nutrients there, they revert back to the dormant spore state.

The infection of herbivores (and humans) proceeds as follows: the spore is located and engulfed by scavenger cells of the immune system specialized to deal with invaders. Inside the scavenger cell, the spore turns into a bacillus, multiplies, and eventually bursts the cell, releasing bacilli into the bloodstream. There they release a protein toxin which has macrophages as its principal target. The toxin has two components: edema factor and lethal factor. Edema factor inactivates macrophages so that they cannot participate in the fight against the infection; lethal factor causes the macrophage to make TNF-alpha and interleukin-1-beta, both normal components of the immune system used to induce an inflammatory reaction. The excessive inflammation throughout the body ultimately leads to septic shock and death.

The virulence of a strain of anthrax is dependent on its enclosing capsule and its toxin; the two are determined by different genes.

Spores of this bacteria can be used in biological warfare. US Army personnel are now routinely vaccinated prior to active service in places where biological attacks are considered a threat. The anthrax vaccine, produced by BioPort Corporation, contains no live bacteria, and is approximately 93% effective in preventing infection. Anthrax vaccination is one of many factors suspected of causing Gulf War syndrome.

British tests in 1942 contaminated Gruinard Island in Scotland with anthrax spores, and rendered it unusable for the following 48 years. An accidental release of anthrax in a research lab at Fort Detrick in Frederick, Maryland in the United States led to the permanent sealing of a building with plastics and glues.

The simplest method of obtaining an anthrax sample would be to get it from an animal killed by anthrax; this source is rare in developed countries but common in underdeveloped countries.

Cultivating anthrax spores takes minimal equipment and about a first-year collegiate microbiological education. However, to make an aerosol form of anthrax suitable for biological warfare requires extensive training and highly developed equipment.

The 2001 anthrax attacks occurred over the course of several weeks beginning on September 18, 2001. Letters containing anthrax bacteria were sent to five media offices and two US Senators. Five people died as a result. To date, no individual or group has been charged with the attacks themselves.

After the devastating September 11, 2001 attacks there was immediate speculation of linkage between the two events. It is alleged that a "skin lesion" found on Ahmed al-Haznawi, one of the September 11 hijackers who sought treatment in Fort Lauderdale, Florida, was cutaneous anthrax. However, the anthrax letters were mailed after September 11. It is difficult to see how the hijackers were responsible for the anthrax attacks unless there were surviving comrades who continued their terrorist campaign.

The FBI claims the anthrax attack was a result of domestic terrorism. The Justice Department has labeled a former government scientist Dr. Steven Hatfill as a "person of interest" in the case, but has not brought charges. Hatfill has maintained his innocence and is suing the government and the news media for ruining his life.

The letters that were mailed contain at least two different grades of anthrax material. However, all of the anthrax within the letters were of the same strain. This strain, known as the Ames strain, was first researched at the Army's Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, Maryland. The Ames strain was then distributed to at least fifteen bio-research labs within the US and six overseas.

The first set of anthrax letters are believed to have been mailed from Trenton NJ on September 18, 2001, exactly one week from the attacks on September 11. Five letters are thought to have been mailed at this time and addressed to media outlets, mostly in the New York City area. The recipients of these letters were ABC News, CBS News, NBC News, and the New York Post — all located in New York City. The other letter believed to have been mailed around this time was addressed to The National Enquirer's old mailing address and later forwarded to their new offices at American Media Inc. in Florida. AMI also publishes a tabloid called Sun where one of the anthrax victims, Robert Stevens, died.

A note was found in the New York Post letter which read, "09-11-01, THIS IS NEXT, TAKE PENACILIN NOW, DEATH TO AMERICA, DEATH TO ISRAEL, ALLAH IS GREAT." Some believe this note represented a benign form of bio-terrorism since medical advice was included in the misspelled precaution: "take penacilin now." It has been suggested the mailer of the New York media anthrax really did not want to harm anyone. A more convincing argument points to the fact that the anthrax in the New York media letters consisted of coarse brown granules and could have caused only skin infections, cutaneous anthrax, and not death.

The second group of anthrax letters were mailed three weeks later on October 9 from Trenton NJ and addressed to two Democrat Senators, Tom Daschle of South Dakota, and Patrick Leahy of Vermont. The two letters had identical notes which read, "09-11-01, YOU CAN NOT STOP US. WE HAVE THIS ANTHRAX. YOU DIE NOW. ARE YOU AFRAID? DEATH TO AMERICA. DEATH TO ISRAEL. ALLAH IS GREAT." The anthrax in the Daschle letter was described as fine powder. In comparison, the Daschle material was ten times denser in anthrax spores when compared to the retrieved New York Post sample. The Daschle anthrax consisted of nearly pure spores, with a concentration of a trillion spores per gram.

The anthrax material in the Leahy letter was similar to the powder found in Daschle letter. However, the Leahy anthrax powder had particles that were smaller and more uniform in size when compared to the Daschle anthrax. Bio-defense experts are having difficulty in reverse engineering the anthrax material found in the Senate letters.

Microb Pathog, 2005 Jan, 38(1), 33 - 40 Epub 2004 Dec 19.
Mouse model characterisation for anthrax vaccine development: comparison of one inbred and one outbred mouse strain; Flick-Smith HC et al.; In order to evaluate the immunogenicity and protective efficacy of anthrax vaccine candidates a suitable small animal model is required . The inbred A/J strain of mouse has been selected as a potential model, and its immune response to immunisation with recombinant protective antigen (rPA) vaccine characterised, by assessment of rPA specific antibody production, and protection against injected challenge, with the unencapsulated STI strain of Bacillus anthracis . Studies were conducted to determine the time required post immunisation to develop a protective immune response, to define the minimum protective dose of vaccine required and to assess the long-term immune response to immunisation . From the results of these studies it was possible to establish that the A/J mouse is a consistent and robust small animal model for rPA vaccine testing . A comparison of the immune response to rPA vaccine immunisation in the Turner Outbred (TO) mouse strain was also conducted . Both inbred and outbred mouse strains displayed a predominantly Th2 biased immune response and showed a comparable antibody response to rPA immunisation . An assessment of protection in the TO mouse against aerosol challenge with the fully virulent strain of B . anthracis, Ames, was also made.

Microb Pathog, 2005 Jan, 38(1), 1 - 12 Epub 2005 Jan 6.
Construction of a rhamnose mutation in Bacillus anthracis affects adherence to macrophages but not virulence in guinea pigs; Bozue JA et al.; Carbohydrate analyses of whole-spore extracts have confirmed the presence of rhamnose in the spore of the fully virulent Ames strain of Bacillus anthracis . A gene cluster containing loci with high homology to the rhamnose biosynthetic genes, rmlACBD, was identified within the B . anthracis chromosome . The first gene of this cluster, rmlA, was inactivated by forming a merodiploid cointegrate using an internal fragment of the gene within the Ames strain of B . anthracis to construct the mutant strain Ames-JAB1 . Carbohydrate analysis of spores from this mutant demonstrated the loss of rhamnose . When assaying for spore infection of macrophages, we detected a significant decrease in the recovery with the Ames-JAB1 strain compared to the recovery with the Ames wild-type strain . When pre-treating macrophages with cytochalasin-D, spores of the mutant were further hindered in recovery, indicating that the spores were not able to bind as well to the macrophages . However, in guinea pigs challenge experiments, no difference in virulence was observed between the mutant and wild-type strains . These results suggest that the incorporation of rhamnose into the spore coat of B . anthracis is required for optimal interaction with macrophages but is not required for full virulence in this animal model.

J Ethnopharmacol, 2005 Feb 10, 97(1), 43 - 47 Epub 2004 Dec 15.
Some pharmacological properties of extracts of Terminalia sericea roots; Moshi MJ et al.; Terminalia sericea Burch . Ex . DC (Combretaceae) extracts are used to treat bacterial infections, diarrhea, and diabetes . Intermediate and polar extracts of the roots exhibited antibacterial activity against Staphylococcus aureus, Escherichia coli, Bacillus anthracis, and Pseudomonas aeruginosa, while the petroleum ether extract was inactive . The extracts were mildly active against Bacillus anthracis and Pseudomonas aeruginosa but exhibited the highest activity against Staphylococcus aureus . They also exhibited antifungal activity against Candida albicans and Aspergillus niger . An 80% aqueous ethanol extract of the roots did not have any effect on blood glucose levels during an oral glucose tolerance test (OGTT), in mice (P>0.05) . With the exception of the dichloromethane and petroleum ether extracts, all the intermediate and polar extracts were toxic to brine shrimps giving LC(50) (95% confidence intervals) values ranging from 5.4 (3.5-8.4) to 17.4 (11.4-26.5) mug/ml, while that of cyclophosphamide, a standard anticancer drug, was 16.3 (10.6-25.2) mug/ml . Further work is in progress to isolate and identify active compounds in the extracts.

Toxicol In Vitro, 2005 Mar, 19(2), 221 - 9
Differential susceptibility of macrophage cell lines to Bacillus anthracis-Vollum 1B; Gutting BW et al.; Bacillus anthracis (BA) is a spore forming bacterium and the causative agent of anthrax disease . Macrophages (Mvarphis) play a central role in anthrax disease . An important step in disease progression is the ability of BA to secrete lethal toxin (LeTx) that kills Mvarphis . LeTx is a heterodimer composed of protective antigen (PA) and lethal factor (LF) . Researchers have shown that Mvarphi cell lines demonstrate differential susceptibility to purified LeTx; for example RAW264.7 and J774A.1 Mvarphis are sensitive to LeTx whereas IC-21 Mvarphis are resistant . Research has also suggested that exogenous factors, including other BA proteins, can influence the activity of LeTx . For this reason, the objective of the current work was to examine if RAW264.7, J774A.1, and IC-21 Mvarphis demonstrated differential susceptibility when cultured with a LeTx-producing strain of BA . Here, we co-cultured Mvarphis with LeTx(+) Vollum 1B (V1B) spores for >15h and assayed for Mvarphi cell death by morphology, trypan blue (TB) staining, neutral red (NR) activity, and lactate dehydrogenase (LDH) activity in the culture media . Following the addition of V1B spores, necrosis ( approximately 50% mortality) was observed in RAW264.7 and J774A.1 Mvarphis at 7.5 and 10h, respectively . By 15h, both RAW264.7 and J774A.1 Mvarphis demonstrated 100% mortality . In contrast, IC-21 Mvarphis, under identical culture conditions, remained viable (98%) and activated throughout the course of the experiment (>24h) . The mechanism of RAW264.7 cell death appeared to involve LeTx because the V1B-induced cytotoxicity was dose-dependently reversed by the addition of anti-PA antibody to the culture media . These observations suggest there is differential susceptibility of Mvarphi cell lines to the LeTx(+) V1B strain of BA . Further development of this in vitro model may be useful to further characterize the interactions between Mvarphis and BA spores.

J Biol Chem . 2005 Jan 11; {Epub ahead of print}
Purified bacillus anthracis lethal toxin complex formed in vitro and during infection exhibits functional and biological activity; Panchal RG et al.; Anthrax protective antigen (PA, 83 kDa), a pore-forming protein upon protease activation to 63 kDa (PA63), translocates lethal factor (LF) and edema factor (EF) from endosomes into the cytosol of the cell . The relatively small size of the heptameric PA63 pore (~12 A) raises questions as to how large molecules like LF and EF can move through the pore . In addition, the reported high binding affinity between PA and EF/LF suggests that EF/LF may not dissociate but remain complexed with activated PA63 . In this study, we found that purified (PA63)7:LF complex exhibited biological and functional activities similar to the free LF . Purified LF complexed with PA63 heptamer was able to cleave both a synthetic peptide substrate and endogenous mitogen-activated protein kinase kinase substrates and kill susceptible macrophage cells . Electrophysiological studies of the complex showed strong rectification of the ionic current at positive voltages, an effect similar to that observed if LF is added to the channels formed by heptameric PA63 pore . Complexes of (PA63)7:LF found in the plasma of infected animals showed functional activity . Identifying active complex in the blood of infected animals has important implications for therapeutic design, especially those directed against PA and LF . Our studies suggest that the individual toxin components and the complex must be considered as critical targets for anthrax therapeutics.

Genome Biol . 2005;6(1):R10 . Epub 2004 Dec 17.
Microarray-based resequencing of multiple Bacillus anthracis isolates; Zwick ME et al.; We used custom-designed resequencing arrays to generate 3.1 Mb of genomic sequence from a panel of 56 Bacillus anthracis strains . Sequence quality was shown to be very high by replication (discrepancy rate of 7.4 x 10(-7)) and by comparison to independently generated shotgun sequence (discrepancy rate < 2.5 x 10(-6)) . Population genomics studies of microbial pathogens using rapid resequencing technologies such as resequencing arrays are critical for recognizing newly emerging or genetically engineered strains.

Appl Environ Microbiol, 2005 Jan, 71(1), 566 - 8
Chlorine inactivation of bacterial bioterrorism agents; Rose LJ et al.; Seven species of bacterial select agents were tested for susceptibility to free available chlorine (FAC) . Under test conditions, the FAC routinely maintained in potable water would be sufficient to reduce six species by 2 orders of magnitude within 10 min . Water contaminated with spores of Bacillus anthracis spores would require further treatment.

Biomed Instrum Technol, 2004 Nov-Dec, 38(6), 476 - 84
Damage of office supply, personal use items, and over-the-counter medical devices after sterilization by ethylene oxide gas, electron beam, and gamma radiation; Lucas AD et al.; After letters containing Bacillus anthracis spores entered the U.S . mail in 2001, a problem emerged regarding how to decontaminate the letters, packages, and personal items in offices that received these letters . The effects of three sterilization methods (i.e . ethylene oxide gas {EO}, electron beam {e-beam} radiation, and gamma radiation) were evaluated for a variety of office supply and equipment, personal use items, and over-the-counter medical devices . No single sterilization method was suitable for all items that could be mailed or found in an office . Damage or discoloration was evident for some items by each sterilization method . There were changes in the color of certain items, such as some of the packaging material, some pacifiers, some of the fabrics, and the nylon stockings after e-beam and gamma radiation . Both e-beam and gamma radiation damaged all film samples . Following EO sterilization and normal aeration, there were a number of samples with high (above 250 microg/g) levels of EO and samples with detectable ethylene chlorohydrin levels . The data would suggest that certain items exposed to EO sterilization must be further aerated prior to use, or discarded . Generic descriptions of products (such as plastics) or grouping of items (such as condoms) were not sufficient to predict what is safe in terms of EO residual levels remaining on an item . Successful decontamination of a wide variety of items will require careful selection of different sterilization methods.

J Infect Dis, 2005 Feb 1, 191(3), 422 - 34 Epub 2004 Dec 22.
Late treatment with a protective antigen-directed monoclonal antibody improves hemodynamic function and survival in a lethal toxin-infused rat model of anthrax sepsis; Cui X et al.; Background . In animal models, treatment with 5H3, a fully human protective antigen-directed monoclonal antibody (PA-MAb), improved survival when administered close to the time of Bacillus anthracis lethal toxin (LeTx) bolus or live bacterial challenge . However, treatment with PA-MAb would be most valuable clinically if it were beneficial even when administered after the onset of shock and lethality due to LeTx.Methods . We investigated the effects of PA-MAb versus placebo administered in rats (n=324) at the time of or 3, 6, 9, or 12 h after the initiation of a 24-h LeTx infusion.Results . In rats receiving placebo, mean arterial blood pressure (MBP) and heart rate (HR) were decreased in nonsurvivors, compared with those in survivors, at 6 h and then worsened further, with lethality first evident at 8 h (median, 16 h; range, 8-152 h) . At each treatment time, survival rates were greater for PA-MAb than for placebo, although improvement was decreased at later treatment times (P=.001, for the effect of time) . Compared with placebo, PA-MAb significantly increased MBP during the 12 h after the initiation of treatment, but the increase was greatest for treatment at 3 h; similarly, PA-MAb significantly increased HR at all treatment times.Conclusion . In this rat model, improvements in outcome due to PA-MAb were significant when it was administered up to 6 h (and approached significance when administered up to 12 h) after initial exposure to LeTx . Clinically, PA-MAb may be beneficial even when administered after the onset of shock and lethality due to LeTx.

Anal Chem, 2005 Jan 1, 77(1), 284 - 9
Autonomous detection of aerosolized biological agents by multiplexed immunoassay with polymerase chain reaction confirmation; Hindson BJ et al.; The autonomous pathogen detection system (APDS) is an automated, podium-sized instrument that continuously monitors the air for biological threat agents (bacteria, viruses, and toxins) . The system has been developed to warn of a biological attack in critical or high-traffic facilities and at special events . The APDS performs continuous aerosol collection, sample preparation, and detection using multiplexed immunoassay followed by confirmatory PCR using real-time TaqMan assays . We have integrated completely reusable flow-through devices that perform DNA extraction and PCR amplification . The fully integrated system was challenged with aerosolized Bacillus anthracis, Yersinia pestis, Bacillus globigii, and botulinum toxoid . By coupling highly selective antibody- and DNA-based assays, the probability of an APDS reporting a false positive is extremely low.

Biophys Chem, 2005 Jan 1, 113(1), 67 - 81
A fractal analysis of pathogen detection by biosensors; Morris BA et al.; A fractal analysis is presented for the detection of pathogens such as Franscisela tularensis, Yersinia pestis (the bacterium that causes plague), Bacillus anthracis, Venezuelan equine encephalitis (VEE) virus, Vavcinia virus, and Escherichia coli using a cellular analysis and notification of antigens risks and yields (CANARY) biosensor {T.H . Rider, M.S . Petrovic, F.E . Nargi, J.D Harper, E.D . Schwoebel, R.H . Mathews, D.J . Blanchard, L.T Bortolin, A.M . Young, J . Chen, M.A . Hollis, A cell-based sensor for rapid identification of pathogens, Science 301 (2003, 11 July) 213-215, T.H . Rider, M.S . Petrovic, F.E . Nargi, J.D . Harper, E.D . Schwoebel, R.H . Mathews, D.J . Blanchard, L.T . Bortolin, A.M . Young, J . Chen, M.A . Hollis, A cell-based sensor for rapid identification of pathogens, Science 301 (2003, 11 July) 213-215 . Science Online, } . In general, the binding and dissociation rate coefficients may be adequately described by either a single- or a dual-fractal analysis . An attempt is made to relate the binding rate coefficient to the degree of heterogeneity (fractal dimension value) present on the biosensor surface . Binding and dissociation rate coefficient values obtained are presented . Due to the dilute nature of the analyte(s) present, in some cases, a triple-fractal analysis is required to adequately describe the binding kinetics . It should be noted, and this is not entirely unexpected, that there is a lot of variation in the original experimental data when dilute concentrations of the analyte were analyzed by the CANARY biosensor {T.H . Rider, M.S . Petrovic, F.E . Nargi, J.D Harper, E.D . Schwoebel, R.H . Mathews, D.J . Blanchard, L.T Bortolin, A.M . Young, J . Chen, M.A . Hollis, A cell-based sensor for rapid identification of pathogens, Science 301 (2003, 11 July) 213-215, Science Online, } . The data analyzed in this manuscript appears smoother since only discrete points at different time intervals were analyzed . The kinetics aspects along with the affinity values presented are of interest and should along with the rate coefficients presented for the binding and the dissociation phase be of significant interest in help designing better biosensors for an application area that is bound to gain increasing importance in the future.

EMBO J . 2004 Dec 16; {Epub ahead of print}
Capsule synthesis by Bacillus anthracis is required for dissemination in murine inhalation anthrax; Drysdale M et al.; Bacillus anthracis, the agent of anthrax, produces a poly-D-glutamic acid capsule that has been implicated in virulence . Many strains missing pXO2 (96 kb), which harbors the capsule biosynthetic operon capBCAD, but carrying pXO1 (182 kb) that harbors the anthrax toxin genes, are attenuated in animal models . Also, noncapsulated strains are readily phagocytosed by macrophage cell lines, whereas capsulated strains are resistant to phagocytosis . We show that a strain carrying both virulence plasmids but deleted specifically for capBCAD is highly attenuated in a mouse model for inhalation anthrax . The parent strain and capsule mutant initiated germination in the lungs, but the capsule mutant did not disseminate to the spleen . A mutant harboring capBCAD but deleted for the cap regulators acpA and acpB was also significantly attenuated, in agreement with the capsule-negative phenotype during in vitro growth . Surprisingly, an acpB mutant, but not an acpA mutant, displayed an elevated LD(50) and reduced ability to disseminate, indicating that acpA and acpB are not true functional homologs and that acpB may play a larger role in virulence than originally suspected.

Arch Microbiol . 2004 Dec 21; {Epub ahead of print}
Flow cytometry analysis of germinating Bacillus spores, using membrane potential dye; Laflamme C et al.; Germination of Bacillus anthracis spores is necessary for the transcription of plasmidic genes essential to the infection . Assessing germination potential is crucial to predict the risk associated with pathogenic Bacillus exposure . The aim of this study was to set up a viability assay based on membrane potential in order to predict the earliest germination event of spores . B . cereus and two strains of B . subtilis were used . The spores were isolated with a sodium bromide gradient . Approximately 10(7) spores were incubated at 37 degrees C in tryptic soy broth (TSB) . Aliquots were harvested at predetermined times and stained with 3,3'-dihexyloxacarbocyanine iodide {DiOC(6)(3)} or with bis-(1,3-dibutylbarbituric acid) trimethine oxonol {DiBAC(4)(3)} . Fluorescence characteristics were obtained using flow cytometry . The earliest detectable activation of membrane potential occurred after 15 min of incubation in TSB using DiOC(6)(3) . Using DiBAC(4)(3), the earliest detectable signal was after 4 h of incubation . Control experiments using carbonyl cyanide m-chlorophenylhydrazone (CCCP)-treated spores did not show any change in the fluorescence intensity over time . Since no membrane potential and no germination were detected in CCCP-treated spores, the activation of membrane potential seems to be associated with germination . DiOC(6)(3) can be used as an early membrane potential indicator for spores . DiBAC(4)(3), by contrast, is not a early membrane potential marker.

J Exp Med, 2004 Dec 20, 200(12), 1647 - 55
Anthrolysin O and Other Gram-positive Cytolysins Are Toll-like Receptor 4 Agonists; Park JM et al.; Exposure of bone marrow-derived macrophages (BMDMs) to low concentrations of Bacillus anthracis lethal toxin (LT), whose catalytic subunit is lethal factor (LF), results in induction of a robust apoptotic response dependent on activation of Toll-like receptor (TLR)4 . A similar TLR4-dependent apoptotic response is observed when BMDMs are infected with live B . anthracis (Sterne strain) . However, TLR4 is considered to be a specific signaling receptor for lipopolysaccharide (LPS), a typical product of gram-negative bacteria, whereas B . anthracis is gram-positive . To understand how B . anthracis can activate TLR4, we analyzed its culture supernatants and found them to contain a potent TLR4-stimulating activity that can also induce apoptosis in macrophages in which the antiapoptotic p38 MAP kinase (whose activation is prevented by LF) was inhibited . Purification of this activity suggested it consists of anthrolysin O (ALO), a member of the cholesterol-dependent cytolysin (CDC) family . We show that recombinant ALO can activate TLR4 in a manner independent of LPS contamination and, together with LT, can induce macrophage apoptosis . We also provide genetic evidence that ALO is required for induction of macrophage apoptosis in response to infection with live B . anthracis and that other CDC family members share the ability to activate TLR4.

J Infect Dis, 2005 Jan 15, 191(2), 278 - 88 Epub 2004 Dec 15.
Protective Immunization against Inhalational Anthrax: A Comparison of Minimally Invasive Delivery Platforms; Mikszta JA et al.; A new anthrax vaccine under clinical investigation is based on recombinant Bacillus anthracis protective antigen (rPA) . Here, we investigated microneedle-based cutaneous and nasal mucosal delivery of rPA in mice and rabbits . In mice, intradermal (id) delivery achieved up to 90% seroconversion after a single dose, compared with 20% after intramuscular (im) injection . Intranasal (inl) delivery of a liquid formulation required 3 doses to achieve responses that were comparable with those achieved via the id or im routes . In rabbits, id delivery provided complete protection against aerosol challenge with anthrax spores; in addition, novel powder formulations administered inl provided complete protection, whereas a liquid formulation provided only partial protection . These results demonstrate, for the first time, that cutaneous or nasal mucosal administration of rPA provides complete protection against inhalational anthrax in rabbits . The novel vaccine/device combinations described here have the potential to improve the efficacy of rPA and other biodefense vaccines.

Proc Natl Acad Sci U S A, 2004 Dec 21, 101(51), 17813 - 8 Epub 2004 Dec 15.
Activation of liver X receptors and retinoid X receptors prevents bacterial-induced macrophage apoptosis; Valledor AF et al.; Microbe-macrophage interactions play a central role in the pathogenesis of many infections . The ability of some bacterial pathogens to induce macrophage apoptosis has been suggested to contribute to their ability to elude innate immune responses and successfully colonize the host . Here, we provide evidence that activation of liver X receptors (LXRs) and retinoid X receptors (RXRs) inhibits apoptotic responses of macrophages to macrophage colony-stimulating factor (M-CSF) withdrawal and several inducers of apoptosis . In addition, combined activation of LXR and RXR protected macrophages from apoptosis caused by infection with Bacillus anthracis, Escherichia coli, and Salmonella typhimurium . Expression-profiling studies demonstrated that LXR and RXR agonists induced the expression of antiapoptotic regulators, including AIM/CT2, Bcl-X(L), and Birc1a . Conversely, LXR and RXR agonists inhibited expression of proapoptotic regulators and effectors, including caspases 1, 4/11, 7, and 12; Fas ligand; and Dnase1l3 . The combination of LXR and RXR agonists was more effective than either agonist alone at inhibiting apoptosis in response to various inducers of apoptosis, and it acted synergistically to induce expression of AIM/CT2 . Inhibition of AIM/CT2 expression in response to LXR/RXR agonists partially reversed their antiapoptotic effects . These findings reveal unexpected roles of LXRs and RXRs in the control of macrophage survival and raise the possibility that LXR/RXR agonists may be exploited to enhance innate immunity to bacterial pathogens that induce apoptotic programs as a strategy for evading host responses.

Biophys J . 2004 Dec 13; {Epub ahead of print}
Anthrax toxin protective antigen: inhibition of channel function by chloroquine and related compounds and study of binding kinetics using the current noise analysis; Orlik F et al.; Protective antigen (PA) of the tripartite anthrax toxin binds to a cell surface receptor and mediates the transport of two enzymatic components edema factor (EF) and lethal factor (LF) into the cytosol of host cells . Here recombinant PA63 from Bacillus anthracis was reconstituted into artificial lipid bilayer membranes and formed ion permeable channels . The heptameric PA63-channel contains a binding-site for 4-aminoquinolones, which block ion transport through PA in vitro . This result allowed a detailed investigation of ligand binding and the stability constants for the binding of chloroquine, fluphenazine and quinacrine to the binding-site inside the PA63-channel were determined using titration experiments . Open PA63-channels exhibit 1/f noise in the frequency range between 1 and 100 Hz, while the spectral density of the ligand-induced current noise was of Lorentzian type . The analysis of the power density spectra allowed the evaluation of the on- and off-rate constants (k1 and k-1 ) of ligand binding . The on-rate constants of ligand binding were between 10(6) and 10(8) M (-1) s(-1) and were dependent on the ionic strength of the aqueous phase, sidedness of ligand addition as well as the orientation and intensity of the applied electric field . The off-rates varied between about 10 s(-1) and 2600 s (-1) and depended mainly on the structure of the ligand.

Microbiol Mol Biol Rev, 2004 Dec, 68(4), 617 - 29
Rabbit and nonhuman primate models of toxin-targeting human anthrax vaccines; Phipps AJ et al.; 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.

J Clin Microbiol, 2004 Dec, 42(12), 5825 - 31
Real-time PCR assay for a unique chromosomal sequence of Bacillus anthracis; Bode E et al.; Real-time PCR has become an important method for the rapid identification of Bacillus anthracis since the 2001 anthrax mailings . Most real-time PCR assays for B . anthracis have been developed to detect virulence genes located on the pXO1 and pXO2 plasmids . In contrast, only two published chromosomal targets exist, the rpoB gene and the gyrA gene . In the present study, subtraction-hybridization with a plasmid-cured B . anthracis tester strain and a Bacillus cereus driver was used to find a unique chromosomal sequence . By targeting this region, a real-time assay was developed with the Ruggedized Advanced Pathogen Identification Device . Further testing has revealed that the assay has 100% sensitivity and 100% specificity, with a limit of detection of 50 fg of DNA . The results of a search for sequences with homology with the BLAST program demonstrated significant alignment to the recently published B . anthracis Ames strain, while an inquiry for protein sequence similarities indicated homology with an abhydrolase from B . anthracis strain A2012 . The importance of this chromosomal assay will be to verify the presence of B . anthracis independently of plasmid occurrence.

Biotechnol Prog, 2004 Nov-Dec, 20(6), 1651 - 9
Production of biologically active Bacillus anthracis edema factor in Escherichia coli; Cooksey BA et al.; Anthrax is caused by the gram-positive spore-forming bacterium Bacillus anthracis . The anthrax toxin consists of three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF) . PA facilitates the translocation of LF and EF into the cytosol of mammalian cells . LF is thought to be a zinc-dependent metalloprotease that results in death . EF is a calmodulin- and calcium-dependent adenylate cyclase that causes edema upon entrance into the cytosol by elevating the cAMP levels in cells . Previous efforts to produce recombinant EF (rEF) in Escherichia coli yielded only 2.5 mg of rEF per liter of culture . In this work, we produced soluble rEF in large quantities in both the periplasm and cytoplasm of E . coli from shake flasks and fermentors . The rEF protein was purified by standard chromatography and yielded >97% pure, biologically active rEF . Yields of purified rEF from medium cell density fermentations resulted in up to 2.38 g/L of highly pure, biologically active rEF protein . These results exhibit the ability to generate gram quantities of active rEF from E . coli.

Appl Environ Microbiol, 2004 Dec, 70(12), 7040 - 5
Evaluation of the Biological Sampling Kit (BiSKit) for large-area surface sampling; Buttner MP et al.; Current surface sampling methods for microbial contaminants are designed to sample small areas and utilize culture analysis . The total number of microbes recovered is low because a small area is sampled, making detection of a potential pathogen more difficult . Furthermore, sampling of small areas requires a greater number of samples to be collected, which delays the reporting of results, taxes laboratory resources and staffing, and increases analysis costs . A new biological surface sampling method, the Biological Sampling Kit (BiSKit), designed to sample large areas and to be compatible with testing with a variety of technologies, including PCR and immunoassay, was evaluated and compared to other surface sampling strategies . In experimental room trials, wood laminate and metal surfaces were contaminated by aerosolization of Bacillus atrophaeus spores, a simulant for Bacillus anthracis, into the room, followed by settling of the spores onto the test surfaces . The surfaces were sampled with the BiSKit, a cotton-based swab, and a foam-based swab . Samples were analyzed by culturing, quantitative PCR, and immunological assays . The results showed that the large surface area (1 m2) sampled with the BiSKit resulted in concentrations of B . atrophaeus in samples that were up to 10-fold higher than the concentrations obtained with the other methods tested . A comparison of wet and dry sampling with the BiSKit indicated that dry sampling was more efficient (efficiency, 18.4%) than wet sampling (efficiency, 11.3%) . The sensitivities of detection of B . atrophaeus on metal surfaces were 42 +/- 5.8 CFU/m2 for wet sampling and 100.5 +/- 10.2 CFU/m2 for dry sampling . These results demonstrate that the use of a sampling device capable of sampling larger areas results in higher sensitivity than that obtained with currently available methods and has the advantage of sampling larger areas, thus requiring collection of fewer samples per site.

Proc Natl Acad Sci U S A, 2004 Dec 7, 101(49), 17061 - 5 Epub 2004 Dec 7.
Inorganic polyphosphate in Bacillus cereus: motility, biofilm formation, and sporulation; Shi X et al.; Chains of inorganic polyphosphate (poly-P) with hundreds of P(i) residues linked by phosphoanhydride bonds, as in ATP, are found in every bacterial, fungal, plant, and animal cell, in which they perform various functions . In the spore-forming Bacillus cereus, we have identified three principal enzymes and genes involved in the metabolism of poly-P, namely, (i) poly-P kinase (PPK), which synthesizes poly-P reversibly from ATP, (ii) exopolyphosphatase (PPX), which hydrolyzes poly-P to P(i), and (iii) poly-P/AMP phosphotransferase (PAP), which uses poly-P as a donor to convert AMP to ADP, reversibly . In the null mutant of ppk, poly-P levels are reduced to <5% of the WT; in the ppx mutant, the PPK activity is elevated 10-fold, and the accumulation of poly-P is elevated approximately 1,000-fold . All of the null mutants of ppk, ppx, and pap showed defects in motility and biofilm formation, but sporulation efficiency was impaired only in the ppx mutant . These enzymes and genes in B . cereus are nearly identical to those in the very closely related pathogen Bacillus anthracis, and, thus, they may provide attractive targets for the treatment of anthrax.

Dermatol Ther, 2004, 17(6), 505 - 12
Therapy of other bacterial infections; Werlinger KD et al.; Cutaneous bacterial disease continues to account for a significant proportion of clinical visits . The continuing emergence of strains that are resistant to available antibacterial agents creates a challenge for dermatologists, who need to keep abreast of current treatment strategies . In this article, antibacterial regimens are presented for skin infections caused by organisms such as Staphylococcus, Streptococcus, Pseudomonas, Neisseria, Haemophilus ducreyi, Treponema pallidum, Bacillus anthracis, Yersinia pestis, Pasteurella multocida, Vibrio vulnificus, Actinomyces, and Nocardia.

Proc Natl Acad Sci U S A, 2004 Dec 7, 101(49), 17246 - 51 Epub 2004 Dec 7.
EST-based genome-wide gene inactivation identifies ARAP3 as a host protein affecting cellular susceptibility to anthrax toxin; Lu Q et al.; The lethality of infection by Bacillus anthracis is largely due to its plasmid-encoded toxins, which consist of a carrier protein, the protective antigen (PA), in combination with either the lethal-factor or edema-factor moiety . During B . anthracis infections, PA secreted by bacteria binds to membrane receptors of susceptible cells, is cleaved proteolytically, attaches to lethal factor or edema factor, undergoes oligomerization and internalization, and transports its toxin partners to acidic endosomes where they are released into the cytosol . To identify specific host functions that mediate these events, we used RNA encoded by a lentivirus-based library of approximately 40,000 human ESTs to inactivate chromosomal genes in a human cell population, and we isolated clones that survived PA-dependent toxin-induced death . This phenotypic screen and subsequent analysis identified ARAP3, which is a phosphoinositide-binding protein implicated previously in membrane vesicle trafficking and cytoskeletal organization, as a mammalian host-cell gene that is essential for normal anthrax toxicity . ARAP3 deficiency produced by antisense expression of an ARAP3 EST impaired entry of PA and its bound toxigenic moieties into both human and mouse cells, resulting in reduced toxin sensitivity . Our results demonstrate the usefulness of antisense EST libraries for global chromosomal gene inactivation, establish the practicality of loss-of-function phenotypic screens for the identification of genomic loci required for pathogen effects in mammalian cells, and reveal an important role for ARAP3 in cellular internalization of anthrax toxin.

Antimicrob Agents Chemother, 2004 Dec, 48(12), 4873 - 7
Beta-lactamase gene expression in a penicillin-resistant Bacillus anthracis strain; Chen Y et al.; Expression of the bla1 and bla2 genes in an archetypal Bacillus anthracis strain is insufficient for penicillin resistance . In a penicillin-resistant clinical isolate, both genes are highly transcribed, but bla1 is the major contributor to high-level resistance to ampicillin . Differential expression of the bla genes is dependent upon strain background.

Antimicrob Agents Chemother, 2004 Dec, 48(12), 4643 - 9
Functional cloning of Bacillus anthracis dihydrofolate reductase and confirmation of natural resistance to trimethoprim; Barrow EW et al.; Bacillus anthracis is reported to be naturally resistant to trimethoprim (TMP), a drug that inhibits dihydrofolate reductase (DHFR), a key enzyme in the folate pathway . A microdilution broth assay established that the MIC of TMP for B . anthracis Sterne is >2,048 but < or =4,096 microg/ml . A putative DHFR sequence was amplified from B . anthracis Sterne genomic DNA . The PCR product was cloned into the Invitrogen pCRT7/CT-TOPO vector, followed by transformation into Escherichia coli TOP10F' chemically competent cells . Plasmid DNA from a clone showing the correct construct with a thrombin cleavage site attached downstream from the terminus of the cloned PCR product was transformed into E . coli BL21 Star (DE3)pLysS competent cells for expression of the six-histidine-tagged fusion protein and purification on a His-Bind resin column . Functionality of the purified Sterne recombinant DHFR (Sterne rDHFR) was confirmed in an established enzyme assay . The 50% inhibitory concentrations of TMP and methotrexate for the Sterne rDHFR were found to be 77,233 and 12.2 nM, respectively . TMP resistance was observed with E . coli BL21 Star (DE3)pLysS competent cells transformed with the Sterne DHFR gene . Alignment of the amino acid sequence of the Sterne DHFR gene revealed 100% homology with various virulent strains of B . anthracis . These results confirm the natural resistance of B . anthracis to TMP and clarify that the resistance is correlated to a lack of selectivity for the chromosomally encoded gene product . These findings will assist in the development of narrow-spectrum antimicrobial agents for treatment of anthrax.

Biochemistry, 2005 Jan 25, 44(3), 1047 - 53
Interaction of the 20 kDa and 63 kDa Fragments of Anthrax Protective Antigen: Kinetics and Thermodynamics; Christensen KA et al.; The action of anthrax toxin begins when the protective antigen (PA(83), 83 kDa) moiety binds to a mammalian cell-surface receptor and is cleaved by a furin-family protease into two fragments: PA(20) (20 kDa) and PA(63) (63 kDa) . After PA(20) dissociates, receptor-bound PA(63) spontaneously oligomerizes to form a heptameric species, which is able to bind the two enzymatic components of the toxin and transport them to the cytosol . Treatment of PA(83) with trypsin yielded PA(63) and a form of PA(20) lacking unstructured regions at the N- and C-termini . We labeled these fragments with dyes capable of fluorescence resonance energy transfer to quantify their association in solution . We kinetically determined that the equilibrium dissociation constant is 190 nM with a dissociation rate constant, k(off), of 3.3 x 10(-)(2) s(-)(1) (t(1/2) of 21 s) . A two-step association process was observed using stopped-flow: a fast bimolecular step (k(on) = 1.4 x 10(5) M(-)(1) s(-)(1)) was followed by a slower unimolecular step (k = 3.5 x 10(-)(3) s(-)(1)) with an equilibrium isomerization constant, K(iso), of 2.1 . The two-step mechanism most consistent with the data is one in which the dissociation of the PA(20).PA(63) complex is followed by an isomerization in the PA(63) moiety. h, i, a. Our results indicate that, following the cleavage of PA on the cell surface, PA(20) is largely dissociated within a minute . A slow isomerization step in PA(63) may then potentiate it for oligomerization and subsequent steps in toxin action.

Biosecur Bioterror, 2004, 2(4), 281 - 93
Risk of occupationally acquired illnesses from biological threat agents in unvaccinated laboratory workers; Rusnak JM et al.; 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.

ILAR J, 2005, 46(1), 15 - 22
Demand for nonhuman primate resources in the age of biodefense; Patterson JL et al.; The demand for nonhuman primates will undoubtedly increase to meet biomedical needs in this current age of biodefense . The availability of funding has increased the research on select agents and has created a requirement to validate results in relevant primate models . This review provides a description of current and potential biological threats that are likely to require nonhuman primates for the development of vaccines and therapeutics . Primates have been an invaluable resource in the dissection of viral disease pathogenesis as well as in testing vaccine efficacy . DNA vaccine approaches have been studied successfully for Ebola, Lassa, and anthrax in nonhuman primate models . Nonhuman primate research with monkeypox has provided insight into the role of cytokines in limiting disease severity . Biodefense research that has focused on select agents of bacterial origin has also benefited from nonhuman primate studies . Rhesus macaques have traditionally been the model of choice for anthrax research and have yielded successful findings in vaccine development . In plague research, African green monkeys have contributed to vaccine development . However, the disadvantages of current vaccines will undoubtedly require the generation of new vaccines, thus increasing the need for nonhuman primate research . Unfortunately, the current biosafety level (BSL)-3 and BSL-4 facilities equipped to perform this research are limited, which may ultimately impede progress in this era of biodefense.

Acad Emerg Med, 2005 Jan, 12(1), 45 - 50
Online bioterrorism continuing medical education: development and preliminary testing; Terndrup T et al.; OBJECTIVE: Education to achieve awareness and competency in responding to incidents of bioterrorism is important for health care professionals, especially emergency physicians and nurses, who are likely first points of medical contact . The authors describe the development of a computer-based approach to initial education, incorporating a screensaver to promote awareness and a Web-based approach to provide initial content competency in the areas of smallpox and anthrax . METHODS: Screensavers were developed and tested on emergency department rotating senior medical students and internal medicine interns . Conceptually, screensavers were designed as "billboards" for attracting attention to the educational domain . Five rotating images sequenced at five-second intervals incorporated a teaser question and an interactive toolbar . An interactive toolbar was linked to a Web site that provided content on smallpox and anthrax for hospital-based specialties (emergency physicians and nurses, infection control practitioners, pathologists, and radiologists) . The content included both summary and comprehensive content as well as free continuing education credits in an online, specialty-specific, case-scenario format with remediation pop-up boxes . RESULTS: Formal testing indicated that the screensaver and Web site combination deployed on computers in the emergency department and the events of the fall of 2001 significantly increased the percentage of correct responses to five standardized bioterrorism questions . Formal evaluation with a randomized trial and long-term follow-up is ongoing . CONCLUSIONS: Screensavers and Web sites can be used to increase awareness of bioterrorism . Web-based education may provide an effective means of education for bioterrorism.

BMC Public Health . 2005 Jan 5;5(1):2 {Epub ahead of print}
Patients' request for and emergency physicians' prescription of antimicrobial prophylaxis for anthrax during the 2001 bioterrorism-related outbreak; M'ikanatha NM Drph Mph et al.; BACKGROUND: Inappropriate use of antibiotics by individuals worried about biological agent exposures during bioterrorism events is an important public health concern . However, little is documented about the extent to which individuals with self-identified risk of anthrax exposure approached physicians for antimicrobial prophylaxis during the 2001 bioterrorism attacks in the United States . METHODS: We conducted a telephone survey of randomly selected members of the Pennsylvania Chapter of the American College of Emergency Physicians to assess patients' request for and emergency physicians' prescription of antimicrobial agents during the 2001 anthrax . RESULTS: Ninety-seven physicians completed the survey . Sixty-four (66%) respondents had received requests from patients for anthrax prophylaxis; 16 (25%) of these physicians prescribed antibiotics to a total of 23 patients . Ten physicians prescribed ciprofloxacin while 8 physicians prescribed doxycyline . CONCLUSION: During the 2001 bioterrorist attacks, the majority of the emergency physicians we surveyed encountered patients who requested anthrax prophylaxis . Public fears may lead to a high demand for antibiotic prophylaxis during bioterrorism events . Elucidation of the relationship between public health response to outbreaks and outcomes would yield insights to ease burden on frontline clinicians and guide strategies to control inappropriate antibiotic allocation during bioterrorist events.

Appl Spectrosc, 2004 Nov, 58(11), 1360 - 3
Mid-ultraviolet light-emitting diode detects dipicolinic acid; Li Q et al.; Dipicolinic acid (DPA, 2,6-pyridinedicarboxylic acid) is a substance uniquely present in bacterial spores such as that from anthrax (B . anthracis) . It is known that DPA can be detected by the long-lived fluorescence of its terbium chelate; the best limit of detection (LOD) reported thus far using a large benchtop gated fluorescence instrument using a pulsed Xe lamp is 2 nM . We use a novel AlGaN light-emitting diode (LED) fabricated on a sapphire substrate that has peak emission at 291 nm . Although the overlap of the emission band of this LED with the absorption band of Tb-DPA (lambda(max) doublet: 273, 279 nm) is not ideal, we demonstrate that a compact detector based on this LED and an off-the-shelf gated photodetection module can provide an LOD of 0.4 nM, thus providing a basis for convenient early warning detectors.

Nature, 2004 Dec 16, 432(7019), 901 - 4
Public health vaccination policies for containing an anthrax outbreak; Brookmeyer R et al.; Concern about biological weapons has raised questions about the most effective public health policies to contain an anthrax outbreak . We developed a probability model to predict the impact of different anthrax antibiotic and vaccination policies . An anthrax outbreak can be significantly contained by minimizing the delay until initiation of antibiotic prophylaxis . However, even if mass distribution of antibiotics is completed within six days of the initial exposure, then at most about 70% of cases can be prevented . Post-exposure vaccination will not significantly increase that prevention rate if adherence to antibiotic regimens is similar or higher than that attained in the 2001 US outbreak . However, post-exposure vaccination can be useful either in shortening the duration of a prolonged antibiotic regimen, in the event of an antibiotic-resistant strain, or if antibiotic adherence rates are very low . Here we show that a mass pre-exposure vaccination programme for the general population would require very high population coverage rates to significantly increase prevention rates from that achieved with targeted and rapid post-exposure prophylaxis programmes.

J Occup Environ Med, 2004 Oct, 46(10), 1065 - 75
Disability among U.S . Army personnel vaccinated against anthrax; Sulsky SI et al.; This study was conducted to examine whether U.S . Army personnel receiving > or =1 dose of anthrax vaccine adsorbed (AVA) between March 1998 and February 2002 were at higher risk of disability than unvaccinated personnel . We studied a historical cohort study of 716,833 active-duty soldiers (154,456 vaccinated) followed for 4.25 years to determine rates of evaluation for disability discharge . Cox proportional hazards models compared estimated risk of evaluation for disability, accounting for occupation and sociodemographics . Adjusted hazard ratio (HR) and 95% confidence interval (CI) was 0.96 (CI = 0.92-0.99) . Separate adjusted HRs for men, women, permanent and temporary disability, musculoskeletal and neurologic conditions were similar, ranging from 0.90 to 1.04 . Latency assumptions did not affect results . Anthrax vaccination does not increase risk of disability . This finding may be partially the result of factors influencing selection for vaccination or vaccine tolerance.

Fam Community Health, 2004 Jul-Sep, 27(3), 232 - 41
Theoretical perspectives on public communication preparedness for terrorist attacks; Wray RJ et al.; The experience of federal health authorities in responding to the mailed anthrax attacks in the Fall of 2001 sheds light on the challenges of public information dissemination in emergencies . Lessons learned from the Fall of 2001 have guided more recent efforts related to crisis communication and preparedness goals . This article applies theories and evidence from the field of communication to provide an orientation to how public health communication can best contribute to the preparedness effort . This theoretical orientation provides a framework to systematically assess current recommendations for preparedness communication.

Biosecur Bioterror, 2004, 2(3), 175 - 85
A bitter pill to swallow: nonadherence with prophylactic antibiotics during the anthrax attacks and the role of private physicians; Stein BD et al.; To generate recommendations for improving adherence to public health advice during public health crises, we conducted semi-structured interviews with employees at the Brentwood Road Postal Facility and on Capitol Hill to identify key themes associated with decisions to adhere to recommended antibiotic prophylaxis during the 2001 anthrax attacks . Factors used in deciding to adhere to recommended prophylactic antibiotics and concerns about the official response were similar in Brentwood and Capitol Hill employees, and in adherent and nonadherent participants . All participants used multiple sources of information and support as they weighed the risk from anthrax against the advantages and disadvantages of antibiotics . We found that nonadherent participants were commonly following the advice of private physicians, whereas adherent participants commonly described ongoing support from multiple sources when discussing their decisions . Our findings highlight the need for better integration between the public and private health care systems during public health crises and the importance of equipping private physicians for their key role in aiding decision-making during a public health crisis . Special attention also should be given to enhancing support and information from multiple sources throughout the entire period of risk.

Biosecur Bioterror, 2004, 2(3), 157 - 63
Diagnosing smallpox: would you know it if you saw it?
Woods R, McCarthy T, Barry MA, Mahon B.
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.

G Ital Med Lav Ergon, 2004 Oct-Dec, 26(4), 353 - 63
{Indoor air quality and occupational health, past and present}; Maroni M; The expression "indoor pollution" of residential, office and public buildings appeared for the first time in western societies toward the end of the '60s to indicate a complex phenomenon as important to public health as that of the "outdoor air pollution" or even more so . The demonstration of the toxic effects of passive smoking, radon, and other chemical and biological pollutants present in office and residential environments has prompted a wide spectrum of research into health risks, has led to position-taking by national and international authorities, and has given rise to a new scientific multi-disciplinary field of research, with respective international associations, scientific journals, and international conferences attended by thousands of participants . In Italy, since 1988, several scientific conferences have been organised and these have led to institutional initiatives such as the Commission set up in 1990 by the Italian Environment Ministry and the Commission set up in 1998 by the Italian Ministry of Health . The latter produced a Prevention Plan for Health Protection and Promotion in Indoor Environments which, for the first time, tackles indoor air pollution in a systematic way and provides an overall picture of the health and economic impact of the main illnesses related to indoor pollution on society . Decree 626/94 has also been affected, in some way, by these new scientific findings and in art . 33 has produced an update of the old art . 9 of the Decree 303/56 . The attention to the subject of indoor air quality, in addition to that of offices and commercial buildings, has turned in more recent years to special environments such as schools and hospitals, resulting in the production of important publications . The modern frontier of research on indoor and outdoor air hazards is represented by fine air particulate matter . A large number of worldwide epidemiological studies have revealed that the daily variation in fine and ultra-fine particle air concentration in urban areas is associated with the simultaneous daily variation in the morbidity and mortality of the general population . The particle-linked increase in mortality has been attributed to respiratory and cardiovascular toxic effects, but the mechanisms by which urban air particles (indoor and outdoor) induce worsening of respiratory and cardio-vascular diseases are so far unknown and are the subject of intense investigation . Workers employed in the tertiary sector (offices, trade, banking, hospitals, schools, etc.) now account for 80% of the Italian labour force and the occupational physician is increasingly requested to assess the risk and monitor the health status of tertiary sector workers . These working environments are believed to be healthy and lacking in specific health risk factors, but such a belief is often only the result of the limited knowledge that employers, workers and the physicians themselves have about these environments and the results of international research studies over the last forty years. f, b. This issue is surely at the centre of the interest of our discipline and of public health throughout the developed western world and represents an ongoing challenge for the occupational physician, with new research topics and new problems to deal with . Recent issues include SARS and the defence of buildings and the air of working environments against terrorism attacks, such as the use of anthrax dust.

Berl Munch Tierarztl Wochenschr, 2004 Nov-Dec, 117(11-12), 508 - 24
{Strategies of vaccination against anthrax}; Beyer W; Apart from live spore vaccines with a certain amount of residual virulence for various animal species, there are two acellular protein vaccines for immunoprophylaxis against anthrax in humans . For ethical reasons there are no experimental data available on the efficacy and duration of the immunity they induce in men . Their efficacy was evaluated in laboratory animals, mainly rabbits and rhesus monkeys . Furthermore, it is well known that these vaccines elicit only partial protection in guinea pigs and almost no protection in mice against a challenge with fully virulent spores of Bacillus (B.) anthracis . Other disadvantages are the high amount of boosters necessary to elicit and to maintain a protective immune response, the variability in the composition of bacterial culture supernatants used for production, and the appearance of clinically relevant side effects . Therefore, there is ongoing work worldwide to improve the existing vaccines by substitution with recombinant antigens and to develop new vaccines on the basis of recombinant bacterial or viral live vectors, DNA-vectors, and by addition of new adjuvants . Special attention is given to supplementing the existing toxoid-vaccines with an anti-bacterial component.

Clin Infect Dis, 2004 Dec 15, 39(12), 1842 - 7 Epub 2004 Dec 15.
Screening for inhalational anthrax due to bioterrorism: evaluating proposed screening protocols; Howell JM et al.; Eleven known cases of bioterrorism-related inhalational anthrax (IA) were treated in the United States during 2001 . We retrospectively compared 2 methods that have been proposed to screen for IA . The 2 screening protocols for IA were applied to the emergency department charts of patients who presented with possible signs or symptoms of IA at Inova Fairfax Hospital (Falls Church, Virginia) from 20 October 2001 through 3 November 2001 . The Mayer criteria would have screened 4 patients (0.4%; 95% CI, 0.1%-0.9%) and generated charges of 1900 dollars . If 29 patients (2.6%; 95% CI, 1.7%-3.7%) with >or=5 symptoms (but without fever and tachycardia) were screened, charges were 13,325 dollars . The Hupert criteria would have screened 273 patients (24%; 95% CI, 22%-27%) and generated charges of 126,025 dollars . In this outbreak of bioterrorism-related IA, applying the Mayer criteria would have identified both patients with IA and would have generated fewer charges than applying the Hupert criteria.

J Am Acad Dermatol, 2004 Nov, 51(5 Suppl), S137 - 42
Rickettsialpox: report of three cases and a review; Saini R et al.; Rickettsialpox is a rare mite-borne rickettsiosis that is encountered in urban populations in the eastern United States and throughout the world . It is characterized clinically by an eschar, fever, and a papulovesicular eruption . Both of these cutaneous manifestations may be mimicked by infectious diseases that have been designated as bioterrorist agents by the United States Centers for Diseases Control and Prevention: the former by anthrax, and the latter by smallpox . It is thus important for clinicians to be familiar with rickettsialpox . We report 3 cases and review the epidemiology, clinical and laboratory findings, differential diagnosis, and management of this disease.

Radiats Biol Radioecol, 2004 Sep-Oct, 44(5), 544 - 6
{Susceptibility of irradiated animals to extremely dangerous pathogens (literature review)}; Expanded interaction fingerprint method for analyzing ligand binding modes in docking and structure-based drug design; Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, UKAn expanded interaction fingerprint method has been developed for analyzing the binding modes of ligands in docking and structure-based design methods . Taking the basic premise of representing a ligand in terms of a binary string that denotes its interactions with a target protein, we have expanded the method to include additional interaction-specific information . By considering the hydrogen-bonding strength and/or accessibility of the hydrogen bonding groups within a binding site as well as their geometric arrangement we aim to provide a better representation of a ligand-protein interaction . These expanded methods have been applied to the postprocessing of binding poses generated in a docking study for 220 different proteins and to the analysis of ligands generated by an automated ligand-generation algorithm for the anthrax oedema factor . In the docking study, the application of the interaction fingerprint method as a postprocessing tool resulted in an increased success rate in identifying the crystallographic binding mode . In the analysis of the ligands generated for the anthrax oedema factor, the incorporation of additional interaction-specific information resulted in a more intuitive and comprehensive analysis of automated ligand-generation output.

Ann Ig, 2004 Jan-Apr, 16(1-2), 407 - 18
{Considerations on the evolution of the concept of zoonoses}; Mantovani A et al.; The existence of diseases common to humans and animals has been known since the beginning of history . The disease transmission from animals to people was first observed on rabies ("the mother of all zoonoses") and later on in occupational diseases (anthrax, glanders, "mange"); in time, also certain food-borne infections were included in this category . The microbiological era has first brought along the concept of infection, then that of zoonoses, which has extended and now numbers some 200 infections . The World Health Organization has been interested in zoonoses since its foundation . Zoonoses represent an important issue for public health, fundamental in veterinary public health, whose functions are here listed . This discipline is concerned with all health problems associated with direct and indirect relationships between humans and animals, including also non-communicable ones . It has been proposed, therefore, to extend the term zoonoses as follows: "Any detriment to the health and/or quality of human life deriving from direct or indirect relationships with (other) vertebrate, or edible or toxic invertebrate animals" . Contributors (e.g . Blancou) suggest to remove "edible or toxic "from the definition . Examples of zoonoses are provided according to the extended concept.

J Public Health Manag Pract, 2004 Sep-Oct, 10(5), 406 - 12
Combining the benefits of decision science and financial analysis in public health management: a county-specific budgeting and planning model; Fos PJ et al.; State public health agencies are charged with providing and overseeing the management of basic public health services on a population-wide basis . These activities have a re-emphasized focus as a result of the events of September 11, 2001, the subsequent anthrax events, and the continuing importance placed on bioterrorism preparedness, West Nile virus, and emerging infectious diseases (eg, monkeypox, SARS) . This has added to the tension that exists in budgeting and planning, given the diverse constituencies that are served in each state . State health agencies must be prepared to allocate finite resources in a more formal manner to be able to provide basic public health services on a routine basis, as well as during outbreaks . This article describes the use of an analytical approach to assist financial analysis that is used for budgeting and planning in a state health agency . The combined benefits of decision science and financial analysis are needed to adequately and appropriately plan and budget to meet the diverse needs of the populations within a state . Health and financial indicators are incorporated into a decision model, based on multicriteria decision theory, that has been employed to acquire information about counties and public health programs areas within a county, that reflect the impact of planning and budgeting efforts . This information can be used to allocate resources, to distribute funds for health care services, and to guide public health finance policy formulation and implementation.

Proc Natl Acad Sci U S A, 2004 Nov 30, 101(48), 16756 - 61 Epub 2004 Nov 30.
Evidence that translocation of anthrax toxin's lethal factor is initiated by entry of its N terminus into the protective antigen channel; Zhang S et al.; Entry of the enzymatic components of anthrax toxin {lethal factor (LF) and edema factor} into the cytosol of mammalian cells depends on the ability of the activated protective antigen (PA63) component to form a channel (pore) in the membrane of an acidic intracellular compartment . To investigate the mechanism of translocation, we characterized N-terminally truncated forms of the PA63-binding domain of LF (LFN) . Deleting 27 or 36 residues strongly inhibited acid-triggered translocation of LFN across the plasma membrane of CHO-K1 cells and ablated the protein's ability to block PA63 channels in planar lipid bilayers at a small positive voltage (+20 mV) . Fusing a H6-tag to the N terminus of the truncated proteins restored both translocation and channel-blocking activities . At +20 mV, N-terminal H6 and biotin tags were accessible to Ni2+ and streptavidin, respectively, added to the trans compartment of a planar bilayer . On the basis of these findings, we propose that the N terminus of PA63-bound LF or edema factor enters the PA63-channel under the influence of acidic pH and a positive transmembrane potential and initiates translocation in an N- to C-terminal direction.

J Mol Biol, 2004 Nov 26, 344(3), 739 - 56
Acid-induced unfolding of the amino-terminal domains of the lethal and edema factors of anthrax toxin; Krantz BA et al.; The two enzymatic components of anthrax toxin, lethal factor (LF) and edema factor (EF), are transported to the cytosol of mammalian cells by the third component, protective antigen (PA) . A heptameric form of PA binds LF and/or EF and, under the acidic conditions encountered in endosomes, generates a membrane-spanning pore that is thought to serve as a passageway for these enzymes to enter the cytosol . The pore contains a 14-stranded transmembrane beta-barrel that is too narrow to accommodate a fully folded protein, necessitating that LF and EF unfold, at least partly, in order to pass . Here, we describe the pH-dependence of the unfolding of LF(N) and EF(N), the 30kDa N-terminal PA-binding domains, and minimal translocatable units, of LF and EF . Equilibrium chemical denaturation studies using fluorescence and circular dichroism spectroscopy show that each protein unfolds via a four-state mechanism: N<-->I<-->J<-->U . The acid-induced N-->I transition occurs within the pH range of the endosome (pH 5-6) . The I state predominates at lower pH values, and the J and U states are populated significantly only in the presence of denaturant . The I state is compact and has characteristics of a molten globule, as shown by its retention of significant secondary structure and its ability to bind an apolar fluorophore . The N-->I transition leads to an overall 60% increase in buried surface area exposure . The J state is expanded significantly and has diminished secondary structure content . We analyze the different protonation states of LF(N) and EF(N) in terms of a linked equilibrium proton binding model and discuss the implications of our findings for the mechanism of acidic pH-induced translocation of anthrax toxin . Finally, analysis of the structure of the transmembrane beta-barrel of PA shows that it can accommodate alpha-helix, and we suggest that the steric constraints and composition of the lumen may promote alpha-helix formation.

Mil Med, 2004 Oct, 169(10), 833 - 8
Health effects of anthrax vaccination in the Canadian forces; Hunter D et al.; OBJECTIVE: The objective of this study was to determine whether anthrax vaccine resulted in adverse health effects in Canadian Forces members 8 months after vaccination . METHODS: A quasi-experimental, retrospective chart review was undertaken for two groups within the Canadian Forces, one group that received anthrax vaccination and another that did not . Information on symptoms, diagnoses, and injuries for 848 persons for which there were approximately 35,000 chart entries was abstracted from charts over a 4.5-year period and was coded using the International Statistical Classification of Diseases and Related Health Problems, 10th edition . RESULTS: The chart retrieval rate was 84% . The mean number of chart entries per person was higher in the comparison group (43.4) than in the vaccine group (38.2) . No statistically significant differences were seen in the percent change before and after vaccination in the number of chart entries for specific diagnoses and symptoms for the vaccine group compared with the comparison group. h, b. Visual inspection of the time trend in rates showed no unexplained increases in the rate of diagnosis and symptoms in the vaccine group after vaccination . CONCLUSION: This study found no evidence that the anthrax vaccination resulted in an increase in adverse health effects in the 8-month period after vaccination.

Vaccine, 2004 Dec 2, 23(3), 411 - 7
Media coverage of anthrax vaccination refusal by Australian Defence Force personnel; Ackermann D et al.; BACKGROUND: During February 2003 a number of Australian sailors were returned home from their deployment to the Persian Gulf after refusing anthrax vaccination . This paper examines the media coverage of this episode as a case study in how controversies about vaccine safety escalate . METHODS: Frame analysis of articles from major Australian newspapers (n=83) and transcripts of radio and television news and current affairs programs (n=22) to identify the main supportive and oppositional themes used in reportage and media debate . FINDINGS: Initially, the major news frames were supportive of the vaccine refusing soldiers, and conveyed a sense of distrust of the government's actions . These initial themes were rapidly re-framed and new dominant discourses appeared . First, sailors went from brave whistleblowers to being portrayed as deserters and cowards . Second, proponents shifted from their portrayal as faceless regulators to personal risk takers embodied in a well-respected Major General having the vaccine . Third, the voluntary nature of the vaccine was emphasised, thus dousing the flames of implied coercion . CONCLUSION: Marked shifts in the representation of vaccine opponents and proponents possibly contributed to the rapid diminishment of media interest in the story.

Przegl Lek, 2004, 61(3), 177 - 80
{Anthrax as a bioweapon}; Szafraniec S et al.; The risk of biological and chemical terrorism is growing according to availability of modern biotechnologies and financial resources . The most potent biological weapon mentioned in the last decade is anthrax . The number of naturally acquired infections in humans is constantly reduced, however endemic areas are located in South and Middle Americas, South Europe, Asia and Africa . In any case of infection laboratory confirmation is indicated . Primary basic testing is available in all microbiology laboratories . In the current situation the medical and epidemiological infrastructure and microbiology diagnostics must be prepared for early and rapid coordinated action in order to detect and respond to biological and chemical attacks.

Eur J Cell Biol, 2004 Jul, 83(6), 281 - 8
Effects of dynamin inactivation on pathways of anthrax toxin uptake; Boll W et al.; Internalization and traffic to acidic endosomes of anthrax lethal factor (LF) and protective antigen (PA), bound to the anthrax toxin receptor (ATR), is required for LF translocation into the cytosol, where it can elicit its toxic effects . Dynamin is required for clathrin-mediated endocytosis, and long-term disruption of dynamin function blocks internalization of PA . We have used LFn-DTA, a surrogate of LF consisting of the N-terminal domain of LF fused to the catalytic subunit of diphtheria toxin, to differentiate the effects of acute and long-term block of dynamin function on LFn-DTA toxicity . Both forms of interference reduce LFn-DTA toxicity only partially, consistent with alternative routes for LFn-DTA endocytosis . In contrast, a long-term block of dynamin activity results in a further interference with LFn-DTA toxicity that is consistent with an altered endosomal environment, probably an increase in endosomal pH.

Ann Fam Med, 2004 Sep-Oct, 2(5), 438 - 44
The primary care differential diagnosis of inhalational anthrax; Temte JL et al.; PURPOSE: Inhalational anthrax is an extremely rare infectious disease with nonspecific initial symptoms, thus making diagnosis on clinical grounds difficult . After a covert release of anthrax spores, primary care physicians will be among the first to evaluate cases . This study defines the primary care differential diagnosis of inhalational anthrax . METHODS: In May 2002, we mailed survey instruments consisting of 3 randomly chosen case vignettes describing patients with inhalational anthrax to a nationwide random sample of 665 family physicians . Nonrespondents received additional mailings . Physicians were asked to provide their most likely nonanthrax diagnosis for each case . RESULTS: The response rate was 36.9% . Diagnoses for inhalational anthrax were grouped into 35 diagnostic categories, with pneumonia (42%), influenza (10%), viral syndrome (9%), septicemia (8%), bronchitis (7%), central nervous system infection (6%), and gastroenteritis (4%) accounting for 86% of all diagnoses . Diagnoses differed significantly between cases that proved to be fatal and those that proved to be nonfatal . CONCLUSIONS: Inhalational anthrax resembles common diagnoses in primary care . Surveillance systems for early detection of bioterrorism events that rely only on diagnostic codes will be hampered by false-positive alerts . Consequently, educating frontline physicians to recognize and respond to bioterrorism is of the highest priority.

Ann Fam Med, 2004 Sep-Oct, 2(5), 434 - 7
Rapid assessment of agents of biological terrorism: defining the differential diagnosis of inhalational anthrax using electronic communication in a practice-based research network; Temte JL et al.; PURPOSE: Early detection of bioterrorism requires assessment of diagnoses assigned to cases of rare diseases with which clinicians have little experience . In this study, we evaluated the process of defining the differential diagnosis for inhalational anthrax using electronic communication within a practice-based research network (PBRN) and compared the results with those obtained from a nationwide random sample of family physicians with a mailed instrument . METHODS: We distributed survey instruments by e-mail to 55 physician members of the Wisconsin Research Network (WReN), a regional PBRN . The instruments consisted of 3 case vignettes randomly drawn from a set describing 11 patients with inhalational anthrax, 2 with influenza A, and 1 with Legionella pneumonia . Physicians provided their most likely nonanthrax diagnosis, along with their responses to 4 yes-or-no management questions for each case . Physicians who had not responded at 1 week received a second e-mail with the survey instrument . The comparison group consisted of the nationwide sample of physicians who completed mailed survey instruments . Primary outcome measures were response rate, median response time, and frequencies of diagnostic categories assigned to cases of inhalational anthrax . RESULTS: The PBRN response rate compared favorably with that of the national sample (47.3% vs 37.0%; P = not significant) . The median response time for the PBRN was significantly shorter than that for the national sample (2 vs 28 days; P < .001). d, g, c. No significant differences were found between the PBRN and the Midwest subset of the national sample in the frequencies of major diagnostic categories or in case management . CONCLUSIONS: Electronic means of creating differential diagnoses for rare infectious diseases of national significance is feasible within PBRNs . Information is much more rapidly acquired and is consistent with that obtained by conventional methods.

J Public Health Manag Pract, 2003 Sep-Oct, 9(5), 427 - 32
Building academic-practice partnerships: the Center for Public Health Preparedness at the Columbia University Mailman School of Public Health, before and after 9/11; Morse SS; The Center for Public Health Preparedness at the Columbia University Mailman School of Public Health is part of a national network of academic centers established by the Centers for Disease Control and Prevention to strengthen links between public health practice and academe, especially for public health workforce development . Since its inception in Fall 2000, the Center has been working in partnership with the New York City Department of Health & Mental Hygiene (DOHMH) on planning and competency-based training in emergency preparedness (including bioterrorism and infectious diseases) and evaluation . Initial programs with DOHMH included development of a 3-hour orientation to basic emergency preparedness for their workforce . In the wake of 9/11 and the anthrax events, Center members gave over two dozen presentations at community forums, seminars, and clinical rounds, and over 100 press interviews, provided lay language information through community forum presentations and the School's Web site, and developed a database of volunteers for surge capacity . Subsequent programs include bioterrorism response training for clinicians and emergency medical services personnel, incident command for public health, and a study of evacuation from the World Trade Center on 9/11.

J Public Health Manag Pract, 2003 Sep-Oct, 9(5), 357 - 60
Persistence of a mock bio-agent in cross-contaminated mail and mailboxes; Cole LA; Among the 22 confirmed or suspected cases of anthrax during the bioterrorism incidents in 2001, all but two seemed traceable to spores from threat letters . Although no anthrax spores were found in the environments frequented by two females who died of the disease, a suspicion persists that the deaths were somehow related to the mail . This study assesses the spread and persistence of a mock biological agent from a source-letter to other mail and to a receiving mailbox . Successive placement and removal of letters were found to reduce the number of residual bio-agent particles in a mailbox . This suggests that a sweeping action is taking place that can affect the quantity of bio-agent remaining . The exercise supports the possibility that the two females were exposed to spores on mail that had been in their mailboxes although no trace of spores could later be found in their boxes.

N J Med, 2004 Sep, 101(9 Suppl), 45 - 50; quiz 50-2
Communicable-disease surveillance in New Jersey; Bednarczyk M et al.; The DHSS and federal agencies have expanded their surveillance efforts to improve existing methods of reporting notifiable communicable diseases and to include additional data sources that might provide a more comprehensive view of disease activity in New Jersey . Currently, the DHSS is evaluating these efforts and recognizes several issues that need to be addressed, including: assessment of the timeliness, completeness, and accuracy of surveillance data; validation of surveillance data through comparison with hospital uniform billing data; characterization of the sensitivity of alerts through examination of reasons for identified aberrations in disease activity; evaluation of DHSS staffs and LINCS epidemiologists' follow-up efforts in response to alerts; evaluation of cumulative data trends to determine patterns in baseline disease activity (e.g., variations in disease activity attributed to seasonality); development of methods to integrate data from all surveillance efforts to provide timely, comprehensive, and coordinated summaries of disease activity and to distribute these summaries regularly to all New Jersey public health partners to better inform public health and clinical management; and development of a coordinated multi-agency response plan in conjunction with adjacent states . Though the DHSS hopes that these surveillance efforts will contribute to the early detection of sentinel events that might represent possible bioterrorist or emerging infectious disease threats, the DHSS will also need to engage the medical community more fully in surveillance activities . In previous experiences, astute clinicians were responsible for the identification of the first cases of West Nile virus, anthrax, and SARS . Therefore, to further ensure the success of its surveillance efforts, the DHSS will also need to continue educating clinicians about its surveillance activities and the importance of timely reporting of patients with illness patterns that might suggest an unusual infectious disease outbreak associated with bioterrorism or emerging infectious diseases.

J Med Biogr, 2004 Nov, 12(4), 202 - 9
Thomas Morison Legge (1863-1932): the first medical factory inspector; Waldron T; After a brief career in public health, Thomas Morison Legge was appointed to become the first medical factory inspector, in 1898, and remained in post until his resignation in 1927 . During his tenure in office he became the leading authority on lead poisoning and anthrax; he resigned when the government refused to ratify the White Lead Convention . Subsequently he became the first medical adviser to the Trades Union Congress.

Occup Med (Lond), 2004 Oct, 54(7), 489 - 93
The history of woolsorters' disease: a Yorkshire beginning with an international future?
Metcalfe N.
Woolsorters' disease was a feared industrial disease associated primarily with Yorkshire's textile industry of the nineteenth and early twentieth centuries . Early occupational health methods were attempted locally before concerted national efforts produced legislative measures . When its link with anthrax was established, attention in prevention focused upon chemical disinfection methods . Together, these factors were instrumental in decreasing the incidence of woolsorters' disease . However, by the beginning of the Second World War, the lack of treatment options for anthrax meant that the bacterium was experimented upon as a potential war-winning weapon . Today, woolsorters' disease and other industrial manifestations of anthrax are extremely rare, but the increasing threat of bioterrorism means that the international dread and historical lessons of this significant condition should never be forgotten . Consequently, this paper reveals the history of woolsorters' disease in order to remind those involved in occupational medicine today of the dread it caused both physicians and workers in previous generations.

Osiris, 2004, 19, 220 - 33
Poisoned food, poisoned uniforms, and anthrax: or, how guerillas die in war; White L; Many people believe that Rhodesia, struggling to maintain minority rule in Africa, used chemical and biological weapons against African guerilla armies in the liberation war . Clothes and food were routinely poisoned, and Rhodesian agents, perhaps in concert with global forces of reaction, caused the largest single outbreak of anthrax in modern times . Oral interviews with traditional healers and Rhodesians' confessional memoirs of the war suggest that deaths by poisoning or disease were not so straightforward, that guerillas and healers and doctors struggled to understand not only what caused death but also what kind of death a poisoned uniform or poisoned boot was.

Arch Intern Med, 2004 Oct 11, 164(18), 2012 - 6
Antibiotics for anthrax: patient requests and physician prescribing practices during the 2001 New York City attacks; Hupert N et al.; BACKGROUND: Little is known about patient encounters with primary care physicians and prescribing practices during the 2001 US anthrax attacks . METHODS: We retrospectively reviewed the electronic medical record of outpatient telephone and clinic visits at a large primary care practice in New York City from September 11 to December 31, 2001, to identify physician- and patient-related factors that were associated with prescribing antibiotics for anthrax prophylaxis . RESULTS: Average daily patient volume from October to December was higher in 2001 (221.2 patients per day) compared with 2000 (199.1; P<.01) and 2002 (215.8; P = .14) . Patient-initiated discussion about anthrax or smallpox were involved in 244 patient contacts with 63 physicians, including 92 (0.6%) of 14917 telephone contacts and 152 (1.0%) of 15 539 office visits . Fifty patients (21%) requested antibiotics or vaccines and 52 (22%) received antibiotics: 39 received ciprofloxacin; 12, doxycycline; and 1, both drugs . Independent predictors of receiving anthrax prophylaxis included requesting medication (odds ratio {OR}, 8.1; 95% confidence interval {CI}, 3.5-18.6), reporting powder or workplace exposure (OR, 4.5; 95% CI, 2.1-10.0), having an abnormal physical examination finding (OR, 3.9; 95% CI, 1.4-11.0), and being asymptomatic (reporting any illness symptoms was associated with an OR of 0.3 {95% CI, 0.1-0.6}) . CONCLUSIONS: Primary care physicians played an important and heretofore underdocumented role in responding to the 2001 anthrax attacks . Prescription of prophylactic antibiotics for anthrax was uncommon and appears to have been selective among concerned patients . These results highlight the importance of including primary care physicians in community-wide bioterrorism response planning.

J Mol Biol, 2004 Oct 29, 343(4), 971 - 84
The DxDxDG motif for calcium binding: multiple structural contexts and implications for evolution; Rigden DJ et al.; Calcium ions regulate many cellular processes and have important structural roles in living organisms . Despite the great variety of calcium-binding proteins (CaBPs), many of them contain the same Ca(2+)-binding helix-loop-helix structure, referred to as the EF-hand . In the canonical EF-hand, the loop contains three calcium-binding aspartic acid residues, which form the DxDxDG sequence motif, and is flanked by two alpha-helices . Recently, other CaBPs containing the same motif, but lacking one or both helices, have been described . Here, structural motif searches were used to analyse the full diversity of structural context in the known set of DxDxDG-containing CaBPs, including those where the structural resemblance of a given DxDxDG motif to that of EF-hands had not been noted . The results obtained indicate that the EF-hand represents but one, among many, structural context for the DxDxDG-like Ca(2+)-binding loops . While the structural similarity of the binuclear calcium-binding sites in anthrax protective antigen and human thrombospondin suggests that they are homologous, evolutionary relationships for mononuclear sites are harder to discern . The possible scenarios for the evolution of DxDxDG motif-containing calcium-binding loops in a variety of non-homologous proteins suggested loop transplant as a mechanism perhaps responsible for much of the diversity in structural contexts of present day DxDxDG-type CaBPs . Additionally, while it can be shown that existence of a DxDxDG sequence is not enough to confer a conformation suitable for calcium binding, local convergent evolution may still have a role . The analysis presented here has consequences for the prediction of calcium binding from sequence alone.

J Biomol Struct Dyn, 2004 Dec, 22(3), 253 - 65
Three-dimensional model of the pore form of anthrax protective antigen . Structure and biological implications; Nguyen TL; Although pore formation by protective antigen (PA) is critical to cell intoxication by anthrax toxin (AT), the structure of the pore form of PA (the PA63 pore) has not been determined . Hence, in this study, the PA63 pore was modeled using the X-ray structures of monomeric PA and heptameric alpha-hemolysin (alpha-HL) as templates . The PA63 pore model consists of two weakly associated domains, namely the cap and stem domains . The ring-like cap domain has a length of 80 A and an outside diameter of 120 A, while the cylinder-like stem domain has a length of 100 A and outside diameter of approximately 28 A . This provides the PA63 pore model with a length of 180 A . Based on experimental results, the channel in the PA63 pore model was built to have a minimum diameter of ~12 A, depending on side chain conformations . Because of its large size and structural complexity, the all-atom model of the PA63 pore is the end-stage construction of four separate modeling projects described herein . The final model is consistent with published experimental results, including mutational analysis and channel conductance experiments . In addition, the model was energetically and hydropathically refined to optimize molecular packing within the protomers and at the protomer-protomer interfaces . By providing atomic detail to biochemical and biophysical data, the PA63 pore model may afford new insights into the binding mode of PA on the membrane surface, the prepore-pore transition, and the mechanism of cell entry by anthrax toxin.

J Biol Chem, 2004 Dec 10, 279(50), 52473 - 8 Epub 2004 Dec 10.
Involvement of domain II in toxicity of anthrax lethal factor; Liang X et al.; Anthrax lethal factor (LF) is a Zn2+ -metalloprotease that cleaves and inactivates mitogen-activated protein kinase kinases (MEKs) . We have used site-directed mutagenesis to identify a cluster of residues in domain II of LF that lie outside the active site and are required for cellular proteolytic activity toward MEKs . Alanine substituted for Leu293, Lys294, Leu514, Asn516, or Arg491 caused a 10-50-fold reduction in LF toxicity . Further, whereas pairwise substitution of alanine for Leu514 and either Leu293, Lys294, or Arg491 completely abrogated LF toxicity, pairwise mutation of Leu514 and Asn516 resulted in toxicity comparable with N516A alone . The introduction of these mutations reduced LF-mediated cleavage of MEK2 in cell-based assays but altered neither the ability of LF to bind protective antigen nor its ability to translocate across a membrane . Interestingly, direct in vitro measurement of LF activity indicated that decreased toxicity was not always accompanied by reduced proteolytic activity. l, c, j. However, mutations in this region significantly reduced the ability of LF to competitively inhibit B-Raf phosphorylation of MEK . These results provide evidence that elements of domain II are involved in the association of LF into productive complex with MEKs.

Expert Opin Biol Ther, 2004 Oct, 4(10), 1661 - 7
DNA vaccines against anthrax; Galloway DR et al.; DNA vaccination is vaccination at its simplest . Due to renewed interest in vaccination against anthrax and other biothreat agents, a genetic immunisation approach offers attractive possibilities for rapid, responsive vaccine development . DNA vaccination against anthrax is an active area of research showing promising results at present, which in the short-term and in the future could form the basis for new advances in multi-agent vaccine development . The anthrax 'model' constitutes an important experimental system for genetic immunisation technology development.

J Forensic Sci, 2004 Sep, 49(5), 961 - 7
Stable isotope ratios as a tool in microbial forensics--Part 2 . Isotopic variation among different growth media as a tool for sourcing origins of bacterial cells or spores; Kreuzer-Martin HW et al.; Since the anthrax attacks of 2001 the need for methods to trace the origins of microbial agents has become urgent . The stable isotope ratios of bacteria record information from both the nutrients and the water used to make their culture media and could potentially be used to provide information about their growth environment . We present a survey of carbon (C), nitrogen (N), and hydrogen (H) stable isotope ratios in 516 samples of bacteriological culture media . The observed variation was consistent with expected isotopic variation in the plant and animal products upon which the media are based . The variation is sufficient to translate into substantial isotope variation in cultures grown on different batches of media, and thus to allow investigators to determine whether seized media could have been used to produce seized bioweapons agents.

Am J Infect Control, 2004 Oct, 32(6), 345 - 54
Respiratory protection against bioaerosols: literature review and research needs; Rengasamy A et al.; Research on respiratory protection against biologic agents is important to address major concerns such as occupational safety and terrorist attack . This review describes the literature on respiratory protection against bioaerosols and identifies research gaps . Respiratory protection is a complex field involving a number of factors, such as the efficiency of respirator filter material; face-piece fitting; and maintenance, storage, and reuse of respirators . Several studies used nonpathogenic microorganisms having physical characteristics similar to that of Mycobacterium tuberculosis to analyze microbial penetration through respirators . Some studies showed that high-efficiency particulate air (HEPA) and N95 filters provided a higher level of protection than dust/mist (DM) and dust/mist/fume (DMF) filters . Flow rate and relative humidity appear to alter the level of penetration of microorganisms through respirator filters . The relationship between microbial penetration through respirator filters and the aerodynamic diameter, length, or other physical characteristics of microorganisms remains controversial . Whether reaerosolization of bioaerosol particles should be a concern is unclear, given the fact that one study has demonstrated significant reaerosolization of 1- to 5-microm particles loaded onto respirator filters . Respirator maintenance, storage, and decontamination are important factors to be considered when reusing respirators . The respiratory protection against biologic warfare agents such as anthrax in military and civilian situations is described.

Biologicals, 2004 Jun, 32(2), 62 - 9
Evaluation of an anti-rPA IgG ELISA for measuring the antibody response in mice; Little SF et al.; A recombinant protective antigen (rPA)-based enzyme-linked immunosorbent assay (ELISA) was developed to measure the serological response of female A/J mice after inoculation with the new rPA-based anthrax vaccine . Several fundamental parameters of the ELISA were evaluated: specificity, precision, accuracy, linearity, and stability . Experimental results suggested that the quantitative anti-rPA IgG ELISA could be used to measure antibody levels in female A/J mice and may be useful as a potency assay to monitor consistency of manufacture of a rPA-based vaccine for planned clinical trials.

Am J Public Health, 2004 Oct, 94(10), 1667 - 71
The pitfalls of bioterrorism preparedness: the anthrax and smallpox experiences; Cohen HW et al.; Bioterrorism preparedness programs have contributed to death, illness, and waste of public health resources without evidence of benefit . Several deaths and many serious illnesses have resulted from the smallpox vaccination program; yet there is no clear evidence that a threat of smallpox exposure ever existed . The anthrax spores released in 2001 have been linked to secret US military laboratories-the resultant illnesses and deaths might not have occurred if those laboratories were not in operation.The present expansion of bioterrorism preparedness programs will continue to squander health resources, increase the dangers of accidental or purposeful release of dangerous pathogens, and further undermine efforts to enforce international treaties to ban biological and chemical weapons . The public health community should acknowledge the substantial harm that bioterrorism preparedness has already caused and develop mechanisms to increase our public health resources and to allocate them to address the world's real health needs.

Pharmacoepidemiol Drug Saf, 2004 Dec, 13(12), 825 - 40
Safety of anthrax vaccine: an expanded review and evaluation of adverse events reported to the Vaccine Adverse Event Reporting System (VAERS); Sever JL et al.; PURPOSE: To assess the safety of a licensed anthrax vaccine (AVA) given to more than 500,000 US military personnel, through review and medical evaluation of adverse events (AEs) reported to the Vaccine Adverse Event Reporting System (VAERS) . METHODS: AEs were summarized by person, vaccine lot, type, frequency and impact . A Delphic approach was used to tentatively assess causality in an effort to detect serious AEs (SAEs) or other medically important AEs (OMIAEs) possibly attributable to AVA . RESULTS: The Anthrax Vaccine Expert Committee (AVEC) reviewed 1841 reports describing 3991 AEs (9.4 reports/10,000 doses of AVA) that were submitted to VAERS from 1Q1998 through 4Q2001 . One hundred forty-seven reports described an SAE or OMIAE, of which 26 were tentatively rated as possible, probable or certain consequences of vaccination (injection-site reaction {12}, 'anaphylactic-like reaction' {5} and eight other systemic AEs {1-2 each}) . CONCLUSIONS: This review produced no evidence for an unusual rate of any SAE or OMIAE attributable to AVA . It supported an earlier impression that AVA may cause significant local inflammation and should be administered over the deltoid rather than the triceps to avoid direct or compression injury to the ulnar nerve . The subjects of VAERS reports tended to be older than all recipients of AVA . Females generally had and/or reported AEs more often than males, but transient articular reactions were surprisingly more common in males . Variations in the frequency or severity (as judged by hospitalization and/or loss of duty) of reported AEs did not suggest a significant problem with (1) a particular lot of AVA, (2) recurrent AEs after multiple doses or (3) vaccination of persons with a concomitant illness or those given other vaccines or medications .

Int J Biochem Cell Biol, 2005 Jan, 37(1), 142 - 54
Membrane type-1 matrix metalloproteinase (MT1-MMP) protects malignant cells from tumoricidal activity of re-engineered anthrax lethal toxin; Rozanov DV et al.; Protective antigen (PA) and lethal factor (LF) are the two components of anthrax lethal toxin . PA is responsible for interacting with cell receptors and for the subsequent translocation of LF inside the cell compartment . A re-engineered toxin comprised of PA and a fusion chimera LF/Pseudomonas exotoxin (FP59) is a promising choice for tumor cell surface targeting . We demonstrated, however, that in vitro in cell-free system and in cultured human colon carcinoma LoVo, fibrosarcoma HT1080 and glioma U251 cells membrane type-1 matrix metalloproteinase (MT1-MMP) cleaves both the PA83 precursor and the PA63 mature protein . Exhaustive MT1-MMP cleavage of PA83 in vitro generates several major degradation fragments with an N-terminus at Glu40, Leu48, and Gln512 . In cultured cells, MT1-MMP-dependent cleavage releases the cell-bound PA83 and PA63 species from the cell surface . As a result, MT1-MMP expressing cells have less PA63 to internalize . In agreement, our observations demonstrate that MT1-MMP proteolysis of PA makes the MT1-MMP-expressing aggressive invasive cells resistant to the cytotoxic effect of a bipartite PA/FP59 toxin . We infer from our studies that synthetic inhibitors of MMPs are likely to increase the therapeutic anti-cancer effect of anthrax toxin . In addition, our study supports a unique role of furin in the activation of PA, thereby suggesting that furin inhibitors are the likely specific drugs for short-term therapy of anthrax infection.

Mil Med, 2004 Aug, 169(8), 575 - 9
Mass violence and early mental health intervention: a proposed application of best practice guidelines to chemical, biological, and radiological attacks; Ritchie EC et al.; Based on past episodes, there will be psychological sequelae to chemical, biological, and radiological attacks . Some of the psychological morbidity should be able to be ameliorated through planning and appropriate early intervention . Key components of early intervention are illustrated following a hypothetical scenario of a bomb and anthrax threat near the Pentagon . Many of these components, such as monitoring clear, consistent messages about health risks, are provided by physicians or politicians, not mental health providers, but have a serious impact on the mental health of the population . We hope that this scenario and the principles of response will prove useful to planners of emergency preparedness and responders in the case of an actual attack.

Biophys J, 2004 Dec, 87(6), 3842 - 9 Epub 2004 Dec.
Protein translocation through anthrax toxin channels formed in planar lipid bilayers; Zhang S et al.; The 63-kDa fragment of the protective antigen (PA) component of anthrax toxin forms a heptameric channel, (PA63)7, in acidic endosomal membranes that leads to the translocation of edema factor (EF) and lethal factor (LF) to the cytosol . It also forms a channel in planar phospholipid bilayer membranes . What role does this channel play in the translocation of EF and LF? We report that after the 263-residue N-terminal piece of LF (LFN) binds to its receptor on the (PA63)7 channel and its N-terminal end enters the channel at small positive voltages to block it, LFN is translocated through the channel to the opposite side at large positive voltages, thereby unblocking it . Thus, all of the translocation machinery is contained in the (PA63)7 channel, and translocation does not require any cellular proteins . The kinetics of this translocation are S-shaped, voltage-dependent, and occur on a timescale of seconds . We suggest that the translocation process might be explained simply by electrophoresis of unfolded LFN through the channel, but the refolding of the N-terminal half of LFN as it emerges from the channel may also provide energy for moving the rest of the molecule through the channel.

Microbes Infect, 2004 Jul, 6(9), 835 - 43
Knock-on effect of anthrax lethal toxin on macrophages potentiates cytotoxicity to endothelial cells; Pandey J et al.; Herein we report the knock-on cytotoxic effect of lethal toxin (LeTx) on human umbilical vascular endothelial cells (HUVECs) . HUVECs were treated either directly with LeTx or indirectly with LeTx conditioned medium (LeTxCM) prepared from RAW264.7 macrophage cells . Cytotoxicity assays were done on HUVECs and A549 cells using LeTx . HUVECs were more susceptible to LeTx (61-74% survivals) as compared to A549 cells (83-94% survivals, P < 0.005) . However, LeTxCM from RAW264.7 further potentiated killing of HUVECs (37% survival) compared to the LeTxCM from A549 cells (up to 70-100% survivals) . LeTxCM challenge induced an apoptotic cell death in HUVECs, and this was confirmed by reduction of BCL-2 levels to 54% . Protective antigen (PA) binding to macrophage cell line RAW264.7 > HUVECs >> A549 cells . Thus, we postulate that after the initial prodormal phase of pulmonary entry, LeTx causes not only significant direct damage to macrophages and endothelial cells, but also mediates additional indirect damage to endothelial cells mediated by a knock-on effect of LeTx on macrophages that causes apoptotic cell death in endothelial cells.

Ann Ig, 2004 Jul-Aug, 16(4), 603 - 12
{Use of anthrax as biological weapon}; Rizzo G; We describe the epidemiology of anthrax and the several human disease aspect, by spores penetration through the skin or by inhalation or ingestion, and the action of exotoxin secreted by B . anthracis . We detail pulmonary anthrax that could derive from intentional release of endospore as biological weapon . Laboratory diagnosis of Cereus group with cultural, FDA and PCR methods are reported . Environment disinfection, vaccination, antibiotic prophylaxis, passive antibody administration and treatment of anthrax are