Anthrax Insights

ANTHRAX INSIGHTS

David N. Rahni, Ph.D.

Professor of Analytical Chemistry

Adjunct Professor, Environmental Law and Dermatology (NYMC)

PACE UNIVERSITY

Pleasantville, New York 10570-2799

Member of the Editorial Board, FBI’s Forensic Science Communications

Bacillus anthracis (anthrax) has a unique place in medical history. The name Bacillus anthracis derives from the Greek word for coal, anthrakis, since the disease manifests itself as black, coal-like lesions on human skin. It was the first bacterium discovered by Robert Koch in 1877 exhibited then for the first time to cause infectious disease. This led to the production of the first vaccine by Louis Pasteur in 1881, when he employed attenuated live B. anthracis to protect sheep against anthrax. Species of the genus Bacillus are gram-positive, and form endospores that are strictly or facultatively aerobic. Most of the nearly one hundred Bacillus species are ubiquitous mostly in soil but also in water, and they are mostly encountered in medical bacteriology as airborne germs; Bacillus anthracis, the cause of the disease anthrax is an exception to the general manifestation of the Bacillus genus due to its increased risk. Anthrax has increasingly been used as a terrorist tool since the September 11th terrorist crimes in the US. Hence, early detection and deterrence of biological agents such as anthrax, as well as its containment and control, decontamination, and medical treatment for those infected is of extreme priority. In environmental forensics while employing the techniques of DNA finger printing (Mitochondrion and Plasmid) and other key bio-molecular marker strategies, law enforcement should also be in a position to trace back to the source of such agents for criminal prosecution purposes. NSF has now earmarked grants to private research firm to decipher the genetic DNA map of Bacillus anthracis to be able to trace out whether various specimen found in different locations are of the same origin. Acomprehensive search ofMEDLINE databases from January 1966 to September 2001 using the Medical Subject Headings anthrax, Bacillus anthracis, biological weapon, biological terrorism, biological warfare, and biowarfare led to the conclusion that there is an immense increasing interest on the part of medical, clinical, scientific, public health management and law enforcement communities on the these topics, particularly within the past decade.

For instance, a working group comprised of representatives from major academic medical centers and research, government, military, public health, and emergency management institutions and agencies, who met to develop consensus-based recommendations for measures to be taken by medical and public health professionals following the use of anthrax as a biological weapon against a civilian population, exemplifies such emergent efforts (Journal of American Medical Association, 1991; 281:135-1745). Specifically, consensus recommendations were made regarding the diagnosis of anthrax, indications and justifications for vaccination, therapy for those exposed, post-exposure prophylaxis, decontamination of the environment, and additional research and development needs.Of the numerous biological agents that may be used as weapons especially against civilians, the Working Group on Civilian Biodefense has identified a limited number of organisms that could cause disease and deaths in sufficient numbers to cripple a city or region. Not surprisingly, anthrax was designated as one of these most serious diseases.High hopes were at one juncture vested in the Biological Weapons and Toxins Convention, which prohibited offensive biological weapons research or production and was signed by most countries. However, Iraq and the former Soviet Union (currently Russia), both signatories of the convention, have subsequently acknowledged having offensive biowarfare programs; a number of other countries besides the U.S. are also believed to have such programs or certain stockpiles of these agents, as may have some autonomous terrorist groups. The possibility of a terrorist attack using bioweapons would be especially difficult to predict, detect, or prevent; thus, it is among the most feared terrorist scenarios, since the technology of production of such biological agents is textbook based in most cases.

For centuries, anthrax has caused disease in animals and, uncommonly, serious illness in human throughout the world. In fact, the use of biological agents in clandestine warfare is deemed to date back to a few millennia. Dropping covertly dead animals in adversaries’ water supply such as in wells and “qanat” (underground aquifers) has been practiced as far back as there is any historical accounts of human clashes. In antiquity, it was believed that the dead corpse’s soul would “hunt” the water, thereby making it lethally poisonous. In retrospect, however, it actually turned out the corpse was the hospitable source of nutrients where the proliferation of deadly microorganisms like B. anthracis (anthrax), Colostridium botulinum (botulism), Yersinia pestis (plague), and viral smallpox, cholera (Vibrio cholerae), typhoid fever (Salmonella typhi) and dysentery were the actual culprits behind the endemic epidemics that at times annihilated an entire city in a short time. Research on anthrax as a biological weapon began more than 80 years ago. Today, at least 17 nations are believed to have offensive biological weapons programs; it is uncertain how many other countries or clandestine terrorist groups are actively engaged in the field. Iraq has acknowledged producing and weaponizing anthrax. This is not surprising as Iraq not only used chemical lethal agents against Iranian civilians, but also against its own Kurdish minority in the north in the late 80’s. There is henceforth, sufficient evidence to conclude that certain autonomous terrorist groups are actively working on the acquisition, production and illegal application of biological and chemical agents worldwide.

Epidemiology:

Naturally occurring Anthrax is generally an enzootic disease of worldwide occurrence. An enzootic disease is endemic to a population of animals, i.e., its occurrence changes little over time. Anthrax in contrast, as an epizootic disease attacks large number of animal species, similar to a human epidemic. Domestic hoofed herbivores such as sheep, goat, cattle, camels, horses, lamas, and antelopes are the usual victim of anthrax. For instance, during 1945 alone, one million sheep died of anthrax infection in Southwest Iran alone, with a simultaneous widespread cutaneous human anthrax. Domestic animal vaccination since then has dramatically reduced the animal mortality rate. It could, however, be transmitted to human via contact with such infected animals. Human contact with anthrax leads to the sub-cutaneous inoculation of spores through incidental skin abrasions. The inhalation of spore-laden dust in the range of 1-10 μm in particular, will lead to the less frequent, nonetheless, more dangerous pulmonary infection; in occupational hazard, this latter cases is also referred to as “wool-sorter’s disease. B. anthracis spores may remain viable for years, and similar to Clostridium botulinum (responsible for foodborne botulism) are highly resistant to physical and, or chemical disinfecting agents. In the U.S. a widespread veterinary vaccine has minimized the sources of the disease. Contaminated imported agricultural goods on occasion, has resulted in the quarantine of goods from endemic areas.

Pathogenesis:

B. anthracis is comprised of a capsule made up of polymers of D-glutamic acid, which is not immugenic in itself. This envelope is antiphagocytic, albeit crucial for full virulence. The bacterium also excretes two plasmid-coded exotoxin eliciting protective antibodies: edema factor, which is a calmodulin-dependent adenylate cyclase that causes the elevation of intercellular cAMP, responsible for the severe dark edema as observed in B. anthracis infections; and, lethal toxin which is auxiliary to additional adverse effects.

Clinical Considerations:

1. Cutaneous anthrax: Approximately 90 percent of human cases of anthrax are of cutaneous category. For instance, between 1944 and 1994, 224 cases of cutaneous anthrax were reported in the US; the number was substantially higher in other less developed regions of the world. Upon skin contact with the organism or its spores that germinate, an itchy papule that resembles an insect bite is induced within 1-2 days, It rapidly leads to a painless ulcer 1-3 cm in diameter, black severely swollen “malignant pustule” with a characteristic necrotic (dying) area in the center which eventually crusts over. The organism may invade the general circulation via regional lymph nodes. Even though some cutaneous based anthrax cases remain localized and ultimately heal spontaneously, the overall mortality rate in untreated cutaneous anthrax is about twenty percent. Deaths are rare with appropriate local and oral antimicrobial therapy, however.

1. Pulmonary anthrax: There were 18 reported cases of inhalational anthrax cases in the United States between 1900 to 1978, mostly from special risk groups, i.e., those who directly worked with domestic animals and products like wool; it is noteworthy to cite two of the 18 infected were laboratory workers. The initial symptoms may resemble a common cold or flu-type symptoms. These symptoms can worsen resulting in serious breathing and convulsion problems, while causing detrimental breathing in some cases when untreated in time. After typically several days, but in rare circumstances after prolonged delays of up to 60 days, the disease fully manifests itself. Also referred to as “woolsorter’s disease” in the past, pulmonary anthrax is caused by inhalation of spores in the dimension range 1-10 μm. There are industrial micro-encapsulation technologies that would lend itself to the stabilized production of spores in such detrimental dimension range. The naturally occurring spore colonies are generally much larger than the above range. When developed as a biological weapon, the organism is encapsulated in envelopes that are sufficiently small to increase their penetrating effectiveness via the nasal canal and the pulmonary system. Pulmonary anthrax is characterized by progressive hemorrhagic lymphadenitis, i.e., inflammation of the lymph nodes. The mortality rate, if left untreated, approaches one hundred percent. Based on primate data, it has been estimated that the LD 50 (lethal dose sufficient to kill 50% of persons exposed to it) for human is 2500 to 55,000 inhaled anthrax spores.

1. Gastrointestinal anthrax: Although very rare, this form of anthrax manifests itself by the ingestion of spores through eating inadequately cooked meat or unsanitized fruits and vegetables. The gastrointestinal disease form of anthrax is characterized by an acute inflammation of the intestinal tract. Initial signs of nausea, loss of appetite, vomiting, and fever are followed by abdominal pain, vomiting of blood, and severe diarrhea. Intestinal anthrax results in death in 25% to 60% of cases when untreated in time. Gastrointestinal anthrax is more frequent in animals, however.

Laboratory Identification:

B. anthracis is readily recovered from clinical specimens. It is, however, often present in massive numbers in the form of colonies. This organism appears under the microscope as blunt-ended bacilli, occurring singly, in pairs, or more often as long chains. They do not sporulate in clinical samples, but do so on culture medium. The spores are oval and centrally located. On blood agar, these colonies are large, grayish, non-hemolytic with irregular boundaries. Gram-positive stain of culture smear showing non-staining spores. Contrary to many other Bacilli, B. anthracis is non-motile, and encapsulated in vivo. A direct immunofluorescence assay aids in the identification of the organism. Extreme caution should be taken to prevent transmission via aerosols when handling this organism in the laboratory.

Treatment and prevention:

The symptoms of disease vary depending on the mode of contraction, but symptoms usually occur within 7 days. Direct person-to-person spread of anthrax is extremely unlikely to lead to a contagious manifestation of the disease. Communicability is not, therefore, a concern in managing or visiting with patients with inhalational anthrax. The only way to acquire infection of the disease anthrax is to be directly exposed to large numbers of spores of the microbe. Naturally occurring B. anthracis is sensitive to antibiotics: ciprofloxacin, penicillin, and doxycycline. Careful investigation must be followed to assess the possible resistivity of a specific organism that may have been mass-produced through genetic engineering, adaptation and, or other biotechnology techniques for the mere terrorist applications. A cell-free vaccine is available for those in high- risk occupations. Because anthrax is considered to be a potential agent for use in biological warfare, the Department of Defense (DoD) has had mandatory vaccination of all active duty military personnel who might be involved in conflict since the 1991 Desert Storm and Desert Shield Operations in Persian Gulf against Iraq. The vaccine is reported to be 93% effective in protecting against anthrax. The anthrax vaccine is manufactured and distributed by BioPort, Corporation, Lansing, Michigan. The vaccine is a cell-free non-infectious sterile ultra-filtrate (less than 0.05 μm at minimum) form of the culture of an attenuated strain of Bacillus anthracis that contains no dead or alive bacteria. The final product contains no more than 2.4 mg of aluminum hydroxide as adjuvant base, a substance that when injected with an antigen, serve to enhance the immunogenicity of that antigen. Anthrax vaccines intended for animals should not be used in humans. Pregnant women should be vaccinated only if absolutely necessary. The immunization consists of three subcutaneous injections given 2 weeks apart followed by three additional subcutaneous injections given at 6, 12, and 18 months. Annual booster injections of the vaccine are recommended thereafter. Mild local reactions occur in 30% of recipients and consist of slight tenderness and redness at the injection site. Severe local reactions are infrequent and consist of extensive swelling of the forearm in addition to the local reaction. Systemic reactions occur in fewer than 0.2% of recipients.

Epitomizing, however, it is the ultimate better understanding of the exact macro-molecular toxins interactions in the cell and as released by the B. anthracis that would truly relieve us of the dangers associated with anthrax. Upon diagnosis of the infection stage, large amount of toxins presumably comprised of three proteins, has already been released independently into the blood stream to be reunited in cells. Antimicrobial drugs are not any longer effective against the control of these already circulating toxins in the body at such juncture. This endeavor falls in the realms of the emerging field of proteomics, an area where recent findings could help design drugs to neutralize in vivo presence of anthrax toxins. Looking back at the twentieth century, one might summarize the three scientific breakthroughs: atom, computer and gene, the latter of which has now mapped out the up to one hundred thousands human genes, which in turn is comprised of up to one billion nitrogenous pairs on the forty six chromosomes. A harmonic integration of these three breakthroughs will be the main theme of the current century, in which proteomics, the science of identification and quantitation, structural elucidation, mechanistic functions, etc. of up to a billion proteins is on the scientific community agenda. The two research groups at Harvard Medical School and University of Wisconsin (R. J. Collier & A.T. Young, Nature, November 8, 2001, 415: 15-26) have striven to tackle that goal for anthrax. They just reported the first anthrax protein called protective antigen, binds to a cell receptor and facilitates the entry into the cell of the other two proteins, which in turn while serving concurrently as hydrolytic enzymes impair the body’s defense mechanism. One hypothesis postulates finding a novel method of eliminating the reunion of these two enzymes as a means of disease prevention and control. Notwithstanding, the structural elucidation and molecular based mechanism of action of such protein toxins are the pre-requisites toward the ultimate eradication of not only anthrax but also of other similar biological borne diseases. Such exciting multi-disciplinary approach, should ultimately pave the way toward deciphering the actual effect at the molecular level. Another possibility might be to carry out molecular modeling of these enzymes after their structures are fully elucidated and based on x-ray diffractions among other spectroscopic techniques. Then, utilizing combinatorial chemistry one should then be able to model a suitable competitive inhibitor that binds to the enzyme, thereby incapacitating it swiftly. Alternatively, if one could pinpoint the exact intracellular receptor to which the enzyme binds, one should then strive to block that receptor site with benign antagonists. For now, it is important to note that because of the resistance of endospores to chemical disinfectants, autoclaving is the most reliable means of decontamination. Gamma irradiation of suspicious objects coupled with strong magnetic fields is finding increasing merit, nevertheless, despite its extreme cost and retrofitting schemes. Upon sufficiently suspecting the presence of a contaminated object, the ventilation system must immediately be turned off, the section closed off, and the area evacuated. Law enforcement authorities must be contacted at once. A list of those individuals who were in the immediate contaminated area should be reported to the authorities for their immediate medical evaluation, monitoring and possible treatment. Federal and State Departments of Health and Human Services and Center for Disease Control among other agencies maintain excellent sources of information accessible via Internet and phone systems. Finally, other biological agents such as smallpox are far more dangerous than anthrax due to their immediate epidemic characteristics. An integrated vaccination program, possibly in coordination our allies for a common stockpile is, therefore, warranted.