ANTHRAX: Bacillus anthracis

There’s nothing quite like a mail-order disease to propel a pathogen into notoriety. In 2001, several envelopes containing spores of the bacteria Bacillus anthracis were shipped across the United States, infecting those exposed with anthrax. This was hardly the first time B. anthracis cropped up as a prominent pathogen or agent of biological warfare; the bacterium has a sordid history as a major player in biological warfare in the past century, along with a biblical history of outbreaks.

Mail Surprise

Meet the Microbe

Anthrax is caused by the bacteria Bacillus anthracis, whose name derives from the Greek word for coal (“anthrakis”) due to the black, dead tissue that develops at the site of infection. The bacterium reproduces rapidly within host cells as an obligate pathogen, and cannot survive outside of its host. When exposed to adverse conditions, B. anthracis forms a spore. These spores can exist for decades in the absence of any nutrients and similarly stressful conditions – after an animal dies of an anthrax infection, anthrax spores will persist in the dirt where the body decomposed for upwards of 70 years. When these spores are ingested by a grazing animal or get caught in a cut in the flesh of a passing creature, they will germinate and the bacteria will begin reproducing within their new host[5,8,12,13].

bacterium

Anthrax

Anthrax spores are found in soil samples from every continent except Antarctica. As most animals are susceptible to the disease, anthrax is best known as a worldwide disease of wildlife and livestock[5,14]. Animal infections tend to occur in unsuspecting herbivores that ingest the spores while grazing. The scale of annual wildlife deaths is unknown but likely large; in 1945 over a million sheep died in Iran due to anthrax infections[5].

deer

The impact of anthrax on the human population is still significant, with 20,000 – 100,000 cases occurring annually worldwide[13]. Most human cases are caused by handling infected animals or animal products, such as hide and wool. The largest known human outbreak occurred in Zimbabwe between 1979-1985, with approximately 10,000 cases[12]. In the mid 1800’s anthrax became known as woolsorters’ and ragpickers’ disease in Europe due to infections caused by inhaling spores in textile factories[6]. Generally, humans are considered moderately resistant to respiratory infections of anthrax. Factory and mill workers were found to inhale between 600-1300 spores during their eight hour shifts and showed no infection at these exposure levels – most of the spores are removed from the lungs by a wondrous structure in our airway known as the mucociliary escalator system[12].

The anthrax infection is spread via the spores of B. anthracis. The disease varies in pathology and severity depending on the method of infection. The most common way of ‘catching’ anthrax is through a break in the skin, which causes cutaneous anthrax. If consumed and spores are absorbed through a mucosal membrane in the gut, gastrointestinal anthrax develops. When inhaled, the disease is aptly known as inhalation anthrax. In rare cases during the late stages of an infection, anthrax meningitis can develop with a mortality rate near 100%[12,13].Types

 

Tools of the Trade

The key to B. anthracis’s pathogenic success is the unique virulence factors it employs. The bacteria contain two virulence plasmids (circular loops of DNA) called PX01 and PX02. A loss of either of these plasmids creates an attenuated strain, highlighting their importance. PX01 contains 3 toxins, or a tri-toxin/tripartite toxin, which cause bleeding, swelling, and cell death. PX02 encodes the genes necessary to create a capsule around the bacteria that prevents host immune cells from destroying it via phagocytosis[7,9].

The tri-toxin in PX01 is made up of the edema factor (EF), lethal factor (LF), and protective antigen (PA). EF and LF are responsible for the actual toxicity; EF contains an adenylate cyclase (protein) that impairs host defenses and causes swelling, while LF is a zinc-dependent protease (another protein) that causes the host’s macrophages immune cells to burst. Neither of these toxins would have any effect without PA, which delivers the toxins into host cells by recognizing target cells, binding to them, and creating a pore through which the toxins can enter[1,2].

Pathogenesis of Anthrax

pathogenesis

Once the spores of B. anthracis gain entry into a host body, they are ingested by macrophages (white blood cells) at the site of infection. Inside a macrophage, the spores survive by an unknown mechanism and germinate into active bacteria that multiply, encapsulate themselves in the protective coating encoded on PX01, and induce lysis in the macrophages, causing them to burst and release the multiplying bacteria. Once in the blood stream, the bacteria’s tri-toxin enters and wreaks havoc in further host cells. The LF toxin may cause macrophage cells to produce proteins that trigger inflammation and cell death, with the surplus of these proteins in the blood leading to sepsis and death. Anthrax may also target endothelial cells that line body cavities, blood vessels, and lymph vessels. Once these cells are destroyed, leaking fluids and cells can cause sepsis, shock, and liver failure[3,10,12].

anthrax_eschar

The course of the disease varies based on the method of acquisition. Cutaneous anthrax makes up approximately 90% of human anthrax cases. Within 2-3 days of exposure, a small pimple will develop at the site of infection. On the 5th or 6th day, this pimple will ulcerate and dry into the characteristic black eschar. The eschar is painless and will grow slightly, surrounded by swelling. In most cases, the eschar will resolve over the following 2-6 weeks. If untreated, 20% of patients will develop sepsis and die. If patients use antibiotic treatment, this mortality rate drops to below 1%[12].

 

Gastrointestinal anthrax is the rarest human form and can be acquired by eating the undercooked meat of infected animals. If this presents as abdominal anthrax, the spores usually incubate for 2-5 days before the characteristic black eschar forms on the internal wall of the intestine. The early symptoms include nausea, vomiting, anorexia, and fever; these may develop into severe abdominal pain, vomiting blood, and bloody diarrhea eventually leading to sepsis and death. The non-specific presentation can lead to underdiagnoses, resulting in a high mortality. Depending on how early treatment starts, mortality ranges from 25% to 60%. Gastrointestinal anthrax can also present as oroesophageal anthrax, with symptoms including a sore throat, difficulty swallowing, fever, and swelling of tissues and cervical lymph nodes[12].

gastro anthrx

If spores are inhaled, inhalation anthrax may develop. The lethal load for humans is approximately 10,000-20,000 spores. These spores are transported into the lungs where they enter the lymphatic system and are transported to the lymph nodes. The bacteria spread and multiply in both the blood and lymph, causing severe sepsis. In industrial exposure cases (with a lower spore-load than cases of bioterrorism), the infection initially presents with flu-like symptoms including a fever, fatigue, cough, and muscle pain. The acute illness presents 48 hours later with shortness of breath, high-pitched breathing, skin discoloration due to a lack of oxygen, rapid breathing and heart rate, and a buildup of fluids around the lungs. These symptoms worsen, leading to disorientation, coma, and death. Half of patients develop meningitis. The mortality rate of this type of anthrax was believed to be higher than 95%, but antibiotic treatment and medical support increase the chance of survival: in the 11 cases of inhalation anthrax in the US, the mortality rate was 45%[12].

Anthrax meningitis can occur toward the end stage of any of these infections. It is characterized by the appearance of blood in the cerebrospinal fluid along with a loss of consciousness and death. The mortality rate of this nearly 100%[13].

History

History

The earliest references to anthrax are arguably the biblical Fifth and Sixth Egyptian plagues. We lack analyzable scientific evidence of these plagues, so any conclusions are conjecture at best. If they occurred, the two plagues could be connected: the fifth as a disease of livestock and the sixth a plague of humans through contact with livestock. The latter plague is known as the Plague of Boils which could hardly be more ambiguous when it comes to ancient plagues, but the description could possibly fit cutaneous anthrax. Historical medical records trace appearances of anthrax symptoms back to 1491 BC in Mesopotamian and Egyptian civilizations.

The Romans were also familiar with Anthrax, with references of the disease found in the famous writings of Homer and Virgil. Homer’s Iliad describes plagues infecting domesticated animals before spreading to soldiers, setting one of the first historical records to be about 850BC. Virgil’s Georgics eight centuries later described a disease of “feverish blisters and foul sweats” to be caused by handling and wearing hides or fleeces, once again suggestion cutaneous anthrax.

Further outbreaks are noted in Europe in the 14th and 17th centuries, including the 1613 epidemic known as the Black Bane that decimated over 60,000 humans and animals.

In 1863, Bacillus anthracis was first observed in the infected blood of a sheep by Casimir-Joseph Davaine. Shortly after, in 1876, Robert Koch proved that the bacteria was the causal agent of anthrax. Impressively, it was only five years later when Louis Pasteur produced a vaccine for livestock (incidentally, it was the first effective vaccine of any bacterial agent!).

Biological Warfare

biowarfare

Anthrax is one of the more popular and potentially devastating agents of biological warfare. The World Health Organization estimates that a release of 50kg worth of dried anthrax spores via aerosilzation over 2 hours in a city of 500,000 would result in 95,000 deaths and 125,000 further citizens incapacitated. The strain on medical resources would rapidly result in a breakdown of resources and infrastructure[12]. Such an attack would be odorless, invisible, and not too unfeasible: the Department of Defense reports that 3 defense employees with minor technical skill and no expert knowledge of bioweapons manufactured an simulant of B. anthracis in less than a month for only a million dollars[5]. Unsurprisingly, this bacterium is considered a select agent and is a hot topic in biodefense research.

The potential of anthrax as a weapon was first realized in World War I. Germany and German sympathizers in the US weaponized it for use against animals, including attacks on US livestock. In 1916, Scandinavian rebels were supplied anthrax by Germans to use against the Imperial Russian Army in Finland[11,15].

By the time World War II rolled around, anthrax was being investigated and utilized against humans. In the 1930’s, Japan’s Unit 731 in Manchuria began testing anthrax and other biological agents on prisoners of war, resulting in over 10,000 deaths. This branch of the Japanese Kwantung Army also infected Chinese cities with B. anthracis along with Yersinia pestis (the Black Death). The Former Soviet Union had a large production program for anthrax under its offensive bioweapons program that lasted for decades[5]. The British and USA also both had offensive biological warfare programs. In the United States, anthrax bombs were first produced in 1942[12]. In England, anthrax received special attention in 1942 and 1943, during which the English tested anthrax bombs on the island of Gruinard. It took over 40 years and 280 tons of formaldehyde to decontaminate the island to the point where travel restrictions could be lifted in 1989. The British plans also included Operation Vegetarian, for which they produced over 5 million anthrax infected cattle cakes to drop in Germany in an effort to decimate their meat stocks (and anyone who ate the infected cows). Luckily, the war was won before the Brits could turn Germany into a vegetarian nation. cows

 

Despite pressure to dismantle offensive biological warfare programs,  research continued decades after the war. In the 1960’s, the US military experimented with aerosol dispersion over the Pacific Ocean and determined that a 32 mile long line of released anthrax would travel over 60 miles before it lost infectiousness[5]. Following these tests, international pressure rose to dismantle such offensive programs and, in 1969, President Nixon signed an executive order stopping all production of biological weapons in the United States. Unfortunately, this was hardly the end of weaponized anthrax.

In 1979, the largest outbreak of inhalation anthrax occurred in the former Soviet Union. A military facility on Sverdlovsk that was conducting research accidentally released anthrax spores, leading to 66-106 casualties[12]. Just over a decade later in 1993, Iraq acknowledged producing and weaponizing anthrax to the UN Special Commission and Amun Shinrikyo, a Japanese cult infamous for sarine gas attacks, dispersed aerosols of anthrax throughout Tokyo (luckily, the strain they used was not particularly infectious to humans)[5]. In 2001, letters containing anthrax spores were mailed to media offices and senators in the United States, causing five deaths and 17 further infections[12].

Today, over 17 countries are still believed to have active, offensive programs of biological warfare.

Treatment

The mortality of an anthrax infection can be greatly reduced using antibiotics. If naturally acquired, a treatment course lasts approximately 7-10 days. In the cases of aerosolized bioterrorism, a 60-day course is recommended. Vaccinations for anthrax do exist, but unfortunately they lack standardization, are extremely costly, and require repeated dosing with unpleasant side effects. Over two million soldiers in the US military were vaccinated between 1998 and 2008 due to fears of weaponized anthrax[12].

c-schmitz-closed-envelope-clip-art

References

[1] Abrami, L et al. “Anthrax toxin triggers endocytosis of its receptor via a lipid raft-mediated clathrin-dependent process.” Journal of Cell Biology. 2003. 160(3).

[2] Bradley, K et al. “Identification of the cellular receptor for anthrax toxin.” Nature. 2001. 414.

[3] Dixon, T et al. “Early Bacillus anthracis-macrophage interactions: intracellular survival and escape.” Cellular Microbiology. 2000. 2(6).

[4] Hongbin, L et al. “Formation and composition of the Bacillus anthracis endospore.” Journal of Bacteriology. 2004. 186(1).

[5] Inglesby, T et al. “Anthrax as a biological weapon, 2002. Updated recommendations for management.” JAMA. 2002. 287(17).

[6] Jernigan, J et al. “Bioterrorism-related inhalation anthrax: the first 10 cases reported in the United States.” Emerging Infectious Diseases. 2001. 7(6).

[7] Leppla, S. “The anthrax toxin complex.” Alouf J, Freer JH, eds. Sourcebook of bacterial protein toxins. London: Academic Press 1991:277–302.

[8] Manchee, R et al. “Formaldehyde solution effectively inactivates spores of Bacillus anthracis on the Scottish Island of Gruinard.” Appl Environ Microbiol 1994. 60.

[9] Makins, S-I et al. “Molecular characterisation and protein analysis of the cap region, which is essential for encapsulation in Bacillus anthracis.” Journal of Bacteriology. 1989. 171.

[10]Moayeri, M et al. “Bacillus anthracis lethal toxin induces TNF-α-independent hypoxia-mediated toxicity in mice.” Journal of Clinical Investigation. 2013. 112(5).

[11] Redmond, C et al. “Deadly relic of the Great War.” Nature. 1998. 393.

[12] Spencer, R. “Bacillus anthracis.” Journal of Clinical Pathology. 2003. 56.

[13] Swartz, M. “Recognition and management of anthrax – an update.” New England Journal of Medicine. 2001. 345(22).

[14] Van Ert, M et al. “Global genetic population structure of Bacillus anthracis.” Plos One. 2007. 5.

[15] Wheelis, M. “Biological sabotage in World War I.” Geissler E, van Courtland Moon JE, eds. Biological and toxin weapons: research, development and use from the Middle Ages to 1945. Oxford: Oxford University Press. 1999:35–62.

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