anthrax

Anthrax: villain or misunderstood?

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Stewart holds a different colony of anthrax in his lab. Stewart’s work with anthrax and other similar organisms focuses on understanding the tough protein shell of the bacteria’s spores that enable the pathogen to survive in soil for extended periods of time, even hundreds of years. | photo by Phillip Sitter, Bond LSC

By Phillip Sitter | MU Bond Life Sciences Center

For a tiny spore, anthrax holds a lot of danger and promise.

If you found yourself wondering about more than its safety in the lab, we have a answers to a few persistent questions.

What makes anthrax dangerous, and how does it spread? How common are infections of it in nature? If we have antibiotics that already treat it, beyond finding new and better ones why study the organism?

Bovine slayer and bio-weapon

Anthrax bacteria are highly resilient organisms. Like only a few other bacteria in nature, they produce spores with protein-shelled casings that lay dormant in soil for long periods of time, waiting to be taken in by grazing animals like cattle and sheep.

Once they enter the nutrient-rich environment of an animal’s bloodstream — exactly how the bacteria gets there after entering an animal’s mouth is unknown — spores germinate inside the white blood cells that absorb them. As anthrax reproduces rapidly inside its host, it releases toxins that quickly kill the infected animal. That sometimes happens in just a matter of hours according to farmers’ accounts, said George Stewart, Bond Life Sciences Center scientist, medical bacteriologist, McKee Professor of Microbial Pathogenesis and chair of Veterinary Pathobiology at MU.

When infected animals die the bacteria are exposed to oxygen in air that penetrates the decomposing body and new spores escape as the dead host decays. The newly-produced spores are deposited back into the soil, where they wait in a state of suspended animation for as long as it takes to be ingested by another grazing animal, sometimes decades to a hundred years.

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“Most of these plates are anthrax, but they’re all non-virulent,” Stewart says of the various bacterial colonies on the counter in his lab. Non-virulent strains are safe to handle with the precautions of a BSL-2 lab: gloves, a lab coat and eye protection. The Bond LSC does not have a BSL-3 or higher facility to study more dangerous pathogens like virulent strains of anthrax. That work is done at the Laboratory for Infectious Disease Research, or LIDR. | photo by Phillip Sitter, Bond LSC

Sporadic, natural outbreaks of anthrax can happen almost anywhere in the world except Antarctica, as the spores have been found to exist worldwide, Stewart said. In the United States, outbreaks in cattle and bison usually happen in the Plains and West in states like Colorado and Wyoming — anywhere that cattle or sheep have been raised or their wildlife equivalents graze, lending to the descriptor from Stewart of “anthrax belt” for the states stretching from Texas to the Dakotas.

During the Cold War, the U.S. and the Soviet Union, among others, were attracted to anthrax for their respective biological weapons programs because of the hardiness of the organism and high lethality rates of untreated gastrointestinal and, especially, pulmonary anthrax infections in humans. Today, Americans’ are probably most familiar with anthrax from the mailing attacks of 2001, when letters containing weaponized anthrax spores were delivered to the offices of media outlets and politicians, infecting 22 people and killing five. 

Hero in a half-life shell?

Despite their grim reputation, anthrax spores hold a lot of potential for Stewart.

The outermost layer of the protein-shell structure of a spore holds particular interest — how it’s made, what proteins it’s composed of and the function of those proteins. Studying it could not only help find future anthrax vaccines and therapies, but also be used for other applications.

Scientists have coated spores of a close biological relative of anthrax, Bacillus thuringiensis, with plant growth-promoting and anti-insect enzymes and other proteins to treat crops. Its durable structure in the form of the spore’s protein shell attach to these enzymes and remain longer in the environment, making them more effective, Stewart said.

This long biological half-life even presents potential for bio-remediation work — using natural organisms to cleanse the environment of toxins and pollutants. In the case of B. thuringiensis, this might include cleaning up soil from the herbicide atrazine and the notorious pollutant dioxin, a by-product of various industrial processes including incineration, smelting and the production of paper pulp and some herbicides and pesticides.

According to collective findings of the National Institutes of Health and Environmental Protection Agency and others, atrazine is probably not a human carcinogen but can cause genetic damage in animals and is still acutely toxic to people at high enough levels. According to the World Health Organization (WHO), dioxin causes cancer and other ill human ailments.

Pollutants that contaminate soil can eventually leech into groundwater and also enter the food supply through grazing animals. That makes organisms adapted to living in soil like B. thuringiensis perfect candidates as potential carriers of agents used to clean up soil pollutants like atrazine and dioxin.

However, Stewart said that there is too much public stigma to use anthrax for similar applications, “which is too bad because Bacillus anthracis actually works a little better in these applications.”

Rethinking the anthrax image 

So is anthrax a dastardly bio-villain or a misunderstood hero?

The truth is neither. Anthrax bacterium is just another living thing.

As a predator of other living things, needing to feed to survive and reproduce, it goes about its life cycle without any conscious agenda. It has no malice, and unlike a wolf, shark or hawk it doesn’t have a brain so it doesn’t even have instinct, only genetic code to mindlessly live by.

How we interact with anthrax is largely dependent upon us humans.

We have the vaccines to prevent it and antibiotics to treat it if it makes us sick. After that, we can harness and modify anthrax’s natural power for ill as a weapon, or maybe to do some good in the environment. So long as we have the technology to study and manipulate organisms like anthrax, the potential for both scenarios will always be there.

Meanwhile, at MU Stewart will continue to study anthrax, searching for its secrets in order to better understand it, to better serve the rest of us.

Under the hood

The safety behind studying deadly disease
By Phillip Sitter | MU Bond Life Sciences Center

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George Stewart, McKee Professor of Microbial Pathogenesis and Chair of Veterinary Pathobiology holds up a colony of Bacillus anthracis in his lab. The strain of anthrax he holds is non-virulent, and is therefore safe to handle under BSL-2 precautions as opposed to BSL-3 for virulent strains that cause disease in humans. | photo by Phillip Sitter, Bond LSC

You’ve seen it before in the movies.

Sweaty scientists put on their full-body, spacesuit-like get-ups to stave off a potentially extinction-level outbreak and at least one scientist invariably gets infected with the deadly agent of disease.

While popular culture propagates this sense of peril, in reality bio-containment labs are designed with safety in mind.

“People tend to think bio-containment facilities are dangerous, mostly from movies I think, but the history is actually spectacular,” said George Stewart, a medical bacteriologist, a Bond Life Sciences Center scientist, McKee Professor of Microbial Pathogenesis and Chair of Veterinary Pathobiology.

For Stewart — whose lab works on the basic science behind anthrax — no one is “under the gun because of big outbreaks, not with the pressure you see in movies.”

But one type of pressure is an important part of bio-containment lab safety. Air pressure differences maintain certain labs at lower pressure compared to the rooms and hallways around them, ensuring that air will only flow in toward a lab and not out, keeping any airborne pathogens trapped inside.

“They know if everything is done properly, it’s perfectly safe for them and even safer for everyone else [outside a lab],” Stewart said of the safety features, procedures and systems of bio-containment lab safety in place at facilities like those at the Bond LSC and elsewhere at MU.

Stewart said he is vaccinated against anthrax. “Whether I have protective immunity or not, I don’t know.”

 

“Almost like you were working underwater”

The powerful capabilities of anthrax and other lethal pathogens call for particular safety precautions for scientists.

Stewart looks like he’s straight out of the movie Contagion when donning the full-body suit for his more dangerous research in a Bio-Safety Level (BSL) 3 facility at MU’s Laboratory for Infectious Disease Research (LIDR). In the trees on the eastern fringes of campus, the specialized building is where he and other researchers study diseases animals can transmit to humans, including plague, Brucella, tularemia and Q fever, and mosquito-borne diseases like dengue, chikungunya and now Zika virus. The safety protocols and systems at a BSL-3 lab like the LIDR facility Stewart described reflect the likely transmission by aerosols of the human pathogens inside.

After passing through security access to the building and the labs inside, Stewart enters an ante room off of a hallway. The air pressure in this room and the lab beyond it is such that air will only flow in toward the lab, and not out and away.

Anything that goes into the lab only leaves if it is autoclaved, disinfected in a steel machine using pressurized steam that “essentially kills everything, even heat-resistant spores,” so that means Stewart changes clothes, removes his watch, phone and any other personal items.

Next come layered scrubs and a water-proof Tyvek suit with booties and a hood that cover everything but his hands and face. Two layers of gloves take care of his hands, but shielding his face is a bit more technical.

A plastic face cover with a Tyvek hood shrouds over Stewart’s shoulders. Inside, a pump fills the hood with positively-pressured filtered air – this has the inverse effect of the negative pressure of the rooms and keeps air flowing out away from his face and not toward it.

Everything inside the lab and the building is about redundancies like that. A final safety measure is that all work on pathogens take place in bio-safety hoods – HEPA-filtered cabinets.

Stewart said it can be difficult to hear with the air filter systems blowing, so every move by researchers is calculated and announced. Colleagues take their time in handing off equipment to one another, so as to avoid torn gloves.

It’s “almost like you were working underwater as two divers,” Stewart said about working in the BSL-3 lab with a colleague.

“Everything is orchestrated in a very intentional way.”
Only dangerous when dry

Anthrax isn’t always lethal, so the scene is quite different inside a BSL-2 lab at Bond LSC where Stewart studies non-virulent strains.

BSL-2 labs study infectious agents that can cause disease in humans, but are usually treatable. Researchers only need lab coats, gloves and eye protection in these labs and all waste must be autoclaved. Here anthrax and other colonies of organisms are stacked in covered Petri dishes and handled without any Tyvek or air pumps.

The Anthrax bacterium researched here is missing a specific plasmid, a DNA molecule essential for virulence that protects the anthrax bacteria from white blood cells that attack them.

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Stewart holds a colony of anthrax and says that it is impossible to see a visually recognizable colony like this naturally in soil. | photo by Phillip Sitter, Bond LSC

On top of that, all samples in this lab are wet, and anthrax spores are only dangerous as aerosols when dry. Before 2001, Stewart said virulent strains of anthrax were only labeled BSL-2 agents for this reason.

The anthrax letter attacks that year not only changed some of the organism’s lab classifications, but also interest in it. Prior to 2001, Stewart said there was not a lot of funding available for anthrax-focused researchers, a small and tight-knit community. Even though the attacks did spur an increased investment of government money into the field at the time for defense against anthrax as a bio-weapon agent, almost 15 years later Stewart said that funding is more or less back at pre-2001 levels, “perhaps marginally better.”

There are now only a handful of anthrax-dedicated labs in the U.S., Stewart said, trying to name them off as he counted his fingers. The Bond LSC has not had any lab higher than a BSL-2 plus for years now, not since the BSL-3 research moved to LIDR, Stewart said.

Several local residents called when LIDR was under construction and asked questions and voiced concerns about the facility and the work to be done there, Stewart recalled. However, Stewart said he and his colleagues gave the callers honest answers, and he has not heard of any pushback since.

Stewart sees bio-containment labs as positive technological achievements in the study of disease – without them, many advances in treatment would never have been possible. In terms of the work done at facilities at MU and in the Bond LSC, Stewart said “we have the facilities, we have the equipment, we have the training,” to ensure the safety of researchers inside the labs, and even more so everyone else on the outside.

Anthrax is not contagious and responds well to antibiotics, despite concerns in the scientific community Stewart shared that there is a possibility antibiotic resistance could be intentionally engineered into anthrax.

Stewart could only think of a couple of cases when lab workers got infected with the organism through mishaps, and those were at USAMRIID – the United States Army Medical Research Institute for Infectious Disease at Fort Detrick, Maryland – when anthrax was produced there in very large quantities for research of it as a bio-weapon during the Cold War.

When you spend a lot of your time working with potentially lethal pathogens though, what do you tell your doctor when you come in with flu-like symptoms? Stewart said that not only does he and any of his colleagues disclose to their doctor the organisms that they work with, but doctors at MU already know exactly what organisms are being researched inside the LIDR labs. As a precautionary measure for their own well-being in case accidental infection did occur in a lab after all, Stewart or another colleague working with anthrax who turned up sick would receive antibiotics just to be safe – “there are standard operating procedures for everything.”

He does not want to make light of the dangerous organisms he works with, but inside the BSL-3 facility at LIDR, Stewart said that breathing in HEPA-filtered air all day there does do wonders for his hay fever.

Stewart couldn’t help but share a chuckle with that one. Laughter might be the most un-containable thing in nature.

Quicker anthrax detection could save millions of dollars, speed bioterror response

Anthrax bacteria is a rod-shaped culture. Most common forms of transmission are through abrasions in the skin and inhalation.

Anthrax bacteria is a rod-shaped culture. Most common forms of transmission are through abrasions in the skin and inhalation.

 

Imagine researchers in hazmat suits moving slowly and deliberately through a lab. One of them holds up a beaker. It’s glowing.

This light — or the absence of it — could save millions of dollars for governments and save the lives of anthrax victims.

Scientists at the University of Missouri Laboratory of Infectious Disease Research proved a new method for anthrax detection can identify anthrax quicker than any existing approach.

When the “bioluminescent reporter phage” — an engineered virus — infects anthrax bacteria, it takes on a sci-fi-movie-type glow.

George Stewart, a medical bacteriologist at MU’s Bond Life Sciences Center, and graduate student Krista Spreng, observed the virus against a variety of virulent strains of bacillus anthracis, the bacteria causing anthrax disease.

“For this technique, within a few hours, you’ll have a yes or no answer,” Stewart said.

The research, funded by the USDA, was published in the Journal of Microbiological Methods in Aug. 2013. David Schofield at Guild BioSciences, a biotech company in Charleston, S.C, created the reporter phage.

This new method could save a significant amount of money associated with the decontamination of anthrax from suspected infected areas.

Expensive clean-up from the 2001 “Letter attacks”

With the country on high-alert following Sept. 11, 2001, a slew of bioterrorists mailed anthrax letters, filled with a powder that if inhaled could cause death.

Numerous Post Offices and processing facilities were closed and quarantined.

The clean-up bill for the 2001 Anthrax Letter attacks was $3.2 million, according to a 2012 report in Biosecurity and Bioterrorism: Biodefense Strategy, Practice and Science.

Theoretically, the new detection method would alert of a negative result potentially five hours into clean-up efforts instead of two or three days into expensive decontaminating.

Current methods take anywhere from 24 hours or longer to produce a definitive answer for anthrax contamination.

A five-hour benchmark

Stewart said from contamination levels expected from a bioterrorism threat, a positive answer could be found in five hours. If contamination levels were higher, results would come back much more quickly.

Prior to this bioluminescent reporting phage, experts used techniques that were culture based or PCR (polymerase chain reaction) based. Both methods, require additional time for a definitive answer, a minimum of 24 to 48 hours, Stewart said.

“Normally to identify whether an organisms is present, you have to take the material culture, the organism and all the bacteria that might be present in the sample,” Stewart said. “You have to pick colonies that might be bacillus anthracis and do chemical testing which takes some time.”

From a bio-threat standpoint, breathing in anthrax, is the highest concern for public health and homeland security officials and has the highest fatality rate among forms of anthrax.

“If you have a situation and need a quick yes or no answer, this is a tool that will help that,” Stewart said.

Terrorists have used a powder form of anthrax, which has been slipped into letters of political persons and media. A person is infected when an anthrax spore gets into the blood system, most commonly through inhalation or an abrasion on the body, according to Centers of Disease Control and Prevention.

For low levels of contamination, the bioluminescent reporter phage would still detect the presence of the bacteria, but it would take longer.

“This method will be as quick as any of the others and quicker than most,” Stewart said.

The bioluminescent-detection method can detect low levels of anthrax bacteria and rule out false positives. The added benefit to this reporting system is its ability to show that anthrax bacteria are present and it’s alive, Stewart said.

What’s next?

The next step in the bioluminescent reporter phage is getting it approved so a product can be produced and branded. The agency that would warrant the stamp of approval would depend on the eventual use of the phage — food-related testing would likely go through the Food and Drug Administration, Stewart said.

When that happens, a product would not necessarily require a formal lab — it would need a place where cultures could grow at 37 degrees.

“Samples could be collected, brought back to the state public health lab for example and then the testing could be done within a few hours of the collection of the samples and you would have a result,” Stewart said.

The last anthrax attack was in 2001, but the possibility of one happening again, Stewart said, remains a driver for proactive research.

“In the years since the postal attacks, we haven’t had any bona fide anthrax attacks,” Stewart said. “That doesn’t mean it’s not going to happen — we have to be prepared for when it does occur again.”

Stewart’s research on anthrax bacteria and detection methods recently appeared in the August 2013 edition of The Journal of Microbiological Methods.