Biopreparat-MKNAOMI

Anthrax: An introduction to the genetics of Bacillus anthracis

2010-04-25T08:59:00.000-07:00

The central problem in writing about this is that the subject quickly becomes highly complex, and the level of background knowledge needed to understand issues can start becoming exponential. Unfortunately, if there’s one subject where popular science writing has failed to inform the public, it’s in the area of biochemistry. For every hundred books on physics or biology, there might be – at most – one on biophysics, biochemistry and molecular biology. Hence, the following might seem overly basic or overly complex, depending on one’s background knowledge.

Perhaps the relative lack of popular science writing in this area is because most of the action goes on outside a normal scale – here, we are in a world bounded on one end by quantum mechanics (at the detailed level of enzyme action, say), on the other by classical physics (at the scale of cells), and whose character is necessarily defined by evolution and ecology. This is the world of the microbial cell, the oldest form of life on the planet.

Pathogenic bacteria represent but a tiny fraction of overall microbial diversity – and are a special subclass in that the completion of their life cycle requires a host of some kind – in the case of anthrax, a mammalian host. This is in contrast to their closest ecological relations, the harmless and even beneficial microbes that inhabit the digestive tracts of all animals, assisting with food digestion and nutrient uptake (while also meeting their own nutritional needs).

The basic functional elements of any bacterial cell can be divided into the genome, the proteome, and the overall cellular structure – cell walls, cell membranes, and various internal structures of varying complexity. The genome is the information database, the proteome consists of the tools and materials produced using information stored in the genome, and the result of the proteome in action is the overall cellular structure and activity. Technically, information about the genome is easier to come by then information about the proteome, due to the new ease of DNA sequencing. This is why the study of the proteome has lagged well behind the study of the genome.

Microbes (like us) are constantly using their cellular system to sense their local environment, and when that environment changes, signals are sent back to the genome (via the proteome) which lead to new rounds of gene expression, an altered proteome, and hence an altered cellular activity and/or structure. This is clearly seen in the lifecycle of Bacillus anthracis – as it transitions from a spore to a vegetative cell in a host and back to a spore again.

Plants, animals, fungi, bacteria – all operate on this fundamental level. Obviously, complex multicellular creatures have even more complicated genome-proteome relationships than do bacteria – but for pathogenic bacteria, which have to survive within multicellular creatures, they must adapt to that complexity in order to be successful as pathogens. As we shall see, this is also true for Bacillus anthracis. Hence, some limited discussion of the human immune system is also required to understand anthracis.

In this (not-for-profit, informational-only blog) I’ve relied on the excellent work done by the web site www.theprotein.lounge.com, in particular on graphics they produced.

Pictured here is the initial stage of inhalational anthrax infection – airborne spores are first inhaled into the lungs. The infectious dose is greatly dependent on the particle size – large clumps of spores are far less likely to make it down into the alveoli of the lungs, and are more likely to be trapped in the mucus lining the upper respiratory tract. On the other hand, individual isolated spores fall into the 1-5 micron size range and can easily penetrate the lungs. The spore material used in the 10/9 Daschle-Leahy anthrax letters (and possibly the 9/18 letters as well) was chemically treated in such a manner that the spores were prevented from clumping together – which greatly increased the infectious potential. This is no simple trick, but rather a highly sophisticated approach drawing on modern knowledge of nanotechnology.

One across the lung barrier, spores are collected by macrophages (white blood cells) in the lung lymph nodes. The bacterial spores germinate within these macrophages and begin to grow.

Like any other bacteria, most of Bacillus anthracis’ genetic information is encoded on a single circular chromosome (x million base pairs?). However, anthracis keeps the major virulence factors (proteins that play critical roles in infection) on two smaller plasmids, which are (like the chromosome) circular loops of double-stranded DNA. Every time anthracis replicates itself by cellular division, all three genetic elements are copied, and thus each daughter cell gets a full complement of genetic information – and this is now taking place within a macrophage in a lung lymph node.

After using the macrophage as their first stage incubator, the newly formed cells burst out and begin to spread through the blood stream to other lymph nodes and tissues. At this point, an immunized mammal is already mounting a full-scale immune response to deal with the invader, allowing it to wipe out the bacteria before they can move on to their next, deadly stage: toxic protein synthesis. In other words, it’s a war against time – and in the non-immunized mammal, that race is typically won by the bacteria – and that’s what happened to the five victims of the anthrax letter attacks.

From the blood stream the bacteria reach many major lymph nodes, and once there, they begin ramping up protein synthesis, using the genome as the informational template. The plasmid, pX01, contains genes coding for three proteins, named lethal factor, edema factor, and protective antigen (lef, cya, pag), as well as a regulatory gene/factor, AtxA. It is 174,000 DNA base pairs in length (and contains many other gene sequences besides these three). The second plasmid, pX02, contains genes coding for enzymes that produce a poly-D-glutamic acid capsule. This crosslinked-chemical capsule helps protect the bacteria from the scavenging action of certain white blood cells. We can now picture the invader, safely enclosed in a protective membrane coat (not a spore coat, though), while actively pumping deadly toxins into the bloodstream and lymph:

Pictured here is the binding of the first factor, protective antigen, to its cellular receptor, which of course plays some other role in human biology – in essence, as with viral infections, the receptor is hijacked by the toxin. It is only called “protective” because this is apparently the main protein recognized by an anthrax-vaccinated human immune system, and that in turn allows the immune system to launch a pre-emptive assault before the bacteria has a chance to establish itself in large numbers.

Once the protective antigen binds to the receptor, it is cleaved in two by furin enzymes that are naturally present, creating an active binding site on the receptor-bound residue. Since the receptors are mobile in the lipid membrane, drifting around, they eventually encounter one another, creating the heptamer (seven identical units bound in a specific arrangement) seen on the right.

The toxic proteins produced from these gene sequence operate together as a function unit in order to cross the protective cellular barrier. The seven-membered structure first binds the lethal factor and the edema factor, and then translocates them across the cell membrane into the interior of the host cell via the process of endocytosis – again, a normal cellular process that Bacillus anthracis has hijacked for its own purpose. The package has now been delivered to the cellular interior.

Pictured here is the tiny lipid bubble that contains the heptamer complex. This complex now releases the toxic proteins that it carried into the intracellular mileau.

These proteins have two major effects – one binds to ATP, the major intracellular energy carrier, hydrolyzing it all the way to cyclic AMP, which acts as a signal to the white blood cell host to stop ingesting bacteria. The other destroys intracellular proteins that play key roles in maintaining the cell cycle, leading to cellular suicide – again, a normal biological process that B. anthracis has manipulated to devastating effect. In short, these toxins disrupt essential cellular processes, triggering cell death and the release of cellular contents to the extracellular space (lymph/blood), where Bacillus anthracis cells – growing as filaments – absorb the nutrients, and continue their explosive replication and toxin production.

Eventually, the host animal dies and the nutrient supply is exhausted – at which point an entire new set of B. anthracis genes are activated, leading each vegetative cell to create an internal spore structure within which the genetic material is securely protected from damage. The entire corrupt mass of fluids, dead cells and spores then leaks out of the dead animal into the soil, where the spores will lie dormant, sometimes for decades, until another host comes along – typically a grass-eating ruminant of some kind or other.

Now that we’ve considered the physiology of anthrax (and a bit of the ecology), we can consider how one might use the genetics of B. anthracis in a forensic examination.

The first thing to note about the genetics of Bacillus anthracis is the remarkable genetic homogeneity among all known isolates – but what is an isolate? That’s actually a non-trivial question, particularly in relation to the new microbial forensics techniques. It’s been known for a long time now that the artificial media cultures that microbiologists use to collect their isolates actually select, evolutionarily speaking, from among the possible isolates.

Bacillus anthracis is not as finicky as many other microorganisms, however, and if anthracis bacteria are streaked across a solid medium (sheep blood agar), isolated colonies will grow up on the surface – as round little colonies all descended clonally from a single parent cell. However, different strains of anthracis are very hard to distinguish from one another. The most common bacterial genotyping method, analysis of the ribosomal backbone coding sequences (16S and 23S rDNA) are barely able to distinguish Bacillus anthracis from its relative, Bacillus cereus. Hence, a great deal of effort has gone into developing better genetic methods for distinguishing different strains.

Here, we'll cover the efforts made in the 1990s, which should give one enough background to understand the subject of the next post, the whole genome methodology applied later.

Initial efforts began with variable number tandem repeats – short series of sequences repeated in a series within a gene (Jackson et al. 1997) – and a different method known as amplified restriction length polymorphism (Keim et al. 1997). That’s vaguely similar to the human genetic fingerprinting methods used in modern forensics. (If only Sherlock Holmes could see us now... we’ve gone from identifying bloodstains by chemical means, to extracting the human fingerprint from those bloodstains.) However, this was still a very low-resolution method for distinguishing among strains, relative to today. For example, the Sterne, Ames and Vollum strains of anthrax were indistinguishable via the VNTR methods used even though Sterne lacks the pX02 plasmid. AFLP methods were more effective, even though B. anthracis isolate similarities were very high (97%).

These methods did allow researchers to place B. anthracis in relation to its closest relatives, Bacillus cereus, Bacillus mycoides, and Bacillus thuringiensis (some GMO crops controversially express thuringiensis insect toxins in their tissues – which could plausibly result in negative ecological and health effects, as some studies indicate). Hence, as of about 1999, the AFLP method was the state-of-the-art for distinguishing B. anthracis isolates (Keim et al. 1999).

Soon, however, the advent of whole-genome sequencing would lead to new and better possibilities. With B. anthracis, the pX01 plasmid (181,654 bp) from the Sterne strain was the first large-scale sequencing effort in this area. Suddenly, huge amounts of data began to roll in from these methods – and organizing and making sense of this data became the central challenge. This was also critical data for determining all the variable regions of the B. anthracis genome.

Keep in mind, that back in pre-whole genome era, the goal was mainly to trace naturally occurring outbreaks of anthrax back to their source regions in order to better understand the epidemiology of the disease – how it spread from host to host, in other words. If you consider the biology of anthrax, as described above, then the following might make sense:

“The high lethality of anthrax, with a concurrent massive and efficient multiplication over a few days, and the absence of chronic anthrax infections provide little opportunity for host immunological selection for altered types. When B. anthracis is not growing in a host organism, it is thought to be quiescent as spores and, hence, perhaps evolving at a reduced rate.” – Keim et al. 1997

Here, another key point emerges – when B. anthracis is grown on sheep-blood agar or similar media, it doesn’t really need its set of lethal proteins in order to survive – that work has already been done for it by the microbiologist. Over time or in large cultures, therefore, the rate of mutation may increase and those mutations may be tolerated to a greater extent then in the natural infectious cycle.

The first reported application of these genetic methods to biowarfare-related issues focused on the infamous Sverdlovsk anthrax outbreak in the Soviet Union. (Jackson et al. 1998) Preserved tissues from the Sverdlovsk victims were subjected to DNA extraction and polymerase chain reaction amplification. Their most remarkable finding was that each victim had been infected with multiple strains of anthrax. That would in turn indicate that the Sverdlovsk bioweapons facility had mixed isolates up during the production process, or was working with seed cultures that contained a wide variety of isolates.

This fact is worth keeping in mind – thirty years after Sverdlovsk, the issue of impure seed cultures has become a very critical one in the analysis of the 2001 anthrax letter attacks. The Sverdlovsk result was later verified by a detailed PCR study based on the pag gene, encoding the protective antigen protein. (Price et al. 1999)

This business of multiple strains infecting the same host is critically important in diseases such as swine flu, but it seems very rare with B. anthracis under natural conditions. The reason it matters is that multiple strains can swap their genetic material within a host (this applies to viruses like swine flu as well as bacterial pathogens) – which, incidentally, is one of the main epidemiological dangers involved with factory farming pigs, cows, chickens, etc.

What can we say in conclusion? By the late 1990s genetic methods for typing anthrax strains were gradually becoming more sophisticated, but they were seriously hampered by the low genetic diversity of the gene regions examined. The next major technological breakthrough - whole genome sequencing - would change all that.

The question then becomes, was that approach correctly applied in the anthrax investigation, or not? To answer that will require another long post, so let's leave it at that for now.

However, there is one other issue that some might be unaware of - namely, that anthrax was the first disease ever conclusively proven to arise from the action of a microbe. That was the famous discovery of Robert Koch, who was the first to cultivate the microbe in isolation, show that it infected mice and guinea pigs and sheep, and demonstrate that sporulation allowed it to survive extremes of heat and cold. Paul de Kruif described this in his book, "Microbe Hunters", published in 1926 - here is a short excerpt of some interest on sporulation:

"To himself Koch muttered guttural curses. "Other microbes have doubtless gotten into my hanging-drop," he grumbled, but when he looked very carefully he saw that wasn't true, but when he looked very carefully he saw that wasn't true, for the shiny little beads were inside the threads - the bacilli that made up the threads have turned into these beads! He dried this hanging-drop, and put it away carefully, for a month or so, and then as luck would have it, looked at it once more through his lens. The strange strings of beads were still there, shining as brightly as ever. Then an idea for an experiment got hold of him - he took a drop of pure fresh watery fluid from the eye of an ox. He placed it on the dried-up smear with its months-old bacilli that had turned into beads. His head swam with confused surprise as he looked, and watched the beads grow back into the ordinary bacilli, and then into long thin threads once more..."

This is of interest for several reasons - but I'd like to hear the FBI and Battelle microbiologists explain how the spores could end up coated with silica by any "natural" process - as the silica would have to be taken up into the microbial cell and incorporated into the spore as it was forming inside the vegetative cell - and there's zero evidence for any such process.

I also wonder what Koch would have to say about those who would use these spores as a weapon against other human beings...

A Review of the MSNBC Interview with Dr. Steven Hatfill

2010-04-18T10:01:00.000-07:00

MSNBC’s Today Show had Steven Hatfill on the other day for his first interview since he settled with the Department of Justice over persecution and harassment. While informative, the interview didn't cover many topics. Hence, I've transcribed the MSNBC interview and added some commentary - plus a personal note at the end.

The photo collage, I hope, explains what Dr. Hatfill went through and the levels of stress and intimidation he suffered as a result of the FBI's effort to respin the anthrax attacks as the work of a "lone wolf". From the top left clockwise, you have the Oct 9 Daschle letter, the five victims of the anthrax attacks (Joseph Curseen, Thomas Morris, Ottie Lungren, Robert Stevens, Kathy Nguyen), an autopsy photo of the effects of anthrax on the human brain (from Sverdlovsk), two images of the FBI media circus at Hatfill's home on June 25, 2002.

From the bottom right, we have images from the August 1 2002 press conference labeling Hatfill a "person of interest." First is FBI Director Mueller and DHS Secretary Chertoff, a more recent picture of Mueller being grilled before Congress, Ashcroft leading the press conference, and the 2002-2006 FBI lead on the case, Richard Lambert (now at Oak Ridge TN). The two people above Lambert were also at the press conference, not sure who they are, would very much like to know. Finally, we have the two of the reporters who went after Hatfill based on "secret sources"- Nicolas Kristoff of the New York Times, and Toni Locy of USA Today.

The MSNBC interview did not touch on the issue of media culpability in the harassment of Dr. Hatfill, however. This is a curious omission by MSNBC, as Hatfill also brought cases against these and other reporters. The federal judge in those cases, Reggie B. Walton, said in 2008 that "There's not a scintilla of evidence to suggest Dr. Hatfill had anything to do with it." In the end, the reporters were not required to reveal their sources on their Hatfill allegations - and no federal official has ever been held accountable for the Hatfill witch hunt.

The MSNBC interview also did not mention the fact that the FBI team that went after Hatfill was not the original FBI team assigned to the case - that team was dismissed early in 2002, and the head of the investigation was forced into early retirement. Immediately afterwards, the new FBI team headed by Lambert apparently dropped all investigations into Battelle Memorial Institute and Dugway Proving Ground, and instead focused on harassing Dr. Hatfill - with, as evidence has shown, no real cause whatsoever.

Not only that, the MSNBC report glosses over the multi-faceted evidence that also exonerates the FBI's final suspect, Dr. Bruce Ivins - namely, the fact that anthrax spores from Detrick were shipped to other locations, including Dugway Proving Ground and Battelle Memorial Institute, and more importantly, that the high-tech spore processing method used to create the powdered spores was not available to anyone at Fort Detrick. The technical details clearly show that neither Ivins nor anyone else at Detrick had the means to create this material - and certainly, neither did Saddam Hussein nor Al Qaeda. This only leaves the biological threat assessment program run by the DIA, the CIA and Battelle Memorial Institute in the late 1990s (into 2001?) as the only plausible source of the attacks. Regardless, Here is the transcript of the interview:

-begin segment-

MATT LAUER: We're back now at 7:40 with the man wrongly pursued by the FBI in connection with the anthrax attacks that terrified this nation in the weeks and months after 9/11. Several members of Congress and media outlets including NBC received tainted letters. Five people died, seventeen others were sickened.

Now, for the first time, Dr. Steven Hatfill is speaking out about what he was forced to endure. Dr Stephen Hatfill had worked as a scientist at the United States Army Medical Research Institute of Infectious Diseases at Fort Detrick Maryland, which holds stores of anthrax.

When the FBI started investigating the deadly attacks, it looked at more than a thousand people. He wasn't surprised to be one of them.

MATT LAUER: Did you volunteer to take the polygraph, or did they ask you to?

STEVEN HATFILL: They asked if I would - I said sure.

MATT LAUER: You had no - that would be a moment that would get my blood going a little bit, or at least my heartbeat going.

STEVEN HATFILL: No, I think this is just standard...

MATT LAUER: No reservations about it whatsoever?

STEVEN HATFILL: No, why?

MATT LAUER: What kind of questions did they ask you - any of them jump out at you?

STEVEN HATFILL: Sort of mundane things, really.

MATT LAUER: They didn't come right out and say, "Did you kill people with anthrax?"

STEVEN HATFILL: No, actually I got upset with them - with all these mundane, you know, "Did you ever cheat on a test?" type things - why don't you just ask me? And they said, yeah, okay, and that was the end of it.

Actually, that's normal polygraph technique - they have to establish a baseline. However, any trained person can beat a polygraph test, and they are also inadmissible in courts.

But unlike the others, he became the focus of the investigation after two outside sources said he fit the profile of the anthrax killer. For the next five years, his life would be turned upside down.

DAVID FREED [Atlantic Monthly] : Imagine being the fox chased by the hounds, and I think you begin to get an idea of what it was like for Steven Hatfill.

On June 25th 2002, Hatfill's name became very public. The FBI said they wanted to take some tests at his home. Thinking he had done nothing wrong and had nothing to fear, he agreed.

STEVEN HATFILL: Well [said the FBI], we'd like to do some swabs. It will be very discreet, quiet...

MATT LAUER: You signed the consent form?

STEVEN HATFILL: Sure.

MATT LAUER: You got home later that day?

STEVEN HATFILL: No, I walked out in the parking lot and there were already news cameras - filming me walking to the car - and they were taking me back to the apartment, and the whole news thing was out there, and the helicopters and all this.

MATT LAUER: Didn't you say to one of the FBI agents, "How did this get leaked?" and then that person said to you, "Someone must have leaked this to the media?"

STEVEN HATFILL: I can't remember the exact comment but I said "You know, what's going on here?" and [the FBI replied] "Well, it got leaked." What a performance.

MATT LAUER: When you say, "What a performance" they were lying to you?

STEVEN HATFILL: I'm watching this on television and there are guys in HazMat suits. This is - what a show. And apparently that's what it was - a show.

Note here that the goal of this "show" was to distract attention away from the FBI's previous work, which had pointed towards the involvement of private contractors linked to the U.S. biological threat assessment program. As noted, the first thing FBI agent Richard Lambert did when he took over the case was to drop all such investigations, and instead focus on Hatfill and Fort Detrick.

A show that was watched by Americans on live TV - with no concrete evidence. Steven Hatfill was now public enemy number one.

MATT LAUER: Can you try to explain to me what happens over the next several years? You are now under surveillance constantly by the FBI and under the scrutiny of the media.

STEVEN HATFILL: It just never seemed to end.

MATT LAUER: When they say they're following you everywhere you go, is it like in a bad movie where they're in a van peeking out of a side window - or are they just obviously...

STEVEN HATFILL: No, they're right behind you.

MATT LAUER: Making it clear to you, "We are watching every move you make."

STEVEN HATFILL: It just became a way to harass. You would go into a restaurant and they'd sit down on either side of you.

MATT LAUER: How did you react to that?

STEVEN HATFILL: You keep thinking, well this will end. Somebody will, you know, come to their senses.

In August 2002, Attorney General John Ashcroft took the unprecedented step of naming Hatfill a "person of interest" in the anthrax killings.

As the news conference images indicate, by that time the Amerithrax 1 / Amerithrax 2 FBI team was out of the picture, and the new team had taken over. Days later, the first real break in the anthrax case appeared - the identification of the Princeton mail box where the spores were sent from.

ASHCROFT: "He's a person that - that the FBI's been interested in..."

The scrutiny cost Hatfill his job and every aspect of his privacy.

MATT LAUER: You can't go anywhere without them following you. You can't have a candid phone conversation because you have to assume they are listening to every word you say...

STEVEN HATFILL: I think that by the time the FBI come and see you, I think your phone's already been done. Which - you know - if the law says they can do that, then fine.

MATT LAUER: How long did this disaster last for you?

STEVEN HATFILL: It lasts forever.

MATT LAUER: What about friends? When the spotlight was the brightest on you, did you lose friends, were people afraid to associate with you?

STEVEN HATFILL: No, what upsets me is that I don't know of any law that permits the FBI to go by your closest friends and say, "You're not to associate with Dr. Hatfill. You're not to see him, or talk to him - you know, what they're trying to do is socially isolate you as part of the stress.

MATT LAUER: Trying to make you snap?

STEVEN HATFILL: Yeah - essentially.

This was the exact technique that the third FBI team used to harass Bruce Ivins and his family, although they never named him a "person of interest" until after his alleged suicide. Richard Lambert had been removed from the case in 2006, after failing to drive Hatfill over the edge.

In 2007, the FBI was able to track the deadly strain of anthrax used in the attacks to a supply kept by Dr. Bruce Ivins, who worked at the same Army lab as Dr. Hatfill. As investigators closed in, Ivins took his own life. His family maintains his innocence. "Case closed" says the FBI.

MATT LAUER: Given what you've told me, Steven, about your now complete lack of confidence in the Justice Department and the investigation they conducted surrounding you - do you think they got it right now?

STEVEN HATFILL: I have no way - I haven't seen the data, obviously the FBI haven't felt it necessary to share anything with me. I have talked to some senior scientists that I trust and respect, I have to take their opinion that, uh, yeah. Other than that I, I can't say.

Not very convincing at all - why no discussion of the chemical additives, the high purity, and the fact that the "unique genetic fingerprint" from the Detrick Ames spore repository was found at several other institutions that had received spores from Detrick for use in testing the U.S. military's anthrax vaccine in research animals? As far as I can determine, all such "Ames anthrax vaccine challenges" were done at Battelle Memorial Institute in West Jefferson Ohio - or possibly at Dugway Proving Ground in Utah.

MATT LAUER: What's your feeling about the way the Justice Department treated you?

STEVEN HATFILL: I learned a couple things. The government can do to you whatever they want. They can break the laws - federal laws - as they see fit - and you can't turn laws on and off... and the privacy act laws were put in place specifically to stop what happened to me. I used to be somebody who trusted the government, and now I really don't trust anything.

MATT LAUER: You settled with the Justice Department - a legal settlement - for a lot of money. This may sound trivial to you, but did they ever apologize?

STEVEN HATFILL: Somebody phone me up and say, "We're sorry?"

MATT LAUER: "Yeah - we screwed up?"

STEVEN HATFILL: No, they don't do that.

MATT LAUER: The falsely accused have often asked the MATT LAUER, where do I go to get my reputation back? Where does Steven Hatfill go?

STEVEN HATFILL: I was fortunate that about halfway through this mess, I had a band of brothers, and they never left my side. I still work with them to this day. Patriots, soldiers, highly decorated men. And that gives you the strength, just to be in their company, to carry on.

MATT LAUER: Sounds to me like you wouldn't have made it without them.

STEVEN HATFILL: No, I doubt it.

While he was never formally cleared after being labeled a person of interest, the government's multimillion-dollar settlement seems to speak for itself. You can read more about Hatfill's story in the May issue of the Atlantic Monthly.

MEREDITH VIEIRA: So chilling, how easily that can happen.

MATT LAUER: He said he used to drive his car, they would be right behind him and just follow him everywhere, and they would even pull him over for routine traffic stops, just give him tickets for ridiculous things. His life was really turned upside down.

MEREDITH VIEIRA: Well you can see it still is - when he talks about it, there's the pain in his face...

MATT LAUER: It's true.

-end segment-

Hatfill was hardly the only person subjected to surveillance and harassment over this case. Soon after I began looking into the details of the anthrax attacks and possible government malfeasance related to the biological threat assessment program, I found quite a few odd characters tailing my every move. My approach, however, was to attempt to engage these characters in conversations, and to follow them around as well, surreptitiously eavesdrop on their cell phone conversations and so on. On one occasion I listened while a co-worker at a job instructed his associate on the phone to get all of my email records (he thought I was doing something else, but I had snuck back to the office and heard the whole thing). This same character later offered to sell me cocaine & LSD, and attempted to engage me in a discussion about the feasibility of assassinating President Bush - I kid you not. At the same time, another old acquaintance began begging me to find someone who had LSD for sale! Needless to say, I put two and two together and immediately quit that job, and cut off contact with those acquaintances.

I haven't bothered to conduct a further investigation into such matters, but I did take several further protective steps - I cut off all contact with old friends, I severely limited all family contacts, and began more direct engagement with various political, law enforcement and military agencies. This was not the expected response, I imagine - but believe me, it was quite effective in getting these creeps off my back. It has changed my life a bit, however... but that's something I can deal with.

Of course, it's no comparison to the kind of public drubbing Hatfill was given - but I know how he feels. I more or less expected the harassment, once I decided I had to warn people about the government's biowarfare programs, and was in some sense prepared for it. I didn't expect people to try and set me up with criminal charges, however - that was a surprise.

Certain individuals, who I will not name, also played their part by tipping me off to what was going on - and to them, I owe a great deal of confidential thanks. There are plenty of decent people in this country, I can assure you.

In all fairness to these snoops, I am a recipient of a National Science Foundation Graduate Student Fellowship in microbiology, and as a result of my investigations I probably know as much about the manufacture of biological weapons as anyone who hasn't seen all the classified documentation. For example, I'm quite certain that if given the equipment and materials I requested, I could produce biological weapons identical to those in the Daschle letter. That would however require a full-scale BSL3/4 laboratory - something better than anything Saddam Hussein could come up with, millions of dollars in highly specialized equipment, and a lot of time and money. That's what it would take to produce the material employed in these attacks.

Obviously, this creates a certain problem - how much should I tell people? In this regard, I follow in the footsteps of the Ted Taylor, the nuclear weapons scientist who set out to warn the world about diversion of nuclear materials for homemade bombs - he only used information that was already available in the public domain. This seems to me to be the responsible approach.

However, if our scientific institutions are going to support this kind of thing - if our government is going to refuse to sign on to an updated Biological Warfare Convention - and if the biological threat assessment programs are going to escape scrutiny for their role in these attacks - well, that's a problem.

It's a lot bigger problem than many people suspect, I'm afraid. These attacks were conducted with what is essentially "old-school" technology - meaning no genetic manipulation of the strain employed, the nanotech spore processing system aside. If antibiotic resistance genes had been incorporated, for example, far more people would have died - and if they had put smallpox in with the anthrax? We'd have had a major epidemic on our hands.

There's only one conclusion here, however: The U.S. government cooked up illegal biological weapons in defiance of the Biological Warfare treaties we signed on to, and then, somehow, those biological weapons were turned against the American public in a deliberate (and successful) effort to create fear and terror.

That's not acceptable, and neither is the FBI's coverup of this astonishingly flagrant criminal behavior. It is even looking like our National Academy of Sciences is participating in this - and that's even more outrageous. The FBI can at least claim scientific ignorance - but the National Academy?

I'd have expected this behavior from the Soviet Academy of Sciences, who worked overtime to cover up the Sverdlovsk anthrax incident in the old Soviet Union - but is that what we've become?

Introduction to the National Academy of Sciences Anthrax Letter Committee

2010-04-16T19:36:00.000-07:00

I got around to transcribing the introductory remarks from the NAS Committee, which is working under contract with the FBI to review the validity of their scientific methods, the application of those methods, and their conclusions. All the members of the committee can be seen in the photo.

However, the NAS Committee has seen fit to close all sessions to the public since the September 24-25 meeting, even to the extent of not even naming the witnesses that they've called. Why?

They claim that they are concerned about the release of classified material... welcome to the secret trials of Dr. Bruce Ivins. This is not at all surprising, considering the history of this case. This flies in the face of claims of "openness" and "transparency" made by the Committee Chair, Alice P. Gast.

The affiliations of the committee members makes for some interesting reading, too:

Here is the official list from the NAS web site - although they left out quite a bit of material, which I added in:

Alice Gast (Chair), President, Lehigh University

Gast is a past director of research at MIT during the period when biowarfare research funding was exploding. MIT has a very large "Biodefense Systems Group."

David Relman (Vice Chair), Professor of Medicine and Microbiology and Immunology, Stanford School of Medicine, Stanford University

Stanford University is a member of the Pacific Southwest Regional Center of Excellence For Biodefense and Emerging Infectious Disease research.

Arturo Casadevall, Chair, Department of Microbiology and Immunology; Leo and Julia Forchheimer Professor of Microbiology and Immunology; Professor, Department of Medicine, Albert Einstein College of Medicine

-Casadevall is also the Deputy Director of the Northeast Center of Excellence For Biodefense and Emerging Infectious Disease research. - and why does the NAS not include this title in their summary?

Nancy Connell, Professor of Medicine, University of Medicine and Dentistry of New Jersey

- The University of Medicine and Dentistry of New Jersey is a member institution of the Northeast Biodefense Center. These "Centers of Excellence" were part of the massive expansion in biowarfare-related research funding that the anthrax attacks engendered.

Thomas Inglesby, Chief Operating Officer and Deputy Director of the Center for Biosecurity, University of Pittsburgh Medical Center; Associate Professor of Medicine and Public Health, University of Pittsburgh Schools of Medicine and Public Health

As the director of this institution, he must be aware that there are charges that these attacks were intended to alarm the public and create a causus belli for a massively expanded biowarfare research program - a program that funds his institution. Tagging "lone wolf" Bruce Ivins as the culprit would go some way towards protecting this funding train. Not only that, the "biowarfare scenarios" he has promoted avoid any discussion of actual the likelihood of a terrorist organization acquiring such weapons - they're just designed to raise fear and increase funding.

Murray Johnston, Professor of Chemistry, Department of Chemistry and Biochemistry, University of Delaware

I see no direct conflicts of interest here - but this guy is an expert in mass spectrometry, and should have asked more questions about why no stable isotope analysis were conducted.

Karen Kafadar, James H. Rudy Professor of Statistics and Physics, Indiana University

Indiana University is a member of the Great Lakes Regional Center of Excellence For Biodefense and Emerging Infectious Disease research.

Richard Lenski, Hannah Distinguished Professor of Microbial Ecology, Department of Microbiology and Molecular Genetics, Michigan State University

Michigan State University is a member of the Great Lakes Regional Center of Excellence For Biodefense and Emerging Infectious Disease research.

Richard Losick, Harvard College Professor; Maria Moors Cabot Professor of Biology; Howard Hughes Medical Institute Professor in the Faculty of Arts & Sciences, Harvard University

Harvard University is a member of the New England Regional Center of Excellence For Biodefense and Emerging Infectious Disease research.

Alice Mignerey, Professor, Department of Chemistry and Biochemistry, University of Maryland

The University of Maryland has extensive links to the Middle Atlantic Regional Center of Excellence For Biodefense and Emerging Infectious Disease research. It's so excellent, dude...

David Popham, Professor, Department of Biological Sciences, Virginia Tech

Virginia Tech is in the institute that hosts the Middle Atlantic Regional Center of Excellence For Biodefense and Emerging Infectious Disease website.

Jed Rakoff, Judge, U.S. District Court for the Southern District of New York

Why put a judge with zero scientific background (other than baseball) on a scientific review panel, other than as a PR stunt?

Robert Shaler, Professor, Biochemistry and Molecular Biology Department; Director, Forensic Science Program, Pennsylvania State University

Pennsylvania State University is a member of the Middle Atlantic Regional Center of Excellence For Biodefense and Emerging Infectious Disease Research.

Elizabeth A. Thompson, Professor, Department of Statistics, University of Washington

The Unversity of Washington is the lead institution in the Pacific Northwest Regional Center of Excellence For Biodefense and Emerging Infectious Disease Research.

Kasthuri Venkateswaran, Senior Research Scientist, Jet Propulsion Laboratory, California Institute of Technology

Caltech is associated with the University of California, Irvine's massive grant for the establishment of the Pacific-Southwest Center of Excellence For Biodefense and Emerging Infectious Disease Research - the "largest grant in UCI history", delivered in 2005.

David Walt, Robinson Professor of Chemistry and Professor of Biomedical Engineering; Howard Hughes Medical Institute Professor, Tufts University

Tufts University is a member of the Great Lakes Regional Center of Excellence For Biodefense and Emerging Infectious Disease Research, and is doing work with botulinum toxin among other things.

The audio begins fairly abruptly, but the speaker is Alice Gast, the Chair:

-begin segment-

....Chair of the National Academies Committee on the scientific approaches used during the FBI's investigation of the 2001 Bacillus anthracis mailings and along with committee vice-chair Dr. David Relman, I'd like to welcome you to our second meeting.

The National Research Council has agreed to evaluate the scientific foundation of the specific techniques used by the FBI during its investigation to determine whether these techniques met appropriate standards for scientific reliability and for use in forensic validation, and whether the FBI reached appropriate scientific conclusions from its use of these techniques.

In instances where novel scientific methods were developed for purposes of the FBI investigation itself, the committee will pay particular attention to whether these methods were appropriately validated. The committee will review a range of scientific information including study plans, results, analysis, and reports documenting findings in connection with the 2001 Bacillus anthracis mailings.

In assessing this body of information, the committee will limit its inquiry to the scientific approaches, methodologies, and scientific techniques used during the investigation. I would also like to make it clear and remind you that what the committee will not do - the committee will only review and assess the scientific information related to the investigation - the committee is not charged, nor is it constituted, to address other aspects of the investigation not related to science.

Most importantly, the committee will not address the identity of the perpetrator of the anthrax mailings. The purpose of this meeting today is for the committee to continue its information gathering efforts - the committee will conduct additional information gathering over the next several months and will deliberate thoroughly before writing its draft report. Moreover, once the draft report is written it will go through a rigorous external review by experts who are anonymous to the committee, and the committee will then respond to this review with appropriate revisions that satisfy the NRC's report review committee, and the chair of the NRC, before the report is issued as a National Academies document.

The report will describe the committee's work, finding and conclusions from the review and assessment of the information provided by the FBI, and other experts appearing before the committee. The National Research Council is committed to an open scientific process, and as per NRC's contract with the FBI, is committed to producing a final report that will be available to the public in its entirety.

Now, a little aside here - she says that "The National Research Council is committed to an open scientific process..." This must be why the NRC closed all sessions after this one to the public, even to the extent of refusing to publish a list of witnesses?

It is important to note that the committee with no preconceived notions and no premature conclusions. It is this committee's task to gather and analyze the information that will allow it to formulate findings and conclusions to respond to its charge. The fact that committee members may make comments or ask probing questions should not be interpreted by anyone as anyone taking a position on anything at this point, nor should remarks made by committee members be construed to be representing the position of the NRC.

Committee members, all of us being academics or scientists or judges, typically ask probing questions, some more probing than others, in these information gathering sessions that may or may not be indicative of their personal views. I want to note that this is an open on the record session - interested individuals and members of the press may be attending as observers, I may ask them if they'd like to introduce themselves, so we'll know who is here.

Again, this is quite strange - since when are spectators at a public proceeding asked to identify themselves? There is also a serious lack of gravitas in the tone of these proceedings, incidentally.

However, this is not a press conference, and neither the committee nor the speakers will be entertaining questions from the floor. Reporters who would like to talk to the committee are kindly asked to touch base with us at the end of this session.

As noted in previous transcripts, the questions asked of the speakers were hardly probing - and the most important questions went unanswered.

This afternoon we will hear from a panel of scientists:

Dr. Rita Colwell, from University of [Maryland] College Park and John Hopkins University

Dr. Stephen Schuster, University of Madison in Dentistry of New Jersey

Dr. Paul Keim, Northern Arizona University

Dr. Patricia Worsham, United States Army Research Institute for Infectious Diseases

They will present approaches, findings and results of the scientific analysis used in the investigation. One final point, if anyone would like to submit information to the committee for its consideration, you may provide that information via the National Academy's current project systems website, at www.nationalacademies.org

So thank you very much for coming, I'd like to first ask my committee members to introduce themselves. [see above]

Well, thank you. I've asked Rita to start us out with her overview of the scientific investigations, to change the order a bit from the agenda. Thank You.

-end segment-

By the way, when you think about biological warfare research, you should really think about this picture of dogs, not the one at the top of the page:

This is what is going on inside the gigantic new biodefense complex that has been created as a result of the anthrax attacks - a $5 billion a year cash cow for a handful of well-connected private firms, as well as a new source of funding for academic departments with few scruples.

In case you think this is some kind of exaggeration, allow me to explain with a few quotes from "The Biology of Doom: The History of America's Secret Germ Warfare Project" by Ed Regis:

1) "The 8-Ball [testing chamber] ran through a sizeable population of Camp Detrick test animals. The anthrax trials alone consumed more than 2,000 rhesus monkeys."

This was not done to save lives, either - not by a long shot. The same goes for the modern biowarfare game.

2) "At the time of the first operational test of the M33/Brucella munition, therefore, the total indoor and outdoor population of this enemy city on the desert plain would be 3,230 guinea pigs."

3) "By the end of the M33 operational suitability trials, a total of 11,628 guinea pigs had been attacked by Brucella bombs at Dugway Proving Ground within the space of two months. As an Army Chemical Corps General remarked years later, "Now we know what to do if we ever go to war against guinea pigs."

4) "Between August 1943 and December 1945, seventeen different species of animals had been utilized at Detrick. The final tally was 598,604 white mice, 32,339 guinea pigs, 16,178 rats, 5,222 rabbits, 4,578 hamsters, 399 cotton rats, 225 frogs, 166 monkeys, 98 brown mice, 75 Wistar rats, 48 canaries, 34 dogs, 30 sheep, 25 ferrets, 11 cats, 5 pigs and 2 roosters..."

That was just when they were getting started, too. There is no "polio vaccine" excuse for this research - all they were trying to do was to quantify how toxic their new bioweapons were.

The complete transcript of Dr. Joseph Michael's testimony before the NAS, Sept 25, 2009

2010-03-11T19:59:00.001-08:00

From the genetic analysis, introduced by Keim & Worsham, we move on to the physical analysis of the attack materials as found. One major difference is that while testimony from the early days of the genetic analysis is included, this is not so for the physical analysis. The original analytical work done at USAMRIID and AFIP by Geisbert & Mullick among others is, for whatever reason, apparently not part of the NAS Committee Review.

One odd thing about Sandia is that prior to this, they had no experience with microbial samples - they mainly worked with semiconductors, nuclear reactor materials, etc. Why would the FBI ask a novice in this area to analyze samples that were mainly prepared elsewhere - and from what source? What happened to those samples between the letter and their arrival at Sandia, and why isn't that part of the NAS inquiry?

Dr. Michael also states in his testimony that there were no weaponized Ames samples to look at from Dugway, and that "no one was weaponizing Ames" - in marked contrast to reports that Dugway was doing precisely that as part of their biological threat assessment work in the late 1990s. It's logical that they would have used Ames, if the resultant powder was to be tested on animals for 'efficacy' (aka virulence), so they could compare it to previous benchmarks. Ames is the standard anthrax vaccine challenge strain, used to test vaccinated animals for immunity in drug trials. (It replaced the Vollum strain in the 1980s - Vollum ended up in Saddam Hussein's hands c. 1985)

For those and other reasons, the following testimony is less than convincing. The most obvious issue is failure to do adequate method validation with standards, particularly with ion beam sample preparation, which could easily have blown off a large percentage of the silica in the samples - especially near the surface.

An equally confounding issue is the sample preparation, which was largely done elsewhere - and why did they get the Leahy powder in the form of glassy clumps (see below)? They also had a "weaponized sample" for comparison - but the means of weaponization is not reported. You would think, for a new forensic method, they'd have a lot more standards - another oddity.

This also raises another question: what happened to all the material in the letters? Were archival samples preserved under lock and key in secure locations, or is it "all gone"?

The media analysis of this has been almost nonexistent. Analysis is out, kowtowing to the grand poo-bahs is in - like this remarkable quote from the New York Times editorial, written by an apparently anonymous author, Feb 27, 2010:

The F.B.I.’s conclusion rests in large part on pioneering laboratory techniques.... The National Academy of Sciences will complete a review of that lab work in coming months. But the techniques were devised with the aid of some of the country’s most sophisticated scientists, so they are presumably reliable.

Appeal to authority is a logical fallacy when it is the mainstay of your argument. Why? Here's a good discussion:

If there is a significant amount of legitimate dispute among the experts within a subject, then it will fallacious to make an Appeal to Authority using the disputing experts. This is because for almost any claim being made and "supported" by one expert there will be a counterclaim that is made and "supported" by another expert. In such cases an Appeal to Authority would tend to be futile. In such cases, the dispute has to be settled by consideration of the actual issues under dispute. Since either side in such a dispute can invoke experts, the dispute cannot be rationally settled by Appeals to Authority.

With that in mind, read on.

-begin transcript-

Chair: Just to remind you that this is an open session on the record, and we do have members of the public and the press with us, and I would like to remind them that the committee's charge is focused on the scientific methods and approaches used during the FBI's investigation and I would also like to remind everyone that they should not infer any opinions or preconceived ideas based on the questions or nature of the discussion coming from committee members, and we're very pleased to welcome our two outside speakers this morning.

Dr. Joseph Michaels from Sandia National Laboratories will be speaking first.

Testimony begins:

So today I'd like to tell you about some of the work we did in support of the FBI in this case. I'd like to acknowledge my colleague Paul -inaudible- he did much of the transmission electron microscopy work in our laboratory... what button do I push here to advance my slides?

Okay - I'll talk louder - very good. Just by way of introduction, what I'll be talking about - first of all, a few of the tools that we use for elemental microanalysis, in our laboratory, in this study. A few words on spectral imaging, because it's a new concept for a lot of people, and how we do it in the laboratory. Then I'll start talking about the attack materials, the Leahy and the New York Post materials with our scanning electron microscope, then I'll talk about Leahy, New York Post, and Daschle with STEM and time-of-flight SIMS, and then I'll answer the question of whether the powders are unique with respect to elemental signature.

- It's unclear what is meant by "unique with respect to elemental signature." The content of silicon is one issue - the presence of trace elements (tiny amounts of iron, tin, etc.) is another - and the isotopic composition of the material as a whole (carbon, oxygen and nitrogen isotopes in particular) is a third. No isotopic composition work was done, however - or at least, no such efforts were ever reported. Nevertheless, the best hope for a "unique fingerprint" is from the isotopic ratios - so why wasn't it done?

First of all, a little thing to mention on signature statistics. We really don't have much to say about variability within the bulk material. Our samples came - the powders that we got were on the orders of milligrams, so it's a small part of the entire amount that was mailed, so really within the bulk material I can't say much. Did we get a representative sample? I'm hoping we did.

More to the point, did they get samples of the powder in their native state, or had they been autoclaved first?

We can talk about variability between fields of view in our microscopes, and then we talk about the variability of signals from individual spores. So I'll be talking about these two variables, in general.

Just a quick review, if nobody has ever seen a scanning electron microscope or a transmission electron microscope here's our SEM in our laboratory, we use it as a test bed for various detector systems, this is the SEM, this is a new type of x-ray detector that we're developing, this is another x-ray detector over here, another x-ray detector over there, so we get lots of signals off this microscope. And over here is our STEM, it's a scanning transmission electron microscope, and I'll tell you a little bit about that in a minute. So, just by way of review again, a STEM or scanning transmission electron microscope, currently we're running about six nanometer image resolution on a modern piece of equipment. We can do microanalysis, that means we can determine elemental signatures down to about the one micron level, we can detect elements down to beryllium, we can do some diffraction work, and really the nice thing about an SEM is you can take whatever you want to look at, sprinkle it on a stub, and image it. The downside to the SEM is of course we can focus our probe to a nice small spot, but really when it interacts, these high energy electrons, with sample, gives us a resolution of about a micron or so in elemental.

Keep in mind that a high resolution image of an artifact produced during the sample prep process is not of much value.

The STEM, on the other hand, the scanning transmission electron microscope, it's a high resolution tool, we can image down to about 0.2 nanometeres with it, in terms of microanalysis we can expect to see about one-two nanometer spectral resolution - so that means if we have a feature that's one to two nanometers in size, we can analyze it. Again, the elements limited to atomic numbers greater than beryllium, difraction, and in this case we need some sample prep, so we need electron-transparent samples.

Generally in this study we're going to make use of the characteristic x-rays generated by the sample - by the electrons when they interact with the sample. So how did we approach this problem? One of the things we've been working on out at Sandia for many years is, how do we characterize materials. It's very important to the nuclear weapons complex, in terms of aging and reliability of the, uh, systems we build. And one of the areas we worked in is how do we comprehensively survey compositions and chemistries in large sample areas?

This is the same basic method used at the Armed Forces Institute of Pathology - and that study showed a very large silicon peak, which fits with the reported "cream or bone" color of the anthrax letter spores. (Anthrax spores are dark brown in their native state).

So, in the old days, I'll say, here's just a picture of some spores - if I wanted to do a compositional analysis, I put a beam here, well that's sort of interesting, I'd move my beam over here, and maybe that's another interesting spot, and then you put it here. So we're doing things very subjectively. We're putting the beam where we think interesting features appear. Now that computers have caught up, become faster, larger capacity - what we need are two-dimensional distributions of chemical phases. And that's why we came up with this concept of chemical component images. And what we do is we make a spectrum image of our sample, and the way we do that is we take our electron beam and we scan it pixel by pixel over our sample. In the old days we'd monitor one chemical signal at a time, in the modern days now we collect the entire x-ray spectrum from that sample from every pixel, so we end up with this nice data cube, where we have spatial dimensions in the x and y, and in depth we have our energy spectrum. So, that gives us an x-ray, a data cube. And so we can go back now and interrogate any pixel we like for any sort of elemental signatures within that data.

The bad thing about this is we end up with tens of millions of pieces of data out of this, and going through all this by hand, it becomes quite tedious. So we began working on statistical tools to analyze these data, these are talked about in these two patents here, notice they were filed June 1st of 2001, and then there's a number of papers where we use this to analyze these various materials of interest to us at Sandia.

So what we do is, here's our data cube, and we use this statistical tool, and what it comes up with in the end is the component images that make up that sample. So, it makes the associations for us, and just to give you an example here, this isn't from the case, this is just a test sample we did, we took a variety of materials like alumina, some iron-cobalt nanoparticles, and some other materials, mixed them all together, and said can we tell them apart?

So what you see here is, the red is the carbon support that we put it on, green is the alumina particles, blue is the iron cobalt, and the cyan is an outlier, it's one we didn't expect to see in there, but it's a calcium sulfur silicate type of material. So we can use this tool to scan the whole area, we don't have to subjectively put our beam here, there and everywhere to decide what we're going to see.

Now, the following section points to a major flaw in their methodology:

A little bit about sample prep, for these tools. The blue were steps either performed at USAMRIID or NBFAC. The green are what we did at Sandia. We're not equipped to handle live anthracis, so everything that came to our lab had to be irradiated or fixed or killed in some manner. So, those steps were done at USAMRIID. For the scanning electron microscope we just take the dry powder, we access the sample, dust it onto a grid or a stub support in a disposable glove bag, and then we can image those directly, either using uncoated materials in the variable pressure SEM or doing some conductive coating if needed to be. We'll talk about the rest later.

"Irradiated or fixed or killed in some manner" is hardly definite. What precisely was done to each sample, and why didn't they compare the effects of autoclaving (which is what the Battelle scientists did with their samples on Oct 17 or 18, 2001) versus irradiation (which is what the USAMRIID scientists did - it's a far less destructive method). Indeed, one could wonder whether Battelle's autoclaving of the samples was a deliberate effort to destroy evidence...

So, what do weaponized materials look like, or materials treated for flow improvement? What we have on the - over here is an SEM image of, I think this is Bt, treated with silica nanoparticles, and you can see this fluffy sort of appearance you can sort of get an idea that there might be a spore there, and over here, and they're coated with this very fine coating of silica. When we look at that in our EDS, our energy dispersive spectrometer, you can see here that we see carbon, oxygen and a huge silicon peak, and just a little calcium associated with the spore bodies themselves. And really, when you coat these particles with silica, it's pretty obvious - in the x-ray spectrum. If we do a spectral image of that, so we scan at fairly low mag, you can see this is a 100 micron bar, these are clumps of spores, not individuals. You can see the various components that come up, you can see the silicon and oxygen, associated with the clumps of spores here, there are other things in here, you can see some magnesium phosphorous type material, and of course some calcium and phosphorous as well - so we see this distribution of elements, but we're not getting any idea what's spatially located inside individual spores- so just sort of an overall look at the data.

The above points towards another serious flaw - how was their "weaponized" material prepared? Was it prepared at Dugway, for example? Did they compare different kinds of weaponization methods, using surrogates? Did they examine what happened to such material after being autoclaved or carved up with their ion beam?

In terms of the attack materials, we had powders in my labs from both Leahy and the New York Post - this is what they looked like. Over here is the low-mag SEM of the New York Post material, and over here is the low-mag SEM of the Leahy material. And you notice in the New York Post are these fairly large chunks - this is a 100 micron bar, so these chunks are quite large - and in fact, they're very hard little pieces of material. In fact, we like to break them up to see more of the internal structure of these clumps. And you can take a pair of tweezers and clamp them on there and kind of click them, to make these particles break apart - so they're very hard, uh, little particles.

This flies in the face of every eyewitness report on the material - which dispersed "like a cloud of smoke" or "like steam from a teapot." How did it turn into glassy chunks? In the autoclave?

When we look at higher magnification, you can see little individual spore bodies, but again, we don't see that fluffy silica coating that we expect from a standard weaponized, uh, sort of material. Again, down here was Leahy material, it was smaller clumps, but we still saw clumps. This is a nice picture, this happened to show up on a book cover, if you've ever seen the book Microbial Forensics. And I was surprised when I bought the book and saw the picture I took in my lab on the cover three years ago. It was a little surprising.

What precisely is "standard weaponized material," again?

At any rate, at higher mag here, we can see the fact that those spore bodies here again are not covered with silica particles, they look as what you'd expect spores to look like - untreated spores. If we put our electron beam on here and do elemental analysis, what we see are some interesting things. First of all, here is the Leahy letter material, and we see carbon, oxygen and a very small silicon peak here. If we do quantitative analysis and again this may be termed semi-quantitative analysis, or just bad quantitative analysis, I put an error bar of plus or minus 50%, because of the nature of the material is not amenable to good quantitative analysis in the scanning electron microscope, as far as bulk characterization is concerned, we sort of get in the 1.2 to 2.3 weight percent range silicon. And if we looked in the New York Post we see the same sort of spectra, not very different from this one up here - and again, the silicon is 1.2 to 1.5 weight percent - not very much.

Nowhere in this does Sandia report what "untreated spores" look like. Appartently, they never even looked at any untreated dried spore material - another basic flaw in the methodology.

QUESTION: [inaudible] the range?

That would be a relative error - yeah, it's not very good. And again, that's because when we do quantitative analysis, our first assumption is that the electron beam intercepts material that's all the same composition, and then we can do our correction factors. In this case, we are hitting void space, we're hitting the calcium rich spore center, we're hitting the silicon material, so it's really hard to do good quantitative analysis.

That's worth repeating - at this micro-nanoscale, their quantitative analysis is poor. Not only that, they're not used to looking at biological materials - this is a materials science lab that seems to spend most of its time characterizing metals, semiconductors, nuclear materials and the like.

QUESTION: I just want to make sure that you're not saying it's 1.2 to 1.5 plus or minus 50%

No, no - relative. Right, right. And again, both samples look very similar in this case, and they look very different from the previous material that I showed you that was weaponized.

QUESTION: Calcium, though, was really low? -[inaudible]- how much calcium was there?

Right there, I'm seeing about three to six weight percent - I don't think that's low, from what I've seen in the literature, actually, in terms of weight percentage. What's interesting is, if we change the electron voltage on our microscope, as we lower the voltage we become more and more surface sensitive, okay, because the electron beam doesn't penetrate as deeply. What you see over here is a result at 20 kV, 15 kV and 5 kV, and these are estimates of the range which the electron beam penetrates at those accelerating voltages, so this is like 3.3 microns, um, we see that as we go to lower voltages, so we're getting more and more surface sensitive, the silicon signal goes from up here and goes down to this black line. So that's a really preliminary indication that the silica and the silicon and oxygen, and I shouldn't say silica, because we don't know the stoichiometry of it, is not on the outside of the spores.

Again, their quantitative analysis is poor - and what is he saying? There's silica inside the spores? Another possibility is that when they sliced their samples up with the ion beam, they blew off the surface silicon - so when they turn up the analytical beam, they see the silicon that didn't get blown off. That's more evidence of artifacts.

Just looking through the literature, here's and EDX spectrum I dug up from this report, and you can see here, carbon, oxygen, a little silicon, phosphorous, and if you go back, very similar x-ray spectra. And again, this paper didn't indicate that they had added silicon anti-foam or things like that to the preparation, so the spectra are not inconsistent with what has been seen before.

Yeah, I actually dug that old paper up - and you know what? They speculate that silicon vacuum oils could have contaminated their preps. That's the best "prior evidence" they could come up with.

If we look at the Leahy material using the spectrum imaging approach, here's our SEM image that's ten micron bar, so again these are clumps of spores, we see that in the spore material, we see the calcium, sulfur, phosphorous, silicon and a few magnesium and sodium and oxygen, and they're all mixed together, so we don't have the resolution to separate out those different materials, different elements at this point, and again we see the support material.
So it is indicating that there is silicon in there, and oxygen, but at this point we don't know where. We have a hint that it might be on the inside.

That hint is spurious.

Just to summarize the SEM work, we know that silicon is present in both of these samples, the ones we have powders of, again microanalysis tells us it is there, but it's not quantitative. The low kV work shows us that it is probably on the inside of the spores, again, the spectral imaging really helps us out with some of these - with some of this information.

Repetition of an unsubstantiated claim... get used to it.

At this point, we knew we needed to go to a higher resolution tool, so this is where we started to apply the scanning transmission electron microscope, and this will tell us more about what's inside the spore bodies. And again now, we took a couple different approaches to specimen prep, again, these were done at USAMRIID or NBFAC later on, I think they're standard, sort of, preps.

You know, this is completely unprofessional - you have to compare different preps, and the specific details matter - a lot. "Standard sort of preps?" What exactly does that mean? Autoclaved first?

One thing is that microbiologists love to use these heavy metal stains so they can see things in bright field mode, we'd prefer not to have them, the uranium in uranyl acetate and the lead citrate, the lead in the lead citrate, cause all kinds of contamination peaks in our spectrum, so we prefer to leave that out. We eventually convinced them not to do that. And we still get plenty of image contrast in the STEM mode, the dark field stem mode.

The other approach we applies was sort of unique. We took the dry powder, we mounted it on a stub, and we took it to a tool called a focused ion beam, dual beam SEM, and made thin samples that way. And this is a tool I'll talk to you a little bit about later. It's interesting, this is some of the first work ever applied to a biological sample back in 2002, and unfortunately we couldn't publish it an now there's been lots of papers on the subject, so this is sort of old hat now. But this was a perfect way to make nice thin TEM samples of even single spores if you'd like to.

A quick look at Google Scholar reveals that artifact generation with the use of ion beams is a serious problem when dealing with biological samples.

So, back to our weaponized Bt surrogate - here's a bright-field TEM images of just the spores sprinkled on a grid. Okay, so this is our first approach, to sprinkle them on a grid and take a look here's the spore body, here's all this silica nanoparticles all around it - so that's pretty obvious, if we then go to a uh, thin section, and ultramicrotome thin section, you can see the spores here and then the silica particles all around the outside of the spore body.

If we do some spectrum imaging of that, here's a spore, here's some of that silica nanoparticles, and if we do the spectrum imaging approach, we now see that the calcium is in the spore body, we see the green phase, uh, as indicated on this spectrum over here, is the silicon and the oxygen, and we see some other things, some calcium, phosphates and things like that in the actual ah, preparation. And this has been published in this paper here.

If we go back now and look at the weaponized material, here's our image, and now here's a component image showing you where this component is, silicon and oxygen, we see that all this fluffy material around the outside of the spores, is located on the outside of the spores itselves. Okay, so that's the traditional weaponization approach - everything is on the outside of the spore.

Again, the method of sample prep is not mentioned - and the only powders Sandia got were chunky and glassy - which indicates that these were the autoclaved samples, not the powders in their native state. Not only that, they didn't get ANY of the Daschle material - only as pre-prepped and mounted samples. Who did that work?

If we now start to look at the attack materials, using the same technique, now this is an ultramicrotome section, this one happened to be fixed and stained, so we do see peaks from lead and uranium in it, so here's an image showing all the stain elements, you have your lead, your uranium, your osmium, this is the actual STEM annular dark-field image, and this is the image we worked from mostly because it gave us enough contrast to see what we are looking at, and we didn't need to do bright field in this case.

They based their analysis on a single image of a pre-prepped slide? What?

When we looked at this, we can see here's the silicon and the oxygen component, if you look closely it's this material here, and that appears to be located in or on the spore coat, not on the outside of the exosporium. And down here is a colorized image showing the different materials shown here, the green is the silica and oxygen, the red is some of the stain elements in the exosporium, and of course the spore body itself.

QUESTION: Can you go back to the weaponized spores? Can you see if it's the exosporium?

Well, if you look the exosporium is out here, and here is the silica - okay?

If we look at the Leahy letter material, a little bit higher magnification, this is again a fixed and stained section, here we see the silicon component, and out here we see the exosporium with the red is the stain, it's got lead, uranium, carbon a bunch of other elements in it, osmium - and down here underneath the exosporium we see the green layer, and that again is the silicon and oxygen associated with the spore coat.

So, they see silicon and oxygen associated with the spore coat, and this is the Leahy material - which had been autoclaved. And the quantitative analysis is poor, as well...

QUESTION: Were the preparations for the surrogate Bt silica and these Leahy samples - were they prepared exactly the same way?

I believe so.

QUESTION: Irradiated then fixed?

Yes. And in fact I didn't say this but we did get samples of the Bt irradiated and not irradiated to start with, compared the two images and really saw no difference from the irradiation.

How did "I believe so" turn into "Yes?"

QUESTION: Joseph, I was trying to understand that sample prep - did they take powdered material and then suspend it and redry it? It looks like there were dehydration steps.

I believe that's what they did - and you'd have to ask somebody from -

Now, he's changed his story again - irradiated? I believe so. Suspended and re-dried? I believe so. You get the sense that this guy has never worked with biological materials in his life before this.

QUESTION: Formaldehyde fixing? They never looked directly at dry samples straight out of the envelope?

Well, we did, but they had to be irradiated first - and I'll show you - that's why we had to do the focused ion beam work - so we're on to two different specimen techniques to compare the data from. So, I'll show you that in a moment.

QUESTION: But these have been in suspension and then re-dried?

I believe that's correct.

Well, that's a good way to reduce the surface silicon content, I imagine... I find this discussion surreal.

Just to bring that up right now, this is the focused ion beam tool, I don't know how much you are familiar with these tools - what we do in the focused ion beam tool is we combine the scanning electron microscope with an ion column, and the ion column is there because we can focus it to a fine spot, and we can micromachine materials with it. So we can actually watch what we are doing with the scanning electron microscope, while we remove nano-scale amounts of material from the sample. It's quite a great tool - it started off twenty years ago in the semiconductor industry and now they make these small dual beams that fit in your laboratory, and if you have $1.8 million dollars you can have one. Sounds not like a lot of money when you say it fast.

Anyway, in specimen prep, this happens to be some pictures that we took when we were preparing a sample from a clump of spores, a large clump of spores, so we wanted to make a sample across here for transmission electron microscopy so we used our focus ion beam to remove this material and remove this material, and then gradually thinned this sample down to electron transparency on the order of 100 nm or less, and here's our finished sample, we used the ion beam to cut it free, on all three sides, we manipulate that out of the hole, put it on a carbon support cell, and then we can look at that in the TEM.

This whole preparation technique takes about an hour, so we can prepare samples quite quickly this way - which is great if you have a semiconductor fab, because they want to know right away what's wrong with their material, or their process, and in this case it it's very nice to prepare samples quite quickly as well. So what do those samples look like? This is one of our first attempts, we just picked a couple of spores, and honestly we could STEM one spore if we wanted to, it's really straightforward. These are actually large features for what we work with.

So, here's a cross-section - we lay some platinum down on top of that, to protect the sample when we make the section, to give it a little bit of mass, and this is the annular dark field image so you can see the spores themselves, we can see the spore coat, and if you look closely there is some indication of the exosporium up here and up here.

What happens when we do the chemical analysis, microanalysis of that? Now we start to see the sample elemental distributions as we expect, we see calcium in the center of the spore, and you can see that here in the spectrum, here's the calcium peak, there''s the oxygen, so that's located in the center, out on the spore coat, again, we see this silicon and oxygen, in or on the spore coat. So, we are seeing the same information using two different preparation techniques - the traditional ultramicrotomy and the less traditional focused ion beam technique.

Let's repeat that: "We see this silicon and oxygen, in or on the spore coat." That directly contradicts FBI claims that no chemical additives were employed, doesn't it? (Bruce Ivins didn't have access to such chemical additives, or to the equipment needed to apply them to individual spores.)

One other point - now we don't have all those other elements involved, the lead, uranium, osmium, we now pick up other trace elements - so here we see tin and iron, out here in the spectrum, and that was consistent in all the spores we looked at from either the, all three mailing materials we had, this is just the blow-up of them. It's not a lot, it is a fairly low amount - of tin and silica - of tin and iron, I should say.

Note that this could be used to distinguish the Detrick Ames spores from the letter spores - but did they ever look at the RMR flask spores? The ones that the FBI claims Ivins harvested and used in the anthrax attacks? What if the RMR flasks spores had no tin or iron?

We did want to verify that with another technique, so we took it to our time-of-flight SIMS, this is a secondary ion mass spectroscopy technique, and indeed when looked at that, it doesn't have the spatial resolution we'd like to have, but it does have enough resolution that we can see the tin, and the ion, in our SIMS spectra. So, this was verification of what we had seen earlier.

QUESTION: Were those previous spectra on the previous slide, were those actual -[inaudible]- spectra, or were those multivariate factors or whatever?

These are the multivariate data that corresponds to this...

QUESTION: And so then, the second study then just reconfirms that your optical method is...

Correct. And we can actually go back now, since we do have a spectrum image, we have the original data from every pixel in this image, we can say now, let's sum all these pixels from around here and see what that spectrum looks like. And again, that reproduces this spectrum quite nicely. So there's a couple internal checks that we do here to make sure everything's working correctly.

QUESTION: Joe, can you give us a just a rough idea of how many spores were looked at with this degree of detail?

Um... on the order of thousands by the time we were done - so we looked at quite a large number of spores, to get the statistics, and I was going to get to that in the next couple of slides, because there's two signatures here that I'd like to leave you with.

One is the fact that we see silicon and oxygen on the spore coat, the other is, every spore doesn't seem to have this signature - so to produce that statistic, we had to look at a lot more data.

Sample processing artifacts?

Okay, here's the New York Post - these are the only two samples that we had powders of that we could do the thin prep of, I think they ran out of Daschle material, or didn't want to share it with us, and you can see again the same thing, calcium, in the spore body itself, silicon and oxygen co-located with the, ah, spore coat, and again we see the iron and tin signature - so that sort of links the two attacks together, maybe, they're very similar on a spore-by-spore basis, if you look at single spores. This is the thin prepared section, looking at many spores, and what you see here again is the same result, we see the calcium in the spores and we see the silicon and the oxygen on the spore coat.

What's interesting here, and what I want to point out to you, is not every spore - if you look at - I don't know, there's a couple in here like this one here - not every spore shows the same silicon-oxygen signature on the spore coat. This was the same result we got from the ultramicrotome sections as well.

QUESTION: What relationship between the amount of calcium and silicon and oxygen in the spore coat?

I didn't see any, in our studies.

QUESTION: When you just said it relates to two preparations, to the exclusion of other preparations of spores...

We had the samples the FBI sent us, which were ultramicrotomed and prepared traditionally, and then we had our focused ion-beam prepared sections. Both gave us the same answer as far as the silicon and the oxygen signature - minus the fact that in some cases with the staining and the osmium, we couldn't see the small features like the iron and tin.

QUESTION: Did you do a control that wasn't these samples? To see what elemental signatures they showed?

We looked at quite a few samples before this, and quite a few samples after this for the FBI that did not show any of these signatures. So...

QUESTION: Did you have an example of that?

Something that doesn't show anything,I didn't bring any of those with me, but we can show you blank pictures...

QUESTION: Many of the preparations don't contain silica?

Most of the ones we looked at don't contain silicon, or the iron and tin, in the same location, co-located, making that association between the two.

QUESTION: And the other samples you looked at, these were the repository samples?

Ah, you'll have to ask the - all samples came to us blind, after the fact we learned what these were, and a few others, and I'll show you...

He doesn't even know if he was ever given samples from the RMR flasks to look at!

QUESTION: The Post material was quite different from the Leahy material...

Well, physically it's different - but it's not different on the spore - if you handed me a spore from the Leahy and a spore from the New York Post, I couldn't tell you which one it came from.

QUESTION: Yeah, that's exactly what I'm getting at.

Right. And - this was another point I wanted to make, and along those lines - I'll make it later - is, if this silica had something to do with the material's ability to be aerosolized, one wonders why is the Leahy clumped up in big clumps, and the other ones are not, since they all look the same? So to me that's sort of an indication that this has nothing to do with aerosolization or intentional, uh, acts to make it more aerosolizable.

QUESTION : Right, but -[inaudible] is very interesting -[inaudible]- spores - in other respects, preparations - it conceivably could have been prepared differently?

Yeah, we were able to tell the FBI most of that - probably March 2002.

QUESTION: Early on, when you showed your low resolution data, you had the estimate of the amount of silicon. I assume that in the Leahy letter, that actually as I recall matches what they found by bulk analysis. Is that generally what you have found, and did you - when you received samples, did you have all the information as well, the low resolution - was roughly in the same ballpark as the STEM, or...

We had no information other than, "here's your sample, tell us about it" - so we knew nothing - and in fact, I only heard that number, what their bulk analysis was, was at the ASM meeting this winter. That was the first time I heard their actual numbers. Umm... one point I'd like to make that I think was important, Tom Geisbert at USAMRIID sent us on this chase to find vegetative cells during the spore forming process - he said, there's a few in there, in a grid, and go look for them. Well, it took me three days to find one, at this level, but here is a mother cell in the process of forming a spore, and if we do the chemical analysis what I'd like to point out, we are already seeing silicon and oxygen on that spore coat, in the mother cell. Okay, so...

These "vegetative cells" could have been a Bacillus subtilis contaminant, something picked up during the growth process. Or, they could have been anthrax strains deficient in the ability to sporulate - or simple vegetative anthrax cells that hadn't had time to finish sporulating before being harvested. None of that seems to fit with the Detrick RMR flasks.

QUESTION: What else could be....

Well, I believe that this, and I've been told this to be true, this is the cell..

QUESTION: It's not an exosporium?

Well, I don't believe so because there's another chain down here...

QUESTION: That thing down there - well, I don't know what that is.

Well, isn't that the section through a string of these things? You remember, this is a thin section.

QUESTION: -[inaudible]- A chain of cells halfway through the process?

And even then we see the silicon and oxygen signature on that spore, okay? We only found one of these, since I spent three days finding this one, and we sort of stopped at that point.

QUESTION: So the fact that you see the silicon inside a vegetative cell is what really allows you to conclude that this is probably a natural process?

One, one of the things, sure, right, I believe that's true - and by natural, we can argue what natural means all day long.

This may be the most ridiculous statement in the entire talk - possibly.

QUESTION: Such as mineralization?

Right, right, umm... Just a comparison of the Leahy material, the Daschle, and the New York Post - from - In terms of the silicon and oxygen signature, again, the tin and iron, and here we see the iron but our tin is obscured by, I think it's an osmium peak, and again there's that point - the silicon and oxygen layer does not seem to prevent the New York Post powder from clumping, okay.

Well, that's because it was autoclaved, most likely.

QUESTION: Joseph, you mentioned in passing anti-foam agents that were used. Could silicon-based...

I believe they are silicon-based...

QUESTION: What kind of materials are those and what kind of molecular weights... do you know what they are?

You'll have to ask someone that grows bacteria.

QUESTION: Yeah, we got a few of those... We used them long ago but we don't use them now but I don't remember what the form of the silicon is.... Could be a silane of some sort.... But there are small molecules, silicon based materials that... If you're shaking large cultures of bacteria, you tend to get foam building up... Especially in fermenters, where like, you're bubbling air through... It could be a surfactant like material... Yes, that's an interesting point. It suggest that growing them -[inaudible]-

Damn it, didn't any of them read the "reference paper" this was based on? They suggest that it was the silicon vacuum oil in the centrifuge that may have contaminated their samples.

I think we looked at a lot of samples, with various preparations - and sometimes they mentioned they used anti-foams and sometimes not - and it wasn't - we couldn't correlate this sort of appearance with the use or not use of the anti-foam.

QUESTION: So, along the same lines, have you looked into any other Bacillus species, spores?

Yeah, I've looked in the literature...

QUESTION: -[inaudible]- have seen all this, kind of silicon, -[inaudible]- similar spores, have you seen those things?

We certainly don't grow our own spores and our own samples, so we were dependent upon whatever the FBI gave us to look at.

And that's a major problem, isn't it?

QUESTION: -[inaudible]- You have not seen anything else?

Right. I have not. But I do have, and I'll show you some literature, from the eighties, when people have done this sort of experiment on other species and shown the same sort of things.

QUESTION: I'm reading ahead - it says there tin is somewhat -[inaudible]- by the stain. Are there other elements that might have been obscured by the process?

When we actually made our - that was just referring to the Daschle material, because Daschle only came to us as pre-prepped ultramicrotomed fixed and stained sections. So they always came with the uranium, the osmium, the lead, the tungsten - all these nice heavy elements that the biologists love to stick in their samples that we microanalysts hate to see, because they have this huge range of characteristic peaks that obscure lots in the spectrum. I think, you know, as an aside, you microbiologists just might want to consider STEM instead of bright-field TEM because in STEM, you can see lots of the features without staining, a little aside.

-[inaudible]-

The question of spore count came up, how many spores we looked at, this is just our estimate of what the error bar would be if we looked at a total of N spores, and X showed a particular chemical feature, what would be our confidence, our 95% confidence level. And you can see if we look at ten spores, we really have a difficult time putting a confidence level on there that is small. up a hundred spores and a thousand spores, we bring that confidence level way down, so with this data in mind we went off and started doing more statistics, and this is the result of that - this is the number of spores we imaged, the number with the Si-O on the spore coat, or in the spore coat, and this is the percentage. So, the top one's Leahy, the next one's Daschle, and the next one's New York Post.

So all those three materials had quite high numbers of spores that showed the silicon and oxygen signature. When we looked at other materials - this is a sample produced I believe at Dugway, we saw things like this, 26%, 11% and 29%, this is RMR-1030, I'm sure you are familiar with that flask, now that was another one grown at USAMRIID, it showed about 6% of the spores with the same signature, and when we get down here to RMR-1029, we saw no silicon signature on the spore coat.

Again, they did not prepare these samples themselves. Who did? And doesn't this contradict the earlier claim about samples coming to them blind?

QUESTION: So,um -inaudible- level 0402055 level 258 - are those - do we know much about those specimens?

I have some of the data on how they are grown, I do not know what levels 2, 5 and 8 mean. I believe they were from - I think what they were trying to do was take a capsule and section different parts through the material that's been spun down into it, but I'm not sure of that. You'd have to ask the people who made the sample.

QUESTION: I thought you were saying that you hadn't seen any other spore preparations that had silicon...

I think the question was other species?

QUESTION: Oh, I thought earlier, I thought Richard had asked you were there any other spore that showed a silicon coat, and you said you looked at thousands...

Well, maybe I misunderstood the question.

QUESTION: Maybe I misunderstood.

We have seen, as you can see here, and I'll show you some of this data. We did see other samples with similar - on a spore basis, similar appearance and similar signatures, but again when you start to look at the statistics, they're not the same sort of account, okay.

QUESTION: Just so I understand - the RMR-1029, the presumption is that was the source - or some of those three samples at the top - so that would imply that those were a culture, to get them to the point where they actually had some kind of silicon -[inaudible]-

That would be my understanding of it was well.

QUESTION: Because the primary culture or the stock culture did not have any - anything - but that was a liquid culture, that presumably had to be washed and dried and prepared, right?

I would expect that to be true.

Just an image from RMR-1030, here's the spores, a little bit dark, but you can again see the silicon and oxygen signature on the spore coat, very much like we've seen before. This is that sample from Dugway, this is the one that had different levels. Again, here's our sample, and here's the spores that had different - that had the silicon and oxygen on the spore coat, and again, here is the same signature that we saw. And again, note the variability here. We see a couple spores in this field and we see a lot of spores in this field that have the silicon and oxygen on the spore coat. And as far as I can find out, this is Ames grown via fermentation at Dugway using a L-D media. That's all the information I had about that sample.

Another sample that came to us in May 2008 was quite fascinating to me, these are the numbers, I was told the sample was to be described as 'evidence.' That's all they gave me, and that's all they would give me about this sample, but I think this is a fascinating series because you can see up here, I should also add we've developed better techniques so we get better statistics over the past eight years - when we first started doing this, it took us two or three minutes per spore, to actually do this analysis, now we're down to about 0.2 seconds per spore, so now we can really start to build up statistics. And that was with a nice grant from DHS that we managed to pull that off.

If you look at this series of samples, though, we've analyzed quite a few spores, the interesting thing is here, this would be a great sample to mine for conditions to see why it went from 18% of the spores with the silicon and oxygen signature, down to 1.2. And there were others in this series that had nothing, in that batch of samples. So, just to bring this to your attention, this is a great sample that you might want to pull the string on a little bit if you can.

QUESTION: Pull the string - what are we pulling for?

I don't know anything about these or where they came from. Okay - and if they know the growth conditions, you may be able to associate this change in silicon content, or silica - the number of spores with silica - with the growth conditions.

QUESTION: It could also be a calibration curve, where two things are being mixed, to see if it scales linearly?

I don't know what it was.

QUESTION: Many of these also have the tin and iron signatures?

I don't think we ever saw tin and iron again in these. We've seen it in other sample's that we've gotten from a DHS study that we published in that - the reference I gave previously when we looked at the weaponized material. In some cases we've seen tin, and iron, in those materials as well. And again, that's documented in one of the publications we've put out, and I think it's in your publication list.

QUESTION: I've probably confused myself - so the - the Dugway, and one of the -[inaudible]- the actual Leahy -[inaudible]- even though the preps were superficially different, it was a common chemical signature, that was the tin?

The tin and the iron and the silicon, and the oxygen, on the spore coat.

QUESTION: You've shown silicon in other, unrelated spore preparations, so it's not distinctive to the, uh, materials that were used in the attack.

That's correct.

QUESTION: But the tin?

The tin was very distinctive in this case. And that was...

QUESTION: So the tin...

and the iron, they're very small peaks, they're not...

QUESTION: The tin, and the iron, were found uniquely, or distinctively, in those preps and not unrelated preparations.

JM : Although we did, like I said, we have seen tin in some other preps that we got from other laboratories, in an unrelated study for this, or a sort of related study, but again those samples though didn't have the silicon and the oxygen in the same way,so.

QUESTION: So if you take all of these...

JM: These were unique.

QUESTION: ...elements, you've got a certain profile...

JM: Correct.

QUESTION: ...that is characteristic of the attack preparations, and not other preparations that you've seen?

JM: That's correct.

QUESTION: Does this technology allow you to get a sense of the ratios of isotopes, so that - what I'm thinking of is, with the minor elements, from different parts of the country with different isotope compositions, that it may be possible for example to track down something? - [inaudible]-

Great question!

JM: One of the problems is that the amounts of the material are quite small, so you can go back and look at that SIMS data, and you can see the isotopic ratio in the tin peak, for example. It looks very much like a naturally occurring - what you'd expect from nature for tin. Trying to geolocate from that data would be really tough. It wasn't something we thought about at the time because the signal was so low. It really doesn't lend itself to that sort of work - and it's not the right tool to use for that anyway. There are better tools to do that sort of analysis in.

Translation: No, we didn't do any isotopic analysis. Remarkable, considering that that would be the best "fingerprint" option - and you need tiny amounts of material to do this. You don't look at the tin, you look at the carbon and oxygen. Seems like a high level of ignorance here.

QUESTION: -{inaudible]- the silde of RMR-1030 and RMR 1029 - the level, 2, 5, 8, 40255? You said that was from Dugway?

JM: Yes.

QUESTION: - And was that examined under SEM or STEM to see whether that was silicon on the coat or in the exosporium?

JM: That was - this data right here. So, again, if we look closely it's on the spore coat.

QUESTION: And on those samples, did you find tin and iron?

JM: No we did not.

QUESTION: The tin and iron were close to the detection limits, I recall?

JM: That's correct.

QUESTION: How close to the limit? -{inaudible]-

JM: Actually, um, down here you can clearly see their peaks. I'm guessing that these peaks are three or four times our detection limit.

QUESTION: Would you see those routinely on different spores? -[inaudible]- like silicon?

JM: If the silicon and oxygen were co-located with the spore coat, we saw the tin and the iron. The reverse was not true.

QUESTION: And the Ames strain, which you just showed us a moment ago, -[inaudible] - we know that never had silica? Or do we know anything about what that ever had?

JM: I don't know. That's the number, and you can query the FBI on that question. My presumption is it didn't, because they gave me this information about it, and they didn't say it had been treated in any specific way.

QUESTION: Did you have the opportunity to do the same assessment on multiple specimens that had been weaponized?

JM: Yes, we did. And that's - of course, it wasn't Ames, because nobody's weaponizing Ames, it was always a surrogate. Just a comment about how we could get all these large numbers of spores, in this example. We transferred the STEM technique from the expensive STEM instrument to the slightly less expensive SEM instrument, developed a new type of X-ray detector during the eight years we were doing this, and this was under DHS funding, and that enabled us to do extremely large fields of view, you can see a couple hundred spores in one image, then we can go ahead and find the ones that contain silicon on the spore coat, and count those up quite easily, and here's a blow-up of what one of them would look like. We went back and validated this technique against our previous STEM work with the same samples and got the same answers, within statistics. So this was a nice added thing that we added with DHS funding, and this allowed us to go from two or three minutes per spore down to a couple tens of a second per spore in analysis time.

Dugway was weaponizing Ames for use in the biological threat assessment program - that's clear enough.

Just a couple comments on previous studies that are out there... so this is some work done by Johnston back in 1980, previous generation piece of equipment, not quite as high-resolution as what we have today, here's their specimen prep technique, and here's their background signal, this is an X-ray spectrum, here's the spore coat, and we go right there and there's a nice silicon signal on the spore coat, and again, resolution allows that to go over to the cortex, and we don't see that signal in the core. So again, this is an example from Bacillus megatherium. So, it's a different species where they actually saw silicon on the spore coat. There was another study, this is the Stewart study, back in July of 1980. These are their images...here is their silicon signature... here is the spectrum... we actually had this sample in our laboratory, and were able to go back and analyze this same sample... and again we saw this same result, just a little higher resolution. So there are multiple examples from the literature where they found silicon. On both of these examples, none of them mentioned any silicon antifoam agents, that I could find.

QUESTION: Is there any -[inaudible]- why there's -[inaudible]- in the same preparation?

JM: Yeah, they made the statement that the variation in silicon, both within and between spores, they saw this same variation that we saw, and they marked it down as an observation, and they also did two different kind of preps... and saw the same result again. So they were saying it wasn't the prep technique... maybe pickup from glassware, or something like that. Something in the environment. And both people made the same sort of observation - both papers I cited. So I'd like to stop here. I think the conclusions are:

*The New York Post, Leahy and Daschle materials are basically indistinguishable, elementally, at the spore level, and they both have a similar fraction of the spores with the silicon and oxygen in or on the spore coat.

*We found the silicon and oxygen signature on an endospore in the New York Post material, which leads me to believe that this is something that is happening during the spore formation process, not something that's added later on as an additive, once the spores have been formed.

*I believe that the letter powders are not unique. We've seen examples in the literature and examples from other samples grown for the FBI that show the silicon and oxygen signature - they do differ, though, in the percentage of spores that have the silicon and oxygen on the spore coat. And finally, I think that STEM and SEM are really the two tools that really helped us out in this study. I'll be happy to answer any questions.

QUESTION: That was really good.

JM: Thank you.

QUESTION: It looks like you've done a lot of work on addressing the sampling statistical problem with respect to the portion of the sample that you're looking at - lots of numbers of spores of the particular portion that's being analyzed - but, because some of these samples are grossly heterogeneous, I'm wondering if there were also efforts to sample different, distant parts of "the sample"? And then, the same kind of statistical, intra-portion analysis of lots of spores from that section as well?

JM: We didn't make any specific effort to do that. We were given I think 1.4 milligrams of Leahy material and about 14 milligrams of New York Post and were told to be careful how we used it... So, that is a subsample, so we really can't say much about what was in the vial... But the samples that the FBI had prepared at USAMRIID or NBFAC presumably came from another part of the vial, because we had that part in our lab. So, maybe that an indication that these are fairly consistent throughout the bulk of the material.

QUESTION: Although I guess we can't say. It may be that certain parts of a heterogeneous sample are more amenable to either picking and processing, etc. so it may be biased in a way.

JM: It may, but that's going to be a tough one for us to answer.

QUESTION: Related to that, -[inaudible]- sample preparation? do you generally do that on most samples, or just a few samples...

JM: We did that on a few samples, we didn't do that on most of them. Generally, one of the reasons we didn't do it on most of them was because they came as ultramicrotomed thin sections - so we didn't have a lot of powders that came to the lab. Most everything came already prepared for us.

QUESTION: So when you did that, it was mostly ion beam -[inaudible]- attack samples, and then a few samples early on?

JM: That's right. We did a few samples early on as well. I think we probably did have some powders early on of Bt and didn't see signatures of.

-end transcript-

The complete transcript of Dr. Patricia Worsham's testimony before the NAS, Sept 24, 2009

2009-12-14T13:32:00.000-08:00

Dr. Patricia Worsham and her research group at USAMRIID initially identified morphological variants that were either present at low numbers in the attack material, or arose during the course of cultivation of the attack material. These variants were selected solely by visual inspection of colonies that grew up on Bacillus anthracis growth medium - so this is a classical old-school microbial study, very similar to the kind of work done in, say, the 1950s.

For comparison, I would refer those interested to work such as this:

CRITERIA FOR THE IDENTIFICATION OF BACILLUS ANTHRACIS
JOSHUA M. LEISE, CHARLES H. CARTER, HAROLD FRIEDLANDER, AND
SAMUEL W. FREEID
United States Army Chemical Corps, Fort Detrick, Frederick, Maryland
Received for publication October 24, 1958

The basic point is that without the associated genetic analysis, this (rather primitive) work would never be admissible in a court of law. The curious thing about it is that there was apparently almost no communication between the Worsham group that isolated the morphs and the people who developed the genetic assays for the morphs, along the lines described by Dr. Paul Keim in his testimony.

According to an article in Nature News,

Patricia Worsham and her colleagues had noticed differences in shape, colour, and rate of spore formation even within a single anthrax culture. Ravel's team identified the genetic mutations associated with four variants and developed an assay for one of them, called Morph E. Researchers at Commonwealth Biotechnologies in Richmond, Virginia, and the Midwest Research Institute's Florida Division in Palm Bay created assays for three other variants.

Note that the Commonwealth Biotechnologies has a clear financial interest in seeing biological warfare research funding expanded (via a press release):

Since 1999, Commonwealth researchers have managed or participated in awarded biodefense grants exceeding $20,000,000 in value. CBI also operates a secure BSL-3 containment facility able to receive and work with select agents as defined by the Centers for Disease Control...

The Midwest Research Institute is a similar entity, closely linked to the Battelle Memorial Institute (via a press release):

MRI is a nonprofit research organization whose research encompasses areas such as energy, national security and life sciences. It had consolidated operating revenue of $387.8 million in fiscal 2009, which ended June 30. It has 1,800 employees across the country.

MRI also jointly manages at least one DOE lab in a joint partnership with Battelle Memorial Institute.

So, the take-home message here is that unlike the assays for the Ames variants, developed at Northern University by Paul Keim, under one roof at a public university with extensive validation and multiple refereed publications, the morph assays were developed in isolation from one another, with three of them created at secretive private institutions closely allied to Battelle Memorial Institute, one of the few institutions known to have access to both the Ames strain as well as cutting-edge spore purification and aerosolization capabilities, developed under contracts with Dugway, the CIA and the DIA. Unlike with the Ames work, there seems to be a serious lack-of-transparency issue here - one apparently glossed over by the both the committee and by Dr. Worsham.

Why didn't the FBI instead use the best-equipped and best-validated lab - the Keim lab- the one that had developed the Ames assays, all under stringent, court-admissible guidelines? Why did they farm the assays out to multiple private entitites? And why wasn't any of this brought up in the following testimony on the identification of the morphs?

All that the Chair of the NAS committee said was that "we'll get to them eventually" - but who, precisely is "them" - the complete list, that is? And when is "eventually"? Why were they not present to give supporting (or not) evidence?

The original audio that this was transcribed from is available at the NAS website:
http://www.nationalacademies.org/newsroom/nalerts/20090925.html

Slides were shown with this presentation, but as with other testimony, the NAS did not see fit to post them online. There are a lot of issues here - but one main question pops up in the very first statement: what are the FBI restrictions, and how can the NAS committee do its work under "limited release"?

Transcript begins:

Dr. Patricia Worsham: One thing I probably should mention before I start is that I am under limited release from my non-disclosure agreement which means - I just wanted to mention I'm under limited release from my non-disclosure agreement.

So, anyway, in fall of 2001 the Leahy letter was brought to the United States Army Research Institute of Infectious Diseases, to the Special Pathogens Unit, where the FBI had asked them to analyze the material. As part of that analysis, it was plated on sheep blood agar, and in one case the plates were left in the incubator a couple of extra days, and Terry Abshire? who is an excellent microbiologist and has a good eye, noticed that there was something unusual about the plates, and in discussion with other people in their division an in ours, she recalled that my lab had published a paper some years before describing certain variants that arise during laboratory culture of Bacillus anthracis.

So, this is not the same plate that Terry had, but this is very similar. This is the more typical kind of Bacillus anthracis colony - it's gray, it's pitted, you tend to find some medusa forms, but you notice these sort of creamy yellow-colored colonies here - that's not the way Bacillus anthracis typically looks. But it is very characteristic of the variants that we had described years earlier that were defective in sporulation. This is just a close-up of some of them.

So, Terry made this observation that after long-term incubation of plates - they're not nearly as obvious after 24 hours or so - the samples contained more than one colony type, and that these aberrant types were stable, that if you picked them and streaked them out for isolation they maintained that unusual phenotype. They didn't revert back. So the question is, was this a mixed culture or was this an artifact produced by the long incubation? Because in our experience it is this kind of long incubation that gives you this aberrant morph type - and if it is a mixed culture, can we use that phenomenon, that thing, to trace the origins of the sample?

So I haven't published all of this, but this is work before the FBI, so I can say whatever I want. Stable variants can be isolated from infected animals - I've seen them come out of guinea pigs, I've seen them come out of vaccinated animals, by phage selection and during laboratory culture. Not all of these are oligosporogenic or asporogenic, there are various kinds of variants you can see. The type and the frequency of the variants appears to be strain-dependent - and one thing that I had not noticed several years before this is the Ames strain is one of the strains that tends to throw off strains that are defective in sporulation - and some of the most common variants found in laboratory culture are oligosporogenic - that is, they are not asporogenic, they are capable of making spores, but they make fewer spores under conditions where Bacillus anthracis would normally make spores, or they will only make spores under certain conditions.

So in my experience what causes the in vitro selection of oligosporogenic strains? In my hands, it was repeated passages of overgrown liquid cultures. So if you have a culture, and the organism is running out of nutrients, and the majority of the population begins to sporulate, they stop dividing. If you have a mutant in the population, that is not getting the signal to sporulate, and continues to replicate, it forms a larger percentage at the end of that culture, than it started. And if you continue to do this, it will eventually take over the culture. If you pick from the edge of colonies on a plate - particularly an old plate - that is, if you have a colony and that colony has reached a state where it would ordinarily start sporulating, sometimes what you'll see are fingers coming out from the side of the colony, and these are mutant that have popped up, that are continuing to replicate under conditions where the parent has stopped growing and started to sporulate - and if you prepare stocks from old cultures, plate or broth, you tend to have this sort of an issue.

The phenotype is that they're generally larger, a more spreading colony morphology, you lose some of that characteristic medusa formation, there's a slight cream yellow colony pigment on sheep blood agar, so they're not as grey as typical Bacillus anthracis and they are somewhat hemolytic, which is why when I first stumbled across these I was convinced that they were not Bacillus anthracis. If you put them on sheep blood agar and leave them for a couple of days, they will be hemolytic, or you can sometimes observe it in younger cultures by say, taking a 20-hour blood agar plate and putting it in the refrigerator for a week. When you come back, and this is probably the sulfhydryl-based hemolysin that anthracis has.

This reminds me that the Congo Red issue is how I initially found these variants many many years ago. And that is, I was interested in looking at virulence in surface proteins, and one of the ways - I'm from a gram-negative background - that we often look for changes in the surface properties of an organism, is by binding of dyes. And so I incorporated Congo Red into the growth media with Bacillus anthracis and I began to find that the wild-type formed pink colonies but then these sort of white outgrowths would appear - and those were actually oligosporogenic. They have decreased amounts of the surface array protein, EA1, there's a brown pigment that they make which is a byproduct of catechol metabolism, and there's diminished as I said spore production. I was interested in these from a virulence perspective, I tested the vegetative cells of some of these strains for virulence, and they really weren't impaired compared to the wild-type, and that was pretty much the end of my love story with these strains for quite a while, because they didn't seem to be important to virulence, and I had to move on.

QUESTION: In your Canadian Journal of Microbiology paper, you pinned one of these down to -inaudible- which is part of the set of genes that drive entry into sporulation.

Correct.

QUESTION: Did you characterize any other mutants or did they generally prove to be response regulators, or the relay, or components of the relay, or did you not go beyond that?

I have a large collection in storage in the freezer, which I've never gotten back to. I think it would be an interesting thing to do. I'm guessing from the phenotypes that some of them are other early events.

QUESTION: They look like early blocks...

The spoO series, a lot of them look like that, but I haven't done the genetic studies to prove that. This is pre-FBI.

So, our goal was when the FBI came and asked us to work on this, and I should mention that Sargeant First Class Melissa Hunter was a big part of this - we worked together on this project to determine if the original samples contained more than one stable variant of Ames or other strains or other organisms - to purify and characterize the variants present in the original samples and submit them for genetic analysis - and identify any recognizable phenotypes that might lead to genetic fingerprinting of variants and potential identification of source material. Part of the challenge involved with this is that the very conditions that show us the difference between wild-type and variant are the ones that encourage spontaneous development of this type of mutant, and that was a difficult thing to get around.

So, as you grow a culture long-term, comparing variant and wild-type, it becomes easier and easier to see the difference between them, but on the other hand you run the risk of selecting novel mutations along the way, because that's what promotes this kind of mutation. So, is it a variant in the original population of spores, or is it a variant produced by spontaneous mutation favored over the wild-type, under these growth conditions - and in this case, the variants, they would have been artifacts, and not present in the original sample.

So, our solution to this was to minimize the number of generations in the laboratory between germination of the evidentiary material and storage of archival samples. We performed the genetic analysis, or I should say the people who did the analysis did it on material cultured directly from the archival samples - and we stored a second set of samples, and I'll explain this a little bit more in a minute - after another round of laboratory passage, it gave us more material to work with, in terms of doing phenotypic studies.

So - the spore samples I was given among other samples to work with - powdered material from the Daschle and the Leahy and the Post -these were resuspended in sterile water, and then plated on sheep blood agar, at 37 degrees Centigrade, at 37 degrees with CO2, and at 26 degrees. And part of this was just an attempt on my part to find some way of finding the best growth conditions for distinguishing between wild-type and variant in that population, and I was kind of hunting, and that's why there's so many parameters there.

We picked colonies, and froze half of the colony directly into glycerol, and that went into the freezer as the archival stock. The other half of the colony went to a streak plate, and that's what we froze as what we call the S1 subculture one, the freezer stock. The archival stock was what was used to send sample material for genetic analysis, and the S1 was generally used for - I'll explain the pick plates in a minute - Congo Red, new sporulation medium, L-D? medium, and I'll explain this too, and gamma phage.

I described the Congo Red, that is the wild-type tends to be somewhat salmon colored and shiny, whereas the variants, if they're oligosporogenic, will be white to cream colored, flat and spreading. New sporulation medium is a solid medium, in this case a slant, and what you are looking for is production of spores, and morphology. L-D is a classic medium for growing spores, in the laboratory, and that's what we have used - it's a liquid medium, if I didn't mention that.

Genetic analysis is probably going to be talked about by the people from TIGR, gamma phage is a bacteriophage specific for Bacillus anthracis. And so it was also important to make sure that whatever we were pulling out of these samples was Bacillus anthracis before we went further with other analysis.

So as I said we took the powdered material that was diluted in sterile water, and diluted it to get between 500 and 100 colony-forming units per mL, so then when we spread a 100 microliters, we would have had between ten and fifty colonies, so that we would get sufficient separation, to be able to look at each colony clearly. The plates were incubated under various conditions, and we looked after between 18 and 22 hours for variants, and I'll show you a picture of one of those plates in a minute. We picked both typical and atypical colonies for minus 70 stocks, and the primary subcultures. We found during this process that even blood agar has variations, so the homemade blood agar that we make in our tissue culture center at USAMRIID actually worked better for differentiating at this time point than the commercial Remel? blood agar. But Remel? has its place too, as we'll see in a minute.

So this is what one of these plates looks like at about 20 hours. It's definitely not as easy to distinguish between wild-type and mutant as it was on that first set of plates that I saw, so we did a lot of squinting - as you can see down here, this one is slightly yellowish, that one is a little yellowish, so it took a lot of eye exercise to look at the sheer number of plates we went through, looking at variants in different samples.

QUESTION: What is the pigment?

The pigment? That particular yellow pigment, I don't know for sure if it's the same brown, the diffusible pigment that we see in other situations, but that particular pigment is a catechol degradation product.

QUESTION: So it's a melanin-type?

Not melanin per se, but it's more like the siderophore catechols, I think, when they're oxidized, they turn brown. That's the brown pigment I believe, because - oops - is this me? I have a bad effect on computers.

Where was I - pigment - thank you. The yellow pigment in the colony per se, I don't know if it's the same thing as the brown pigment, it may or may not be. The brown pigment is easier to visualize in liquid culture than it is in solid medium.

So this will show you, this is one of the S1 plates, half of the colony was taken from that original plating and plated on blood agar, and this would be wild-type here, and you can kind of see the medusa forms in this area - and this is one of the mutants, this is one of the variants - so you can see overall, the colonies are larger, it's hard to tell the height of the colonies from this angle, but they're more flat and spreading, and you can kind of see in this area where there is some pitting and sort of a shiny appearance, and that's very characteristic of Bacillus anthracis as it starts to sporulate, and you don't see that...

QUESTION: Are all of the morphotypes defective in sporulation to some extent? Did you actually quantify...

All of the ones that I examined for effects in sporulation were defective in sporulation, and I'll show you different degrees in a minute - there are different categories, and it's interesting.

This is just a close-up, you can see a little better the difference in the morphologies and the pitting here as this begins to go into sporulation that you don't see over here. Pick plates are something else that we invented as part of this process - we were looking for a way to differentiate between variants. It looked like we had found ways to differentiate between wild-type and variant, but how many different kinds of variants did we have? And after a number of attempts we came up with a microbiological methodology where we used fine-tipped pipet tips to pick colonies, variant and wild-type, into a pattern with control strains, so that we could compare morphology directly alongside control samples. Otherwise it's too hard to shift your eyes from plate to plate. The plates were incubated at 48 hours and 37 degrees, and then incubated for up to two weeks to assess hemolysis, and in this case we found that the Remel blood agar produces better results than the homemade, and the difference here is in the blood agar base, they're slightly different in peptone versus tryptone, you wouldn't think it would make that big of a difference, but it does.

So here's a close-up of a pick plate. Here's wild-type Bacillus anthracis down here, this is the Ames ancestor strain, which we call 1981, this is one of the morphs, this is morph A, so you can see it's much more spreading, it has a moist bull's eye appearance in the center. Another morph that we saw originally in the Leahy - again, this is wild-type down here, this one does not have the bull's eye appearance, it does not have the moist center, it tends to be more pigmented underneath. I think that this is a result of more hemolysis and that may actually be the cause of some of that yellow pigment as well, I have no proof of that, but the strains that tend to be more hemolytic also tend to have more of that yellow pigment.

This is a different type of morph, um, this is wild-type and this is a morph that is smaller, I call this one opaque originally, it forms small very dense colonies that also tend to be a little bit different in color. This is what a pick plate looks like after 48 hours. These are the ones after 48 hours. This is after it's been refrigerated. So, there is a pattern that we put on each one of these plates - this is the wild-type strain, this is what we call the A morph, the first one I showed you on that pick plate, this is the B morph, and this the C/D morph, they're really identical in terms of morphology. And these are two picks from the same colony that I'm assessing - so, these on the outside are the controls, and these are the testers. And so in this case, what we did is we picked with a toothpick or actually a fine tipped pipet tip, from a single colony, and dipped it twice into the agar, so we have two - sort of look off up here - and this is a different I should say this is wild-type it looks most like this one. In this case there is wild-type, there's wild-type, there's the A morph, the B morph, the C/D morph, and it's kind of hard to tell because the hand is behind there, but hemolysis is the same between all of these, and this is a B morph, in this case. This was also tedious.

So, just as a little summary of some of the morphs that we looked at. The colors varied from grey-white to yellow and various versions of gray and yellow. The wild-type is medusa, it binds Congo Red well, it forms compact pitted growth areas on pick plates, it is shiny and pitted on new sporulation medium, which indicates that it is sporulating, and if you look at a subculture of that you can see the spores. It makes a little bit of pigment on new sporulation medium, but not much. Sporulation on L-D is excellent at 37 degrees, and it also makes spores at 28 and I'll get to that in a minute.

The A morph, the one you saw that had the bull's eye and the sort of moist center to it, is the largest of the morphs that I identified in these samples. It is slightly hemolytic, there is no pitting on NSM, lots of pigmentation on NSM, and this is one of the ways to sort of also divide out this morph from the others. It's a poor sporulator on NSM, it's a poor sporulator on L-D, it's a poor sporulator on sheep blood agar, and the temperature doesn't make any difference.

QUESTION: So can you explain the scale? Four plus - what does that mean?

Four plus in this case would be almost entirely spores.

QUESTION: Four plus is then - five would be?

I guess it comes back - I'm trying to think of my clinical days - there was nothing bigger than four plus. I don't remember where that came from.

QUESTION: Okay, and is plus minus greater or smaller than one to two plus?

It's smaller. It means that you might ocassionally see a spore, but not all of the time.

QUESTION: So, one percent or something like that?

Yes.

QUESTION: So, two would be noticeably defective?

Noticeably defective Yeah - we were not trying to quantitate this per se...

QUESTION: I understand. So all of these morphs were defective in sporulation?

All of them were defective in sporulation. The interesting one was the opaque, which although it didn't resemble the others in the least, in terms of morphology it didn't fit all the criteria that I was looking for, I just happened to notice this small unusual organism and thought it might be something other than Bacillus anthracis - ah this one is also defective in sporulation but it's temperature sensitive. That is it will sporulate at low temperature, but not at 37. And that - that's a totally different class of morph than I had ever seen before.

QUESTION: You say that, it's only under low temperature - so what would be the degree? Is it four plus?

As I recall, it was only about three plus. It wasn't a hundred percent - but there was a definite temperature effect.

So, just as a summary, these morphs sporulate better on blood agar than then do on L-D, and the E morph, the opaque morph, sporulates more efficiently at room temperature than at 37.

QUESTION: So, organisms that are defective in sporulation, as a population goes and sporulates, you can imagine that those may go through one or a couple more divisions, right? So, as you set up sporulation preps, poor sporulators may be significantly favored.

I don't have a lot of experience with preparing spores, per se, in my background, I have done some of it - but I would say that the conditions in which I saw these things arise most often are conditions that the organism should not have been subjected to. If someone is ignoring their cultures and not being very careful about the way that they're working with them, I would say that's the kind of situation where you tend to get these things popping up.

QUESTION: So, sporulation is the result of some sort of stress - you run out of something, or you're not happy or something - so if you have a problem with it, with sporulation, you keep going.

You keep going up to a point, and then they die out.

QUESTION: So, then they die out. But if someone keeps adding media, you could imagine this population become...

Yes - you can't leave them too long or what you see is- if you put them on sporulation medium, and you leave them to the point that they're truly out of nutrients, you'll see huge long filaments, very thin filaments, just before the culture dies. So it's - I think that, under conditions where you let it go on for two days instead of one day, then you subculture it - that's a situation where you might be more likely to see these than if you let it go twelve hours.

QUESTION: And you have seen, in your experience, the same morphs come up?

I can't tell you they are the same morphs, because we haven't done any genetic analysis. Things that look the same, and that's why we even started looking at sporulation - they looked enough like it, that.

QUESTION: Did you do microscopy, to see whether these morphs were blocked early in sporulation - or use some other criteria to judge?

I would love to do that, but this was sort of a small-scale operation here - ah - there are a lot of studies that would be interesting to do. I think that would be fun.

QUESTION: That was never done?

No. I didn't do it.

QUESTION: You say they look the same. Is that just on the initial plate, or did you take any of those things that pop up and subjected them to these other tests?

Okay, let me see if I understand the question. Isolates that I had from years before?

QUESTION: Years before, or if you take your Ames ancestor strain...

And do it again now?

QUESTION: Go to it two days...

I haven't done that study. Yeah, there are a lot of interesting approaches you could use with this, but I think thats - at the time, it seemed more like a basic science interest question, that was more interesting to me than to anyone else.

QUESTION: If I follow you correctly, the fact that you recognized these morphotypes, in some ways - I don't want to put words in your mouth, but you correct me where I'm wrong - you had seen some of these things before?

Yes.

QUESTION: And they in fact looked similar to things you had seen before?

Yes.

QUESTION: Okay, that's very important, because that means some degree of reproducibility, in the ability of these things to come up.

I don't know how these things specifically arose - I can just say how I've known them to arise in the past in my hands.

QUESTION: And the ones you've seen before are from Bacillus anthracis?

Correct - and not all strains of Bacillus anthracis do this.

QUESTION: The Ames strain does?

The Ames strain does, New Hampshire does, Vollum does not, and it actually - if we can go into scientific theory here - if you look at a whole series of Bacillus anthracis strains on Congo Red, the ones that are the darkest, sort of salmon-colored, that are picking up the dye the best, are the ones which tend to be the most virulent, and also tend to throw these morphs off the most. So I think that dye uptake is indicative of something about the ability to sporulate.

QUESTION: Just going in that direction, the Ames does, and the others don't.

Some others do.

QUESTION: Some others do, some do, some don't - but the morphs that you've seen before of the Ames, just to your general approximation, are any of the morphs that are in the RMR-1029 new, or have you seen them all before?

I don't think I ever looked at any of my old morphs as much as I looked at these. I don't think there was ever the level of intensity. As I said when I first worked with those strains, I was looking for some kind of correlation with virulence, and when I didn't find it they went to sit in the freezer and they never came back out.

QUESTION: Were any of the older morphs that you have just stashed away, has anybody ever suggested doing genetic analysis on those compared to...

They were submitted to the repository and they would have been screened along with everything else.

QUESTION: So even the morphs were screened, and were any of them among the ones that came up positive for one, two or three of the genetic tests?

I don't know. I did all my work blind.

QUESTION: And the ones who know the answer to that question?

The FBI has never told me specifically whose samples came up positive.

QUESTION: By specifically, I mean the genetic identity, whether these phenotypic similarities reflect genetic identity - not whether they are the actual material that...

And then how often those particular mutations pop up.

QUESTION: Absolutely - you're very clear. [multiple voices, inaudible] of a mechanism for selection, potentially, which is this little correlation, now when we heard anecdotal evidence that these things can be recognized on a plate but we don't know whether [multiple voices, inaudible] they were a genotype and they would have turned up positive and independently...

I will say that the one thing I can sort of say to that is, um, that - and this is what Dr. Losick had referred to in terms of the earlier paper on the asporogenic strain that I isolated that is a vaccine production strain, that isolate has a deletion in spoOA and it is a rather large deletion, and so I did get excited when I saw that, and go back and screen a number of these strains to see if it had that same deletion, and none of them did. So, at least that particular one is not a hotly reoccurring event, say like the pigmentation deletion locus in Yersinia pestis which happens routinely in a specific way.

QUESTION: I was wondering how many Ames morph variant isolates would you have frozen away, would you say, from all this past work?

I'm guessing between fifty and a hundred. And I don't know that they are all unique, because they've not been analyzed, other than they were sent to the repository and presumably they've been screened in that respect.

QUESTION: So, they were sent to the repository.

We had to send every Ames strain we had to the repository, yes. It didn't matter whether it was typical Ames or not - anything that had Ames on it went to the repository.

Alright, so we did, as we went along, analyze a subset of repository samples for variants, but as you can see from the flowchart, this is a really tedious way to go about this, and you almost need the same pair of eyes to look at everything - and when you have a thousand plus samples it really seemed to be impractical to go about it that way. So certain variants were selected that had been isolated from the evidentiary material, and they were submitted for genomic analysis in order to facilitate molecular genetic assays.

So here's just some of that early work having isolated an A type and a B type along with wild type from the Leahy, the Daschle and the Post. So, basically the way that this went down - I initially screened the Leahy and I pulled out a number of variants from that, and then I went back to the Daschle and Post and tried to find similar morphotypes in those samples, and the good news is I know wild-type when I see it, and the B morphs turned out well - all three of those had the same SNP. The A morphs were a lot more complicated, in that they all have a lesion in the same locus, but they were three different lesions - so there are hot spots in the chromosome that lead to identical or seemingly identical phenotypes, but they are not genetically identical. This sort of crosses over into Jacques Ravel and David Rasko's work, so I'm not going to go into it too much, but in all three spore samples we found this is a SNP between spoOF and and open reading frame, it's probably affecting initiation of sporulation - it looks like it is an early block from what little I've done.

Opaque is interesting, it can either be a 21 base pair deletion or a 9 base pair deletion, and it is actually in PX01. This is the only morph we've found that localizes to one of the Bacillus anthracis chromosomes. And this appears to be some kind of response regulators.

QUESTION: So it could be feeding in to the phospho-relay.

It could be - and the interesting thing is that because it's on that plasmid, and so many things on that plasmid are controlled by temperature, that does make you wonder about the temperature sensitivity of this phenotype. That would be fun to pursue. The A - numerous duplication types - it's not clear what the role of this is - it's located near a 16S rRNA, an open reading frame, a polysaccharide deacetylase - maybe Jacques and David will have more input on that by now, but it's never been clear why these strains are so affected in sporulation - and the CD morphs all in the same open reading frame - since they're histidine kinase, but all different kinds of things, SNPs and indels and presumably these are acting in phosphorylation of spoOF and possibly spoOA.

QUESTION: So it looks like at least two of those have multiple different genetic basis...

Yes.

QUESTION: ...even within the same gene...

Yes.

QUESTION: ...and I'm a little confused by this paranthetical notation, "all but one unique"?

That means that there was one amorph type found in multiple samples.

QUESTION: All the others were only found in one sample?

That's the last I've heard - now, I don't know how many more they've sequenced, if they have, but there were an incredible number of A types of all different genotypes.

QUESTION: Say that one more time? That's, that's... the morph was.. so saying the genotype was only found...

So, so the phenotype...

QUESTION: The genotype of the morph when subjected to sequence analysis...was more different...

Okay, so let's define what we mean by morph. Morph is phenotype - so, same phenotype, same locus, different types of mutation. Many different kinds of mutation.

QUESTION: So, do you think that - so, when you were doing these experiments, at the beginning you sort of raised the possibility that if you had to distinguish between those that had been outgrowth on your plates where you were characterizing the phenotype versus those that you thought were present in the admixture, if there is any way to relate that to whether you're one off?

Well, but the genetic analysis was done only on material directly from that first culture plate, so it wasn't from subcultures.

QUESTION: So, spores were plated out...

Yes.

QUESTION: ...in multiple colonies...

Yes.

QUESTION: ...and things that had this A-like phenotype were picked and then separately analyzed and each one...

Yeah. Can I draw - would that help? So, you can't really say it's an A morph when it first comes out, so you have four plates - you put int on plates and you pick what looks like aberrant colonies and some wild-type to. But all you can say at this point is that that looks a little bit different than the others - it's a little bit more yellow, a bit bigger, a bit flatter. Half of this colony goes into the -70. Half of it goes onto another plate. That plate is then -[inauduble]- That is the S1. So, most of the phenotypic studies, if I did repeats for example, using this material, I would use this, but this is what was submitted for genetics. Did that make sense?

QUESTION: They then did the DNA extractions directly from that -70 stock?

No, no they didn't do it directly from that -70, it was done as a primary overnight culture.

QUESTION: An overnight culture?

Well, like, from a freezer vial to a plate, and harvested the next day.

QUESTION: How are the cultures purified? Just kind of squished on a plate and gathered up?

Right, right.

QUESTION: Any reason they put it on a plate and not just do a 5 mL overnight?

Well, I was the one who decided that, probably. I would say in general you don't want to go from a glycerol stock to liquid, or I don't. It's easier to tell what kind of growth you are getting on a plate, it's less stressful on the organism, because they're more concentrated in one place, they can modify the microenvironment, so in general I never go from a -70 to a liquid culture.

QUESTION. Alright - so then, as a test - then, if you went back, and looked at the cells on the right that got streaked out and put in the freezer, do you see the same mutation - the identical sequence?

I didn't do that, so I...

QUESTION: That was never done?

I don't know if it was done or not. I prepared a lot of samples, I don't know what was done with all of the samples, I...

QUESTION: That would be a good test!

I can only tell you what I've done because some of it was outside my purview. I - we did harvest material from these and these also but I can't tell you exactly what was done with them.

QUESTION: They still exist?

The FBI has the samples.

QUESTION: So, either the frozen culture or the -70 culture - if you restreaked, do you see any reversion -[inaudible]-

No.

QUESTION, Always, it is an -[inaudible]-

Right, the only -[inaudible]- I've seen is - nd it's not reversion, if you take something that's, and this is going to be a -[inaudible]-, that's intermediate in genotype, that's only partially defective in sporulation, and you subject it to the same miserable conditions that causes mutants to pop up, you'll push it to where it makes fewer and fewer spores. So, I think you're getting secondary mutations in that case that pushes it more towards the asporogenic type. Does that make sense? But I've not seen them revert to wild-type.

QUESTION: It may not be to wild-type, it may be from A to B.

I've never seen them go in that direction - I've seen with other samples C-type things go to A or B. That is, it's somewhat productive, but if you put it under the right conditions it can be -[inaudible]-. And I think, Jacques and David can probably answer some more of these questions.

So, what did we conclude? That more than one stable variant similar to previously characterized strains was identified. The appearance of the variants as single isolated colonies after only eighteen hours of incubation suggested that the original samples contained a mixed population, and I think that there have been other studies that the FBI has done that perpetuates that idea, and a subgroup of those variants were submitted for sequencing, if you leave the plates longer, you see additional variants, but as we said, if you incubate the plates longer it's not clear why you're seeing those additional variants.

I also did a little bit of comparison of random colony picking versus, ah, the enrichment that I've described to you where you look for variants on a plate - just to see if it was really doing any good to look for them, or whether it was just as good to go randomly. So, the protocol enriched for most variants over the wild type. So if I picked colonies that looked like variants as opposed to picking them randomly, I did much better at finding variants, the exception being the opaque variant, E, in which case I was better off picking randomly to find that one, than in spotting it, and that has something to do with the bias in the visualization of that particular mophotype. We always got some colonies that we picked that we thought looked aberrant, but which turned out not to be - I think part of this is just the physical difficulty of looking at plates in this way. Colonies on the edge of the plate always look a little funny. Colonies that are coming from spores that are probably late to germinate are smaller and harder to assess, and those in close proximity to another colony sometimes look different as well. So that's one of the limitations of this.

So we isolated stable variants from evidentiary material. It was facilitated by an enrichment scheme based on morphology, we characterized them, placed them into classes based on phenotypic traits, and in many cases those with the same classes and similar though not necessarily identical genetic lesions, and in some cases the same variant - I should say some, rather than say - in some cases, the same variant genotypes were found within more than one evidentiary sample. Questions?

And actually while I'm thinking about it, I'll answer your question about Ames. The reason that Ames was a popular strain comes back to a paper from Steve Liddle back in the early 1980s I believe showing a group of Bacillus anthracis strains and their ability to overcome certain vaccines, and Ames was one of the ones that appeared to be vaccine refractory to a certain extent - that is, you couldn't protect animals as well against Ames - and that's what led it to be a popular challenge strain for vaccine studies. The other problem with Vollum, which had been used in the past, is that there - at least in my experience - there are a lot of Vollums out there, and no two of them look alike. So Ames had a good history, it had not been passed in the laboratory a great deal, and that's why it was a popular strain. Yes?

QUESTION: I know this is pure speculation on my part, but did you say that qualitatively, the portions of the different morphs in the different samples corresponded to your qualitative grading of their sporulation propensity?

I don't have a lot of experimentation to prove that, but I will say the ones that tended to be not totally defective in sporulation, that seemed to be in that intermediate area, were more likely to be seen than the ones that were severely affected. But I don't have the statistics to back that up.

QUESTION: I have a feeling that this wasn't something you directly did, but in working, to the best of your knowledge, in working with the design of the genomic tests to screen for these variants, did they consider whether those tests would have reacted to some of the similar but not identical mutants that you found -in other words, if they're involving rearrangements and you're designing a primer across those, could you tell if it was an 8 base pair deletion, versus a 12 base pair deletion?

I believe that they will answer that - I'm sure that - they're coming to speak, correct? Jacques? Rasko? Somebody?

CHAIR: Yes - we'll get them eventually, yes.

They were - then answer to your question is as far as I know yes, they can tell the difference - but they need to answer that question themselves.

QUESTION: They knew about the different...

Oh, yes. Well, they had to because they were the ones who sequenced them, yes. They were the ones who did the original sequences so they knew of all the variability. Anything else?

QUESTION: Do you know of anyone else in anthrax science who is also working on these morphs? Or is this something that you had come across...

It was pretty obscure so I think I was the only one interested in it for a long time. There's been more interest in them from a sporulation point of view and how it reflects control of sporulation. That's the most interest that I've gotten from it. Thank You.

CHAIR: Thank you very much, I want to see if there are any further questions from anyone else. Dr Keim is still here... - [audio cut off in mid-sentence] -

The complete transcript of Dr.Paul Keim's testimony before the NAS, Sept 24 2009

2009-12-12T15:08:00.000-08:00

Dr. Paul Keim and his research group developed the assays used to distinguish the Ames strain of Bacillus anthracis from other closely related strains. These assays played a critical role in defining the crime scene in the 2001 anthrax attacks, and a survey of the research papers produced by this group indicates a very thorough approach to the subject, as you'll see if you read the transcript.

However, he was not directly involved in the later work regarding 'morphs' - subvariants of the Ames strain.

This testimony is highly technical, but I'll try to parse it out later.

The original audio that this was transcribed from is available at the NAS website:
http://www.nationalacademies.org/newsroom/nalerts/20090925.html

Slides were shown with this presentation, but unfortunately the NAS did not see fit to post them online.

Transcript begins:

Dr. Paul Keim from Northern Arizona University

So, unlike the other speakers, I of course delivered my speech by slides five minutes ago, so we'll see if they actually come up alright.

Thanks very much for invinting me, I will try to encapsulate eight years of hard work into thirty minutes, so if it feels like you're flying at 40,000 feet, it's close - because the details, we've tried to publish, and most of the details of the analysis - not all, but most have actually appeared in the peer-reviewed literature already, and I have a long list of reading material for you to do at home. My understanding is you already have quite a bit of material to read.

So what I intend to do today is to go over what the Ames strain is, what we know about it, how we developed methods for detecting it and understanding it, a little bit about the history of that work, our involvement in the investigation, talk about the validation of assays and how those assays were used in the investigation of this crime.

So we were involved from right at the very beginning - this is a sample that came to my laboratory on a Thursday evening - it was a culture that came out of the cerebrospinal fluid of Mr. Stevens, the first victim. This culture came into the laboratory at about eight o'clock at night, and we spent all night analyzing it with the best methods we had at the time, which we had been developing over several years under funding from the Department of Energy. The next morning we were able to actually call the CDC in Atlanta and the FBI and tell them that this in fact was something that matched the Ames strain.

This was based on a DNA fingerprinting method that involved eight different loci in the genome of Bacillus anthracis that were developed in a pre-genomics era - so this was hard wet-bench laboratory work, it was necessary to develop this technology, and by current standards it seems like the stone age.

So we got a match, but what does that mean? Because at the time, there were lots of issues, and these are issues that are still relevant today, but we've tried to put them at rest over the last eight years.

So, first off, we had to be able to distinguish a natural event from a nefarious event - and to do that, we needed to know if the Ames strain was common in nature, was it your everyday Bacillus anthracis, or anthrax, that was spread around North America and across the United States, or in fact was it rare? Did it have only a domestic distribution in the United States, or did it have in fact a foreign distribution? In fact, at the time when this occurred, we didn't even know where the Ames strain was. At least the vast majority of the scientific community thought it was from Ames Iowa, as we found out later it wasn't.

And then of course what we finally wanted to do with this type of analysis was attribute the material that was in the letters back to a source, and to do so we needed an understanding of exactly what the Ames strain was. So when we started we were doing research tools, and they definitely needed to be validated, much like Dr. Schutzer was just talking about. This validation is something you don't do on a Thursday afternoon after someone dies of anthrax, in fact it takes lots of time.

And finally of course we were very aware that this might end up in court, and in fact we were hopeful that the evidence that we were developing would end up in court, and so we were aware of the -inaudible- we were carrying, we had constant interactions with the law enforcement community as well as the legal community, so we were thinking about admissibility in court at the same time.

So, this is a dendrogram which represented the state of the knowledge in 2001, it was something that I'd published in the journal Bacteriology in 2000, it was a survey of some 400 Bacillus anthracis isolates from around the world - it contained our resolution at that point in time with these eight loci that we were studying was 89 different genotypes, so these were unique types that we were able to resolve out of about 400 different types.

When we came up with the identity being the Ames strain, in fact it appeared to be somewhat robust, because there was only one isolate in this 400 that in fact matched exactly to what we found out of the first victim, and that was the Ames strain, and so this became widely known as genotype 62 - which was not the intention of this paper. Genotype 62 was merely a reference to this particular dataset - but it took on a life of its own, and for many years - it took me a long time to get the scientific community to stop calling it genotype 62, since it was only relevant to this particular technology. It appeared to be pretty robust, but in fact when you looked closer at this - and again, you've got to remember that this was all across the news media, that it was the Ames strain - "they know its the Ames strain" - but again, we weren't really sure what the Ames strain was at that point in time.

And so what we found was if you go back and look at that same paper, we only had 84 isolates from North America, so out of the 419 total, only 84 were from North America - since this is a North American case, this is really the relevant set of isolates that were in this study. Most of those in fact were from Canada, and the Canadians have a lot of anthrax, so we had a lot of isolates from up there - only 32 then were from the United States. Of those 32 then, we had 16 unique types.

So, suddenly our power from 89 unique types shrinks, if you only consider it to be North American, and so we end up with only 16 unique types. So this is like rolling a few sets of dice and coming up with a match - and so you can see that in fact our power to draw the conclusion that the material in the letters as being the Ames strain was really much more limited than was probably being portrayed across the country at that time.

So we spent the next several years in fact building the databases to get more isolates from North America, and building the tools to get better resolution, so that we could distinguish one type of Bacillus anthracis from another. One of the important innovations here was we omovbed from a statistical approach for estimating or relationships to what I'll call a phylogenetic approach, which is really a logic approach.

Bacillus anthracis is a clonally propagated organism, we have very good papers and datasets showing this, and because of that then as mutations occur you end up with a heirarchical arrangement of mutations, so for example you have a mutation here which is nested inside a mutation there - you can then distinguish these things based on the orderly arrangement of mutations in a population. ANd so that's much of what we did over the last seven or eight years is establish these relationships so that we could identify a particular type.

This mutation for example could be used for distinguishing Bacillus anthracis from Bacillus cereus, this mutation could be used for example to identify the Ames strain from all other types of Bacillus anthracis, and that's what I'll show you now.

The regions of the genome that we had been focusing on before in 2001 really occurred in what we call the VNTR loci - these are very rapidly mutating regions of the genome, which were discoverable again in a pre-genomics era by wet bench type experimentation. What we wanted to move to was very slow-evolving but numerous types of variation, or identifiers that we'll call SNPs, or single nucleotide polymorphisms. So there's a whole range of mutation activity in a Bacillus anthracis genome, and we wanted to move from these types to this type.

As Rita was mentioning earlier, really the approach to getting there required whole genome sequencing. In a pre-genomics era, discovering a handful of SNPs across a 5MB genome was essentially impossible - and so it wasn't until we had access to whole-genome sequencing that we were able to do that, and that's what we are looking at now. And so in conjunction with Claire-Fraisure Liggett and Jack Ravell, we were able to identify very important SNPs for identifying the Ames strain from everything else.

Here's an example of the whole genome sequencing tree that came out of this - actually, this became affectionately known as the Rellman strategy, David Rellman wrote a review of our project in Science, and for some reason it took on the moniker of the Rellman strategy for many years after that - but the point is that we were able to sequence genomes and identify SNPs that were very specific - that we were able to identify the very rare and not very numerous SNPs that were able to distinguish the Ames strain from everything else.

With these SNPs in hand, Jack Ravell designed an Affymatrix chip for genotyping, the genotyping chip which ended up with almost 3000 usable loci on it - there's probably only about 6000 or 7000 SNPs in the entire species of Bacilllus anthracis and we ended up with about half of them on this chip.

From this then we are able to generate what is probably the most accurate phylogeny of any single species on earth, and that's Bacillus anthracis. So these are the data that were generated from about 136 different samples, and so we were able to , with very fine accuracy, arrange these isolates across this phylogenetic tree, which we like to think of as the population structure of Bacillus anthracis.

Now, 3000 SNPs is still a lot, and these Affy chips are still somewhat cumbersome to do, so we wanted to move to single SNP or a smaller number of SNP assays. And for that we developed this concept of what we call canonical SNPs - so on a very long branch for example where we might have a thousands SNPs, we would pick one, and we would canonize it, if you will, and canonize it, and use it as a marker for that particular place in the population structure.

With it then, we could use more rapid assays to categorize an unknown as to whether it was part of this population or part of this population. So this is just representing a reduction in the data analysis from a few thousand SNPs down to just a handful, maybe as few as 24, depending on what you question is.

Now, this is the Ames branch itself, each of the little marks along here is a particular SNP, these are the very closest relatives to the Ames strain itself. So the Ames strain is here - again, this genome sequence was generated at TIGR, and from that then we are able to identify the SNPS that are relevant to identifying the Ames strain, and its very closest relatives. Here you'll see some Texas isolates, and then over here you see Chinese, which are the most closely related outside of Texas. This is the Ames strain itself.

The important thing then is these SNPs right here, which we would call the typomorphic? SNPs or strain-specific SNPs for the Ames strain itself. Now, how do we come to the conclusion that they are very specific for the Ames strain? Well, we did a lot of validation involving a lot of different isolates. I'll show you that now.

The assays that we like to work with are real-time PCR assays involving dual-probe competition for the SNP site during the amplification - these curves then are different amounts, so this is one nanogram, this is 10 femtograms - 10 femtograms of DNA in a test tube is about five genome equivalents - and so these assays are actually sensitive to single molecules, and I'll show you some more data on that which is published.

So they are sensitive to single molecules, and they're sensitive to a single nucleotide difference. So the tools that we developed are at the theoretical maximum of what an assay can do - you can't go below one molecule. Actually, we can - we can go to half a molecule, since DNA is double stranded. You give us half a molecule, and we can identify the Ames strain accurately. You've got to put it in a test tube - we don't deal with that side of it - but if you give us the test tube, we can tell you whether it's the Ames strain or not.

We went through extensive validation. We calculated it up a few months ago, and it turned out we ended up doing over 50,000 PCR reactions. 50,000 in this validation study - we looked at magnesium concentrations, both high and low, we looked at inhibitors that you might find in blood or you might find in the environment, we changed the cycling parameters, we changed the reaction components, we looked a near bacterial relatives, we looked at environmental backgrounds, and we did low level detection type validation.

So these are the kinds of things we went through to see if we had an assay, would it ever give us a wrong answer. Would it ever tell us it was the Ames strain when it wasn't, and would it ever tell us it was not the Ames strain when it was? And what we found was there was only one set of conditions which ever did that for us, and that was if we really contrived and changed the conditions of the reaction - thus, if we left out the probe for the non-Ames, we would then see everything as looking like Ames - but otherwise, it was impossible to change the results. We could kill the reaction, but we couldn't change the final result. It would either tell us it was the Ames strain, or it wouldn't work at all.

Again, so the low-level detection, I just want to reiterate that again, here's the 10 femtograms, which is about one and a half genome equivalents, we did a study of about 5,000 different reactions that were done right at the one molecule level. And what was very satisfying was that the results matched a Poisson distribution, perfectly. So if we could get that molecule in the test tube, we would see the result, and if we couldn't get it in the test tube, we wouldn't see any result at all.

QUESTION: David - what kind of matrices were used in order to assess that level of detection?

So, you mean environmental matrices? Yeah, so we took - there's an environmental microbiologist who works in our group, actually works in our university, Edward Schwartzen, he had a set of 60 different DNAs that had been pulled out of about a dozen different soil types, and so in that case what we did is soil extractions and then we did spiking experiments to see if anything that would be left in there would be detected. We also looked for background, that is we looked to see if any of the DNAs of the microbes he was looking at, which were total soil extractions, would give us a result. And we did in fact find some Bacillus cereus, non-Ames Bacillus cereus that would amplify, but nothing came up as Ames.

QUESTION: You're speaking of actual multiple different assays, are you not?

Yeah, and I'm almost at the point where I'm going to tell you what the assays were, but in fact there's a set of five assays, three that we consider the gold standard, so to speak, three of those are chromosomal, one is on PX02, one is on PX01. The PX01 assay is not 100% specific for Ames but it is a very good way to monitor for the PX01 plasmid. That particular assay involves about four other isolates from Texas as well.

QUESTION: And then will you mention how you use the results in conjunction with each other?

In conjunction? Well, they all agree 100%, so is that what you mean?

QUESTION: Well, in theory - but in practice...

Yeah so in practice, the assays do agree - I mean, the only one that would disagree would be the PX01 which is 99.7% specific for the Ames strain - there are three other isolates that are included in that. And so if we go back to the phylogenetic - this tree right here - so for example, these four SNPs are 100% specific, and we have real-time PCR assays for all four of these. Three of those are really good, and one of them doesn't detect down to a single molecule level, so generally we don't use that one, and then we have a PX02 assay that also appears to be 100% specific, and there is also a PX01 assay out here which includes these others. So that's the only one that would be incongruent, again, one of the assays wasn't quite as sensitive as the others in our validation test, so you could see it failing in situations where the other two didn't.

When you're at the Poisson level, when your down there sampling, you can also end up with one assay working and one not due to the sampling of the genome. We don't sample at the spore level, because we've extracted DNA and put in the DNA equivalent, and of course then you end up with a Poisson there. Does that kind of answer that question?

So the environmental background again, the inhibitors that would be there.

So these assays again were validated at the single molecule level, quite extensively so that they - these would be appropriate for environmental sampling and environmental testing. In addition, the first analysis was done by a graduate student under the supervision of a postdoc, and not a particularly good graduate student at that, and so it was important to get the personnel in place for this, and so one of the things that happened over the next few weeks and months - we had FBI-approved SOPs (standard operating procedures), we put into place chain-of-custody, we did proficiency testing on the technicians who were doing the analysis, we had a method for certifying technicians for microbial forensic analysis, instruments of course had extensive controls - probably more controls - I mean, on a 96-well plate we would have on the order of 16 real samples and everything else on that plate would be controls. We would do critical reagent testing before any forensic analysis was done, everything was witnessed and frequently there would be FBI agents witnessing, and then of course lots of blank controls during the analysis itself.

Rita kindly showed this dendrogram before, let me reiterate that these are canonical SNPs and so we have real-time PCR assays across this, again this is representative of the entire species of Bacillus anthracis. By doing a PCR reaction we are able to categorize unknowns into these particular areas and hence we could rapidly run through thousands of samples and tell you whether or not they were part of this group or part of this group or part of another group.

Here's the phylogenetic distribution of Bacillus anthracis across the world, here's that little tree I just showed you up here, there's 1033 samples in this particular analysis, as you can see a lot from North America, but also Europe, China, the Southern part of Africa, and so we know what the phylogenetic distribution across the landscape is. We've done extensive studes on this, and even since this was published in 2007 we've probably doubled the number of isolates that are in this map, especially in North America where we have much better access to material than in for example Russia, where we have almost none.

So let's zoom in on North America which is relevant for this. This is kind of the distribution of anthax across the United States in our collection back in 2001 and 2002. Hot spot up here in the upper Midwest, some over here in Nevada, and of course this cluster down here that proved to be the Ames strain, and then up here in the Canadian wood bison up there. That's kind of where anthrax occurs in North America. If you again look at the distribution of types, you see this very dominant blue type in Canada and the United States. That's what we call the western North America type, I'll tell you more about that in a second. And then there's a very thin sliver, I think it's right in here, which turns out to be the Ames strain group. So the Ames strain is in fact relatively rare in the United States, and only found naturally at least, in the southern part of Texas, along the Rio Grande river - more on that in just a second.

So this goes back to that 2000 850 SNP tree that we did with Jacque Revelle - as you can see, we've done a lot of work here in western North America, and again, the dot is the relevant population for the question that we were asking - if we blow that up, you can see that each is a different isolate that we've genotyped in this region, so again we know a lot about this western North America group, which was the blue group in that previous tree.

If we break that out, we find that there are a number of these nodes or separate genotypes within the western North America group - remember, this is nearly an identical genotype - so we are able to resolve, using subsequent assays, essentially a monomorphic type, essentially a number of different subtypes, importantly by using phylogenetic analysis as opposed to some other type of statistical analysis, we can assign ancestral nodes vs. derived nodes. That becomes very interesting when you look at the mapping of it across North America - it turns out the ancestral nodes map in the far north, up here in Canada, and then it becomes progressively more derived as it moves south.

So we propose a model for this type of Bacillus anthracis in North America involves a north-to-south migration, perhaps coming across Beringia during the last ice age, the Pleistocene or the early Holocene. So that's the type of Bacillus anthracis that's very common in North America, it's not the Ames strain. The Ames strain is down here in this group, very distinct and different - if we blow that up, we again see the Ames strain, we see these Texas isolates that are very closely related, and we see a whole group from China. So the Ames strain, or the Ames strain type, came from China, or that's a reasonable hypothesis, came into North America, and then somehow became established down here in the southern part of the United States, right in this region along here.

So there's a cline, or a separation, between the western North America strain and the Ames strain found down here, which dominates most of the isolates we have in our collection.

QUESTION: The slide that you showed with the circles, just a couple of slides back, that represents Ames or a variety of samples from the soil?

No, almost all of these isolates come out of - sorry - almost all these isolates come out of something that died. In fact, if you go into the environment and try to isolate Bacillus anthracis, even if you know something died there of anthrax, it's very hard.

QUESTION: If you go back to the global one you had, these are all either soil or animals that died, but they don't represent the breadth of Ames in the culture collections in the world?

No, not at all. In fact, this is supposed to represent a natural distribution, so if we know for example that you have the Ames strain in Porton Down, it would only count once. So if you go back to that original study in 2000, where we only found it once, we did not beef up our numbers, so to speak, by saying that we analyzed one from this lab and that lab and that lab - so if we knew that it came from a single progenitor, or at least we had reason to believe, we'd only count it once, and normalize the data.

QUESTION: Why, if Ames is not representative of the North American strains, how was it the strain that became used in the assault and testing?

Yeah, it's a historical happenstance, I mean, Pat can probably answer this better than I, but it came into USAMRIID at a time when they needed a highly virulent strain to replace the Vollum. The Vollum had been used as the vaccine challenge strain for years, you know it was just a time when they needed something that was working much better, and it really probably had more to do with its characteristics at USAMRIID in animal challenges and then the fact that they were the center that was really the leader - they were the scientific leaders in this area - so people started using it as the K12 of E. coli. It was a constant - it was the constant in all these other studies.

So that's the real reason - in fact I think there's some evidence that there are even hotter strains, more virulent strains out there, those data aren't particularly good - differential virulence in Bacillus anthracis is not a field I think has been studied as intensely as needed to be, but there are reports of more virulent strains and less virulent strains.

QUESTION: So is it correct to say that Ames is a strain that is used for testing and validation around the world, or only in the U.S.?

Well, the other laboratory which I know from personal experience that uses Ames a lot would be Porton Down. An after that, there was a lot of interest - in fact, prior to 2001 we were prepared to ship the Ames strain itself to a number of different laboratories, so that they could standardize their animal testing against what was going on in the U.S., and those laboratories decided they didn't want the Ames strain anymore, after 2001 - not that we would have been able to ship it anyway.

So, here's a blowup - I'm sure that you want to get to Pat, so I'll try to hurry along here. Here's a blowup of the region in Texas, the Ames strain itself came from right here, and that's down here, and then you see these other isolates. So when we realized it was the Ames strain, along with Alex Hoffmaster at the CDC, we made a concerted effort to go in and collect these. These aren't just like random strains that got mailed to me, pardon the pun, but they are strains that we had to go out and look for.

In other words to find the relevant population here, to be sure that we could identify the laboratory Ames strain from anything else, then we had to go out and look - and so what we have here, we're talking about a 5 MB genome, and so we have five SNPs here - that's it - five SNPs that differentiate the laboratory Ames from the isolates - and you know, if you're working with Vibrio, not only would these all be the same strain, probably you wouldn't differentiate the entire Bacillus anthracis as a different species at all. So the level of variation we're talking about here is unique in that it's so low. So again, five SNPs out of five megabases differentiate these, and the validation is that all of the Ames strains we've seen in the laboratory actually have these SNPs and are differentiated including the morphs when you get to that as well - and the morphs all contain these SNPs.

And again, this fits with evolutionary theory and dogma that we would expect that all of the derived strains from this carry those characteristics.

Okay - so the way that these SNPs or these assays were used in the investigation, after all that validation, was really to define the crime scene. So my laboratory - in 2001, the federal government was actually lacking in a forensics laboratory where they could also handle things that were BSL-3. So my laboratory became the, at least one of the repositories for the FBI for biosafety level three material that was also evidentiary. So as they collected evidence, for example from the mailbox in New Jersey, or from letters or spores that were collected in the AMI building, or from the Hart Building, all that material came to my laboratory, and we ended up with on the order of 2000 pieces of evidence being stored in our BSL-3 facility, all under chain of custody, in a way that would be admissible in court eventually. And all of that evidence was analyzed with these Ames SNPs to decide what was part of the crime scene, and what wasn't.

So, these spores that were coating the inside of this mailbox, in fact proved to be the Ames strain based upon those definitive SNPs, again we did a five panel assay on material like this, so that we got congruent results in all cases, the only ones again, David, that weren't congruent were the PX01, and those were only when we went to the Texas isolates.

So we defined the crime scene, and the crime scene was of course quite extensive - this is an FBI slide showing where the letters went - you've probably seen it before. Again, all of these were included with these assays, they all were part of that laboratory derived Ames culture, and different from anything seen in nature other than original isolation.

So this was all very important for defining the crime scene. But probably more important and less heralded was in fact our ability to exclude things from the crime scene. So in the last eight years we have numerous times gotten cases of natural outbreaks. The most, kind of one of the ones that was most important at the time was in November 2001 there were a number of cattle that died at the Hewlett Packard ranch outside of San Jose California, and this was a sample that was flown to us on a government jet, and we analyzed this one overnight as well, and it wasn't the Ames strain, it was something totally different. So what it meant then was that the FBI and law enforcement could not focus in on why cattle were dying near San Jose California, but rather focus back on the real crime scene. And again there were many examples of this, I won't go through them all - but another example would be the New York drummer, a very important result early on, I don't know if you remember this case, this is Mayor Bloomberg up here, and he seems to be quite concerned. But this gentleman was making drums from hides that came from Africa, we were quickly able to say it was not the Ames strain and was not part of what we knew - we said - was part of the crime scene. It might have been a different crime going on.

Again in 2001 I got a sample that had originally been collected by the UN, during the UNSCOM inspections of Iraq, and it was an isolate of Bacillus anthracis that came out of the weapons program that the Iraqis were doing in the 80s. And this proved not to be the Ames strain as well. That of course was very important for policy reasons and in the decisions that were being made as to where the crime might be was domestic or foreign. And there are other examples of exclusion that I won't go into.

I told you that I had a reading list for you, and I put this up here more just so that it's in the record for Erica, but we have since 2001 and of course more before, we published 43 different papers on this topic, on Bacillus anthracis, which doesn't include papers about plague or tularemia and so we publish on those as well - but there's a lot of papers. So it's out there, and the point is, in addition since 2001 I have given 120 public lectures on these same topics.

So, our work has been under peer review from the very beginning and that is an important part of the Daubert? critieria - if you are going to go to court, make it admissible. You've got to be out there and let the scientists take their crack at you - and that's something that we have been doing - and we have had to defend our work, and science, and modify our approach to things.

Out of this we came up with a paradigm for how we think you should approach forensic work, and it has to do with formulating hypothesis and then testing those hypothesis - you have to have population genetics to make it relevant, I'll just go back tothe example that Rita gave you before after we seqeuenced the Florida strain - we had a great sequence there, but we didn't know what to compare it to. It didn't mean a whole lot by itself, and we had to go back and develop these population databases - we got a paper in Science, but in reality we needed to do those population genetic studies to make it relevant. once you have that, then you can start to define specific hypothesis - you need to have the relevant population for comparing that, if you know if it's clonal or recombining you can come up with appropriate analysis for coming up with confidence estimation, and we definitely have confidence estimation built into our entire program. And eventually then you hope you come up with some clues or evidence that goes to court.

I want to finish by recognizing some people who were very important to this - Bruce Bedoulie, Mark Wilson, were critical, they were in my lab on a continuous basis. Jacque Revelle and I worked very closely together under the supervision of Claire. Albert Hoffmaster at the CDC has been important for gathering various collections, Beth George - if I was going to criticize the FBI, I will criticize the FBI, one of the biggest problems we had in the past eight years was in their contracting office.

In fact, we weren't able to get money from the FBI to do these analysis until May 2002, and instead it was Beth George and Pete Cintia at the Department of Energy who said, use our money, you've already got a contract in place, use our money and get this done. So I would say that for the next crisis it would be nice if the federal government had a couple of sugar grants out there - to get the money we had to do the work, because I still had to pay salaries, people had to go home and put gas in their car and feed their kids, and patriotism only gets you so far when you have to do things like that - so Beth George was a real hero in getting us the money - and Rita, as she pointed out, those sugar grants really got off the ground fast, and it was important to do.

So, questions now or questions later?

QUESTION: I should like to point out that um, the meetings that we had included speakers.

Right.

QUESTION: Paul Keim and others would present data, so, constant interaction.

Yeah, I was invited to those meetings many times, it's just that they were in Virginia and I live in Arizona. I did attend at least one of them, though.

QUESTION: It's not a forensic question, just a point of interest. So you don't detect anthracis in the environment? Whereas you detect other endospore forming species readily?

Yeah, but you pick one, and I'll say, find one strain and then let's make an assay for that strain, and you would have a hard time finding that one, too.

QUESTION: Well, subtilis is readily found.

Yeah, but subtilis is an incredibly diverse organism. So let's take one isolate from your laboratory, for example, and let's make an assay that is very specific for that isolate and let's go to the environment and see if we can find it, and I would say you would have a hard time finding it. You could find subtilis, but subtilis is kind of like E. coli, you know, it's really diverse. We're talking about - let's use the E. coli example. Bacillus anthracis is a clonal derivative of Bacillus cereus - it is less diverse as a species than E. coli 0157H sub, for example, at least I think it is. Rich may be able to correct me on this one. But that's an example of a very very defined clone that came out of E. coli, and it is still more diverse than Bacillus anthracis, and what we've done then is gone back and found a particular subclone within that. So Bacillus anthracis should probably never have been called a species. So we should call this Bacillus cereus subspecies anthracis.

But because it has such a dramatically different biology than Bacillus cereus - it causes catastrophic disease - classical microbiologists gave it species status - because of its really unique biology. But there is only a small number of genetic differences between it and Bacillus cereus. But your point is well taken - if you go out in the environment and look for something like cereus, you'll find it. You go out in the environment and look for Bacillus anthracis, I don't think you'll find it - and people have tried to do this in places where they think Bacillus anthracis should be. And BioWatch doesn't get any Bacillus anthracis hits, and they've been testing 10,000 samples per year for four or five years. So - it's a very defined, small set of bacteria.

QUESTION: Paul, if you were to, and I don't mean to put this the wrong way, but if you were to step back and say, critique your own work, where do you think the most work still remains to be done? Where are the needs the greatest? If this were to be taken to the next step, or applied tomorrow in the best possible way, what more would you like to see done, where?

Well, first off we were limited by technology - and over the last eight years technology has changed dramatically. Because we were working with Claire and had the support of the genomics community, I don't think that we were too far behind the curve - and I'm also a faculty member at TGEN which is also a genomics group. So we hopped on genomics as fast as we could. We didn't have the populations - we didn't have the samples - so even now, it would be better if we could go in and do a more extensive sampling of Texas and other areas. And the federal government has put all kinds of hurdles in our way - with the select agent rules, and the shipping - you know, we spend 40% of our time, Arturo, working on select agent rules. And that's the problem - it's hard to do this work, because of the regulatory constraints now. And what that means is you have to put twice as much effort into something to get anything back.

The other thing that happened when the select agent rules changed in 2002, there are a number of laboratories that destroyed their collections. The Texas State health labs, for example, destroyed hundreds of isolates that they collected over the years of Bacillus anthracis, and when they destroyed that we basically lost our forensically valid database of populations, and so - and I remember sitting in meetings where people in the federal government said, oh, they won't destroy their collections, they'll just ship them to you. No - that isn't what happened - they destroyed them. I'll name names later.

QUESTION: And I know you're sensitive about this issue - you just raised it - but how in your estimation, how representative are collections of B. anthracis um, elsewhere in the world? How representative is our notion of what is where around the globe?

It's hard to say. What we get is a snapshot in time. For example, what we have is an excellent collection from China - we have almost 200 isolates from China. But if you look at the dates, most of them were actually collected by one expedition to one province. Okay - so it's a snapshot of that one place in that one year. So we don't have good time series - other than maybe the United States right now. We don't have good time series - they don't go back very far - and they tend to focus on outbreaks. And again, we tried to compensate for that, but we don't always know - they have to be considered the best we've got, but also considered suspect at the same time.

QUESTION: You made the split early in your talk between natural and nefarious, and it seems as though you have knocked that one out of the park. Leaving aside any other evidence, it's very clear that this was not a natural outbreak- but part of our charge is related to - given that it was nefarious, the evidence line there, and I'm wondering whether you have any thoughts or
- what do you think we as a committee should be paying particular attention to?

I mean, it's easier to critique my own work than that of others - but what it comes down to, is the committee is going to have to look at the morphs and the frequency numbers very carefully. In my mind, I would really like to know whether those morphs are under selection under the conditions that were used to raise them - I suspect they are - but there are no experiments that I know of for that. Uh - I wonder if you would go out and do 35 batches of spores, at 10 liters, would you see that same repertoire of morphs again? I think you would. I think that those morphs are inevitable given the growth conditions that were there.

Now, that said, how many times has a batch of spores like RMR-1029 ever been produced? Well, I think it's only ever been produced once, you know, at least in that combination.

And, so those are the types of questions I should be asking, is about the selectability of those things.

Rita Colwell COMMENT: I'd like to emphasize that what needs to be done and what should be done is to continue the investment in the sequencing of the genomic work that the various agencies have funding - that funding should continue. It is really important to have multiple genome sequences of the given species, in order to understand the context

Yeah, so the original "Rellman strategy" was to sequence the most diverse isolates - of course, now we're at the other end of the spectrum and you have to convince people that you have to sequence things that are really really close together to tell them apart - so we'll call that the Keim strategy.

So, Rich, the other thing about that is you know, with the morphs, the competition experiments that you do, like with your populations, would be so straightforward and so relevant, you know, as you will or have heard, there are assays for those morphs, you can set up these growth experiments and do the competition and see how they perform versus the wild-type very easily, and I think that will give you a lot more insights to how you end up with that set of morphs inside of something like RMR-1029.

QUESTION: So Paul, earlier you said that Bacillus anthracis may not be a species but would be the tail end of Bacillus cereus, so you have analyzed 2000 strains, so it is pathogenicity as the one critieria that you have made as Bacillus anthracis, or how did you - what kind of markers did you come up with to say this is Bacillus anthracis?

Yeah, well, I got a multipart answer. First off, I would not propose changing the name Bacillus anthracis now. There are too many legal and regulatory complications to that, so we're going to leave it as a species because of the rest of the world. Scientifically, we know where it sits. Our analysis doesn't involve any pathogenicity - so we look at the pattern of the nucleotide variation inside of this, and we come up with a phylogenetic tree, and it fits the criteria for what we would call a monophyletic clade, so it's a single group - we can define SNPs or other markers that say, everything that contains these falls into this group, and legally, regulatory and traditionally we call that Bacillus anthracis.

QUESTION: So it is possible that these strains might not have PX01 and PX02?

Sure, absolutely. Many examples of strains that are now attenuated for lots of different reasons, besides the plasmids, and we would still call them Bacillus anthracis - but we would presume that they were virulent, or that their ancestors were virulent.

QUESTION: Part of our charge is to talk about validation of the methods used - I just feel that I have to ask the question. Early on, versus now - how much catching up did you have to do in validation?

Tremendous, tremendous. I mean, the level of validation that you do for a research paper that goes into Science - especially if it goes into Science, or the Journal of Bacteriology, is very different from what you do for forensics. Things that we absolutely knew were true, we still spent six months and a lot of money to prove. If you add EDTA, if you add humic acid, melony, all these things - you know, one of the objections - Bruce was sure, that if we went to the single molecule level, and we ran it lots of times, sooner or later a stochastic event would occur, that would give you the wrong result, and it never happened. The reason is the stochastic event would have been a polymerase mistake - I used to work in DNA replication - it would have been a polymerase mistake where it would have put in the wrong nucleotide - and that mistake we've measured, many times, and it would have occurred at 10^-5, 10^-6. So we would have had to run a million times to have seen that mistake - and we didn't, we only ran 5000 reactions. But even then, if we had put in a single-stranded piece of DNA - we are actually starting with two, not one - and so it would have looked like a heterozygote - and we've done lots of analysis on mixtures as well.

So, we didn't see it - but that was the type of scientific critique that we were getting and we were responsive to. And again, is someone were to tell you to prove that a polymerase makes a mistake at 10^-6 for a paper that you publish, you'd say forget it - but for this purpose we did it. We did three years on these assays, trying to break them, and all we could do was get them to fail altogether, we couldn't get them to give us the wrong answer.

QUESTION: The investigation obviously focused on mutations that could be correlated with -inaudible-. But there must be mutations that were silent - inaudible - can you think of -inaudible- comparison of these samples by deep sequencing could have -inaudible- the samples, or separated them?

Yeah, in defense - my role in the morphs is really in handling the material, the live material, and extracting DNA. We were quite busy doing other things at the time, so we weren't involved in assay development and validation - I assume you are going to hear from people who did that. So let's assume for a moment - this is not proven - that these morphs actually have a selective advantage under large growth, such as we saw. And so what that means is that if you repeat this experiment, you know, normally we like to compare results against a random model. So let's go out and repeat the experiment a thousand times so that we get a confidence estimation of point zero zero one. Well, I would guess that 99% of the time, you're going to see these morphs again, if you repeat this experiment the very same way.

So they're not a random event - I mean, the event itself is random but the numbers hear are very large - you're dealing with 10^12 or 10^15 spores, and you're dealing with two or more generations, so even rare events are gong to happen, predictably. The question is, why do they become such a large frequency? My guess is, they're actually under selection. So if instead you were to go to silent mutations, like you're suggesting, and there are doubtlessly - when you have numbers this big, every mutation you can imagine has occurred in that population. If instead you use a repertoire, and you can go to very much higher numbers than four, you use a repertoire of neutral and silent mutations, you would come up with something like a distinctive fingerprint for this type of a batch that would not be replicated inevitably if you repeat the experiment again. Rich actually knows more about this than I do, so you can ask him that in closed session.

QUESTION: So the answer is -inaudible- developed turned out to be indels, -inaudible-

Yeah, so I've hear Claire talk about this, so I'll paraphrase what I've heard her say in public. She said they didn't want to use SNPs because they weren't as stable as indels. And the reason she says that, is when you have an indel, and it's the right kind of indel, you've got to qualify that, that piece of DNA comes out of the chromosome and it's gone. So there's really no way for it to come back in - at least, that's a pretty valid assumption for certain types of indels - not all of them. The worry - the way she states the question is, if you have a SNP, it can mutate from a G to an A, it can mutate back. At least, that's the logic that she's used for not using SNPs. Now in a population context, and if in fact those SNPs are indeed related to a morph, or a phenotype that's under selection, you do worry that that selection could reverse and it could go back.

But in the total structure of Bacillus anthracis, we see very very few reversals. You know, we did a study that was published in 2004 in PNAS where we looked at, I think it was 1500 SNPs, and we saw, I think it was four reversals in the entire population - and it turned out those were amino acids and so there was probably some kind of selection going on there - for the most part, we don't see that happening. So you pick your SNPs right so they're really solid, maybe intergenic - they're unlikely to revert back - and plus, you could use many of them - hundreds, thousands, certainly you could do thousands. You could do a whole chip. Now the chip itself would not give you that type of information from RMR-1029, because in that case all those SNPs would have been fixed in the population - you are looking for new SNPs that occurred after the derivation of the Ames strain, in fact after the construction - during the construction of RMR-1029.

QUESTION: Did you say that anyone who took large, multi-liter batches and concentrated them over time, you would create selection pressure to create those morphs?

First off, I'm speculating, make sure that you understand that, I don't actually know - but I do wonder if in fact these morphs are under selection for growth conditions, such as growing ten-liter batches. So if you go back - it's not the concentration part, it's the growing the big batches, and the 35-some batches.

The indels - the other thing about these types of indels is they tend to occur at higher rates than SNP mutations. Most indels occur between directly repeated pieces of DNA or some other structure in the DNA. And in fact, Pat Worsham will talk - she may or may not talk about this, but she's actually studied one locus that creates and asporogenic morphotype that seems to happen - you can see it more than once. And so indels can happen at a higher frequency due to genome structural things - and so they occur at higher frequencies, maybe 10^-7, 10^-8, it depends on the particular one. So you're even more likely to see those in these large populations, and then if they have a selective advantage, they'll come up to a frequency that's easily observable. Now - ask Pat that question later.

QUESTION: I just want to clarify one thing. I liked your process at the -inaudible- investigation, and one of the aspects of it was the development of population - inaudible - what's been done so far?

Yeah. We really relied on the CDC and natural surveillance to get access to these. One of the things that has happened in the changed regulatory environment is that people have not wanted to cooperate - they haven't been allowed to save isolates, you know, there are large ranches for example in that part of Texas that have outbreaks of anthrax on a regular basis, and they won't report it, so I'm not sure the answer is sending in black helicopters to pick up dead cows, but that's the kind of situation we're in, where we have not been able to get access to material to define that - so we only had four or five new isolates from that Ames branch and then we were into China. And so that is - everything says that we've got it nailed - but it would be nice to have a hundred samples instead of five.

QUESTION: Paul this is just for clarification - someone mentioned earlier that there had been some laboratory work looking at stability of some of these mutations. Now I don't know whether the comment was made with respect to morph mutations, or lineage - you know, specific mutations and I am assuming that if it were the latter, they have been looked at experimentally.

Yeah, I don't know anything about the morph stability. I was not involved in that part of the investigation other than tangentially. But the lineage mutations we've validated by looking at, now, 2000 independent isolates around the world and we do see a very very small amount of reversion. So they're very stable, and again the five SNPs we've used for assays have been looked at very closely.

QUESTION: But no experimental work, serial propagation?

Well, I've know Rich for a long time - so we did a Linsky experiment on Bacillus anthracis back in the late 1990s, and we had it sitting in the freezer, and it was done with the Ames strain, in fact, and so the loci we were working with were the VNTR loci, and we had seen a small number of mutations in those loci, because they mutate at such a fast rate. I'm not sure we've ever done the SNP analysis on those, because we really didn't think it was worth the trouble.

QUESTION: Could you repeat that? What mutations did you see?

So the first typing system or fingerprinting system that we used is what's called a VNTR, variable number of tandem repeats...

QUESTION: Oh, I see, you didn't look for SPOs for example?

Now we did not - but you know, I've cracked open vials from ATCC and seen multiple morphs, Pat's the expert on this. But the SPO mutation - I'll tell you, when we selected for Cipro mutants we saw all sorts of SPO mutations - and you know, Cipro is a mutagen as well as an antibiotic, it's a mutagen if you're a bacteria anyway, and so there were a number of SPO-type mutations that did occur, probably due to deletions, since it's a double-strand break mechanism. I think SPO mutations are selected against in nature, of course, because you've got to have that spore for the ecological infective cycle, but...

QUESTION: Not necessarily in a flask?

Not in a flask, not when you're growing 35 ten-liter fermentation batches, or whatever it was.

MODERATOR: Well, thank you for your 121st lecture, here with us, since 2001, on this subject. Once again, our committee has...

-end transcript-

This certainly seems to validate all the work used to identify the strain used as the laboratory Ames strain - but what it does not address is the validity of the "morphs" which form a key element in the FBI claim that Bruce Ivins was the culprit. More on that later.

Introduction to the subject of 20th century biological warfare programs

2009-12-12T14:56:00.000-08:00

The history of biological warfare development by the world's 20th century superpowers has been covered by numerous authors, but the purpose of this blog is to consider how the legacy of those efforts has persisted into the 21st century.

The first topic we will be dealing with is the 2001 anthrax letters, postmarked September 18 2001 and October 9 2001, which resulted in five deaths, multiple hospitalizations, the closure of the legislative branch of the federal government, and mass panic on a nation-wide scale.

Almost a hundred years ago, one of the first people to consider the use of biological warfare noted that while such weapons might be ineffective on the battlefield, they might very well be used to "harass civilian populations" - a prophetic statement indeed.

I'll also try to point to some of the positive efforts being made by individuals and agencies to halt the spread of biological weapons. This blog intends to take a science-based approach to the subject, and will also hopefully serve as a repository of information that will be useful to journalists and others. It is not intended to serve as an arena for discussion - sorry, but I don't have time to moderate comments, and there are other excellent blogs on related subjects that do provide that service.