Saturday, February 28, 2009

NY Times "Postscript" to Scott Shane's article on Ivins of Jan 4, 2009

A two-paragraph article in today's NY Times, sans byline, appears to end discussion of the so-called "chemical signature" said to identify the source of water used to grow the anthrax letter spores:
On Tuesday at an American Society for Microbiology conference in Baltimore, an F.B.I. scientist, Jason D. Bannan, said the water research ultimately was inconclusive about where the anthrax was grown. An F.B.I. spokeswoman, Ann Todd, said on Wednesday that the bureau “stands by the statements” of Dr. Bannan.
It is a postscript to Scott Shane's major article on Bruce Ivins dated January 4, 2009, which reported that a "chemical signature" of the water in which spores were grown pointed to Fort Detrick, Md.


Sandra's ex-boyfriend said...

Dr. Michael reportedly is suggesting Leighton–Doi broth leads to silicon signature observed.   Leighton Doi medium broth was used to grow the virulent Ames provided the Ann Arbor researchers for testing of biocidal and decontamination agent. (It was used in growing the virulent Ames in the cited 1995 Vaccine article which involved an aerosol spore challenge.) The scientists cite the 1995 article in describing how the virulent Ames was prepared in numerous patents, presentations and published articles. The FBI apparently suspects that heat shock was used and had the effect of increasing the permeability of the outer membrane with considerable individual spore variability. Ivins and co-authors also used Leighton-Doi in connection with an article submitted June 2001 and revised October 2001. The FBI questioned Mrs. Ivins about co-authors Mara Linscott and Patricia Fellows.  Dr. Ivins' attorney had told Bruce to stop emailing Mara and Pat.  Mrs. Ivins in her note to him the day he died asked why he was paying his attorney so much if he was not going to take his advice.  Bruce was leaving Mara anonymous gifts. (After being his lab assistant, she had gone on to receive her M.D.)  Other culture mediums, however, result in a spike for silica as illustrated by the Edgewood study and so the issue certainly requires further confirmation and elucidation.  By way of some background, an authoritative source on the chemical composition of spores recommended by by an FBI scientist is the 1969 The Bacterial Spore, with chapter 7 by a Dr. Murrell.

Ed Lake said...

I seriously doubt that Dr. Michael ever suggested that Leighton-Doi leads to the observed silicon signature in the attack anthrax.

He just reported that two known instances of growing Bacillus anthracis using Leighton-Doi resulted in silicon showing up in the spores. It's not known what the anthrax culprit used. And all three instances resulted in silicon being found in different percentages of the spores.

Two examples isn't enough to "suggest" that Leighton-Doi medium leads to finding silicon in spores. It's just a possibility that needs further exploring.

Sandra's ex-boyfriend said...

Ed Lake said that the attack anthrax was "probably" made using L-D. (March 1, 2009 10:24 AM)

You have no basis to disagree with Mr. Lake.

Iron Man said...

Wunschel et al., “Detection of agar, by analysis of sugar markers, associated with Bacillus anthracis spores, after culture,” J Microbiol Methods. 2008 Aug;74(2-3):57-63. Epub 2008 Apr 12, we see that D. Fetterolf of the FBI provided irradiated Ames. The authors explained "determining information about growth characteristics remains a challenging task that has only recently begun to be addressed. It is vital to be able to assay trace contaminants. Furthermore, in chemical analysis of such a precious sample there is only a finite amount of material that can’t be replenished (e.g. by growth or polymerase chain amplification, PCR, as for molecular diagnostics). Markers for agar which is, of course, a constituent of all widely used solid media could be used to differentiate prior growth in liquid media. Other components of media may be indicative of a specific medium (e.g. one containing animal constituents, such as sheep blood agar …)” While I am not a scientist, does the iron point to use of sheep blood agar rather than Leighton-Doi? (see posted metal analysis of L-D). Perhaps in considering the presence of iron, of the 100+ -300 with access, it would be relevant (and it may be highly probative) if some of those used sheep blood agar.

Dr. Michael concluded that the source for the silicon was probably due to its source being in the culture medium -- Ed has no basis for disputing the FBI on this. Wasn't sheep blood agar used rather than Leighton-Doi? And wasn't the high silicon level inside due to the heat shock method used which increased the permeability of the outer membrane with considerable spore variability? Isn't the fact that sheep blood agar used rather than Leighton-Doi exculpatory of Dr. Ivins?

Iron Mistress said...

Dr. Ivins and co-authors used both sheep blood agar and Leighton-Doi.

"On sheep blood agar plates containing heat shocked culture material from both sheep blood agar cultures and Leighton-Doi medium cultures, there was confluent growth." See also 1995 Vaccine article describing method used to prepare the Ames used in testing the Ann Arbor biocidal and decontamination agent that looks like skim milk.

But you are right the indication would appear to be that the attack anthrax had been heat shocked in the course of its preparation. The amount of silicon detected exceeded the numerous samples reported by Murrell in Chapter 7 of The Bacterial Spore. The FBI has refused to give Dr. Michael the AFIP data which suggests that additional silicon was added to the culture medium (see NYPT post data) and then centrifuged. And so Dr. Michael's conclusion and science is sound -- it was in the culture medium. But once the AFIP data kept from Dr. Michael is taken into account it points to the Microdroplet Cell Culture method.

Rusty said...

The study Bruce co-authored with Pat and Mara, revising it October 2001, involved both sheep blood agar and Leighton-Doi

"B. anthracis isolates Ames, Vollum 1B, Zimbabwe and Namibia were each inoculated onto 5% sheep blood agar and incubated overnight at 37 °C. The following day, an inoculum was prepared from each plate by suspending a loopful of growth into 5 ml of phosphate-buffered saline (PBS). Two-liter Erlenmeyer flasks containing 250 ml of Leighton–Doi [10] broth were inoculated with 0.2 ml of the cell suspension and incubated at 37 °C for 3 days with moderate shaking of 100 reciprocating strokes per minute. The spores were harvested by pelleting at 10,000×g, washed twice in sterile water for injection (McGaw, Inc., Irvine, CA), suspended in 1% phenol and stored at 4 °C. Just before challenge, the spores were heat shocked at 60 °C for 45 min. The appropriate dilution for challenge was made in sterile water for injection and prepared in a 0.2 ml dose."

But so were earlier studies submitted in 2000 and 2001.

In vitro correlate of immunity in a rabbit model of inhalational anthrax Vaccine, Volume 19, Issue 32, 14 September 2001, Pages 4768-4773
M. L. M. Pitt, S. F. Little, B. E. Ivins, P. Fellows, J. Barth, J. Hewetson, P. Gibbs, M. Dertzbaugh, A. M. Friedlander

Efficacy of a human anthrax vaccine in guinea pigs, rabbits, and rhesus macaques against challenge by Bacillus anthracis isolates of diverse geographical origin Vaccine, Volume 19, Issues 23-24, 30 April 2001, Pages 3241-3247

P. F. Fellows, M. K. Linscott, B. E. Ivins, M. L. M. Pitt, C. A. Rossi, P. H. Gibbs, A. M. Friedlander

There was no scientific evidence that pointed to Dr. Ivins as opposed to all those who had access to the flask (including any individual who could have gained access surreptitiously, and so the means, motive, modus operandi and opportunity of the crime needs to inform the investigation.

Purvis said...

The FBI has reportedly refused to give Dr. Michael the AFIP data. This compartmentalization has led both young and experienced investigators and scientists alike to lack necessary puzzle pieces and information. FBI Director Mueller imposed the compartmentalization at the same time he failed to take action with respect to the intentionally misleading leaks regarding Dr. Hatfill by the father of the lawyer for Al-Timimi. Director Mueller's judgment, discretion and leadership on those accounts has only proved unsound in hindsight -- when it led to conclusions that are indefensible based on the evidence disclosed to date.

Sandia's Dr. Michael likely has never even been advised of the fuller list of the articles in Ayman Zawahiri's possession at the time he was charting his plan to infiltrate UK and US biodefense through the recruitment of specialists. Here is a list of a small portion of the articles found in his possession. The more interesting ones were not released under FOIA.

Mangold, T, and Goldberg, J. (1999). Plague Wars: The Terrifying Reality of Biological
Warfare. MacMillan, Great Britain.

Morris, EJ. (1955). A selective medium for Bacillus anthracis. J. Gen. Microbiol.

Pearce, TW, and Powell, EO. (1951). A selective medium for Bacillus anthracis. J. Gen.
Microbiol. 5:387-390

Roberts, TA. (1965). Sporulation of Clostridium botulinum type E in different culture
media. J. Appl. Bacteriol 28(1):142-146.

Roberts, TA, and Ingram, M. (1965). The resistance of spores of Clostridium botulinum
type E to heat and radiation. J. Appl. Bacteriol. 28:125.

Semple, AB, and Hobday, TL. (1959). Control of anthrax: Suggestions based on survey
of imported hides. Lancet ii (3 October): 507-508

Thorne, CB, and Belton, FC. (1957). An agar-diffusion method for titrating Bacillus
anthracis immunizing antigen and its application to a study of antigen production. J. Gen.
Microbiol. 17:505-516.

Ajl, SJ, Kadis, S, and Montie, TC. (1970) Microbial Toxins. Academic Press, New York.
Anderson, RM, and May, RM. (1991). Infectious Diseases of Humans: Dynamics and
Control. Oxford University Press, Oxford.

Batty, I and Walker, PD. (1965). Colonial morphology and fluorescent labelled antibody
staining in the identification of species of the genus Clostridium. J. Appl. Bacteriol.

Hodgkiss, W, and Ordal, ZJ. (1966). The morphology of the spore of some strains of
Clostridium botulinum type E. J. Bacteriol. 91:2031-2036.

Knisley, RF. (1966). Selective medium for Bacillus anthracis. J. Gen. Microbiol. 13:456.

Knisely, RF, Swaney, LM, and Friedlander, H. (1964). Selective media for the isolation
of Pasteurella pestis. J. Bacteriol. 88:491-496.

Proceedings of the Conference on Airborne Infection. (1961). Bacteriol. Rev. 25:173-

Smith, H (1988). The development of studies on the determinants of bacterial
pathogenicity. J. Comp. Pathol. 98:253-73.