1. I n t e g r i t y - S e r v i c e - E x c e l l e n c e Headquarters U.S. Air Force DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09 Pathogenic Ecology: Where have all the pathogens gone? Anthrax: A Classic Case Wesley W. Walker, Capt, USAF, BSC Branch Chief, Public Health Readiness & Entomology USAF School of Aerospace Medicine Air Force Research Laboratory

2. DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09 Additional Contributors Johnathan A. Kiel a, Carrie J. Andrews a, Amy De Los Santos a, Roy N. Adams a, Matthew W. Buchholz a, Shelly D. McBurnett b, Vladimir F. Fuentes a, Karon E. Rizner a, and Keith W. Blount a a Biosciences and Protection Divison, Human Effectiveness Directorate, Air Force Research Laboratory, Brooks-City Base, TX b Conceptual MindWorks, Inc., Brooks City-Base, TX

3. DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09 Pathogenic Ecology ☣ Ecology of a pathogen: The natural relationships to animate and inanimate components of the environment that support the sustainment of a pathogen in the environment or prohibit it’s sustainment ☣ Ecology that yields infectious disease: Relationships of animate and inanimate components of the environment with an introduced pathogen that allow for the establishment of disease in a new environment Blount, K.W.; The Occurrence and Movement of Francisella Tularensis McCoy and Chapin Across Landscapes.; PhD Dissertation, Texas A&M University, 2007

4. DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09 4 Original Project: B.a. Fate in the Environment Original Focus: What happens to B.a. after it enters the soil? ☣ Is it washed downward into the ground and preserved? ☣ Does it move across the surface via precipitation and collect in low-lying regions and water basins? ☣ Does the environment cause the organism to genetically change due to “environmental pressures?” ☣ Are different sub-strains of B.a. within a region genetically “related” to each other?

5. DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09 5 B.a. Characteristics: Animal Mortality Spore Potential General rule of 1ml blood per kg body weight Bacillus anthracis spore potential of various mammals

6. DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09 6 Documented Longevity of B.a. “Gruinard Island was heavily contaminated with the spores of virulent Bacillus anthracis during biological weapons trials in World War II. Annual soil sampling from 1946 to 1969 (unpublished data) showed that although the numbers of spores were slowly declining, measurable contamination was likely to persist until well into the 21st century.” Manchee, R. et al.; Formaldehyde Solution Effectively Inactivates Spores of Bacillus anthracis on the Scottish Island of Gruinard; App. & Env. Microbiology; 1994

7. DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09 NEWS RELEASE Texas Animal Health Commission Box l2966 Austin, Texas 78711 (800) 550-8242 FAX (512) 719-0719 Bob Hillman, DVM Executive Director July 2005 FOR IMMEDIATE RELEASE Anthrax Confirmed in Sutton County, Texas Two ranches in Sutton County have laboratory-confirmed cases of anthrax in horses, deer and cattle, and laboratory results are pending for several other sites in the county, where livestock and deer losses have been reported. Although this bacterial disease occurs almost yearly in this region of the state, cases have not been confirmed within Sutton County for more than 20 years. Typically, outbreaks are in Val Verde, Edwards, Kinney and Uvalde counties, but on rare occasions, cases have been confirmed as far south as Starr County, reports Dr. Thurman Fancher, director for Area 6 (West Texas) of the Texas Animal Health Commission (TAHC).

8. DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09 July 2005 Sutton County, Texas DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09

9. DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09 Adult doe (~120 lbs alive) which expired ~10-12 days prior Body cavity had “popped” (+100°F > 2-weeks), allowing B.a. to sporulate Sample swabs taken from nostrils, throat and abdomen All samples POSITIVE for Bacillus anthracis (Ames Strain) Note: With the generally accepted formula of each ml of blood containing ~1E+9 B.a. spores; this deer specimen had the potential to produce ~5.5E+11 (550 Billion) B.a. spores concentrated within a few square feet. B.a. Outbreak Investigaion Sutton County, Texas—July 2005 DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09

10. DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09 Remains mostly intact—skeleton located at original site Soil samples taken on/around site where carcass found Samples were taken under/downhill (>1% grade) of carcass Samples were taken within the first ½” of soil, then a second taken ~1”-1 ½” below the surface. This was based on the assumption that spores exposed to UV light would degrade, whereas spores beneath the soil would be protected. B.a. Recovery Trials Sutton County, Texas—February 2006 DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09

11. DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09 ☣ 32 samples were taken on day 1 (not counting controls) ☣ Additional 32 samples were taken on day two ☣ BONUS: 17 samples from a newly discovered deer and bobcat (both ~6-months post-mortem and within 25’ of the original specimen) B.a. Recovery Trials Sutton County, Texas—February 2006

12. DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09 Soil Sample Results : ALL NEGATIVE ALL NEGATIVE B.a. Recovery Trials Sutton County, Texas—February 2006 DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09

13. DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09 Additional Field Recovery Trials Vickers Ranch—Jim Hogg County, Texas (Jun 06) ☣ Site of the original case of Ames strain B.a. (1981) ☣ Original site was identified and soil samples taken in immediate vicinity ☣ Soil sample results: ALL NEGATIVE Fulbright Ranch—Jim Hogg County, Texas (Jun-Sep 06) ☣ Site of large anthrax outbreak in 1975—100+ cattle died ☣ Pasture was identified and soil samples taken ☣ Burial mound of bull that died from B.a. was sampled (soil) ☣ Front leg bone of bull recovered for PCR analysis ☣ Soil/PCR sample results: ALL NEGATIVE

14. DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09 14 re·search (rē -sûrch) verb: to search again “Sampling showed that the impact of the aerial bomb had caused considerable contamination…” “… a smaller area on the SW coast was sampled. This was the paddock (corral) where sheep exposed to spore clouds were tethered for observation after each trial, and sheep died of anthrax there. Here, fluids rich in anthrax spores spilt from body orifices, producing ground contamination. Sixty samples were taken from this site, but none contained detectable numbers of anthrax spores.” Manchee, R. et al.; Formaldehyde Solution Effectively Inactivates Spores of Bacillus anthracis on Scottish Island of Gruinard; App. & Env. Microbiology; 1994

15. DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09 15 re·search (rē -sûrch) verb: to search again Publication focused on the reoccurring anthrax outbreaks among bison in a Canadian wildlife refuge. “…seasonal conditions modify host resistance, leaving the animals in a weakened immunological state. This hypothesis was developed to explain the apparent lack of B.a. spores recovered from the soil of affected areas.” Dragon, D. et al.; The Ecology of Anthrax Spores: Tough But Not Invincible; Can Vet Journal; 1995

16. DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09 16 Pathogen Ecology: Environmental Influences on B.a. Spore Survival Positive ☣ Alkaline soil (pH > 6.0) ☣ High nitrogen level in soil ☣ Decaying vegetation ☣ Temperature > 15.5  C ☣ Arid soil conditions ☣ Alternate rain/drought Negative ☣ Acidic soil (pH < 6.0) ☣ Soil with good drainage ☣ Sunlight/UV Rays ☣ Competing bacteria ☣ Flooding/dilution ☣ Steep pitched terrain ☣ Diminished vegetation Note: These environmental influences on B.a. are also key environmental influences on plants

17. DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09 Saile E, Koehler TM, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2006, p. 3168–3174 Vol. 72, No. 5 0099-2240/06/$08.000 doi:10.1128/AEM.72.5.3168– 3174.2006 Copyright © 2006, American Society for Microbiology. All Rights Reserved. Alls/Gifford Strain B.a.: Parker, Kiel, Gifford, Alls, US Patent 7,279,320 B1, Oct 9, 2007 Where is B.a. hiding? “In light of our findings, we suggest that investigations of so-called hot zones, areas in which anthrax epidemics in cattle and wild game occur regularly, should include sampling of roots and rhizosphere soil.” “In conclusion, analysis of isolates confirmed the transfer of plasmid pBC16 and fertility plasmid pXO12 between two B. anthracis strains in the rhizosphere of grass plants in our model system.” Pathogen Ecology: Beyond The Animal Model

18. DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09 Enhanced Soybean Plant Growth Resulting from Coinoculation of Bacillus Strains with Bradyrhizobium japonicum “These results showed that B. thuringiensis is a soybean growth promoting bacterial strain whose effects are stable across the various tested experimental conditions.” Pathogen Ecology: Beyond The Animal Model Yuming Bai, Xiaomin Zhou, Donald L. Smith, CROP SCIENCE, October 2003, p. 1774–1781 Vol. 43 Copyright © 2006, Crop Science Society of America. All Rights Reserved. Legume/Rhizobium Nodules are Red. This is due to the production of Leghaemoglobin which sequesters oxygen. This helps to create a low oxygen environment

19. DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09 Legume Inoculation Trials Preparations: 1.Beaker 1=10ml Bacillus anthracis Sterne (ST) strain (vaccine) spores + 10ml LabLab Bradyrhizobium spp. 2.Beaker 2=10ml Bacillus anthracis Alls/Gifford (curlicue Sterne) strain( AG) spores + 10ml LabLab 3.Beaker 3=10ml Bacillus thuringiensis Javelin® strain (BT) spores + 10ml LabLab Bradyrhizobium spp. 4.Beaker 4=20 ml LabLab Bradyrhizobium spp. only-control ☣ 16 plants per treatment (4 samples=beaker 1-4);64 plants total. ☣ Each plant’s root system soaked in respective cocktail for 60 seconds. ☣ Plants potted in 5” pots—~1000ml Miracle-Gro® potting soil 1.5” deep ☣ Watered initially with 100ml H 2 O and as needed thereafter. ☣ Plant growth was documented through digital photographs taken weekly. ☣ After 40 days of growth, one plant (plant #16) of each treatment (BT, ST, AG, & control) was collected for purposes of isolation/recovery of original inoculums.

20. DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09 SampleBAP CT-16-A1 a Numerous soil bacterial-no visible colonies of any of the original inoculums, picked one colony from A b ST-16-A, ST-16-B Moderate amounts of well isolated Sterne-like colonies, picked one colony each from A and B ST-16-CSmall amounts of well isolated Sterne-like colonies, picked one colony from C AG-16-A1, AG-16-A4, AG-16-A5, AG-16-B Suspicious colonies, but not textbook B.a.g., picked three isolates from A and one colony from B BT-16-A1, BT-16-A2, BT-16-A3 Numerous Bt-like colonies, picked two Bt-like and one B. cereus-like type from A BT-16-B1, BT-16-B2, BT-16-B3,Moderate # of Bt-like colonies, picked three colonies from B BT-16-C1, BT-16-C2Few Bt-like colonies, picked two colonies from C Ba Sterne Control Textbook colony morphology for each isolate with no visible contamination Bt Javelin Control Ba A/G Control a CT=control, ST=Sterne, AG=Alls/Gifford, BT= Bt; b A= main root B= root midsection, C= root apex Legume Inoculation Trials

21. DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09 B.t. (Javelin® Strain) O/N ̃ growt h4xC1 F/RPS-4 F/R†Pa F/R2† Caps F/R BT 212F/547 R^ BT 841F/119 7^ Bt spore CTRL ++ n/t --++ BT-16A1 +- n/t --++ BT-16A2 +- n/t --++ BT-16A3 ++ n/t --++ BT-16B1 +- n/t ---- BT-16B2 ++ n/t --++ BT-16B3 ++ n/t --++ BT-16C1 ++ n/t --++ BT-16C2 ++ n/t --++ Legume Inoculation Trials: Bacillus thuringiensis ^ BT primers designed to positively identify Bacillus thuringiensis commercial formulation Javelin (serovar kurstaki); ^L. Masson, Y. Lu, A. Mazza, R. Brousseau, M. Adang, “The CryIA(c) Receptor Purified from Manduca sexta Displays Multiple Specificities”, J Biol Chem 35, 20309-20315, 1995. † denotes primer set specific for pX01 plasmid in Bacillus anthracis

22. DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09 B.a. (Sterne Strain) O/N growth4xC1 F/RPS-4 F/R†Pa F/R2†Caps F/R Ba Sterne spore CTRL ++ n/t +- ST-16A1 ++ n/t +- ST-16B ++ n/t +- ST-16C ++ n/t +- Legume Inoculation Trials: Bacillus anthracis B.a. (Alls/Gifford Strain) O/N ̃ growth4xC1 F/RPS-4 F/R†Pa F/R2†Caps F/R Ba A/G spore CTRL +-+-- A/G-16A1 +-+-- A/G-16A4 +---- A/G-16A5 - n/a A/G-16B - n/a † denotes primer set specific for pX01 plasmid in Bacillus anthracis

23. DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09 Final Summary ☣ Bacillus anthracis may not persist in contaminated soils over long periods of time, but may have found a safe haven in plants ☣ This passes the scientific sanity test ☣ B.t. residing within nodules provides protection to the root system from arthropods ☣ B.a. residing within nodules provides protection to the root system from browsers/grazers. See R. Johnson, Epizootiology and Ecology of Anthrax, USDA, Animal and Plant Health Inspection Service (APHIS), Veterinary Services (VS), Centers for Epidemiology and Animal Health (CEAH), Center for Emerging Issues(CEI), 1971-2001 http://www.aphis.usda.gov/vs/ceah/cei/taf/emerginganimalhealthissues_files/anthrax.epiecol.draft.pdf

24. DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09 Future Studies ☣ Complete disappearance of “stable pathogens” from the environment may warrant a few follow- up questions: ☣ Has the pathogen found a new niche within nature to survive? ☣ Does nature hold the key to an effective “green remediation” of this pathogen?

25. DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09 Parting Thoughts “We still do not know one thousandth of one percent of what nature has revealed to us.” - Albert Einstein “Nature does nothing in vain.” - Aristotle

26. DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. 311 HSW/PA No. 09-244, 1 Jun 09 Contact Information Capt. Wes Walker, USAF, BSC USAF School of Aerospace Medicine Brooks City-Base, Texas 78251 Commercial: 210.536.2589 e-mail: wesley.walker@us.af.mil