Biological Agent Production- part 2, Bacterial

* Adapted from US Dept of Homeland Security, Advanced Chemical and Biological Integrated Response

Basement Biological Terrorists

•What prevents those willing to attempt the effort from succeeding?•Clearly success is possible; what thwarts their efforts?

Obstacles to Home Production

Access to Starting Material

• Remember Larry Wayne Harris– Harris may not be a an actual terrorist threat, but he is

proof that a capable microbiologist can also be “a little nuts”

• Remember state-sponsored BW programs– Large scale production produced and stockpiled tons of BW

• Legitimate research is performed on biological threats every day– Physical plant security, individual integrity, scientific

publications are always at risk

Biological Terrorist vs. Legitimate Facility

Biological Terrorist vs. Legitimate Facility

Biological Agent Production

Large Facilities

Large Facility Interior

Agent Production Hazards

• High volume/quantity• Aerosol hazards (especially with dry agent)• Waste disposal• Pathogenicity (lethal vs. incapacitating)

Safety Equipment

• Biological safety cabinets• Personal protective

equipment (PPE)• Building engineering

– Airflow– Filtration– Dedicated physical support

(lab within a lab)– Decontamination capability – Waste disposal equipment

(steam sterilizers)

Biosafety Cabinets

• Biosafety cabinets (hoods) provide effective containment for moderate to high risk microorganisms

• Glove boxes are a form of biosafety cabinet

Likely Methods of Production

• Bacteria- Liquid -Solid culture media

• Virus/Rickettsia-Cell culture-Animals-Eggs

• Toxins-Extracted from animals or plants-Fermentation for bacterial toxins

Bacterial Production

Bacterial Production

Bacterial Production

Bacterial Production

Bacterial Production – Seed Stock

Bacterial Production – Starter Culture

• The preparation of a bacterial weapon begins by growing a small amount of the SEED STOCK on a petri plate or in a small amount of broth medium.

Bacterial Production – Scale Up

• The bacterial STARTER CULTURE is used to feed larger amounts of liquid medium. Each step produces increasing numbers of bacterial cells.

Biological Toxin Production

• General Features–Extraction processes/fractionization

-Columns, salts, solvents, centrifugation

–Filtration–Possibly drying equipment

Ricin Production

• Castor plants found as agricultural and ornamental plants throughout the United States

• Castor oil is used as a lubricant and in paints, coatings, plastics, antifungal compounds, shampoo, and cosmetics

• 5 seeds = approximately 2 lethal doses

Ricin Production

• Grind seeds and remove oil from pulp (press)

• Remove residual oils • Water extraction of ricin• Concentrate toxin• Mill for proper particle

size (if needed)

Ricin Facts• METHODS OF DISSEMINATION:

– Indoor Air: Ricin can be released into indoor air as fine particles (aerosol).– Water: Ricin can be used to contaminate water.– Food: Ricin can be used to contaminate food.– Outdoor Air: Ricin can be released into outdoor air as fine particles (aerosol).– Agricultural: If ricin is released into the air as fine particles (aerosol), it has

the potential to contaminate agricultural products. • ROUTES OF EXPOSURE: Ricin can be absorbed into the body through

ingestion, inhalation, or eye contact. Ricin can be absorbed through abraded skin or through wounds, but probably not through intact skin, unless it is carried in a solvent that enhances absorption. Ricin can also be transmitted through the skin via small pellets or projectiles designed to carry toxin.

*from CDC.gov

Ricin Facts• Ricin inhibits cells ability to make vital proteins, eventually causing cell death• INHALATION SYMPTOMS: DIFFICULTY BREATHING, FEVER, COUGH, NAUSEA, AND TIGHTNESS

IN THE CHEST. HEAVY SWEATING MAY FOLLOW AS WELL AS FLUID BUILDING UP IN THE LUNGS. LOW BLOOD PRESSURE AND RESPIRATORY FAILURE MAY OCCUR, LEADING TO DEATH. IN CASES OF KNOWN EXPOSURE TO RICIN, PEOPLE HAVING RESPIRATORY SYMPTOMS THAT STARTED WITHIN 12 HOURS OF INHALING RICIN SHOULD SEEK MEDICAL CARE.

• INGESTION SYMPTOMS: VOMITING AND DIARRHEA THAT MAY BECOME BLOODY. SEVERE DEHYDRATION MAY BE THE RESULT, FOLLOWED BY LOW BLOOD PRESSURE. HALLUCINATIONS, SEIZURES, AND BLOOD IN THE URINE. WITHIN SEVERAL DAYS, THE PERSON’S LIVER, SPLEEN, AND KIDNEYS MIGHT STOP WORKING, AND THE PERSON COULD DIE.

• SKIN AND EYE EXPOSURE: RICIN IS UNLIKELY TO BE ABSORBED THROUGH NORMAL SKIN. CONTACT WITH RICIN POWDERS OR PRODUCTS MAY CAUSE REDNESS AND PAIN OF THE SKIN AND THE EYES.

• DEATH FROM RICIN POISONING COULD TAKE PLACE WITHIN 36 TO 72 HOURS OF EXPOSURE, DEPENDING ON THE ROUTE OF EXPOSURE (INHALATION, INGESTION, OR INJECTION) AND THE DOSE RECEIVED.

* From CDC.gov

Recent Ricin Events – F.L. Mirarchi and M. Allswede

• Three US Senate office buildings closed on February 3, 2004, after ricin was found in the mailroom that serves Senate Majority Leader Bill Frist’s office. No injuries were reported.

• On February 4, 2004, as part of the ongoing investigation as to the source of the 2004 ricin attack, the Secret Service acknowledged that ricin had also been found at a White House mail-processing center in early November 2003.

• A vial containing ricin was also found at a post office in Greenville, South Carolina, in October 2003. The envelope, addressed to the US Department of Transportation, was labeled “caution RICIN POISON.” The letter, protesting a proposed federal limit on the number of truckers’ hours behind the wheel to go into effect in January 2004, was signed “Fallen Angel.”

• Officials suspect that the attacks in October 2003 and November 2003 are related because both letters were signed “Fallen Angel” and contained ricin of poor quality. The FBI is currently investigating whether these two earlier attacks are at all related to the ricin found in the Senate mailroom.

• In Minnesota, 4 members of the Patriots Council, an extremist group that held antigovernment and antitax ideals and advocated the overthrow of the US government, were arrested in 1991 for plotting to kill a US marshal with ricin. The ricin was produced in a home lab. They planned to mix the ricin with the solvent dimethyl sulfoxide (DMSO) and then smear it on the door handles of the marshal's vehicle. The plan was discovered, and the men were convicted.

• In 1995, a man entered Canada from Alaska on his way to North Carolina. Canadian custom officials stopped the man and found him in possession of several guns, $98,000, and a container of white powder, which was identified as ricin.

• In 1997, a man shot his stepson in the face. Investigators discovered a makeshift laboratory in his basement and found agents such as ricin and nicotine sulfate.

• In December 2002, 6 terrorist suspects were arrested in Manchester, England. Their apartment was serving as a "ricin laboratory." Among them was a 27-year-old chemist who was producing the toxin.

• On January 5, 2003, British police raided 2 residences around London and found traces of ricin, which led to an investigation of a possible Chechen separatist plan to attack the Russian embassy with the toxin. Several arrests were made.

Staphylococcal Enterotoxin B (SEB)

Staphylococcal Enterotoxin B (SEB)

• Studied and produced by US Military in 1960’s as an incapacitating agent.

• Easily produced and aerosolized• Most exposures result in debilitating symptoms

that persist for up to 2 weeks• Rumored (but never confirmed) that the US

used it during WWII and had planned to use it in a potential Cuban invasion during the Cuban Missile Crisis

Viruses

• Viruses are a group of parasites that consist of very simple entities.

• No more than DNA or RNA inside a protein package. • Will not grow outside a host cell.• In the laboratory, viruses are commonly grown in embryonated

eggs, tissue culture or laboratory animals.• Diseases caused by viruses include:

–Influenza–Common Cold–Ebola–Smalpox

www.csa.com

Virus Production• Three Methods: 1)Infection of embryonated eggs

•Advantages: cost, less expertise required, moregeneralized equipment

•Disadvantages: yield/egg 2)Growth on mammalian cells in tissue culture

•Advantages: higher yield•Disadvantages: cost, greater expertise required, specialized

equipment 3)Growth in live animals

•Advantages: can grow organisms that won’t grow elsewhere, maintain virulence

• Disadvantages: handling, cost, greater expertise required, lower yields for effort

Virus Production

• Embryonated Eggs (Dry Form) –Fertile eggs (12 days) –Virus injected into chorioallantoic membrane –3 day incubation –Membranes harvested (5x108viruses/egg)* –Remove undesirable solids/concentrate –Stabilize and freeze dry

Virus Production

• Tissue Culture –Established cell lines

•Cells grown on plastic plates or in liquid suspension

–Infect cultures, incubate 3 days –Remove cells from plastic plate and rupture them to release virus –Dry

Example Virus Production Results

Summary of Key Biological Agent Production Techniques

Summary, cont.

• Production of biological agents relies upon the ability to produce a live or active product

• Methods include fermentation of bacteria, growth and purification of toxins, and growth of viruses in eggs, tissue cultures, or animals.