1. Introduction and History of
Biological Warfare Agents
Dr Kamran Afzal
Classified Pathologist and Microbiologist

2. Al-Quran 17 : 53
When We Bestow Favours on Man,
He Turns his Back and Holds Aloof.
But when Evil Befalls him,
He Grows Despondent!

3. I. Introduction

4. Types of Biowarfare Agents
 Bacteria
 Cause disease by reproducing
 Single cell organism
 Typhus, anthrax
 Viruses Anthrax
 Multiply only inside host cells
 Sub-microscopic organisms
 Ebola, Chikungunya
Ebola

5.  Rickettsia
 Larger than viruses
 Smaller than bacteria
 From fleas, lice and ticks
 Q-fever
 Toxins
 Poisons from living things
 Botulinum most lethal known : <10-6 g
 But some beneficial uses

6. CDC - Category A Bacteria
Bacterial Agent Disease
Bacillus anthracis Anthrax
Franscisella tularensis Tularemia
Yersinia pestis Plague

7. CDC - Category A Viruses
Viral Agents Disease
Arenaviruses - Viral Hemorrhagic Fevers
Lassa, Junin, Machupo
Filoviruses – Viral Hemorrhagic Fevers
Ebola, Marburg
Variola major Smallpox

8. CDC - Category A Toxins
Toxin Name Disease
Clostridium botulinum toxin Botulism

9. CDC – Category B
Agents Disease
Coxiella burnetti Q fever
Brucella species Brucellosis
Burkholderia mallei Glanders
Ricinus communis Ricin Toxin
(castor beans)
Clostridium perfringens Epsilon toxin
Staphylococcus Enterotoxin B

10. CDC - Category C
Agents Disease
Nipah virus Viral Hemorrhagic Fevers
Hanta viruses Viral Hemorrhagic Fevers
Tick-borne hemorrhagic fever Viral Hemorrhagic Fevers

11. Key Production Techniques - BTW Agents
Type of agent Low-tech production Hi-tech production
Bacteria Batch fermentation, Genetically engineered
production in animals strains, continuous- flow
fermentation
Rickettsiae and Viruses Cultivation in eggs, mouse Culture in mammalian cells
brains, or tissue culture and grown on beads
Protein toxins Batch fermentation and Cloning of toxin gene in
purification of a bacterial microbial host, extraction
toxin, or extraction of toxin of toxin
from a plant or animal source
Non-protein toxins Extraction from plant or Cloning of a series of
animal source genes, each governing
production of one of the
enzymes needed to
complete a step in the
biosynthetic pathway

12. Production of Biological Agents
by Fermentation

13. Biofermentor

14. What Is a Biological Weapon?
 Uses a living organism
or its toxic agent
 Delivery device
 Both conventional and
unconventional means of
delivery
MAPW (Australia)
Bio & Chem Weapons 2006

15. A PRIMITIVE BIOLOGICAL WEAPON
DELIVERY DEVICE - Aerial Bomb
Explosive
Thin fragile aluminium cylinders filled with
nitrogen under pressure to create an aerosol
and release organisms when the bomb lands
MAPW (Australia)
Bio & Chem Weapons 2006

16. Biological Warfare (BW)
 Biological Warfare (BW) is defined as
‘Intentional or threatened use of viruses, bacteria,
fungi, or toxins from living organisms to produce
death or disease in humans, animals, or plants’
 BW agents can cause widespread casualties with
minimal logistic requirements
 BW agents are easy to produce, economical for
deployment, selective to the target
 Cost of 50% casualties per meter square is “US$ 1”
as compared to conventional weapons (US$
2000), nuclear armaments (US$ 800),
chemical agents (US$ 600)

17. Biological Warfare- A deadly mystery
 Man-made Epidemic of unprecedented scale
 Wide spread morbidity
 High mortality with minimum logistics
 Easy availability of agents from:
a. Universities
b. Biological Research Organizations
c. Biological Production Units
d. Clinical specimens
 Easy deployment through simple aerosol devices
 Incubation period of the BW agent

18. Biological Terrorism
 Use of biological agents to intentionally produce
disease or intoxication in susceptible populations
– humans, animals, or plants
‘to meet terrorist aims’
 Biological agents are much deadlier than chemical
agents
 Estimated 10 grams of anthrax could kill as many
people as a ton of the nerve agent Sarin

19. Features of Bioterrorism
 Weapon: Microbe or toxin
 Strike: Premeditated
 Goals: Political, religious, ideological
 Motivation: Fear, disruption, instability
 Molecular Biology and Genetic Engineering have
enabled scientists to increase the virulence, develop
antibiotic resistant strains, and create novel strains for
which population lacks immunity

20. Biological Terrorism? Epidemiologic Clues
 Tight cluster of cases
 High infection rate
 Unusual or localized geography
 Unusual clinical presentation
 Unusual time of year
 Dead animals

21. The Potential of Bioterrorism
Agent Lethal infective doses in 5mls
Cyanide 50
Mustard gas 100
Sarin 5,000
Botulinum toxin 1,000,000
Anthrax 50,000,000,
Tularemia 50,000 x 106

22. Worst case scenario- WHO Estimates, 1970
A release of 50 kg agent in
an area with population
5 million….
 Anthrax
 250,000 cases
 100,000 deaths
 Plague
 150,000 cases
 36,000 deaths
 Tularemia
 250,000 cases
 19,000 deaths

23. Biodefense

24. Threats…
 Naturally occuring outbreaks – pandemic influenza
 Small intentional outbreaks
 Inadvertant outbreaks – research labs
 Large scale attack
 New encounters – Legionella, Monkeypox
 New organisms – SARS, Nipah virus
 Limited
 Anthrax Letters
 Scary, but very small risk to a small number of people

25. Preparation for BW Defense
 Strong Intelligence
 Strict Bio-Security
 Advance Inoculation (Army and Scientists)
 Extended Scientific Research (Public Sector)
 Creation of Awareness (Service and Medical)
 Personal Protection of Masses

26.  Awareness to the use of protective agents/devices like
respirator MOPP Device
(Mission Oriented Protective Posture – battle dress over garment)
 Extension of genetic engineering
 Scientific vigilance through internet
 Continuous monitoring/surveillance in the field of air
bacteriology/virology etc

27. II. History

28. Emperer Barbarossa - Tortona
1155
Used infected dead bodies to poison the enemy’s water supply

29. The Tatars threw plague infected cadavers by
hurling machines into the city of Caffa- Ukraine
1347–1353

30. Siemenowics- a Polish artillery general
1650
He put saliva from rabid dogs into hollow spheres for firing

31. Gen Sir Jeffery Amherst
1754-1767
Offered infiltrated smallpox infested blankets to un
suspected American Indians during French-Indian war

32. Smallpox Pandemic
1775-1782

33. Dr. Luke Blackburn, future governor of
Kentucky - War between the States
1879-83
He attempted to infect clothing with smallpox and
yellow fever and then sell it to unsuspecting Union troops

34. Japanese Tests with BW agents
1932-1945
More than 1,000 of Chinese, Koreans, Mongolian,
Soviet, American, British, and Australian prisoners were
estimated to have died in experiments by the Japanese
with agents causing anthrax, botulism, brucellosis,
cholera, dysentery, gas gangrene, and plague - Unit 731

35. British trials with B. anthracis
were held on Gruinard Island, Scotland
1941-42

36. US Army established BW research
station - Camp Detrick
1943-1969
 Operationalized 7 months later
 By Jan 1944, field station for Horu Island was functional
 By 1969, US Dept of Defense completed study on fol BW
agents
 Incapacitating agents
 Rickettsia, RVFV and VEE virus
 Lethal agents
 Yellow fever virus, Bacillus anthracis, Rickettsia rickettsiae,
Yersinia pestis

37. Umbrella gun to assassinate Bulgarian
exile Georgi Markov - London
1978
A pellet was designed to contain Ricin toxin

38. The Rajneeshee cult
1984
Salmonella in Oregon restaurants - 751 cases

39. Aum Shinrikyo cult- Tokyo Subway
1995
Sarin Gas Attack, Tokyo Subway- 12 killed; 5,000 injured

40. Iraqi Biological Warfare Program
1995
 166 bombs
 100 botulinum toxin, 50 anthrax,
16 aflatoxin
 25 Scud missile warheads
 13 botulinum toxin, 10 anthrax,
2 aflatoxin
 122-mm rockets filled with
 Anthrax, botulinum toxin, and
aflatoxin
 Spray tanks capable of being
fitted to a fighter aircraft or
remotely piloted aircraft, and
spraying 2,000 L

41. Bioengineering “Super bugs”
1998

42. Anthrax Bioterrorism
1998
San Francisco Chronicle, 20 December 1998

43. Anthrax through post - US
2001
22 Cases: 5 deaths, 11 inhalational, 11 cutaneous

44. NBC Events since 1970
March 1995 Sarin
12 Dead, 5500
Affected
May 1995 April
Plague 1997
1984 U235
1972 June 1994 February 1997
Salmonella Sarin
Typhoid Chlorine
200 Injured 7 Dead,
200 Injured 14 Injured,
500
Evacuated 2001 Anthrax
5 dead
??? Injured
1992 June
Cyanide 1996
1984
Botulinum March 1995 Uranium
Ricin
December
1995
1985 April Ricin
Cyanide 1995
Sarin
November 1995
Radioactive
April-June 1995 Cesium
Cyanide,
Phosgene,
Pepper Spray

45. Pakistan’s Stance On Biowarfare
 Pakistan is a signatory nation of
“The Biological Toxin Weapons (BTW) Convention of 1972”
 Signed by 158 nations
 US has rejected enforcement
 Convention abstract:
 The prohibition of the development, production,
stockpiling, and destruction of bacteriologic and toxin
weapons
 Required to submit information annually to the United
Nations concerning facilities where biological defense
research is being conducted

46. Pakistan’s Experience of Bioterrorism
 A total of 230 suspected samples of Anthrax from 194
sources were analyzed for anthrax spores at NIH from
Nov 2001 to March 2002
 71 samples were from clinical specimens
(anterior nares, skin, blood)
 159 were from non-clinical environmental samples
(powders, swabs from inanimate objects, papers,
envelopes, packages, plastics etc)
 The samples were received from Foreign mission, media
organizations, banks, government institutions,
universities, hospitals and individuals

47.  Out of these, 141 samples yielded growth suggestive of
Bacillus species
 On the basis of colony morphology, Gram’s stain and other
preliminary laboratory tests 62 isolates were found
suspicious for B. anthracis, however all the samples were
negative by animal inoculation
 The suspected anthrax parcel/letter bombs in Pakistan
during the investigation period were hoaxes

48. Challenges in Pakistan
 Smart detection, field preparedness
 Would require quick military intervention
 Vaccination drives—cheaper to prevent
 Limited funding: un-smart intelligence
 Collaborative programs—funds not available
 Need dynamic consolidated vision
 Don’t know where to look for

49. Current Stockpiles MAPW (Australia)
Bio & Chem Weapons 2006

50. Conclusions
 For continuous surveillance and monitoring of important
strategic, tactical and containment areas, and with the
rapid advancement in the field of genetic engineering and
biotechnology and possibility of use of genetically modified
BW agents, it is essential to acquire/ use advanced early
detection devices at national level - in addition to gold
standard conventional microbiological methods for rapid
and quick response
 Plan ahead smartly, and be prepared to move quickly and
decisively
 Communication, data integration and timely delivery of
data analysis to decision-makers is crucial

51. Albert Einstein
‘I Know Not, What the Third World War
would be Fought with, but the Fourth
World War will be Fought with Sticks and Stones!’