Lecture 18

Bioterrorism agent classification
Category A: high priority agents

Easily disseminated or transmitted form person to person
High mortaility rates, potential for major public health impact
Might cause public panic and social disruption
Require special action for public health preparedness
Category B
Some don’t replicate
Moderate morbidity rates
enhanced disease surveillance
Category C:
Emerging pathogens
Potential for high morbidity/mortality rates and major health impacts

Yersinia pestis is a gram negative facultative anaerobe that is intracellular and endemic to rodents, especially in S.asia.

Transmitted via fleas
Susceptible when in contact with rodents
Yersinia pestis has complex epidemiology.
Cyclic from wild rodents to infected fleas
Relatively resistant host rodents maintain a low-profile, stable rodent – flea infection cycle, resulting in endemic enzootic plague
Relatively sensitive host rodents die rapidly resulting in epizootic plague
It causes plague
Bubonic form: sudden onset of fever, headache, chills and weakness.
Painful buboes (swelling of the lymph nodes) develops within 2 to 7 days after the flea bite, usually in the groin or axillary areas.
Drainage of nbuboes not reccommemneded anymore due to aerolisation

How does yersinia pestis enter lymph nodes?
Yersinia pestis is phagocytosed by a macrophage.
The bacteria are transported to a lymph node
The bacteria replicate in the phagosome.
Bacteria escape from the phagosome and convert to phagocytosis-resistant encapsulated forms that cause systemic bacteraemia.
Yersinia pestis can survive and replicate in macrophages

Septicaemic plague – fevers, chills, extreme weakness, abdominal pain, shock and possibly bleeding into the skin and other organs.
Skin and other tissue may turn black and die, especially on fingers, toes and the nose
If someone with bubonic plague gets bacteraemic seeding into the lungs, the whole epidemiology changes as it can be spread person to person with no vector – pneumonic plague.
Bubonic and septicaemic plague has a very high death rate.
Pneumonic plague has no record of anyone surviving if no effective treatment
No vaccine, so all we can do is identify people and treat with streptomycin.

Aerosalisation of yersinia pestis spreads disease rapidly and give dangerous form of disease.

Person to person with no vector
Yersinia pestis has been used for biological warfare

Bacillus anthracis is a gram positive, spore forming organisms. – aerobic
Bacillus anthracis has a strnage protein capsule and is one of the few bacteria with it.
The capsule is required for full virulence: it protects the bacterium from complement opsonisation and prevents phagocytosis by macrophages
B.anthracis releases exotoxins composed of 3 proteins:

protective antigen
edema factor
lethal factor

Anthrax toxin binds cell receptor
It is endocytosed into early endosomes
Proetectivew antigen changes shape and forms a ring oike pore in the late endosomal membrane
The pore allows lethal facor and edema factor to enter cytosol
Lethal factor interacts with apoptotic systems and sgoimulates cell death
edema factor alters water homeostasis so you get swelling

there are three forms of anthrax.

Cutaneous anthrax is the most common form of the disease. The exotoxin causes a painless round black lesion on the skin 2 to 5 days after exposure, the result of localized tissue death. Without antibiotic therapy, the bacteria will continue to proliferate and enter the bloodstream, possibly causing death.
Anthrax:

Characteristic black eschar surrounded by a ring of vesiculation
8 days later, the eschar has enlarged, and the surrounding oedema has diminished

Gastrointestinal anthrax, a rare form of the disease, is caused by ingestion of spores from improperly cooked, contaminated meat. B. anthracis matures and replicates within the intestine, causing a necrotic lesion.

Symptoms include vomiting, abdominal pain, and bloody diarrhoea. Mortality rates are high (25% to 60%) if untreated.

Inhalational anthrax (Woolsorter's disease) occurs when the spores are inhaled. The bacteria mature and replicate in the lungs, where the exotoxin is released.
Symptoms, which include fever, cough, malaise, fatigue, and body aches, generally develop within 7 days of exposure.
Respiratory distress and death quickly follow in 95% to 100% of untreated cases

Treatment (2016 onwards): a combination of antibacterials (penicillin, doxocycline, or ciprofloxacin) and a monoclonal antibody that neutralises the toxin
Anthrax spores have been aerosolized for use in biological weapons.

Francisella tularensis, the causative agent of Tularemia
A small Gram negative coccobacillus.
Difficult to isolate (requires cysteine).
Slow growing.
Three main-sub species:
F. tularensis subsp tularensis
F. tularensis subsp holoartica
F. tularensis subsp mediasiatica
and one (very closely related) species: F. novocida

A facultative intracellular bacterium that can enter the body by inhalation, ingestion or through skin lesions.
Macrophage entry: F. tularensis uses type IV pili to bind to the exterior of host macrophages and thus become phagocytosed. Mutant strains lacking pili show severely attenuated pathogenicity.
Neutrophil entry: requires opsonisation by antibodies or complement
Intracellular replication of Francisella tularensis
① F. tularensis is phagocytosed by a macrophage
② The phagosomal membrane is degraded
③ The bacteria replicate in the cytosol
④ cell death releases bacteria
⑤ re-infection

Typical phagocytosis
The phagocytic cup fuses with early endosomes to form an early phagosome.
The phagosome matures by fusing with late endosomes and finally lysosomes to form the phagolysosome.

F. tularensis alters the phagocytic response:
Inhibition of oxidative burst.
Limited contact with early endosomes and late endosomes
No fusion with lysosomes
Destruction of the phagosome membrane
Bacterial replication in the cytosol
F. tularensis: typical routes of infection
Direct contact with infected animals:
2002, USA: Tularemia was diagnosed in wild-caught prairie dogs destined for sale as pets (47/163 prairie dogs died). Screening of individuals who had handled, cleaned or purchased the animals identified a human infection .
Direct contact with products from infected animals:
Furriers, etc. are at risk
Contact/ingestion of contaminated water/soil
And … worst of all: direct inhalation of bacteria via aerosols:
Farmers clearing straw previously soiled by infected rodents
Laboratory based infections
‘Lawnmower disease’ - 2003
Lawnmower disease
2003: A particularly unusual outbreak of respiratory tularaemia : two adolescents ran over the carcass of a dead rabbit and aerosolised it while mowing a lawn in Martha's Vineyard, USA
Respiratory tularaemia: the most acute form of the disease, resulting from inhalation of bacteria.
Incubation period 2-5 days. Signs and symptoms are non-specific:
Typhoidal form: high fever, headaches, chills, septicaemia, retrosternal pain
Pneumonic form: no person-person transmission
Following spread to draining lymph nodes, F. tularensis can spread to the liver and spleen – severe inflammation and tissue damage

F. tularensis: diagnosis, mortality and treatment
Diagnosis is difficult, relying on immunofluorescence and serology.
Isolation is not easy.
If the infecting strain is F. tularensis subspecies tularensis mortality is >30%.
Treatment is typically by administration of doxycycline or ciprofloxacin.
Until recently, a vaccine has been available to protect laboratorians routinely working with Francisella tularensis. This vaccine is currently under review by the US Food and Drug Administration (FDA) and is not generally available in the United States.

Developed as a biological weapon by Japanese, Germans and stockpiled by US Military in late 1960s.
Tons of Francisella tularensis were produced by the Soviet Union as a potential BW agent – the defector Ken Alibek claims that stockpiles were maintained until the 1990s.

Clostridium botulinum and Botulism
Clostridium botulinum, the causative agent of botulism, is a Gram-positive, spore-forming, anaerobic rod.
Spores of C. botulinum are found worldwide in soil, pond, and lake sediments.

Botulinum toxin and spores
The C. botulinum neurotoxin is heat-labile, and is rapidly destroyed at 100°C.
The spores resist boiling for long periods and must be destroyed by autoclaving.
The spores can germinate, grow, and release the potent exotoxin in foodstuffs under anaerobic conditions.
Home-canned vegetables and home-preserved fish are the most common source of botulism.
Mechanism of action of Botulinum toxin
Each botulinum toxin molecule comprises a heavy chain and a light chain, connected by a disulfide bond.
The heavy chain binds the synaptic vesicle protein SV2 and is endocytosed to a low pH compartment.
Here the light chain penetrates the lipid bilayer and destroys its cellular targets (SNARE proteins) proteolytically.

Botulism – paralysed muscles
Symptoms of botulism occur within 18 - 96 hr following ingestion
The toxin, once absorbed through the small intestine acts on the neuromuscular junctions causing muscle paralysis.
Symptoms:
Difficulty in swallowing and breathing, then:
Without administration of the appropriate antitoxin, respiratory paralysis
death follows in 10% -20% of cases.
Antitoxin is effective in reducing the severity of symptoms if administered early in the course of the disease. Most patients eventually recover after weeks to months of supportive care.

Because of botulinum toxin's extraordinary toxicity, it has received attention as a possible agent in bioterrorism and biological warfare. According to the CDC several countries have developed botulinum toxin as an aerosol weapon.
Yersinia pestis, Plague, Black Death
High mortality rate and easy human-to-human transmissibility, plague is considered by the Centers for Disease Control and Prevention to be a likely candidate for weaponisation and use in bioterrorism and biological warfare.
Bacillus anthracis, Anthrax
Longevity of the spores and ease of weaponisation: a proven candidate for use in bioterrorism and biological warfare.
Francisella tularensis, Tularemia
Since it is one of the most infectious pathogens known: a possible agent for use in bioterrorism and biological warfare.
Clostridium botulinum, Botulism
Extraordinary toxicity of its toxin, it has received attention as a possible agent for use in bioterrorism and biological warfare, and has been aerosolised for use as a weapon.