BIOLOGICAL TOXINS
BOTULINUM TOXINS
First described by Mueller (1700s) and Kerner (1800s) in Germany (raw blood sausages)
Nearly destroyed US canning industry in early 1900s
Seven neurotoxins produced by Clostridium botulinum
C. botulinum - spore-forming, strict anaerobe; Found in soil world-wide (occasionally feces)
normally causes disease if ingested, inhaled or if wound is infected by C. botulinum
also infant botulism
toxin is not absorbed through intact skin
toxins are not produced in an acidic environment
Growing C. botulinum produces CO2
Spores resistant – 100oC for at least 3-5 hours
(less resistant in high acid or high salt)
Most toxic known substance
Lethal does for a 70 kg person: 0.9 to 0.15 g IV or IM, 0.9 g inhaled, 70 g ingested
275 times more lethal than cyanide
(therapeutic botox – type A – 0.3% of the estimated lethal Hu dose if inhaled
or 0.005% of lethal oral dose)
History of use as BW
1990 to 1995 – Japan – Aum Shinricky dispersed aerosols containing botulinum toxin 3 times
no casualties - poor techniques for aerosolization
Japan - Unit 731 – fed cultures of C. botulinum to prisoners in Manchuria
USA – produced toxin beginning in WW II
Millions of doses of antitoxin were prepared for D-Day
USSR – tested botox on Vozrozhdeniye Island in Aral Sea
May have spliced toxin gene into other organisms
Iran, Iraq, North Korea & Syria are thought to have produced or are attempting to produce botox
Iraq – admitted to having produced 19,000 L of concentrated botox
(3 times the amount needed to kill the entire human population – inhalation)
had prepared missiles & bombs with botox
estimated: point source aerosol of botox could kill or incapacitate 10% of people within 0.5 km downwind
could contaminate food
Botox – 7 serotypes (A – G) similar, but do not cross-react to antibodies
150,000 daltons proteins cleaved into peptides linked by a disulfide bond
heavy fragment (100,000 MW long chain) binds receptor on presynaptic membranes
of motor neurons the peripheral nervous system and cranial nerves
helps in penetration
light fragment (50,000 MW) – Zn2+ -containing endopeptidase (cleaves proteins)
released into cytoplasm (enters cell by receptor-mediated endocytosis)
blocks the fusion of membrane-bound vesicles of stored acetylcholine
synaptic vesicle containing acetylcholine normally fuses with the axon cell membrane
SNARE proteins assist in this – part of synaptic fusion complex
Type A, C & E – cleave SNAP-25 (synaptobrevin)
synaptosomal-associated protein (25,000 MW)
Type B,D, F & G cleaves syntaxin
prevents release of acetylcholine from terminal membrane of motor neurons
flaccid paralysis – muscles cannot contract
acetylcholine can still be synthesized and stored
Symptoms
Usually within 12 to 72 hours (range – 2 hours to 8 days)
Larger doses & wound botulism have quicker onset of symptoms
Symptoms may be faster when muscles are contracting
` acute, symmetrical, descending flaccid paralysis
always has multiple cranial nerve palsies (bulbar palsies)
blurred vision, double vision, difficulty speaking & swallowing, dry mouth,
weakness, fatigue and constipation (no fever)
Foodborne cases may have GI symptoms before paralysis
alert with flaccid paralysis
death from airway obstructions or paralysis of the diaphragm
[„4 Ds‟ – diplopia, dysarthia, dysphonia and dysphagia]
Botox – therapeutic & cosmetic applications (FDA has recently given approval)
Treat spastic conditions
Three Types of Naturally-Occurring Botulism
Foodborne botulism
Spores are not killed during sterilization process; make toxin in anaerobic conditions
(home canning of foods with neutral pH)
(green beans, corn, peas, smoked fish & vacuum-packed fish in plastic bags)
(restaurant outbreak – sautéed onions; 1977 – largest outbreak from canned jalapenos)
toxin is absorbed by the gut
Heat will inactivate (denature) the toxins – 100oC for 20 minutes (kitchen rule)
(average 9 outbreaks and 31 cases each year in USA)
Wound botulism – rare – fewer than 3 cases each year in USA
C. botulinum grows in anaerobic wound – toxins produced
(42 cases in CA in 1990s – botulism- contaminated black tar heroin from Mexico)
Infant botulism – ingested C. botulinum grows in intestines – toxins
Stomach does not produce enough acid to have low pH
Average 71 cases in USA each year (some associated with honey)
Often in 2nd month; weakness, poor feeding, paralysis (“floppy baby”)
Most recover with supportive therapy alone
(severe cases may be sudden infant death syndrome)
(less than 200 cases of all 3 natural types each year in USA)
Use as a Biological Weapon
Inhalation botulism – inhaling toxin (aerosolized toxin)
has occurred – research with primates (toxins C, D & G)
accidentally in humans – west Germany in 1962 – 3 people were
exposed to aerosols while disposing of rabbits & guinea pigs
(fur coated with Botox A) symptoms in 72 hours
Botox A was isolated from their serum
Toxin cannot penetrate intact skin; not transmitted Hu to Hu
Covering mouth and nose with clothing may help prevent inhalation
Could be stable in untreated water or beverages for many days
Must identify source
If toxin gene inserted in contagious pathogen –
would spread and may have a longer incubation period
Diagnosis
Suspected cases (on clinical symptoms) must be reported to public health department
CDC and 20 other labs can do confirming diagnostic tests
Identify toxin in serum (or other body fluid) and in food (or other source)
Inject into mice (mouse bioassay)
Passive hemagglutination or radioimmunoassay (ELISA?)
Identify toxin serotype with antitoxin in mice studies
Therapy
Antitoxin (proper serotype) prevents progression of paralysis
Cannot reverse paralysis; must administer early (binds circulating toxin)
equine antitoxin from CDC has antibodies for A, B and E
US Army has antitoxin with A-G
If exposed to a large amount of toxin, may need additional antitoxin
(Allergies to equine serum proteins)
Supportive care – ventilators
Paralysis can last for weeks or months -new axons terminals sprout
60% of untreated foodborn cases will die; with proper care - 85oC for 5 minutes
denaturation within 12 hours in air (extremes of temperature & moisture)
decay rate of 1% to 4% every minute
sunlight (UV) deactivates within 1 – 3 hours
water treatments (chlorine & aeration; dilution) – detoxifies in 20 minutes
decontamination is not a concern – soap & water for clothing & skin
0.1% bleach for surfaces
Research – rapid diagnostic test to types of toxin
Have PCR to detect toxin gene
Enzyme analysis of Botox would be faster than mouse bioassay
Military has ELISA test for aerosolized botox
Antitoxin from human instead of horse – monoclonal?
RICIN
Castor bean plant (Ricinus communis) secreted into seeds
Can be made in large amounts: available, cheap, easy to process
Stable as liquid, crystals or dry powder (lyophilized)
BW –a erosolized, injected or used to contaminate food and water
Cannot be absorbed through skin or transmitted Hu to Hu
1978 - Ricin pellet inserted with umbrella Markovc
2003 - Traces of ricin found in London – 6 men arrested
Type II ribosome inactivating toxin (RIP) irreversible
Contains two hemagglutinins and two toxins (RCL III & RCL IV)
Seeds - up to 5% ricin + agglutinins
66,000 MW and each is a dimer with a linking disulfide bond
Toxins – A and B chains linked by disulfide chains
B chain binds surface receptor (gp) – receptor-mediated endocytosis
A chain has endonuclease activity – binds 60s ribosomal subunit
removes an adenine from a 28s rRNA – prevents binding of EF-2
inactivates the ribosome - cell cannot make proteins and dies
1 ricin molecules can inactivate 1500+ ribosomes / minute & kills cell
1 mg may be enough to kill an adult
Ingestion – nausea, abdominal cramps, severe diarrhea; death on 3rd day
Necrosis of GI, spleen and kidneys; organs fail
Deaths have occurred from accidentally eating raw castor beans
Children more sensitive than adults – 1 seed could kill; leaves also toxic
Inhalation – symptoms within 4-8 hours after exposure
weakness, cough, fever, hypothermia - extensive sweating ends symptoms
no documentation of lethal effects of inhalation in humans
in animals – death within 36-48 hours from cardiovascular failure
or pulmonary distress
Injected – hemagglutination at various sites, multiple organ failure; death within 2 days
Diagnosis – standard tests are not helpful
Collapse of vascular system makes it different from other agents
ELISA or histological detection
PCR can be used to detect residual DNA is sample
Therapy – supportive antitoxin & vaccine in development; Will recover if survive 3-5 days
Inactivated with mild bleach, wash skin with soap and water
Mask but no other protective clothing
Inactivated by normal chlorination of water
Ricin has been used to kill cancer cells – attached to monoclonal antibody
Why not toxic to castor plant – stored in vacuole called “protein body”
Synthesized as proricin – into ER lumen then to Golgi complex (processed)
In protein body – cleaved by endopeptidase to form A and B chains – toxic
Inactivated by hydrolysis a few days after germination of seed
SAXITOXIN (STX)
From dinoflagellates (protozoa) and puffer fish Red Tide (fish kills)
Also in mollusks that feed on dinoflagellates ( clams, scallops, oysters)
1987 – Canada – 156 deaths from contaminated blue mussels (elderly)
more common in Europe than in US
Neurotoxin is heat stable, water soluble, & not destroyed by heat (cooking)
Lethal dose for human – 1-2 mg (1000 times more toxic than sarin)
Block nerve transmission – binds sodium channels of nerves
Blocks nerve impulses along axons
Death from respiratory failure
Symptoms can be within minutes (30 minutes to 3 hours)
Numbness of lips, fingers
Diarrhea, vomiting, weakness & paralysis (floating feeling)
Death can occur within 2-24 hours – elderly are more susceptible
Survivors regain normal function within days
Detect with mouse bioassay ( lethal in 15 minutes); ELISA
No antitoxin or vaccine– supportive care; charcoal for ingested
Decontaminate – bleach inactivates toxin
Soap and water, mask
TETRODOTOXIN (TTX)
From puffer fish, other fish, salamanders, frogs, octopus, mollusks
[Egypt in 1400 – 2700 BC]
More than 50 people die each year in Japan – fugu (raw puffer fish)
Causes a tingling sensation; chefs must be licensed
neurotoxin – lethal dose is 5g/kg
blocks the axonal sodium channels (do not bind other ion channels)
both TTX and STX bind opening of sodium channel with a guanidino group
(TTX bind it for 10 seconds while a sodium ion bind it for a nanosecond)
Symptoms within 30 minutes of ingestion (range – 20 minutes to 3 hours)
Nausea, vomiting, vertigo, numbness, muscle weakness, convulsions, paralysis
May be conscious
Death from respiratory failure – 20 minutes to 8 hours
Detect with mouse bioassayor liquid chromatography
No antitoxin supportive care charcoal
4-aminopyridine has been used in animal studies
K+ channel blocker that enhances release of acetylcholine
Animals have been vaccinated & become tolerant
OTHER BIOLOGICAL TOXINS
TETANUS TOXIN
From Clostridium tetani – anaerobic spore-former
150,000 MW protein cleaved into 2 peptides bound by a disulfide bond
binds irreversibly to presynaptic membranes of motor neurons
migrates up nerve fiber to spinal inhibitory neurons
in spinal cord and brain stem
inhibitory cells include glycinergic interneurons and
aminobutyric acid-secreting neurons of the brain stems
works in a way similar to botox
toxin degrades synaptobrevin (protein needed for docking of
neurotransmitter vesicles to presynaptic membrane)
inhibitory glycine and aminobutyric acid are not released from
inhibitory neurons & motor neurons are not inhibited
muscles contract –(lockjaw) muscle spasms & spastic, rigid paralysis
spores germinate in deep wounds
incubation – 4-5 days lockjaw
alert with extreme pain
death from respiratory failure – diaphragm high mortality rates
human antitoxin stops the progression of the toxin
muscle relaxants, sedation & ventilation
effective prophylaxis with tetanus toxoid vaccination (boosters)
GAS GANGRENE TOXINS
12 toxins produced by Clostridium perfringens
lethal, necrotizing, and hemolytic
(gas is produced by growing cells)
alpha toxin - lecithinase and can be aerosolized
cleaves lecithin in cell membranes – vascular leakage, liver damage
theta toxin – hemolytic & necrotizing – not a lecithinase
DNase and hyaluronidase (degrades collagen) also produced
(7 mg lethal for human adult?)
Growth spreads with toxemia and death – stench of necrotic tissue
Treat with antibiotics (penicillin) and surgery
Antitoxin contains antibodies for many of the toxins
Veterinary toxoids available – for epsilon toxin
Enterotoxin made by some strains – food poisoning
35,000 MW nonessential molecule in spore coat
intense GI distress within 6-18 hours
food poisoning is usually self-limiting
STAPHYLOCOCCAL TOXINS
Staphylococcus aureus. – ubiquitous on skin
Enterotoxins – extracellular proteins 23,000 to 29,000 MW
(entero because normally act in the gut)
Staphylococcal enterotoxin B (SEB) 28,494 MW
Food poisoning most common cause - nausea, projectile vomiting & diarrhea
Incubation 1-6 hours (picnic diarrhea) toxins are ingested
Work on vomiting control center in brain
Recover within 8-24 hours; can be lethal
SEB is toxic when inhaled in low doses (<1/100th of GI dose)
Symptoms in 1-12 hours with inhaled toxin; ill for 1-2 weeks
Fever, chills, headache, myalgia & nonproductive cough
GI if some toxin is swallowed (25 mg SEB causes vomiting)
Cannot penetrate intact skin; mask may help
Supportive therapy; no human vaccine for SEB
Vaccine is being tested
Destroy contaminated food, decontaminate with soap & water
Bleach foe surfaces
Toxic shock Syndrome toxin (TSST-1) similar to SEB
1-6 hours after toxin is present
Flu-like symptoms to fever with failure of multiple organs
[1980‟s – synthetic tampons provided perfect growth medium]
enterotoxins are superantigens - T cells stimulated but B cells fail to make antibodies
massive amounts of inflammatory cytokines made - shock
No vaccine – one is in development
MYCOTOXINS
40+ toxins produced by fungi – Fusarium, Mycotectium, Trichoderma & Stachybotrys
most are secondary metabolites (synthesized when nutrients are depleted)
moldy corn toxicosis in animals “yellow rain” in Laos, Cambodia & Afghanistan
tricothecene mycotoxins - small, nonvolatile, hydrophobic proteins (oily)
very stable - can resist autoclaving; partially inactivated by chlorination
T-2 is most widely studied & could be used as BW
[after WWII Russian civilians – contaminated bread – Fusarium T-2 toxin]
inhibit protein synthesis and normal mitochondrial function
impair DNA synthesis & lyse membranes bone marrow suppression
T-2 causes skin & eye damage – blisters soon after exposure
systemic – shock, hemorrhaging and bone marrow dysfunction
ingested – vomiting, bloody diarrhea, hemorrhaging
long-term – bone marrow diffusion & weight loss
start bleeding from every orifice
aerosol – symptoms within hours and death can occur within 12 hours
eye pain & irritation, bloody nasal discharge, bloody saliva
skin may itch, blister and start to die & start sloughing off
die of respiratory distress
no good rapid test for diagnosis or detection
gas-liquid chromatography; antibodies for detecting toxins
metabolites can be detected in urine for 28 days after exposure
[50 to 75% of toxin is eventually voided]
ascorbic acid may be beneficial (it was in animal studies)
experimental antitoxin used in animal studies
dexamethasone within 3 hours can help
charcoal can absorb ingested toxins
wash with soap and water, 10% bleach; mask & protective clothing
ergot – Claviceps purpurea, fungus on rye – hallucinations – blocks release of serotonin
aflatoxin – (LD50 is 0.5 to 10 mg/kg) alkaloid carcinogen – liver cancer